Signal Transduction: A Practical Approach 2nd ed

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Signal Transduction

The Practical Approach Series SERIES EDITOR B. D. HAMES Department of Biochemistry and Molecular Biology University of Leeds, Leeds LS2 9JT, UK

See also the Practical Approach web site at http://www.oup.co.uk/PAS * indicates new and forthcoming titles

Affinity Chromatography Affinity Separations Anaerobic Microbiology Animal Cell Culture (2nd edition) Animal Virus Pathogenesis Antibodies I and II Antibody Engineering * Antisense Technology Applied Microbial Physiology Basic Cell Culture Behavioural Neuroscience Bioenergetics Biological Data Analysis Biomechanics—Materials Biomechanics—Structures and Systems Biosensors Carbohydrate Analysis (2nd edition) Cell-Cell Interactions The Cell Cycle Cell Growth and Apoptosis * Cell Separation

Cellular Calcium Cellular Interactions in Development Cellular Neurobiology * Chromatin if Chromosome Structural Analysis Clinical Immunology Complement * Crystallization of Nucleic Acids and Proteins (2nd edition) Cytokines (2nd edition) The Cytoskeleton Diagnostic Molecular Pathology I and II DNA and Protein Sequence Analysis DNA Cloning 1: Core Techniques (2nd edition) DNA Cloning 2: Expression Systems (2nd edition) DNA Cloning 3: Complex Genomes (2nd edition) DNA Cloning 4: Mammalian Systems (2nd edition)

* Drosophila (2nd edition) Electron Microscopy in Biology Electron Microscopy in Molecular Biology Electrophysiology Enzyme Assays Epithelial Cell Culture Essential Developmental Biology Essential Molecular Biology I and II * Eukaryotic DNA Replication Experimental Neuroanatomy Extracellular Matrix Flow Cytometry (2nd edition) Free Radicals Gas Chromatography Gel Electrophoresis of Nucleic Acids (2nd edition) * Gel Electrophoresis of Proteins (3rd edition) Gene Probes 1 and 2 Gene Targeting Gene Transcription if Genome Mapping Glycobiology * Growth Factors and Receptors Haemopoiesis * High Resolution Chromatography Histocompatibility Testing HIV Volumes 1 and 2 * HPLC of Macromolecules (2nd edition) Human Cytogenetics I and II (2nd edition)

Human Genetic Disease Analysis * Immobilized Biomolecules in Analysis Immunochemistry 1 Immunochemistry 2 Immunocytochemistry * In Situ Hybridization (2nd edition) lodinated Density Gradient Media Ion Channels if Light Microscopy (2nd edition) Lipid Modification of Proteins Lipoprotein Analysis Liposomes Mammalian Cell Biotechnology Medical Parasitology Medical Virology MHC Volumes 1 and 2 if Molecular Genetic Analysis of Populations (2nd edition) Molecular Genetics of Yeast Molecular Imaging in Neuroscience Molecular Neurobiology Molecular Plant Pathology I and II Molecular Virology Monitoring Neuronal Activity Mutagenicity Testing * Mutation Detection Neural Cell Culture Neural Transplantation Neurochemistry (2nd edition)

Neuronal Cell Lines NMR of Biological Macromolecules Non-isotopic Methods in Molecular Biology Nucleic Acid Hybridization Typesetter to balance columns Oligonucleotides and Analogues Oligonucleotide Synthesis PCR 1 PCR 2 *PCR3:PCR In Situ Hybridization Peptide Antigens Photosynthesis: Energy Transduction Plant Cell Biology Plant Cell Culture (2nd edition) Plant Molecular Biology Plasmids (2nd edition) Platelets Postimplantation Mammalian Embryos * Post-Translational Modification Preparative Centrifugation

Protein Blotting if Protein Expression Protein Engineering Protein Function (2nd edition) Protein Phosphorylation Protein Purification Applications Protein Purification Methods Protein Sequencing Protein Structure (2nd edition) Protein Structure Prediction Protein Targeting Proteolytic Enzymes Pulsed Field Gel Electrophoresis RNA Processing I and II * RNA-Protein Interactions Signalling by Inositides Subcellular Fractionation Signal Transduction if Transcription Factors (2nd edition) Tumour Immunobiology

Signal Transduction A Practical Approach Second Edition Edited by

G. MILLIGAN Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow

OXFORD UNIVERSITY PRESS

OXFORD UNIVERSITY PRESS Great Clarendon Street, Oxford OX2 6DP Oxford University Press is a department of the University of Oxford and furthers the University's aim of excellence in research, scholarship, and education by publishing worldwide in Oxford New York Athens Auckland Bangkok Bogota Buenos Aires Calcutta Cape Town Chennai Dar es Salaam Delhi Florence Hong Kong Istanbul Karachi Kuala Lumpur Madrid Melbourne Mexico City Mumbai Nairobi Paris Sao Paulo Singapore Taipei Tokyo Toronto Warsaw and associated companies in Berlin Ibadan Oxford is a registered trade mark of Oxford University Press Published in the United States by Oxford University Press Inc., New York © Oxford University Press 1999 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior permission in writing of Oxford University Press. Within the UK, exceptions are allowed in respect of any fair dealing for the purpose of research or private study, or criticism or review, as permitted under the Copyright, Designs and Patents Act, 1988, or in the case of reprographic reproduction in accordance with the terms of licenses issued by the Copyright Licensing Agency. Enquiries concerning reproduction outside those terms and in other countries should be sent to the Rights Department, Oxford University Press, at the address above. This book is sold subject to the condition that it shall not, by way of trade or otherwise, be lent, re-sold, hired out, or otherwise circulated without the publisher's prior consent in any form of binding or cover other than that in which it is published and without a similar condition including this condition being imposed on the subsequent purchaser Users of books in the Practical Approach Series are advised that prudent laboratory safety procedures should be followed at all times. Oxford University Press makes no representation, express or implied, in respect of the accuracy of the material set forth in books in this series and cannot accept any legal responsibility or liability for any errors or omissions that may be made. A catalogue record for this book is available from the British Library Library of Congress Cataloging in Publication Data Signal transduction : a practical approach / edited by G. Milligan. — 2nd ed. p. cm.—(The Practical approach series ; 209) Includes bibliographical references and index. 1. G proteins—Research—Methodology. 2. Cellular signal transduction—Research—Methodology. 3. G proteins—Receptors —Research—Methodology. I. Milligan, Graeme. II. Series. QP552.G16S54 1999 571.6—dc21 99-26199 CIP ISBN 0-19-963721-0 (Hbk) 0-19-963720-2 (Pbk) Typeset by Footnote Graphics, Warminster, Wilts Printed in Great Britain by Information Press, Ltd, Eynsham, Oxon.

Preface Since production of the first Edition of Signal Transduction: A Practical Approach in 1992 areas covered by the term "signal transduction" have expanded greatly in range and number. Furthermore, the widespread application of molecular biology to this field has revealed almost unimaginable diversity and complexity in such systems with large families of proteins involved in the production and destruction of second messenger molecules and information transfer via kinase cascades and their associated regulatory proteins. With such complexity known to occur and with many isoforms of signalling proteins frequently being co-expressed in individual cells, assays to measure second messenger production and regulation and the phosphorylation status and activity state of key kinases has become even more important. Within a single volume it would be impossible to attempt to cover the range of techniques currently employed in this field and indeed even within the Practical Approach series specific volumes have since been produced which concentrate in considerable detail on different methodologies employed to measure and analyse the production of inositol phosphates and related molecules or intracellular [Ca2+] for example. The current book thus seeks to update and expand the areas covered by the first Edition of Signal Transduction: A Practical Approach and to give a broad perspective of techniques currently in use, with particular emphasis of studies of systems regulated by agonists at G protein-coupled receptors (GPCRs). However, as the overlap of systems modified by GPCRs and many other classes of receptors has become virtually all encompassing then the techniques and approaches described should find broad applicability. Glasgow May 1999

G.M.

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Contents List of Contributors Abbreviations 1. Direct assessment of conformational changes in G protein-coupled receptors, using fluorescence spectroscopy Ulrik Gether and Brian Kobilka 1. Introduction 2. Expression and purification of the B2 adrenergic receptor 3. Fluorescence labelling and spectroscopic analysis of the purified B2 adrenergic receptor 4. Site-selective fluorescent labelling of the B2 adrenergic receptor Labelling of cysteines Site-specific labelling at lysines 5. Concluding remarks References

xix xxiii

1 1 2 6 11 12 15 16 16

2. Probing the structure of receptor-binding sites by the substituted-cysteine accessibility method 19 Merrill M. Simpson, George Liapakis, and Jonathan A. Javitch 1. Introduction

19

2. Applications of the substituted-cysteine accessibility method (SCAM) Systems studied with SCAM SCAM versus site-directed mutagenesis and affinity labelling

19 19 20

3. Critical parameters Reactivity of wild-type background Cysteine substitution Determination of function of cysteine-substituted mutants 4. Chemistry of SCAM Mechanism of reaction

21 21 21 23 24 24

Contents Description of reagents Reaction with the MTS reagents Determination of rates of reaction of the MTS reagent Protection of substituted cysteines by bound ligand 5. Interpretation of results Assumptions of SCAM Interpreting the effects of reaction with the MTS reagents Secondary structure Conformational changes associated with receptor activation

30 30 30 31 32

References

33

3. Post-translational acylation of signal transducing polypeptid.es: palmitoylation of G protein-coupled receptors 1.

35

Michel Bouvier, Ulla Petaja-Repo, Thomas Loisel, and Charlene Belanger Introduction

35

2. Detection of receptor palmitoylation Heterologous expression systems Metabolic labelling Characterisation of incorporated radiolabel

36 37 39 45

3. Kinetics of palmitoylation Pulse labelling Pulse-chase labelling Agonist-mediated regulation of receptor palmitoylation

47 47 49 50

4. Assessing the palmitoylation state of the receptor at the cell surface

51

5. In vitro palmitoylation of synthetic peptides

54

References

56

4. G protein-coupled receptor phosphorylation and desensitization

59

Andrew B. Tobin, Angela Rae, and David C. Budd 1. Introduction

59

2. Techniques in the investigation of GPCR phosphorylation Determination of GPCR phosphorylation in intact cultured cells by immunoprecipitation Identification of GPCR phosphorylation in a crude membrane preparation

60 60 64

Contents 3. Identification of desensitization of phospholipase C-coupled receptors References

5. G proteins and their identification Ian Mullaney 1. Introduction 2. Production of crude plasma membrane fractions for analysis of G proteins 3. Gel electrophoresis of G proteins Mono-ADP-ribosylation of G proteins by bacterial exotoxins Gel electrophoresis of G proteins

4. Immunological methods

66 71

73 73 75 77 77 81

87

Immunization and serum collection Immunoblotting and immunoprecipitation

5. Quantification of G protein a subunits 6. Functional aspects of G protein signalling Determination of GTP hydrolysis in membrane preparations Measurement of receptor-stimulated [35S]GTP-yS binding in membrane preparations

Acknowledgements References

87 91

95 98 99 100

101 101

6. Construction and analysis of receptor-G protein fusion proteins Alan Wise 1. Introduction 2. Methods to study G protein function Second messenger production G protein activation Use of pertussis toxin-resistant G protein mutants

3. Receptor-G protein fusions

103 103 105 105 105 105

107

Background Construction of receptor-G protein fusion proteins

4. Expression of receptor-G protein fusions in cultured cells Choice of recipient cell line Choice of vector Transient expression of receptor-G protein fusions Xi

107 109

110 110 111 111

Contents Preparation of DNA-Lipofectamine mix Cell harvesting and plasma membrane production 5. Assays used for functional characterization of receptor-G protein fusions Background Receptor-promoted binding of guanosine-5'-[-y-35thio]-triphosphate ([35S]GTP-yS) Measurement of GTPase activity Receptor binding studies

112 112 113 113 114 115 117

6. Receptor-G protein fusions as research tools 117 Measurement of agonist-induced guanine nucleotide turnover byGi1a 117 Measurement of agonist efficacy 121 Elucidating the role of N-terminal acylation of Gi1a 124 Study of interactions between the A1 adenosine receptor and multiple Gi-family G proteins 128 Receptor-G protein fusion regulation of effectors 132 7. Summary and future perspectives

134

Acknowledgements

135

References

135

7. Application of the baculoviral expression system to signal transduction

139

Andrew Paterson 1. Introduction

139

2. Sf9 cell culture Media Reviving Sf9 cells from frozen Maintaining monolayer cultures of Sf9 cells Adaptation to suspension culture Freezing Sf9 cells Adaptation to serum-free medium

140 141 141 141 142 143 143

3. The baculoviral life cycle, and constructing recombinant baculoviral vectors Time course of viral infection and the polh locus Construction of a recombinant transfer plasmid Construction of recombinant baculovirus by cotransfection Recombinant baculoviral construction with baculoviral shuttle vector Baculoviral passage and titre

144 144 145 145 152 155

4. Recombinant protein expression and purification Assessing a recombinant virus

156 156

xii

Contents Scaling-up infection of Sf9 cells as monolayers Infecting suspension cultures Harvesting suspension cultures Hypotonic lysis Nitrogen cavitation Purification of recombinant protein Rapid purification with glutathione- or Ni2+/NTA-agaroses Alternative cell lines Protein complexes Alternative promoters Further scale-up of culture volume References

159 159 160 160 161 162 163 166 166 167 167 168

8. Reporter gene systems for the study of G protein-coupled receptor signal transduction in mammalian cells 171 Stephen Rees, Susan Brown and Jenny Stables 1. Introduction

171

2. What is a reporter gene? Construction of a reporter gene Reporter proteins

171 173 177

3. Reporter gene systems for the study of GPCR signal transduction Reporter genes for GPCRs which couple to members of the Gas and Gai G protein families Reporter genes for GPCRs which couple to members of the Gaq/11 G protein family Reporter genes for G protein B-y signalling

178 178 180 184

4. Factors influencing the design of a mammalian cell reporter-gene assay Choice of cell line Choice of expression vectors Choice of expression protocol Choice of reporter gene and reporter enzyme Optimization of reporter assay conditions

189 189 190 190 195 195

5. Preparation of cells for reporter-gene assays Agonist assays Antagonist assays Constitutive activity and inverse agonist assays

197 198 199 200

6. Reporter enzyme assays Firefly luciferase Renilla luciferase and dual luciferase assays

201 201 203

xiii

Contents LuFLIPRase Secreted placental alkaline phosphatase (SEAP) Chloramphenicol acetyltransferase B-galactosidase B-lactamase

205 205 207 210 212

7. Reporter protein assays Aequorin GFP

214 214 215

8. Summary and future perspectives

217

Acknowledgements

217

References

218

9. Adenylyl cyclases and cyclic AMP

223

Maurice K. C. Ho and Yung H. Wong 1. Introduction

223

2. Mammalian expression of recombinant ACs DEAE-dextran/chloroquine mediated transfection Adenovirus-mediated transfection Other methods of transfection

226 226 228 233

3. Measurement of intraeellular cAMP level Metabolic labelling of ATP [3H]cAMP assay Isolation of [3H]cAMP Data collection and interpretation

233 234 235 236 239

4. cAMP-responsive bioluminescence assays using firefly luciferase cAMP-responsive transcription of the firefly luciferase gene PKA-responsive luciferase mutant Indirect cAMP assay using luciferase

239 240 243 244

5. Miscellaneous systems for monitoring AC activity cAMP-responsive transcription of the chloramphenicol acetyltransferase gene Other methods of cAMP-induced enzymatic activity Pigment movement in Xenopus laevis melanophores cAMP-induced inward current in Xenopus oocytes Functional rescue of CYR1 -defective Saccharomyces cerevisiae mutant Functional rescue of the Escherichia coli Acya TP2000 mutant

244

6. Perspectives

248

References

249

xiv

244 246 246 246 247 248

Contents

10. Analysis of the polyphosphorylated inositol lipids of Saccharomyces cerevisiae Stephen K. Dove and Robert H. Michell 1. Introduction 2. Structures and nomenclature of inositol glycerophospholipids 3. Analysis of inositol glycerophospholipids: general considerations 4. Radioactive labelling of the phosphoinositides of yeast cells 5. Extraction of inositol lipids from yeast 6. Resolution and identification of yeast phosphoinositides Analysis of polyphosphoinositides by TLC Deacylation of inositol glycerolipids, and HPLC analysis of the resulting water-soluble GroPInsPns

7. Anion-exchange HPLC analysis of inositol lipid-derived GrojPInsPns from yeast HPLC: sample injection and detection of radioactivity Commercially available standards for HPLC analysis of inositol lipid-derived GroPInsPns

References

255 255 256 257 259 263 266 266 271

275 277 278

280

11. Phosphoinositide 3-kinases K. E. Anderson, L. R. Stephens, and P. T. Hawkins 1. Introduction 2. Methods for identifying a role for the PI3K signalling pathway in cellular events Inhibition of PI3K Constitutively active alleles of PI3K and downstream effectors

3. Measurement of the activation of PI3K 4. Measurement of PI3K lipid products in the cell 32

P-labelling Adherent cells [3H]-inositol labelling of cells Separation and quantitation of radiolabelled PI3K lipid products Mass analysis of PtdIns(3,4,5)P3

5. Summary References

283 283 285 285 286

287 290 291 293 293 294 297

298 299 xv

Contents

12. Phospholipase D and phosphatidylcholine metabolism Kathryn E. Meier and Terra C. Gibbs 1. Introduction 2. PLD assays in intact mammalian cells Metabolic labelling of cells with [3H]-fatty acids Incubation of cells with agonists Extraction of phospholipids from intact cells Thin-layer chromatography Quantification of PLD activity Interpretation of results 3. PLD assays with broken-cell preparations Preparation of cell membranes Fluorescent PLD assay Acknowledgements References

13. Signal transduction by sphingosine kinase Dagmar Meyer zu Heringdorf, Chris J. Van Koppen and Karl H. Jakobs 1. Introduction G protein-coupled sphingolipid receptors Receptor regulation of sphingosine kinase and the role of intracellular SPP 2. How to study involvement of sphingosine kinase in a certain signalling pathway 'Positively': testing the intracellular activity of SPP 'Negatively': inhibiting sphingosine kinase 3. How to study regulation of sphingosine kinase Measurement of sphingosine kinase activity in intact cells Measurement of sphingosine kinase activity in subcellular fractions Final remarks References

301 301

309 311 312 317 318

321 321 321 323 325 325 326 327 328 334 335 335

14. Detection, isolation, and quantitative assay of mitogen-activated protein kinases in intact cells and tissues 337 Neil G. Anderson 1. Introduction 2. General mechanism of activation of MAP kinases

xvi

337 339

Contents 3. MAP kinase substrates 4. Testing for activation of known MAP kinases Cultured cells Other sources

339 340 340 342

5. Assessment of MAP kinase activity by gel electrophoresis and immunoblotting Detection of phosphotyrosine in whole cell extracts Mobility shifts Phospho-specific antibodies

6. Measurement of MAP kinase enzyme activity Assay of MAP kinase in partially purified cell extracts Assay of MAP kinases following immunoprecipitation Assay conditions Other MAP kinase assays

7. Determination of MAP kinase subcellular localization and activation by immunocytochemistry 8. Assay of upstream activators of MAP kinases 9. Chemical inhibitors of MAP kinase pathways References

15. Measuring inositol 1,4,5-trisphosphateevoked 45Ca2+ release from intracellular Ca2+ stores Colin W. Taylor and Jonathan S. Marchant 1. Introduction 2. Preparation, permeabilization, and 45Ca2+ loading of hepatocytes Isolation of rat hepatocytes Cell permeabilization, loading of intracellular stores with45Ca2+, and the effects of Ins(l,4,5)P3 45

Acknowledgements References

343 344 348

349 350 353 355 356

356 358 358 359

361 361 362 362 364

2+

3. Rapid kinetic measurements of Ca release from intracellular stores Rapid superfusion methods Rapid superfusion apparatus Rapid responses to Ins(l,4,5)P3

343

368 368 370 378

382 382

Appendix Index

385 389 XVii


Contributors K. E. ANDERSON

The Babraham Institute, Babraham, Cambridge CB2 4AT, UK. N. G. ANDERSON

Division of Cancer Studies, School of Medicine, University of Manchester, Room G38, Stopford Building, Oxford Road, Manchester M13 9PT, UK. C. BELANGER

Department of Biochemistry and Groupe de Recherches sur le Systeme Nerveux Autonome, Universite de Montreal, P.O. Box 6128, Down-town station, Montreal, Quebec, Canada, H3C-3J7. M. BOUVIER

Department of Biochemistry and Groupe de Recherches sur le Systeme Nerveux Autonome, Universite de Montreal, P.O. Box 6128, Down-town station, Montreal, Quebec, Canada, H3C-3J7. s. BROWN Molecular Pharmacology Unit, Glaxo-Wellcome Research and Development, Stevenage SG1 2NY, UK. D. c. BUDD Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester LE1 9HN, UK. S. K. DOVE Centre for Clinical Research in Immunology and Signalling and School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. U. GETHER

Division of Molecular and Cellular Physiology, Department of Medical Physiology 12-5-22, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark. T. C. GIBBS Department of Pharmacology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425-2251, USA. p. T. HAWKINS The Babraham Institute, Babraham, Cambridge CB2 4AT, UK. M. K. c. HO Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.

Contributors K. H. JAKOBS

Institut fur Pharmakologie, Universitat GH Essen, Hufelandstrasse 55, D45125 Essen, Germany. J. A. JAVITCH

Columbia University College of Physicians and Surgeons, Center for Molecular Recognition, P&S 11-401, 630 West 168th Street, New York, New York 10032, USA. B. K. KOBILKA

Howard Hughes Medical Institute, Division of Cardiovascular Medicine and Department of Molecular and Cellular Physiology, Stanford University Medical School, Stanford, CA 94305-5428, USA. G. LIAPAKIS

Columbia University College of Physicians and Surgeons, Center for Molecular Recognition, P&S 11-401, 630 West 168th Street, New York, New York 10032, USA. T. LOISEL

Department of Biochemistry and Groupe de Recherches sur le Systeme Nerveux Autonome, Universite de Montreal, P.O. Box 6128, Down-town station, Montreal, Quebec, Canada, H3C-3J7. J. S. MARCHANT

Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK. K. E. MEIER

Department of Pharmacology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425-2251, USA. D. MEYER ZU HERINGDORF

Institut fur Pharmakologie, Universitat GH Essen, Hufelandstrasse 55, D45125 Essen, Germany. R. H. MICHELL

Centre for Clinical Research in Immunology and Signalling and School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. I. MULLANEY

Department of Pharmacology, University of Otago, PO Box 913, Dunedin, New Zealand. A. PATERSON

Division of Signal Transduction Therapy, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, UK.

xx

Contributors U. PETAJA-REPO

Department of Biochemistry and Groupe de Recherches sur le Systeme Nerveux Autonome, Universite de Montreal, P.O. Box 6128, Down-town station, Montreal, Quebec, Canada, H3C-3J7. A.RAE

Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester LE1 9HN, UK. S. REES

Molecular Pharmacology Unit, Glaxo-Wellcome Research and Development, Stevenage SG1 2NY, UK. M. M. SIMPSON

Columbia University College of Physicians and Surgeons, Center for Molecular Recognition, P&S 11-401, 630 West 168th Street, New York, New York 10032, USA. J. STABLES

Molecular Pharmacology Unit, Glaxo-Wellcome Research and Development, Stevenage SG1 2NY, UK. L. R. STEPHENS

The Babraham Institute, Babraham, Cambridge CB2 4AT, UK. C. W. TAYLOR

Department of Pharmacology, University of Cambridge, Cambridge CB2 1QJ, UK. A. B. TOBIN

Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester LE1 9HN, UK. C. J. VAN KOPPEN

Institut fur Pharmakologie, Universitat GH Essen, Hufelandstrasse 55, D45125 Essen, Germany. A. WISE Molecular Pharmacology Unit, Glaxo-Wellcome Research and Development, Stevenage SG1 2NY, UK. Y. H. WONG

Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.

xxi


Abbreviations AC AcMNPV ADP AP-1 App(NH)P APS B-gal B2AR B2 BAPTA BEVS BFP BSA BPBt BPC BTK CAM CaM CaMK cAMP CAT cDNA CDTA CFTR CGRP CHO CLM CMV CO2 CRE CREB Cys DAG DBM ddH2O DG DMEM DMSO DTE DTT

adenylyl cyclase Autographa californica multiple nuclear polyhedrosis virus adenosine 5' diphosphate activator protein 1 5' adenylimidodiphosphate ammonium persulphate B-galactosidase adrenergic receptor l,2-Bis(2-amino-phenoxy)ethane-N,N,N',N'-tetracetic acid baculoviral expression vector system blue fluorescent protein bovine serum albumin BODIPY-phosphatidylbutanol BODIPY-phosphatidylcholine (Note: BODIPY is a trademark of Molecular Probes, Inc.) Brutons tyrosine kinase constitutively active mutant calmodulin Ca2+ calmodulin-dependent protein kinase 3',5' -cyclic adenosine monophosphate chloramphenicol acetyltransferase complimentary deoxyribonucleic acid cyclohexanediamine tetraacetic acid cystic fibrosis transmembrane regulator calcitonin gene related peptide Chinese hamster ovary cytosol-like medium cytomegalovirus carbon dioxide cAMP-responsive element cAMP-responivse element binding protein cysteine sn-1-2 diacylglycerol n-dodecyl-B-D-maltoside double-distilled water diglyceride Dulbecco's Modified Eagle's Medium dimethyl sulfoxide dithioerythreitol dithiothreitol

Abbreviations E. coli EC50 EDTA EGTA ELISA ERK FACS FCCP FLIPR fMLP FRET FSH G protein GDP GFP GM-CSF GPCR GRE GRK GroPIns(3,4)P2 GroPIns(3,5)P2 GroPIns(4,5)P2 GroPIns3P GroPIns4P GroPIns5P GroPInsP GroPInsP2 GroPInsPn GST GTP GTPs 3 H-DHA HEK Hepes hGPHa HSV-TK IANBD IBMX ICAM IgG IMAC Ins(l,4,5)P3 IP3

Escherichia coli concentration causing half-maximal effect ethylenediaminetetraacetic acid ethylene glycol bis(B-aminoethyl ether) N,N,N',N'tetracetic acid enzyme-linked immunosorbant assay extracellular signal regulated kinase fluorescence activated cell sorter carbonyl cyanide p-trifluormethoxyphenylhydrazone fluorescence imaging plate reader formylated-met-leu-phe fluorescence resonance energy transfer follicle-stimulating hormone guanine nucleotide binding protein guanosine 5'-diphosphate green fluorescent protein granulocyte macrophage-colony stimulating factor G protein-coupled receptor glucocorticoid-responsive element G protein-coupled receptor kinase glycerophosphoinositol(3,4)bisphosphate glycerophosphoinositol(3,5)bisphosphate glycerophosphoinositol(4,5)bisphosphate glycerophosphoinositol(3)monophosphate glycerophosphoinositol(4)monophosphate glycerophosphoinositol(5)monophosphate glycerophosphoinositolmonophosphate glycerophosphoinositolbisphosphate glycerophosphoinositolphosphates glutathione S-transferase guanosine 5'-triphosphate guanosine 5'-(O-thio)triphosphate [3H]dihydroalprenolol human embryonic kidney N-[2-hydroxyethyl]piperazine-N'-[2-ethane-sulfonicacid] human glycoprotein hormone-a Herpes simplex virus thymidine kinase nitrobenzdioxazol iodoacetamide 3-isobutyl-1-methylxanthine intercellular cell adhesion molecule immunoglobulin G immobilised metal anion chromatography inositol 1,4,5-trisphosphate inositol 1,4,5-trisphosphate xxiv

Abbreviations IPTG IRES IU JNK L-BPBt LPA LPC LSC luc MAP kinase MAP-2 MAPK MAPKK MAPKKK MBP MBS mcs MEK MEM MG MMTV MOI MTS MTSEA MTSES MTSET NAD NEM NFAT NP40 ONPG OPD ORF PA PACAP PAGE PAP PBS PBt PC PCA pCMBS pcor PCR

isopropyl-B, D-thiogalactopyranoside internal ribosomal entry sites international units c-Jun N-terminal kinase lyso-BODIPY-phosphatidylbutanol lysophosphatidic acid lyso-phosphatidylcholine liquid scintillation counting luciferase gene mitogen-activated protein kinase microtubule-associated protein 2 kinase mitogen-activated protein kinase MAP kinase kinase MAP kinase kinase kinase myelin basic protein Mes-buffered saline multiple cloning site MAP kinase/ERK kinase Eagle's minimal essential medium monoglyceride mouse mammary tumour virus multiplicity of infection methanethiosulfonate methanethiosulfonate-ethylammonium methanethiosulfonate-ethylsulfonate methanethiosulfonate-ethyltrimethylammonium nicotinamide adenine dinucleotide N-ethylmaleimide nuclear factor activator of transcription NonidetP40 o-nitrophenyl B-D-galactopyranoside o-phenylenediamide dihydrochloride open reading frame phosphatidic acid pituitary adenylyl cyclase activating peptide polyacrylamide gel electrophoresis phosphatidate phosphohydrolase phosphate-buffered saline phosphatidylbutanol phosphatidylcholine perchloric acid p-chloromercuribenzene-sulfonate p6.9 basic core-associated protein promoter polymerase chain reaction XXV

Abbreviations PDE PDGF PDK PEI PEt PH PDK Pipes PK-A PKB PKC PLA2 PLC PLCB PLD PMSF ppolh p.s.i. PtdIns Ptdlns PtdIns(3)P PtdIns3p PtdIns(3,4)P2 PtdIns(3,5)P2 PtdIns(3,5)P2 PtdIns(3,4,5)P3 PtdIns(3,4,5)P3 PtdIns(4)P PtdIns4P PtdIns(4,5)P2 PtdIns(4,5)P2 PtdIns5P PTX Ro 20-1724 r.p.m. RS RSH RT SAPK SCAM SDS SDS-PAGE SEAP SH2

phosphodiesterase platelet-derived growth factor phosphoinositide dependent kinase polyethyleneimine-cellulose phosphatidylethanol pleckstrin homology phosphoinositide 3OH-kinase piperazine-N,N'-bis[2-ethanesulfonic acid] protein kinase A protein kinase B protein kinase C phospholipase A2 phospholipase C phospholipase CB phospholipase D phenylmethylsulfonyl fluoride polyhedrin promoter pounds per square inch phosphoinositide phosphatidylinositol phosphoinositide(3)phosphate phosphatidylinositol(3)monophosphate phosphatidylinositol(3,4)bisphosphate phosphoinositide(3,5)bisphosphate phosphatidylinositol(3,5)bisphosphate phosphoinositide(3,4,5)trisphosphate phosphatidylinositol(3,4,5)trisphosphate phosphoinositide(4)phosphate phosphatidylinositol(4)monophosphate phosphoinositide(4,5)bisphosphate phosphatidylinositol(4,5)bisphosphate phosphatidylinositol(5)monophosphate pertussis toxin [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone] revolutions per minute ionized thiolate unionized thiolate room temperature stress activated protein kinase substituted-cysteine accessibility method sodium dodecyl sulphate SDS polyacrylamide electrophoresis secreted placental alkaline phosphatase src-homology region two xxvi

Abbreviations SIE SOS SRE STAT SV40 Tiam-1 T. ni TBAS TC TCA TCF TE buffer TEMED TLC TM Tn7 TRE TRH Tris TTBS UAS X-gal

serum-inducible element son-of-sevenless serum response element signal transducer and activator of transcription Simian Virus 40 T lymphoma invasion and metastasis-1 Trichoplusia ni tetrabutylammonium sulphate tissue culture trichloroacetic acid ternary complex factor Tris-EDTA buffer N,N,N',N'-tetramethylethylenediamine thin layer chromatography transmembrane Transposon 7 TPA response element thyrotropin releasing hormone 2-amino-2-(hydroxymethyl)-l,3-propandiol Tween Tris-buffered saline upstream activating sequence 5-bromo-4-chloro-3-indolyl-B,D-galactoside

xxvii


1

Direct assessment of conformational changes in G protein-coupled receptors, using fluorescence spectroscopy ULRIK GETHER and BRIAN KOBILKA

1. Introduction G protein-coupled receptors (GPCRs) represent the largest class of transmembrane signalling molecules. The mechanism by which hormones and neurotransmitters activate intracellular signalling cascades through these homologous proteins is of great interest. While GPCRs all share a common seven-helix transmembrane structure, domains involved in ligand binding are nearly as diverse as the chemical structures of the known agonists. Small molecular weight ligands bind to sites within the hydrophobic core formed by the transmembrane (TM) a-helices, while binding sites for peptides and protein agonists also include the amino terminus, and extracellular hydrophilic loops joining the transmembrane domains (1). This diversity in ligand binding suggests that different classes of receptors may have unique mechanisms of activation. However, when one considers that all these receptors function by activating a highly homologous family of G proteins, it is reasonable to propose that agonist binding is linked to G protein activation through changes in the conformation of the transmembrane domains, and that what we learn about receptor activation from one class of receptors will apply to most, if not all, members of this large receptor family. It is generally assumed that binding of the agonist to the receptor induces a set of finely orchestrated changes in the tertiary structure of the receptor, that are recognized by the associated G protein a-subunit. Many methodological approaches have been applied in the attempt to understand these structural changes that provide the critical link between agonist binding and G protein coupling (2). Until recently models for how GPCRs are activated have been based on indirect evidence; hence, the conformation of the receptor has mostly been inferred from activation of messenger systems and/or from computational simulations (3-7). However, the possibility of establishing purification procedures and applying biophysical

Ulrik Gether and Brian Kobilka techniques in the study of this class of receptors has now allowed novel insight into the molecular mechanisms underlying activation of GPCRs (8-12). Most of these studies have been performed on rhodopsin. There are abundant natural sources of rhodopsin, and its inherent stability makes it possible to produce and purify relatively large quantities of recombinant protein. The elegant use of EPR spectroscopy by Hubbell, Khorana, and co-workers has provided the most substantial insight into conformational changes associated with photoactivation of rhodopsin (8-10). Rhodopsin is also the only GPCR for which direct information about the tertiary structure is available. Projection maps at 8A resolution of bovine and frog rhodopsin, based on electron microscopy of two-dimensional crystals, have provided crucial information about the relative positioning of the transmembrane helices in the seven-helix bundle (13, 14). Recently, we have applied spectroscopic techniques to the B2 adrenergic receptor (B2AR) (11, 12, 15-17). As described in this chapter, we have taken advantage of the sensitivity of fluorescent molecules to the polarity of their molecular environment. A sulfhydryl-reactive fluorescent probe was covalently incorporated into the purified 32AR, and used as a molecular reporter for structural changes occurring following agonist binding to the receptor (11, 12, 16). The background for applying spectroscopic approaches was a wish to develop methods that would allow direct, time-resolved analysis of conformational changes accompanying ligand-induced activation of GPCRs. In contrast to rhodopsin, which is a highly specialized GPCR with its ligand covalently bound, the B2AR is a typical ligand-activated receptor. Developing spectroscopic techniques for the B2AR would thus allow analysis of differences between the conformational states of unbound receptors and receptors bound to different kinds of ligands, including full agonists, partial agonists, neutral antagonists, and inverse agonists. The goal of this chapter is to describe the background and methodology involved in using fluorescence spectroscopic techniques for analysing conformational changes in ligand-activated GPCRs. The currently available data will be reviewed and discussed in the context of the experimental procedures.

2. Expression and purification of the B2 adrenergic receptor Compared with other methods for studying receptor structure, such as crystallography and NMR, fluorescence spectroscopy requires relatively small amounts of pure protein (~10-100 picomoles per assay). However, the total amount of receptor required to perform a series of studies using different ligands can exceed 1 mg. Therefore, spectroscopic analysis requires a reliable method for producing and purifying recombinant receptor protein. We have used the baculovirus/Sf-9 cell system to express the 32AR. Sf-9 cells are easy to maintain, they do not require CO2, and they quickly adapt to growth in

1: Direct Assessment of conformational changes suspension cultures in a standard shaker, using either glass or polyethylene Erlenmeyer or Fernbach flasks (see also Chapter 7). The most prominent problem of the insect cell expression system may be the varying fraction of improperly folded, and thus non-functional, protein (18, 19). In the case of the B2AR, approximately half the synthesized receptor is non-functional (19). The fraction can vary for different proteins; however, high levels of expression of several GPCRs in Sf-9 insect cells have been reported (20, 21). We have expressed a modified form of the B2AR in the baculovirus/Sf-9 cell system (18). The amino terminus contains a cleavable influenza-haemagglutinin signal sequence followed by the Ml antibody 'FLAG'-epitope (18). The signal sequence resulted in an approximately twofold increase in expression (18). At the carboxyl terminus the receptor was tagged with six histidines (SF-hB2-6H) (19). A three-step procedure was developed to purify the receptor from the Sf-9 cells, including an initial nickel chromatography step, followed by anti-FLAG immunoaffinity chromatography, and alprenolol affinity chromatography (19). These three steps together ensure that only fulllength and properly folded receptor are purified. It should be noted that for many purposes the immunoaffinity purification step can be omitted (12). Protocol 1. Receptor purification Equipment and Reagents • pVL 1392 baculovirus expression vector (Pharmingen), or equivalent. • Sf-9 insect cells • Baculo Gold transfection kit (Pharmingen), or equivalent • SF900-11 medium (Gibco) • Alprenolol

• Triple-baffled Fernbach flasks (Bellco Glass Inc.) • Chelating Fast Flow Sepharose Resin (Pharmacia) • Flag™ M1 antibody (Eastman Kodak) • [3H] dihydroalprenolol (Amersham)

A. Expression vector and transfection We express the B2AR under control of the polyhedrin promoter, using the pVL1392 baculovirus expression vector (Pharmingen, San Diego, CA). In our hands this vector consistently results in high and reproducible expression. 1. Epitope-tag the cDNA, encoding the human B2AR, at the amino terminus with the cleavable influenza-haemagglutinin signal sequence, followed by the 'FLAG'-epitope (Eastman Kodak, Rochester, NY), and tag the carboxyl terminus with six histidines (SF-hB2-6H) as previously described (18, 19). 2. Co-transfect the vector, containing the cDNA encoding the modified B2AR with linearized BaculoGold DNA, into Sf-9 insect cells, using the BaculoGold transfection kit, according to the manufacturer's instruction (Pharmingen, San Diego, CA).

Ulrik Gether and Brian Kobilka Protocol 1.

Continued

B. Virus amplification and plaque purification

1. Harvest the virus 4-5 days after the transfection, and amplify once before plaque-purification. 2. The plaque-purified viruses are usually amplified three times to obtain 500 ml of a high titre virus stock (about 1 x 109 pfu). 3. Test each virus stock in small-scale cultures to determine the optimal inoculum for the large-scale infections. C. Culturing Sf-9 cells

1. Maintain Sf-9 insect cells in SF900-II medium (Gibco, Grand Island, NY) supplemented with 0.1 mg ml-1 gentamicin (Gibco) and 5% heatinactivated fetal calf serum (Gibco). The optional addition of serum allows the cells to grow to higher densities (7-8 X 106 cells ml-1). 2. Keep the cell stock in 250 ml polypropylene Erlenmeyer flasks (Corning Costar, Acton, MA) at 27°C in a shaker set at 125 r.p.m. Each flask contains 70-100 ml medium, and the cells are kept at a density varying from 0.5-6 x 106 cells ml-1. D. Infection for purification

1. Seed cells in 2800 ml triple-baffled Fernbach flasks (Bellco Glass Inc., Vineland, NJ), and grow until they reach a density of 5-7 x 106 cells ml-1 in a total volume of 1000-1200 ml of medium. 2. Remove the culture from the incubator, and keep the flask at room temperature for 1.5-2 h to sediment the cells. 3. Aspirate most of the medium carefully, and resuspend the cells in fresh medium plus 1 mM of alprenolol, to a cell density of 5 x 10s cells ml-1. 4. Infect cells by adding virus stock (1:30 to 1:100 dilution). Determine the optimal inoculum for each virus stock by infecting small-scale suspension cultures (20 ml in 125 ml disposable Erlenmeyer flasks). 5. Incubate cells for 48 h at 27°C in a shaker set at 125 r.p.m., and harvest by centrifugation for 10 min at 2700 g. The resulting cell pellets can be kept at -70°C until purification. E. Purification

For our spectroscopic analyses we have used the following purification procedure. This procedure has been described in detail previously (19). 1. Lyse one or two pellets of cells from 1000 ml infected cultures in 10 mM Tris-HCI buffer, pH 7.5, containing 1 mM EDTA, 10 (mg ml-1 leupeptin (Boehringer, Mannheim, Germany), 10 mg ml-1 benzamidine (Sigma,

1: Direct Assessment of conformational changes St. Louis, MO), and 0.2 mM phenylmetnylsulfonylfluoride using 100 ml per pellet.

(Sigma),

2. Centrifuge the lysed cells at 45000 g for 30 min, discard the supernatant, and weigh the pellets. 3. Resuspend the pellets in 20 mM Tris-HCI buffer, pH 7.5, containing 1.0% n-dodecyl-B-D-maltoside (DBM) (Anatrace Inc., Maumee, OH), 500 mM NaCI, 10mg ml-1 leupeptin (Boehringer), 10 mg ml-1 benzamidine (Sigma), 0.2 mM phenylmethylsulfonylfluoride (Sigma), and 10-6 M alprenolol (Sigma). 4. Solubilize the resuspended pellets in a Dounce homogenizer (20 strokes with a tight pestle), and stir at 4°C for 1.5-2 h. Use 10 ml of buffer for each gram of lysed cells. 5. Separate non-solubilized particulate from solubilized protein by centrifugation at 45000 g for 30 min. Add imidazole from a 2.0 M stock solution (pH 8.0) to the supernatant at a final concentration of 50 mM. Add Chelating Fast Flow Sepharose Resin (Pharmacia) (0.5 ml of packed resin per gram of lysed cells), charged with nickel and equilibrated in high-salt buffer (20 mM Tris-HCI, pH 7.5, with 500 mM NaCI and 0.08% DBM), and incubate for 2-3 h at 4°C with gentle rotation. 6. Isolate nickel resin by centrifugation for 5 min at 2000 g. Wash the resin once in four times the column volume of high-salt buffer, load onto a column, and wash with three times the column volume of high-salt buffer, and twice the column volume of high-salt buffer containing 25 mM imidazole. Elute in 1/4-column volume fractions with 200 mM imidazole in high-salt buffer. 7. Assay fractions for receptor-binding activity, and pool peak fractions. 8. Add CaCI2 to the pooled fractions at a final concentration of 2.5 mM. Load the pooled fractions onto an M1 antibody column (Eastman Kodak) (0.2 ml per nmol of receptor), equilibrate in low-salt buffer (20 mM Tris-HCI, pH 7.5 with 100 mM NaCI, and 0.08% DBM), and recycle four times by gravity flow. 9. Wash the column with four times the column volume of low-salt buffer containing 2.5 mM CaCI2, and elute using low-salt buffer containing 1 mM EDTA in 1/4-column volume fractions. 10. Analyse the fractions for receptor-binding activity, and pool peak fractions. These two purification steps can produce almost pure protein (specific activity around 5 nmol mg-1 of protein). However, approximately half is non-functional (19). To separate the non-functional receptor from the functional, we use alprenolol affinity chromatography, which is a standard

Ulrik Gether and Brian Kobilka Protocol 1. Continued procedure for purification of the B2AR (22-24). It is important to note that we have been able to omit the M1 immunoaffinity chromatography in some applications. This results in a specific activity of the purified receptor of about 5-10 nmol mg-1, compared with 10-15 nmol mg-1 for the three-step purification. Approximately 5 nmol of purified protein can generally be obtained from a 1000 ml culture. Protein is determined using the detergent-insensitive Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). Purified receptor is analysed by classical 10% (w/v) SDS-polyacrylamide gel electrophoresis. Note that samples should not be boiled before loading on to the gel, as this may cause receptor aggregation. The receptor is visualized by standard Coomassie blue staining. F. Binding assay The amount of purified B2AR is assessed in binding assays, using 3Hdihydroalprenolol (3H-DHA) as radioligand (Amersham, Arlington Heights, IL). 1. Incubate purified B2AR (10 ml of an appropriately diluted sample) with 10 nM 3H-DHA (10 ml from a 1:100 dilution of 3H-DHA) in a total volume of 100 ml low-salt buffer (20 mM Tris-buffer, pH 7.5, containing 100 mM NaCI and 0.08 % DBM) for 1 h. Determine non-specific binding in the presence of 10 mM alprenolol. 2. Stop the binding assay, and separate free 3H-DHA from bound by loading the binding mixture onto a 2 ml Sephadex G50 (Pharmacia) column (Poly Prep columns, Bio-Rad). 3. Elute columns directly into 20 ml scintillation vials with 1 ml of ice-cold low-salt buffer. 4. Add scintillation fluid, and count in a scintillation counter.

3. Fluorescence labelling and spectroscopic analysis of the purified (32 adrenergic receptor The emission from many fluorescent molecules is strongly dependent on the polarity of the environment in which they are located. Fluorescent labels incorporated into proteins can therefore be used as sensitive indicators of conformational changes and of protein-protein interactions that cause changes in the polarity of the environment surrounding the probe (25-28). Nitrobenzdioxazol iodoacetamide (IANBD) is a highly fluorescent, cysteineselective reagent (26,27). The fluorescence from IANBD increases as the polarity of the solvent decreases, and is more than tenfold stronger in nbutanol and n-hexane than in aqueous buffer (Figure 1A). There is a parallel 6

1: Direct Assessment of confurmutional changes

Figure 1. Fluorescence properties of IANBD and lANBD-labelled p2AR. (A) emission spectra of cysteine-reacted IANBD (0.3 mM) in solvents of different polarity. Excitation was at 481 nm. (B) emission spectrum of lANBD-labelled B2AR (0.15 mM receptor, and 1.2 mol IANBD per mol receptor). Control is emission spectrum of 0.15 mM B2AR, 'labelled' with IANBD prebound to free cysteine instead of free IANBD, to assess possible nonspecific attachment of the probe to the receptor during labelling. Insert: 10% (w/v) SDSpolyacrylamide gel electrophoresis of lANBD-labelied B2AR. Lane 1, 150 pmol IANBDlabelled B2AR; lanes 2 and 3, 150 pmol B2AR preincubated with iodoacetamide before exposure to IANBD (lane 2) and N-ethylmaleimide (lane 3). Left panel of insert: Coomassie blue staining of gel; right panel of insert: gel photographed under UV tight. The weak band with an apparent molecular weight of 32.5 kDa is a degradation product of the receptor. (Reproduced from Gether et al., ref, 11, with permission).

Ulrik Gether and Brian Kobilka blueshift in the emission maximum from 540 nm in aqueous buffer to 530 nm in n-butanol and 510 nm in n-hexane (Figure 1A). Labelling of B2AR purified from Sf-9 insect cells with IANBD revealed a strong fluorescence signal with an emission maximum at 523 nm (Figure 1B). The blueshift in emission maximum, compared with cysteine-reacted IANBD in aqueous buffer, indicates that the modified cysteine(s) are located in an environment that, on the average, is of lower polarity than n-butanol but higher than n-hexane. This would probably involve labelling of one or more of the five cysteine residues that are located in the transmembrane, hydrophobic core of the receptor (see Figure 3). The covalent modification of the receptor was confirmed by SDSpolyacrylamide electrophoresis of the labelled receptor, and the specificity of the labelling was verified by blocking the incorporation of IANBD with the cysteine-specific, non-fluorescent reagents, iodoacetamide and N-ethylmaleimide (Figure 1B, insert). Importantly, the fluorescent labelling did not perturb the pharmacological properties of the receptor, either in terms of agonist or antagonist binding (11). To examine conformational changes induced by agonist binding, we performed time-resolved spectroscopic analyses (11). As illustrated in Figure 2, binding of the full agonist, isoproterenol, to lANBD-labelled B2AR caused a dose-dependent decrease in fluorescence, reaching a maximum amplitude below the extrapolated baseline after 10 min. The response to isoproterenol could be readily reversed by the active (-)isomer of the antagonist propranolol, but not by the less active (+)isomer (Figure 2). The response to isoproterenol was similarly reversed by several other antagonists, including alprenolol, ICI 118 551, pindolol, and dichloroisoproterenol (11). Moreover, the isoproterenol response was dose-dependent and stereospecific (11). Prior to adding ligand, we normally observe a slight but constant decline in baseline fluorescence (Figure 2). This loss of fluorescence over time is probably caused by bleaching of the fluorophore, combined with some loss of protein possibly due to the protein sticking to the inside of the cuvette. The decrease over time was unaffected by addition of 0.1% bovine serum albumin, 10% glycerol, or phospholipids to the cuvette (11); however, precincubation of the receptor in the cuvette for 15 min before performing the experiments minimized (but never eliminated) the constant decline in baseline fluorescence. It should be noted that the decline in fluorescence is unlikely to be due to denaturation of the protein, since a similar loss of fluorescence also was observed with labelled receptor that was intentionally denatured in guanidinium chloride (11). The observed agonist-induced decrease in fluorescence from the IANBDlabelled receptor is most likely to be due to movement of the fluorophore to a more polar environment upon agonist binding. Importantly, the magnitude of the fluorescence changes was found to correlate with the intrinsic biological efficacy of the ligand, as demonstrated by comparing the effect of a series of partial and full agonists on adenylyl cyclase activity with their effect on the magnitude of the fluorescence changes (11). This suggests that the ligand-

1: Direct Assessment of conformational changes

Figure 2. Reversible decrease in fluorescence from the lANBD-labelled B2AR induced by isoproterenol. (A) Control: addition of water (H2O). (B) and (C), Reversal of the response to isoproterenol (ISO) by the active (-)isomer of the antagonist propranolol, (-)PROP (B), but not by the less active (+)isomer, (+)PROP (C). Dotted lines indicate extrapolated baseline. Excitation was at 481 nm, and emission was measured at 523 nm. Fluorescence in all the individual traces shown was normalized to the fluorescence observed immediately after addition of ligand. All traces shown are representative of at least three identical experiments. (Reproduced from Gether et al., ref. 11, with permission).

induced changes in fluorescence are relevant to the receptor activation mechanism. Protocol 2. Labelling of receptor with fluorescent probes Equipment and Reagents . IANBD (Molecular Probes) • Sephadex G50 (Pharmacia) • Centricon-3-filter (Amicon)

' Chelating Fast Flow Sepharose (Pharmacia) ' Bio-Rad DC protein assay kit (Bio-Rad)

Ulrik Gether and Brian Kobilka Protocol 2. Continued A. Fluorescent labelling of the purified B2AR We have used two different protocols for labelling of the purified B2AR (11,12). Results obtained with the two protocols are indistinguishable; however, we recommend the second procedure since it is more effective at removing non-covalently bound fluorophore from the purified protein (11,12). 1. Incubate purified B2AR(1-1.5 nmol) with 10-15-fold molar excess of IANBD (Molecular Probes, Eugene, OR) (150 (mM) in a total volume of 100 ml buffer (20 mM Tris-buffer, pH 7.5, containing 100 mM NaCI and 0.08 % DBM). IANBD is added from a 10 mM stock solution in DMSO. Allow the reaction to proceed for 1 h at room temperature in the dark, and quench by addition of 1 mM cysteine from a 100 mM stock, followed by 5 min of incubation. 2. Remove cysteine-reacted dye using a Sephadex G50 gel filtration column (0.5 cm X 9 cm). Apply the reaction mixture directly to the column, and elute with 2.0 ml of buffer directly into a Centricon-30 filter device (Amicon, Beverly, MA). 3. Concentrate the eluate, containing the labelled receptor, to approximately 50 ml in the Centricon-30 filter device, fay centrifugation for 45 min at 3000 g in a fixed angle rotor (Sorvall SS-34). Either use the labelled receptor directly for the fluorescence spectroscopy analysis, or store it on ice at 4°C. Under these conditions the protein is stable for several days. 4. Bind purified receptor (up to 5 nmol) to a 150 ml nickel-column by recycling by gravity flow six times (Chelating Fast Flow Sepharose Resin from Pharmacia equilibrated in high-salt buffer, 20 mM Tris-HCI, pH 7.5, with 500 mM NaCI and 0.08% DBM). 5. Perform IANBD labelling by recycling 1.0 ml of 0.5 mM IANBD in highsalt buffer several times over the nickel column for 20 min. 6. Remove excess dye by extensive washing of the column with approximately 50 times the column volume of high-salt buffer. 7. Elute labelled p2AR in 50 ml fractions with 200 mM imidazole in highsalt buffer. Assay fractions for protein content, and pool peak fractions. The labelled receptor can be used directly for the fluorescence spectroscopy analysis, or stored on ice at 4°C. Under these conditions the protein is stable for several days. Both labelling procedures result in incorporation of 1.2-2 mol IANBD per mole of receptor, as determined by measuring absorption at 481 nm and using an extinction coefficient of 21 000 M-1 cm-1 for IANBD and a MW of 50 000 Da for the receptor. Protein concentration was determined using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). 10

1: Direct Assessment of conformational changes B. Fluorescence spectroscopy analysis Fluorescence spectroscopy is performed at room temperature using a SPEX Fluoromax spectrofluorimeter, connected to a PC equipped with the Datamax software package. We use the photon-counting mode, and generally an excitation and emission bandpass of 4.2 nm (11, 12, 16). 1. Use 30-50 pmol lANBD-labelled receptor for emission scan experiments. Add 10 ml of receptor to 390 ml buffer (20 mM Tris buffer, pH 7.4, containing 100 mM NaCI and 0.08% DBM) in a 5 x 5 mm quartz cuvette, and mix by pipetting up and down. Set the excitation wavelength at 481 nm, and measure the emission from 490 nm to 625 nm with an integration time of 0.3 sec nm-1. 2. Like the emission scans, perform time-resolved fluorescence spectroscopy using 30-50 pmol of labelled receptor. Add 10 ml of receptor to 490 ml buffer (20 mM Tris-buffer, pH 7.4, containing 100 mM NaCI and 0.08 % DBM) in a 5 x 5 mm quartz cuvette. 3. Preincubate the mixture for at least 10 min in the cuvette before the experiment is started to stabilize the baseline. Both during this period and during the time-scan experiment, keep the mixture under constant stirring using a 2 x 2 mm magnetic stirring bar (Bel-Art Products, Pequannock, NJ). 4. During time-scan experiments, set the excitation wavelength at 481 nm, and measure emission at a wavelength of 525 nm. The time scan is routinely performed over 30 min, and the first addition of ligand is usually done after 5 min. The volume of the added ligands is one percent of the total volume, and fluorescence is corrected for this dilution. The compounds tested in our fluorescence experiments have an absorbance of less than 0.01 at 481 nm and 525 nm in the concentrations used, thus eliminating inner filter effects.

4. Site-selective fluorescent labelling of the B2 adrenergic receptor Studies of NBD-labelled B2AR provide direct evidence for ligand-induced conformational changes, but do not identify the nature of the structural changes. To obtain information about the movement of specific receptor domains following agonist activation, it is necessary to determine the site or sites of NBD labelling in the wild-type receptor that are responsible for ligand-induced changes in fluorescence. This requires modifying the receptor structure to limit the amino acids that are susceptible to chemical modification. The most reactive amino acids are cysteines and lysines. It is possible to direct a fluorescent probe to either cysteines or lysines by choosing the

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Ulrik Gether and Brian Kobilka appropriate chemically reactive fluorophore. However, directing the labelling to a specific cysteine or lysine often requires mutagenesis to limit unwanted labelling sites. In the sections below we discuss site-directed labelling of cysteines and lysines.

4.1 Labelling of cysteines The B2AR contains thirteen cysteines, of which five cysteines are not expected to be available for chemical derivitization. In the extracellular loops, four cysteines (106Cys, 184Cys, 190Cys, and 191Cys) form two disulfide bridges (Figure 3) (29-31), and in the intracellular carboxyl terminal tail 341Cys has been shown to be palmitoylated (32, 33). To identify the cysteine(s) responsible for the agonist-induced change in fluorescence, and thus to establish a system that would allow site-selective incorporation of the IANBD fluorophore, we mutated cysteines in the receptor and generated a series of mutant receptors with one, two, or three cysteines available for chemical derivitization (12). All these mutants displayed minimal changes in pharmacological properties compared with the wild-type, both with respect to ligand binding and functional coupling to adenylyl cyclase (12). However, mutation of several cysteine residues led to a reduction in receptor expression (12). Notably, a mutant receptor with all free cysteines substituted expressed so poorly that purification in sufficient quantities for fluorescence spectroscopy analysis was impossible (12). Ideally, it should be possible to take out all endogenous cysteines, and either reintroduce them one by one, or introduce single cysteines in new positions. Unfortunately this was not possible in the B2AR. Nevertheless, as illustrated in Figure 3 and described below, it is possible to obtain site-specific information from a system where it is not possible to remove all cysteines. The mutant receptors containing one, two, or three of the naturally occurring cysteines were all purified and labelled with the IANBD fluorophore. As expected, the lANBD-labelled mutants all demonstrated emission maxima around 525 nm (12). Time-resolved analysis of the mutants revealed that agonist-induced changes in fluorescence are observed only in receptors in which 285Cys or 125 Cys are present (Figure 3). A mutant lacking only these two Figure 3. The effect of isoproterenol on fluorescence from IANBD labelled wild-type and mutant B2AR (A) 'Snake diagram' of the B2AR. The receptor contains thirteen Cys residues of which five ("Cys, 116Cys, 125Cys, 285Cys, 327Cys) are predicted to be in the transmembrane domain. Three Cys residues are predicted to be in the cytoplasmic regions (265Cys, 375Cys, 406Cys). Five Cys residues are not expected to be available for chemical derivatization (small white circles): four residues (106Cys, 184Cys, 190Cys, 191Cys) form two disulfide bridges (refs 29-31), and in the intracellular carboxy terminal tail, 341 Cys, has been shown to be palmitoylated (refs 32, 33). (B) Bar diagram of changes in fluorescence in response to the full agonist isoproterenol (1 mM) for the wild-type receptor and indicated mutants. The ligand concentration was chosen to ensure

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1:Direct Assessment of conformtational changes

saturation of the receptors, eliminating any influence from different agonist affinities. Excitation was at 481 nm, and emission was measured at 525 nm. Data are given as percent change in fluorescence (mean ' SE, n - 3-6). The percent change was calculated as the change in fluorescence relative to the extrapolated baseline 15 min after addition of ligand. The cysteine-mutants are named according to the cysteines still present in the receptor and available for chemical derivatization. Thus, Cys(285) describes a construct where 2S5Cys is present but where 77Cys, 115Cys,126Cys, 265Cys, 327Cys, 378Cys and 406Cys have been mutated. 106Cys, 164Cys, 190Cys and 191 Cys were excluded from the 'name', since they are not available for chemical derivatization. (Reproduced from Gether et al., ref. 12, with permission).

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Ulrik Gether and Brian Kabilka

Figure 4. Simplified model of the B2AR with indication of predicted movements in response to agonist binding, NBD bound to 125Cys in TM III and 286Cys in TM VI are, according to a molecular model of the receptor, predicted to lie at the protein-lipid interface, oriented predominantly towards the lipid as illustrated (the NBD fluorophore is indicated by F) (16). 285Cys-NBD is predicted to be at the helix VI-VII interface, in a boundary zone between the lipid bilayer (or the hydrophobic tails of the detergent micelle) and the more polar interior of the protein (16). An agonist-induced counterclockwise rotation of helix VI, as indicated by the arrow (seen from the extracellular side), would lead to movement of 285Cys-NBD from the nonpolar environment of the lipid bilayer (or detergent micelle) to the more polar environment of the interior of the protein, explaining the observed changes in fluorescence. This is consistent with spin-labelling studies in rhodopsin, which also suggest a counter-clockwise rotation of TM VI upon photo-activation of rhodopstn (10). In TM III, 125Cys-NBD is predominantly exposed to the lipid bilayer (or detergent micelle) (16). Our data indicate that the extent of lipid exposure is changed in response to agonist binding. This is consistent with an agonist-induced movement of TM III, causing the fluorophore to be exposed to a more polar face of TM IV and/or the more polar interior of the receptor, as indicated by the arrow.

cysteines (Cys77, 116, 265, 327, 378, 406) showed no response to agonist binding (Figure 3). The data suggest that agonist binding to the B2AR promotes a conformational change in the receptor that exposes NBD, attached to 125Cys in transmembrane segment (TM) III and to 285Cys in TM VI, to a more polar environment. We have attempted to predict the actual structural changes, using molecular modelling and computational simulations (12). Most importantly, the simulations demonstrated a significant conformational restraint for the NBD bound to 285Cys -NBD and 125 Cys-NBD (12), This suggests that the 14

1: Direct Assessment of conformational changes change in molecular environment around the bound NBD reflects movement of the transmembrane helix to which it is attached, rather than movement of the NBD relative to the transmembrane helix. As schematically illustrated in Figure 4, our data are consistent with a counter-clockwise rotation of helix III and VI in response to agonist binding. This is consistent with the suggested rigid body movements of the corresponding helices in rhodopsin (10). It is interesting to note that 285Cys in TM VI is situated one a-helical turn below 288pro, which is highly conserved among GPCRs and provides a flexible hinge in this helix (12). It has therefore been proposed that the movement of 285CysNBD to a more polar environment in the protein interior could be directly facilitated by this flexible hinge connecting the binding site with the putative G protein coupling domain at the cytoplasmic end of the helix (12). A few issues should be emphasized when interpreting data from our fluorescence spectroscopy analysis. It is important to note that the amplitude of the fluorescent change is only a rough indicator of the magnitude of conformational change. For example, we cannot assume that there is a linear correlation between change in fluorescence and magnitude of movements. Therefore, the movement of TM III, for example, may not be of the same magnitude as movement of TM VI. It is also necessary to ensure that the fluorescent probe, when incorporated into the receptor, does not interfere with binding of the ligands. In the B2AR this is highly unlikely. Labelling of the receptor with IANBD does not alter agonist or antagonist binding properties (11), as would be expected if the bound NBD was positioned within the ligand binding pocket. The results from mutagenesis studies have also provided substantial evidence that amino acids involved in forming the ligand-binding pocket are on a different side of the transmembrane a-helix and one to two a-helical turns closer to the membrane surface than 125Cys and 285 Cys. Our results show that even though we were unable to remove all IANBDreactive cysteines from the B2AR, it was possible to deduce the sites of labelling that were responsible for the agonist-induced fluorescence changes observed in the wild-type receptor. While true site-specific labelling is not possible with IANBD, this may be possible by labelling the B2AR with larger, more hydrophillic cysteine-reactive probes which are unable to gain access to transmembrane cysteines.

4.2 Site-specific labelling at lysines Lysine and arginine are positively charged amino acids that are important in dictating topology of membrane-embedded segments of proteins. In many cases lysines and arginines are functionally interchangeable; however, only lysine has a primary amine and is therefore much more chemically reactive towards isothiocyanates and succinimidyl esters. All basic amino acids in the B2AR are predicted to reside in the hydrophillic domains, or at the boundaries 15

Ulrik Gether and Brian Kobilka of transmembrane helices. We replaced the sixteen lysine residues of the 32AR receptor with arginine, using standard PCR mutagenesis (17). The resulting receptor, referred to as the OK-P2AR, retains the ligand binding and G protein coupling properties of the wild-type B2AR receptor (17). While the OK-32AR receptor is expressed at lower levels than the wild-type B2AR, it is possible to purify sufficient quantities for fluorescence labelling studies (17). To explore movement of a specific receptor domain, it is possible to add back one of the original lysines, thereby permitting site-specific labelling at the single lysine. It should be noted, however, that the amino terminus is also reactive towards isothiocyanates and succinimidyl esters. Thus, receptors having single reactive lysines will also be susceptible to labelling at the amino terminus. To overcome this problem, we have recently constructed a OKB2AR receptor having a specific proteolytic cleavage site in the amino terminus. This permits removal of unwanted amino terminal fluorophore.

5. Concluding remarks Using a sulfhydryl-reactive and environmentally sensitive fluorescent probe, IANBD, as a molecular reporter, we have been able to characterize and map ligand-induced conformational changes in the B2AR. Future studies will attempt to determine more precisely the type of conformational changes that take place following agonist binding. Specifically, we would like to determine how specific transmembrane domains move relative to each other and to the lipid bilayer. To accomplish these goals, it will be necessary to make use of site-specific labelling techniques in combination with spectroscopic techniques, such as fluorescence resonance energy transfer or EPR spectroscopy, two methods which can be used to monitor changes in distance between two receptor domains. These dynamic studies will complement high-resolution structural information when it becomes available.

References 1. Ji, T. H., Grossmann, M., and Ji, I. (1998). J. Biol. Chem., 273, 17299. 2. Gether, U., and Kobilka, B. K. (1998). J. Biol. Chem., 273, 17979. 3. Samama, P., Cotecchia, S., Costa, T., and Lefkowitz, R. J. (1993). J. Biol. Chem., 268, 4625. 4. Luo, X., Zhang, D., and Weinstein, H. (1994). Protein Eng., 7, 1441. 5. Ballesteros, J. A., and Weinstein, H. (1995). Meth. Neurosci. 25, 366. 6. Fanelli, F., Menziani, M. C., and De Benedetti, P. G. (1995). Bioorg. Med. Chem. 3, 1465. 7. Scheer, A., Fanelli, F., Costa, T., De Benedetti, P. G., and Cotecchia, S. (1996). EMBO J. 15, 3566. 8. Farahbakhsh, Z. T., Ridge, K. D., Khorana, H. G., and Hubbell, W. L. (1995). Biochemistry, 34, 8812.

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1: Direct Assessment of conformational changes 9. Altenbach, C., Yang, K., Farrens, D. L., Farahbakhsh, Z. T., Khorana, H. G., and Hubbell, W. L. (1996). Biochemistry, 35, 12470. 10. Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. L., and Khorana, H. G. (1996). Science, 274, 768. 11. Gether, U., Lin, S., and Kobilka, B. K. (1995). J. Biol. Chem., 270, 28268. 12. Gether, U., Lin, S., Ghanouni, P., Ballesteros, J. A., Weinstein, H., and Kobilka, B. K. (1997). EMBO J., 16, 6737. 13. Unger, V. M., Hargrave, P. A., Baldwin, J. M., and Schertler, G. F. (1997). Nature, 389, 203. 14. Schertler, G. F., Villa, C., and Henderson, R. (1993). Nature, 362, 770. 15. Lin, S., Gether, U., and Kobilka, B. K. (1996). Biochemistry, 35, 14445. 16. Gether, U., Ballesteros, J. A., Seifert, R., Sanders-Bush, E., Weinstein, H., and Kobilka, B. K. (1997). J. Biol. Chem., 272, 2587. 17. Parola, A. L., Lin, S., and Kobilka, B. K. (1997). Anal. Biochem., 254, 88. 18. Guan, X. M., Kobilka, T. S., and Kobilka, B. K. (1992). J. Biol. Chem., 267, 21995. 19. Kobilka, B. K. (1995). Anal. Biochem., 231, 269. 20. Grisshammer, R., and Tate, C. G. (1995). Quart. Rev. Biophys., 28, 315. 21. Tate, C. G., and Grisshammer, R. (1996). Trends Biotechnol, 14, 426. 22. Benovic, J. L., Shorr, R. G. L., Caron, M. C., and Lefkowitz, R. J. (1984). Biochemistry, 23, 4510. 23. Caron, M. G., Srinivasan, Y., Pitha, J., Kociolek, K., and Lefkowitz, R. J. (1979). J. Biol. Chem., 254, 2923. 24. Parker, E. M., Kameyama, K., Higashijima, T., and Ross, E. M. (1991). J. Biol. Chem., 266, 519. 25. Phillips, W. J., and Cerione, R. A. (1991). /. Biol. Chem., 266, 11017. 26. Dunn, S. M. J., and Raftery, M. A. (1993). Biochemistry, 32, 8608. 27. Gettins, P. G. W., Fan, B., Crews, B. C., and Turko, I. V. (1993). Biochemistry, 32, 8385. 28. Cerione, R. A. (1994). In Methods in enzymology (ed lyengar, R.) Vol. 237, p 409. Academic Press, London. 29. Fraser, C. M. (1989). J. Biol. Chem., 264, 9266. 30. Dohlman, H. G., Caron, M. G., DeBlasi, A., Frielle, T., and Lefkowitz, R. J. (1990). Biochemistry, 29, 2335. 31. Noda, K., Saad, Y., Graham, R. M., and Karnik, S. S. (1994). J. Biol. Chem., 269, 6743. 32. O'Dowd, B. F., Hnatowich, M., Caron, M. G., Lefkowitz, R. J., and Bouvier, M. (1989). J. Biol. Chem., 264, 7564. 33. Mouillac, B., Caron, M., Bonin, H., Dennis, M., and Bouvier, M. (1992). J. Biol. Chem., 267, 21733.

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2

Probing the structure of receptor-binding sites by the substituted-cysteine accessibility method MERRILL M. SIMPSON, GEORGE LIAPAKIS, and JONATHAN A. JAVITCH

1. Introduction The substituted-cysteine accessibility method (SCAM) provides an approach to map systematically the residues on the water-accessible surface of a binding site, such as the binding-site crevice of a G protein-coupled receptor (GPCR) which binds ligand within the transrnembrane portion of the receptor. Consecutive residues in putative membrane-spanning segments are mutated to cysteine, one at a time, and the mutant receptors are expressed in heterologous cells. The water-accessible residues are identified by assessing the reaction of charged, hydrophilic, sulfhydryl reagents with the engineered cysteines.

2. Applications of the substituted-cysteine accessibility method (SCAM) 2.1 Systems studied with SCAM Cysteine substitution and covalent modification have been used to study structure-function relationships and the dynamics of protein function in a variety of membrane proteins (1-6). Moreover, charged, hydrophilic, lipophobic, sulfhydryl reagents have been used to probe systematically the accessibility of substituted cysteines in putative membrane-spanning segments of a number of proteins. This approach, SCAM, has been used to map channel-lining residues in the nicotinic acetylcholine receptor (7-9), the GABAA receptor (10, 11), the cystic fibrosis transmembrane conductance regulator (12), the UhpT transporter (13), and potassium channels (14), among

Merrill M. Simpson et al. others. We have adapted this approach to map the surface of the binding-site crevice in the dopamine D2 receptor (15-19), a member of the GPCR superfamily. SCAM can also be used to determine differences in the structures of the membrane-spanning segments in different functional states of proteins, to map electrostatic potential in membrane-spanning domains, and to size a channel or binding-site crevice.

2.2 SCAM versus site-directed mutagenesis and affinity labelling Site-directed mutagenesis is the most common approach to the determination of critical binding site residues. Insights into critical binding domains have also been gained from the construction of chimeric receptors. The interpretation of the functional effects of typical mutagenesis experiments, however, is complicated by the difficulty of differentiating local effects at the site of the mutation from indirect effects of the mutation on protein synthesis, folding, processing, and structure. It is often assumed that functional changes caused by a mutation, such as changes in binding affinity, are due to local effects at the site of the mutation, and not due to indirect effects of the mutation on protein structure. The validity of this assumption is rarely proven for individual mutations, but the structure of the y phage receptor, maltoporin, showed that 50% of the residues, which on the basis of mutagenesis experiments had been implicated in \ phage recognition, are actually buried (20). Thus mutation of these buried residues alters X phage recognition indirectly. Likewise, the crystal structures of several dihydrofolate reductase mutants have demonstrated that a mutation approximately 15 A from the substrate-binding pocket exerts an effect on catalytic activity through an extended structural perturbation (21). Affinity labelling, an alternative approach to the determination of bindingsite residues, has the advantage that the residues identified are likely to be very near the binding site. Disadvantages of this approach, however, include the limited number of affinity reagents available for a particular site, the limited number of residues that can be labelled, and the significant technical difficulties involved in identifying labelled residues. SCAM does not rely on the functional effects of a given mutation, and allows one to determine whether a residue is at the water-accessible surface of the binding-site crevice even when the mutant has near normal function. In contrast to affinity labelling, SCAM can be applied systematically to any binding site, and, because the engineered cysteine is known, the labelled residue does not need to be laboriously identified with protein chemical techniques. Other advantages of the approach include the ability to probe binding sites, by assessing the ability of agonists or antagonists to retard the reaction of sulfhydryl reagents with particular substituted Cys, and the ability to probe the steric constraints and electrostatic potential of sites by comparing the rates of reaction of reagents of varying size and charge. 20

2: Probing the structure of receptor-binding sites

3. Critical parameters 3.1 Reactivity of wild-type background We typically use small, charged, hydrophilic derivatives of methanethiosulfonate (MTS) for SCAM. The function of the protein used as the background for SCAM must not be affected by these polar sulfhydryl reagents. In some cases, such as the [32 adrenergic receptor, endogenous cysteines are not accessible to reaction with the MTS reagents (or reaction causes no functional effect) (22), while in other cases, such as the dopamine D2 receptor, endogenous cysteines are accessible and must first be identified and mutated to other residues (18). We typically substitute serine or alanine for endogenous cysteine, although at particular positions other residues are better tolerated (23). The mutant protein must retain near normal function, and must be relatively insensitive to the MTS reagents. The ideal starting point would be to create a cysteine-less pseudo-wild-type protein with normal expression and function. Such a construct has been possible with the lactose permease (6), the NhaA-Na+/H+ antiporter (24), and a glutamate transporter (25).

3.2 Cysteine substitution Using a wild-type or pseudo-wild-type protein insensitive to the MTS reagents as background, SCAM can now be used to determine the residues that line the water-accessible surface of the protein. Cysteines are substituted, one at a time, for residues in a putative membrane-spanning segment. The mutant receptors are expressed in heterologous cells either transiently or stably. The affinity of ligand binding to intact cells expressing the cysteine-substituted receptors is determined to ensure that the mutant has near wild-type structure. The effect of the MTS reagents on ligand binding is then determined.

Protocol 1. Cysteine substitution Equipment and Reagents • Mutagenesis or PCR kit

Method 1. Generate a series of Cys mutations in the wild-type receptor or the pseudo-wild-type background by site-directed mutagenesis. The oligonucleotides should be designed to incorporate a change in a restriction site, to facilitate screening and verification of mutants after subcloning.

21

Merrill M, Simpson et al. Protocol 1. Continued 2. Screen the mutations by restriction mapping. 3. Confirm mutations by sequencing.

Protocol 2.

Transient transfection of cells

Equipment and Reagents • Dulbecco's modified Eagle's medium/ Ham's nutrient mixture/F-12 (DMEM/F12), containing 3.15 g 1-1 glucose with 10% defined supplemented bovine calf serum (Hyclone)

• Lipofectamine (Gibco) . Optimem (Gibco) * PRSVTag

Method 1. Grow HEK 293 cells in Dulbecco's modified Eagle's medium/Ham's nutrient mixture/F12 (1:1) (DMEM/F12), containing 3.15 g l-1 glucose with 10% defined supplemented bovine calf serum (Hyclone) at 37°C and 5% C02. 2. Co-transfect 35 mm dishes of HEK 293 cells at 70-80% confluence with 1.6 mg of wild-type or mutant receptor cDNA in an appropriate expression vector and 0.4 mg pRSVTag, using 9 ml of lipofectarnine (Gibco) and 1 ml of OPTIMEM (Gibco). 3. Change the medium 5 h after transfection, and again 24 h after transfection. 4. Harvest the cells 48 h after transfection (see Protocol 4).

Protocol 3.

Stable transfection of cells

Equipment and Reagents • DMEM-F12 + 10%BCS (+/- 0.7 mg ml-1 G418 . Lipofectamine (see Protocol 2} • (see Protocol 2) • Optimem (see Protocol 2)

Method 1. Grow HEK 293 cells in DMEM/F12 containing 3.15 mg ml-1 glucose with 10% defined supplemented bovine calf serum (Hyclone) at 37°C and 5% CO2. 2. Transfect 35 mm dishes of HEK 293 cells at 70-80% confluence with 2 mg of wild-type or mutant DNA, in a bicistronic vector expressing the receptor cDNA from a CMV promoter and the neomycin resistance

22


3. 4. 5. 6. 7.

gene from an internal ribosomal entry site (IRES) (26), using 9 ml of lipofectamine (Gibco) and 1 ml of OPTIMEM (Gibco). Change the medium 5 h after transfection. Split the cells into 100 mm plates 24 h after transfection. After another 24 h select in medium containing 0.7 mg ml-1 G418. Change the medium about every 2 days throughout the selection process to remove dying cells. Following selection, maintain the stable cells in medium containing 0.3 mg ml-1 G418.

3.3 Determination of function of cysteine-substituted mutants For further application of SCAM the affinities of the mutants for radiolabelled ligand must be similar to those of the wild-type or pseudo-wild-type receptor to ensure that the structure of the mutant is close to that of the wildtype receptor, and thus that the substituted Cys is an accurate reporter of the accessibility of the wild-type side chain. Affinity for the ligand can be determined by saturation analysis in intact, dissociated cells.

Protocol 4.

Harvesting of transiently or stably transfected cells

Equipment and Reagents • Phosphate-buffered saline (PBS: 8.1 mM NaH2P04, 1.5 mMKH2P04, 138 mM NaCI, 2.7 mM KCI, pH 7.2)

• Binding buffer (140 mM NaCI, 5.4 mM KCI, 1 mM EDTA, 25 mM Hepes, pH 7.4, containing 0.006% bovine serum albumin)

Method 1. Wash the cells with phosphate-buffered saline (PBS: 8.1 mM NaH2PO4, 1.5 mM KH2PO4, 138 mM NaCI, 2.7 mM KCI, pH 7.2). 2. Treat the cells briefly (30-60 s) with PBS containing 1 mM EDTA. 3. Remove the PBS-EDTA, and dissociate the cells in PBS. 4. Pellet the cells at 1000 g for 5 min at 4°C. 5. Resuspend the cells in an appropriate buffer for binding or treatment with MTS reagents.

The ability to substitute cysteine residues for other residues and still obtain functional receptor is central to this approach. In the dopamine D2 receptor, 91 of 96 cysteine-substitution mutants tested to date bound agonist with nearnormal affinity (15-17, 19). These tolerated substitutions were for hydro23

Merrill M. Simpson et al. phobic residues (alanine, leucine, isoleucine, methionine, and valine), polar residues (asparagine, serine, and threonine), neutral residues (proline), acidic residues (aspartate), aromatic residues (phenylalanine, tryptophan, and tyrosine), and glycine. There are several reasons why cysteine substitution may be so well tolerated. Cysteine is a relatively small amino acid with a volume of 108 A; only glycine, alanine, and serine are smaller (27). In globular proteins, roughly half the non-disulfide linked cysteines are buried in the protein interior, and half are on the water-accessible surface of the protein (28). Furthermore, cysteine has little preference for a particular secondary structure (29, 30). A cysteinesubstitution mutant that does not function cannot be studied by SCAM (or by traditional site-directed mutagenesis). The residues that cannot be mutated to cysteine are either accessible and make a crucial contribution to binding, or make a crucial contribution to maintaining the structure of the site and/or to the folding and processing of the protein. The determination by SCAM of the accessibility of the neighbours of a crucial residue may allow us to infer the secondary structure of the segment containing this residue, and thus whether or not it is likely to be accessible as well. If it is not accessible, then the functional effect of its mutation is probably due to an indirect effect on structure.

4. Chemistry of SCAM 4.1 Mechanism of reaction In the membrane-spanning segments of a GPCR, the sulfhydryl group of a native or engineered cysteine will be either at the water-accessible surface of the protein, at the lipid-accessible surface, or in the protein interior. We assume that the surface of the binding-site crevice is part of the water-accessible surface. We further assume that small, charged, hydrophilic reagents react much faster with sulfhydryls facing into the water-accessible surface than with residues facing the lipid or protein interior. Moreover, the thiosulfonate reagents react with the ionized thiolate (RS-) more than a billion times faster than with the unionized thiol (RSH) (31), and only cysteines accessible to water are likely to ionize to a significant extent.

4.2 Description of reagents For such polar sulfhydryl-specific reagents, we use derivatives of methanethiosulfonate (MTS): positively charged MTS ethylammonium (MTSEA) and MTS ethyltrimethylammonium (MTSET), and negatively charged MTS ethylsulfonate (MTSES) (Figure 1) (32). These reagents differ somewhat in size with MTSET > MTSES > MTSEA. The largest, MTSET, fits into a cylinder 6 A in diameter and 10 A long. The MTS reagents form mixed disulfides with the cysteine sulfhydryl, covalently linking -SCH2CH2X, where X is NH3+, N(CH3)3+, or SO3+, The 24


Figure 1. The structure of the methanethiosulfonate derivatives, and their reaction with cysteine.

MTS reagents are specific for cysteine sulfhydryls, and do not react with disulfide-bonded cysteines or with other residues. MTSET is a quaternary ammonium with a fixed charge, and MTSES is fully ionized at neutral pH. Both are membrane-impermeant (24, 33). MTSEA, however, is a weak base and has been reported to enter membrane vesicles readily (33); it presumably crosses the membrane in the unprotonated state. Thus, although MTSEA should react very much faster with ionized thiolates and should, therefore, only react with water-accessible cysteines, the reagent may gain access to these residues by passing through the membrane and then reacting from the cytoplasmic side. Therefore, a residue which is water-accessible from the cytoplasmic side, but not from the extracellular side of the membrane, should not react at an appreciable rate with extracellularly applied MTSET or MTSES, but might react with MTSEA, although the cytoplasmic reducing environment would be expected to scavenge much of the cytoplasmic MTSEA and thereby reduce the apparent rate of reaction. The hydrophilic, negatively-charged, organomercurial p-chloro-mercuribenzene sulfonate (pCMBS) also has been used to probe the accessibility of substituted cysteines in membrane-spanning segments of a number of membrane proteins (13, 24, 34). Protocol 5. Storage and preparation of the MTS reagents Reagents • 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA), MW 236.2 . [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), MW 278.2

• sodium (2-sulfanatoethyl) methanethiosulfonate, MW 233.2 (Toronto Research Chemicals, Toronto, ON, Canada)

25

Merrill M. Simpson et al. Protocol 5.

Continued

Method 1. Store the stock reagent desiccated at 4°C, and keep a frequently used stock desiccated at room temperature (RT). 2. The frequently used stock can be replenished from the 4°C stock after waiting for the desiccator to warm to RT before opening. 3. Weigh an appropriate amount of the desired MTS reagent. 4. Immediately before use dissolve the MTS reagents in water.a ' At pH 7 and 22°C, the MTS reagents rapidly hydrolyse with a half-time of 5-20 min (35). Thus, the MTS reagents hydrolyse quickly in buffer. If it is necessary to dissolve the reagents or perform an intermediate dilution in buffer at physiological pH, this should be done immediately before starting the reaction. At lower pH and lower temperature, hydrolysis is appreciably slower. A solution in distilled water is stable for several hours at 4°C.

4.3 Reaction with the MTS reagents Reaction can either be detected directly or indirectly, by measuring the effect of reaction on a functional property of the protein. Because of the very small quantities of protein produced in most heterologous expression systems, we cannot rely on the direct detection of reaction. Instead, we use the irreversible modification of function to assay the reaction (Figure 2). Additionally, reaction with a cysteine near a binding site should be retarded by the presence of inhibitor and/or substrate.

Protocol 6. Reaction of MTS reagents with substituted cysteines Equipment and Reagents • MTS reagant (see Protocol 5) • Binding buffer (see Protocol 4}

• Glass fibre filters (grade 934AH) (Brandel, Inc.)

Method 1. Add 5 ml of the appropriate concentration (10x) of freshly prepared MTS reagent into aliquots (45 ml) of cells suspended in binding buffer (140 mM NaCI, 5.4 mM KCI, 1 mM EDTA, 25 mM Hepes, pH 7.4, containing 0.006% bovine serum albumin).a 2. Mix cells and reagent, and incubate at room temperature for 2 min. 3. Dilute the cell suspensions 10-20-fold in binding buffer to slow the reaction.b 4. Use 200 ml aliquots to assay for radioligand binding as appropriate.

26

2: Probing the structure of receptor-binding sites 5. Separate the bound from free radioligand by filtration through glass fibre filters. 6. Count the binding samples by liquid scintillation spectrometry. 7. The fraction of inhibition can be calculated as follows: 1- [(specific binding after MTS reagents)/(specific binding without reagent)] a For screening, use final concentrations of 2.5 mM MTSEA, ImM MTSET, and 10 mM MTSES to normalize for the intrinsic reactivities of the reagents with sulfhydryls in solution (32). b Do not attempt to stop the reaction with a reducing agent such as dithiothreitol or 2mercaptoethanol, as these will reduce the newly formed disulfide bonds between the MTS reagents and the substituted cysteine. The reaction should also not be quenched with solutions containing free sulfhydryls, as these can undergo disulfide exchange with the newly formed disulfide bond. Thus the concentration of the MTS reagents must be decreased by dilution, centrifugation, or filtration.

4.4 Determination of rates of reaction of the MTS reagent For the Cys mutants which are inhibited by an MTS reagent, the rate of reaction can be calculated by determining the effect of reaction at several different concentrations of reagent. The extent of reaction is taken to be the extent of inhibition of binding after a fixed time, typically 2 min, with 5-6 concentrations of reagent. The fraction of initial binding, Y, is fitted to (span*e-kct) + plateau, where k is the second-order rate constant (inM-1S-1),c is the concentration of MTS reagent, t is the time (120 s), plateau is the residual binding at maximal concentration of the MTS reagent, and (span + plateau) = 1. A number of factors, including steric constraints and electrostatic potential, can contribute to differences in the rates of reaction of different substituted Cys, or of a given substituted Cys with different reagents (35). An affinity of the reagent for a site near the substituted Cys can also increase the rate of reaction by boosting the local concentration of reagent. We have observed this with MTSEA at particular positions in the D2 receptor, presumably because of an affinity of the ethylamine moiety for the dopamine binding site (17). We have also observed this with MTSET at a number of positions, and this effect can be explained by an interaction of the hydrophobic cation of MTSET with nearby aromatic groups in the receptor, which increases the local concentration of MTSET (15, 19).

4.5 Protection of substituted cysteines by bound ligand Reaction with MTS reagents of substituted cysteines at or near the binding site should be retarded in the presence of bound ligand. To assess the ability of substrate or inhibitor to retard the rate of reaction of the MTS reactions, cells are incubated in the presence or absence of ligand, and then MTS reagents are added in the continued presence or absence of ligand. 27

Merrill M. Simpson et al.

Figure 2. Schematic representation of the reaction of the MTS reagents with a cysteine accessible in the binding-site crevice. The membrane is represented by a stippled rectangle; the binding-site crevice is indicated within the plane of the membrane, and the oval represents ligand. SSEX represents -SSCH2CH2X (where X is NH3-, N(CH 3 ) n ', or SO3-), which is covalently linked to the water-accessible cysteine sulfhydryl. In the bound state, represented in the upper right panel, ligand is reversibly bound within the bindingsite crevice. In the unbound stale, represented in the upper left panel, the binding site is unoccupied. After irreversible reaction with MTSEX, represented in the middle left panel, ligand binding is altered, as is shown in the bottom left panel. The cysteine sulfhydryl facing lipid or the interior of the protein does not react with MTSEX. Ligand retards the rate of reaction of receptor with MTSEX (middle right panel), thereby protecting subsequent ligand binding (bottom right panell.

Protocol 7.

Protection of substituted Cys by bound ligand

Equipment and Reagents • MTS reagant (see Protocol 6) . Binding buffer (see Protocol 4)

• 96-woll multiscreen plate with GF/B filters (Millipore)

Method 1. Pre-wet with 100 ml binding buffer a 96-well multiscreen plate containing GF/B filters (Millipore). 2, Remove the buffer by filtration under vacuum. 28

2: Probing the structure of receptor-binding sites 3. Add dissociated cells (100 ml) and 50 ml binding buffer, containing ligand or buffer, and incubate for 20 min at RT. 4. Add the appropriate MTS reagent (50 ml of 4x)a in the continued presence or absence of ligand, and incubate for 2 min at RT. 5. Stop reaction by removing the reagents by filtration. 6. Add 250 ml binding buffer per well. Gently mix the plate for 5 min on an orbital shaker at RT and then filter. 7. Repeat step 6 three times.b 8. Add 100 ml binding buffer, and 50 ml of cold ligand in buffer (for the determination of non-specific binding) or buffer (for the determination of total binding) per well. 9. Shake the plate for 5 min at RT to resuspend the cells, and add 100 ml of radioligand per well. 10. Incubate as appropriate, separate the bound from free radioligand by filtration, and wash twice with cold wash buffer. 11. Punch the filters into vials, and measure the radioactivity by liquid scintillation spectrometry.c 12. Protection is calculated as 1 - [(inhibition in the presence of ligand)/ (inhibition in the absence of ligand)]. a

It is important to note that this protocol examines the ability of a reversible reaction to slow an irreversible reaction. Thus, too high a concentration of reagent or too long a time of reaction will obscure the presence of protection. To facilitate determination of a change in the rate of reaction, the concentrations of sulfhydryl reagent should be chosen to produce, in the absence of ligand, approximately 70% of the maximal effect. The concentration of protecting ligand should be approximately 1000-fold greater than its K1 in each particular mutant. b The protecting ligand should be relatively hydrophilic to facilitate complete removal prior to the determination of residual binding. Controls testing for inhibition by residual protecting ligand without MTS reagent should be included. c The hydropure membrane support in Millipore 96-well filter plates avidly binds many small molecule radioligands. Thus, it is imperative to determine if the radioligand used binds to the hydropure backing. If so, the glass fibre filters must be counted without the backing. This is facilitated by complete drying of the filters prior to punching.

A decrease in the rate of reaction of a substituted Cys with an MTS reagent in the presence of ligand is most simply explained by steric exclusion. Not every residue which is protected, however, need contact ligand, as the ligand might protect residues deeper in the binding-site crevice by binding above them and preventing access of the MTS reagents deeper in the crevice. It is also difficult to rule out indirect conformational changes in the mechanism of protection. Such conformational changes are less likely to be caused by an antagonist than by an agonist, but are nonetheless still possible. 29

Merrill M. Simpson et al.

5. Interpretation of results 5.1 Assumptions of SCAM To interpret the results of SCAM we make the following assumptions: (1) the highly polar MTS reagents react much faster at the water-accessible surface of the protein than in lipid or in the protein interior; (2) in membrane-spanning segments, access of highly polar reagents to side-chains is only through the binding-site crevice; (3) the addition of -SCH2CH2X to a cysteine at the surface of the binding site alters binding irreversibly, and, reciprocally, (4) that for substituted cysteines that line the binding site, antagonists or agonists should retard the reaction with the MTS reagents.

5.2 Interpreting the effects of reaction with the MTS reagents The effects of the addition of -SCH2CH2X to the engineered cysteine could be a result of steric block, electrostatic interaction, or indirect structural changes. Thus, reaction could inhibit or potentiate binding. Regardless, an irreversible effect is evidence of reaction, and, therefore, of the accessibility of the engineered cysteine. This can be illustrated in the dopamine D2 receptor by the mutation of Asp108 (17). Mutation to cysteine of this residue at the extracellular end of the third membrane-spanning segment reduced the receptor's affinity for antagonist binding about threefold. Reaction of the positivelycharged MTSEA or MTSET at this position significantly inhibited binding. In contrast, reaction of the negatively charged MTSES restored the negative charge at this position, and shifted the affinity towards that of wild-type receptor, thereby increasing occupancy and potentiating binding. The fact that reaction can potentiate function necessitates care in experimental design; a potentiation of binding could be missed by measuring binding at too high a ligand concentration relative to the KD (36). In the dopamine D2 receptor, we have observed that reaction of MTSEA at certain positions has a much greater effect on the binding of particular ligands: for example, reaction of MTSEA with the highly reactive endogenous cysteine, Cys118, caused only a negligible decrease in the affinity for the antagonist haloperidol, but caused a 3000-fold decrease in the affinity for the antagonist sulpiride (37). If reaction of a substituted cysteine with an MTS reagent results in a negligible effect on the affinity of binding, it might be inferred that the residue was inaccessible, resulting in a false negative determination. Thus, while it seems unlikely that a residue forming the surface of the binding-site crevice could be covalently modified by the addition of the charged -SCH2CH2X without interfering with binding, such a result is possible. Moreover, a residue which is water-accessible might not react with the reagents due to steric factors. These potential complications demonstrate the importance of systematically mutating to cysteine consecutive residues along 30

2: Probing the structure of receptor-binding sites an entire membrane-spanning segment; while mutation of any individual residue might be subject to potential misinterpretation due to silent reaction or steric factors, this is unlikely to be a systematic problem affecting the overall pattern of accessibility of multiple residues in a membrane-spanning segment.

5.3 Secondary structure To infer a secondary structure, we must assume that, if binding to a mutant is not affected by the MTS reagents, then no reaction has occurred, and that the side chain at this position is not accessible in the binding crevice (as discussed above, however, this may not universally be so). In an a-helical structure one would expect the accessible residues to form a continuous stripe when the residues are represented on a helical net. For example, in the third membrane-spanning segment of the dopamine D2 receptor, the pattern of accessibility is consistent with this membrane-spanning segment forming an ahelix with a stripe of about 140° facing the binding-site crevice (Figure 3) (17). In contrast, in a B-strand, one would expect every other residue to be accessible to the reagents. More complex or irregular patterns of accessibility can be more difficult to interpret, but these findings can also be rather informative, and suggest

Figure 3. Helical wheel (left) and helical net (right) representations of the residues in and flanking the third membrane-spanning segment of the dopamine D2 receptor, summarizing the effects of MTSEA on [3H]YM-09151-2 binding. Reactive residues are represented by squares, where the fill indicates the range of the second-order rate constants in M-1S-1 for reaction with MTSEA: solid squares = k > 20; hatched squares = 20 > k > 10; striped squares = 10 > k > 3; open square = 3 > k > 1. Small open circles indicate that MTSEA had no effect on binding. The solid circle indicates no binding after cysteine substitution. D108 and 1109 are represented outside the a-helix in the loop from the second membrane-spanning segment.

31

Merrill M. Simpson et al. the presence of kinks, twists, and dynamic changes in the structure of the membrane-spanning segments (15,16,19, 22).

5.4 Conformational changes associated with receptor activation Conformational changes in a protein may result in changes in the accessibility of substituted cysteines as assessed by their rates of reaction with polar sulfhydryl-specific reagents. For example, residues lining the channel of the nicotinic acetylcholine receptor change in accessibility upon activation of the receptor and opening of the channel (7, 8). Similarly, it should be possible to determine changes in the accessibility of residues in GPCRs in different functional states. To identify activation-induced structural changes in the residues forming the surface of the binding-site crevice, we sought to determine the relative accessibilities of a series of engineered cysteines in the resting and activated receptor. Agonist cannot be used to activate receptor, however, because the presence of a ligand within the binding site would interfere with access of the MTSEA to the engineered cysteines. Alternatively, the activated state of the receptor can be achieved by using a constitutively active mutant (CAM) receptor as a background for further cysteine substitution. A CAM receptor is intrinsically active, and has a higher affinity for agonist than does the wildtype receptor (38). The high affinity state for agonist is typically associated with the activated receptor-G protein complex. That agonist affinity is higher in the CAM even in the absence of G protein suggests that the structure of the binding site of the CAM is likely to be similar to that of the agonist-activated wild-type receptor binding site (or more easily isomerizes to the active state). Thus, we can compare the resting and active forms of the receptor by determining the accessibility of substituted cysteines in the binding-site crevice in these two states, using wild-type receptor and a CAM as background constructs. MTSEA had no effect on the binding of agonist or antagonist to wild-type P2 receptor expressed in HEK 293 cells. This suggested that no endogenous cysteines are accessible in the binding-site crevice. In contrast, in the CAM B2 receptor, MTSEA significantly inhibited antagonist binding, and isoproterenol slowed the rate of reaction of MTSEA (22). This implies that at least one endogenous cysteine becomes accessible in the binding-site crevice of the CAM B2 receptor. Cys285, in the sixth transmembrane-spanning segment (TM6), is responsible for the inhibitory effect of MTSEA on ligand binding to the CAM (22). The acquired accessibility of Cys285 in the CAM may result from a rotation and/or tilting of TM6, associated with activation of the receptor. This rearrangement could bring Cys285 to the margin of the binding-site crevice, where it becomes accessible to MTSEA. Such a movement of TM6 upon receptor activation is 32

2: Probing the structure of receptor-binding sites consistent with the results of fluorescence spectroscopy studies in the B2 receptor (39, 40) and spin-labelling studies in rhodopsin (41), and suggests that the substituted-cysteine accessibility method in a CAM background is a powerful approach for probing conformational change in these receptors. The membrane-spanning segments not only form the binding-site crevice, but also constitute the transduction pathway from the binding site to the intracellular loops which interact with G protein. Agonist binding in the crevice must, therefore, alter the conformations or orientations of at least some of the membrane-spanning segments. As described above, we have identified a cysteine in TM6 which becomes accessible in a CAM background. By extending this method, it should now be possible to map the activation-related changes in accessibility of all the residues that form the surface of the bindingsite crevice.

References 1. Todd, A. P., Cong, J., Levinthal, F., Levinthal, C, and Hubbell, W. L. (1989). Proteins, 6, 294. 2. Altenbach, C., Marti, T., Khorana, H. G., and Hubbell, W. L. (1990). Science, 248, 1088. 3: Jakes, K. S., Abrams, C. K., Finkelstein, A., and Slatin, S. L. (1990). J. Biol. Chem., 265, 6984. 4. Careaga, C. L., and Falke, J. J. (1992). Biophys. J., 62, 209. 5. Pakula, A. A., and Simon, M. I. (1992). Proc. Natl. Acad. Sci. USA, 89, 4144. 6. Jung, K., Jung, H., Wu, J., Prive, G. G., and Kaback, H. R. (1993). Biochemistry, 32, 12273. 7. Akabas, M. H., Stauffer, D. A., Xu, M., and Karlin, A. (1992). Science, 258, 307. 8. Akabas, M. H., Kaufmann, C., Archdeacon, P., and Karlin, A. (1994). Neuron, 13, 919. 9. Akabas, M. H., and Karlin, A. (1995). Biochemistry, 34, 12496. 10. Xu, M., Covey, D. F., and Akabas, M. H. (1995). Biophys. J., 69, 1858. 11. Xu, M., and Akabas, M. H. (1993). J. Biol. Chem., 268, 21505. 12. Akabas, M. H., Kaufmann, C., Cook, T. A., and Archdeacon, P. (1994). J. Biol. Chem., 269, 14865. 13. Yan, R. T., and Maloney, P. C. (1995). Proc. Natl. Acad. Sci. USA, 92, 5973. 14. Pascual, J. M., Shieh, C. C., Kirsch, G. E., and Brown, A. M. (1995) Neuron, 14, 1055. 15. Fu, D., Ballesteros, J. A., Weinstein, H., Chen, J., and Javitch, J. A. (1996). Biochemistry, 35, 11278. 16. Javitch, J. A., Fu, D., and Chen, J. (1995). Biochemistry, 34, 16433. 17. Javitch, J. A., Fu, D., Chen, J., and Karlin, A. (1995). Neuron, 14, 825. 18. Javitch, J. A., Li, X., Kaback, J., and Karlin, A. (1994). Proc. Natl. Acad. Sci. USA, 91, 10355. 19. Javitch, J. A., Ballesteros, J. A., Weinstein, H., and Chen, J. (1998). Biochemistry, 37, 998. 20. Schirmer, T., Keller, T. A., Wang, Y. F., and Rosenbusch, J. P. (1995). Science, 267, 512. 33

Merrill M. Simpson et al. 21. Brown, K. A., Howell, E. E., and Kraut, J. (1993). Proc. Natl. Acad. Sci. USA, 90, 11753. 22. Javitch, J. A., Fu, D., Liapakis, G., and Chen, J. (1997). J. Biol. Chem., 272, 18546. 23. Ferrer, J. V., and Javitch, J. A. (1998). Proc. Natl. Acad. Sci. USA, 95, 9238. 24. Olami, Y., Rimon, A., Gerchman, Y., Rothman, A., and Padan, E. (1997). J. Biol. Chem., 272, 1761. 25. Seal, R. P., and Amara, S. G. (1996). Soc. Neurosci. Abstr., 22, 1575. 26. Rees, S., Coote, J., Stables, J., Goodson, S., Harris, S., and Lee, M. G. (1996). BioTechniques, 20, 102. 27. Creighton, T. E. (1993). Proteins: structures and molecular properties, W.H. Freeman and Co., New York. 28. Chothia, C. (1976). J. Mol. Biol, 105, 1. 29. Levitt, M. (1978). Biochemistry, 17, 4277. 30. Chou, P. Y, and Fasman, G. D. (1977). J. Mol. Biol, 115, 135. 31. Roberts, D. D., Lewis, S. D., Ballou, D. P., Olson, S. T., and Shafer, J. A. (1986). Biochemistry, 25, 5595. 32. Stauffer, D. A., and Karlin, A. (1994). Biochemistry, 33, 6840. 33. Holmgren, M., Liu, Y., Xu, Y., and Yellen, G. (1996). Neuropharmacology, 35, 797. 34. Yan, R. T, and Maloney, P. C. (1993). Cell, 75, 37. 35. Karlin, A., and Akabas, M. H. (1998). In Methods in Enzymology (ed. P. M. Conn.). Vol. 293, p. 123. Academic Press, London. 36. Javitch, J. A. (in press). In Methods in Enzymology (ed. S. G. Amara.). Vol. 296. Academic Press, London. 37. Javitch, J. A., Fu, D., and Chen, J. (1996). Mol. Pharmacol, 49, 692. 38. Samama, P., Cotecchia, S., Costa, T., and Lefkowitz, R. J. (1993). /. Biol Chem., 268, 4625. 39. Gether, U., Lin, S., Ghanouni, P., Ballesteros, J. A., Weinstein, H., and Kobilka, B. K. (1997). EMBOJ., 16, 6737. 40. Gether, U., Lin, S., and Kobilka, B. K. (1995). J. Biol. Chem., 270, 28268. 41. Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. L., and Khorana, H. G. (1996). Science, 274, 768.

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3

Post-translational acylation of signal transducing polypeptides: palmitoylation of G protein-coupled receptors MICHEL BOUVIER, ULLA PETAJA-REPO,THOMAS LOISEL, and CHARLENE BELANGER

1. Introduction In recent years, a number of proteins involved in signal transduction have been found to be acylated by fatty acids or prenyl derivatives. Three major classes of fatty acylation have been characterized. These are: (a) the Nterminal myristoylation of glycine residues through amide linkage; (b) the prenylation of cysteine residues by the formation of thioether links to farnesyl or geranylgeranyl moieties; and (c) the palmitoylation which occurs through the thioesterification of cysteine residues. In contrast to myristoylation and prenylation, which typically occur co-translationally, palmitoylation is a genuine post-translational modification (1). The chemical reactivity of sulfhydryls and thioester linkages have led several investigators to propose that protein palmitoylation may represent a reversible modification which could undergo dynamic regulation. In fact, rapid turnover of the covalently attached palmitate has been reported for several proteins, and this turnover is modulated by external stimuli (2-4). Palmitoylation has been found to be particularly prevalent for proteins involved in processes such as cell adhesion, cell growth, and signal transduction, raising the intriguing possibility that it could play regulatory roles in these events. Palmitoylated proteins include p21ras (5), GAP-43 (6), the endothelial nitric oxide synthase (7), several tyrosine kinases belonging to the p60src family, such as p56lck (8), p59fyn, p55fgr, and p56hck (9), as well as many G protein a-subunits (10), their cognate receptors (11), and the G protein-coupled receptor kinases (12, 13). Palmitoylation of G protein-coupled receptors (GPCRs) was first demonstrated for the visual receptor rhodopsin (14). Indeed, as early as 1984, it was

Michel Bouvier et al. observed that bovine rhodopsin could covalently incorporate palmitate following incubation of rod outer segments with [3H]palmitate. Four years later, physico-chemical analysis of a C-terminal peptide fragment derived from bovine rhodopsin led to the identification of cysteine-322 and -323 as the main palmitoylation sites for this protein (15). Primary sequence comparison revealed that at least one of these two cysteine residues is conserved in a similar position within the carboxyl tail of most GPCRs. Such a high level of conservation led several groups to investigate whether palmitoylation could represent a general modification with functional consequences for this class of signalling polypeptides. Although the occurrence of palmitoylation has been shown for a number of hormonal GPCRs, including the B2- and the a2-adrenergic (16, 17), the Dl and D2 dopamine (18, 19), the serotonin 5-HT1B and 1A (20, 21), the thyrotropin-releasing hormone (22), the V2 vasopressin (23), the m2 muscarinic (24), the lutropin-choriogonadotropin (25) as well as the endothelin receptors A and B (26, 27), and the glutamate metabotropic-4 receptor (28), progress in studying additional receptors and the dynamic regulation of this modification has been hampered by a number of technical difficulties, some of which are inherent to the nature of the GPCRs. For example, the low level of expression of most hormonal GPCRs makes it difficult to assess the palmitoylation state of endogenously expressed receptors. Studying their palmitoylation is also complicated by the fact that, contrary to most other palmitoylated proteins which are soluble (the palmitoylation being proposed as essential for their targeting from the cytosolic fraction to the inner face of the plasma membrane), the receptors are integral plasma membrane proteins that are fairly hydrophobic, thus complicating their purification and handling. Finally, the sparse knowledge concerning the enzymes and molecular processes that control palmitoylation has complicated the development of standardized in vitro palmitoylation assays. In this chapter, we describe a number of experimental approaches and protocols that have been developed over the last ten years or so to circumvent some of the problems described above, and which allow the study of various aspects of GPCR palmitoylation.

2. Detection of receptor palmitoylation One of the major difficulties in directly studying any post-translational modification of GPCRs is the low amount of these proteins that can be isolated from tissues or cell lines naturally expressing them. This problem is particularly important when considering palmitoylation, because of the relatively low specific activity of [3H]- or [14C]palmitate, the tracers used to detect palmitoylation, in comparison with the 32Pi used to detect phosphorylation, for example. One approach that we and others have selected to circumvent this problem has been to use heterologous expression, allowing various levels 36

3: Post-translational acylation of signal transducing polypeptides of over-expression. In addition, to facilitate purification of sufficient quantities of receptor, these systems permit the study of selected receptor subtypes in chosen genetic backgrounds.

2.1 Heterologous expression systems Two major heterologous expression systems have been used to study the palmitoylation of GPCRs: baculovirus-insect cells, and mammalian cells. 2.1.1 Insect cells In the last few years, recombinant Autographica californica nuclear polyhedrosis viruses (baculovirus) have been successfully used to direct high-level expression of many GPCRs in insect cells, in most cases using Spodoptera frugiperda (Sf9) cells (see also chapter 7). Proteins expressed using this system have largely been found to maintain their normal characteristics, including specific post-translational modifications such as palmitoylation and phosphorylation. Receptor levels reaching 20-100 pmol mg-1 of protein have been reported for many GPCRs using the insect system. This high level of expression, rarely reached in mammalian heterologous expression systems, has been perceived as a major advantage for the study of palmitoylation, and many studies have used the insect system to facilitate the detection of the palmitoylated receptor. As an example, the first demonstration of [3H]palmitate incorporation into the human B2-adrenergic receptor required more than 6 weeks of autoradiography, following metabolic labelling of mammalian cells expressing 2 pmol of receptor per milligram of membrane proteins (16). The use of Sf9 cells expressing 10-50 times higher levels significantly reduced the time required to visualize the modification. However, since gene expression occurs through the infectious cycle of the virus, it is transient, the cells dying from the infection several days later. Defining a time window that allows for high levels of expression, while maintaining close to 100% cell viability, is therefore of primary importance for these systems. The optimal time of infection should be determined for each recombinant baculovirus but, in most cases, maximal levels of expression are found at 48 h post-infection, a time at which the proportion of viable cells, as assessed by their ability to exclude Trypan Blue, is usually greater than 90%. Also, to maintain a reproducible level of expression from one experiment to the next, virus stocks must be properly maintained and propagated, and their titres verified regularly. Additional details about baculovirus and Sf9 cells can be found in O'Reilly et al. (29) (see also Chapter 7). i. Construction of the recombinant baculovirus. Typically, to prepare the recombinant baculovirus encoding the GPCR of interest, the coding region of the baculovirus polyhedrin gene is replaced with that of the selected receptor. For this purpose, the receptor DNA fragment is ligated into a baculovirus recombination vector such as pJVETLZ at the NheI site, yielding the pJV-receptor vector. Transfer of the receptor and B-galacto37

Michel Bouvier et al. sidase coding sequences from pJV-receptor to the baculovirus genome is then achieved by homologous recombination with the linear AcMNPV genome, by cotransfection in Sf9 cells using a Cationic Liposome Mediated Transfection kit (Invitrogen). Recombinant baculoviruses are purified by plaque assay, using the B-galactosidase detection system (30). Confirmation of receptor expression can then be assessed by radio-ligand binding assay or Western blot analysis. The availability of commercial systems to clone and purify recombinant baculovirus, such as the Bac-to-Bac kit (Life Technologies) makes the generation of these viruses relatively easy. ii. Culture and infection of Sf9 cells. Sf9 cells are grown in Grace's insect medium (Gibco) supplemented with fetal bovine serum (FBS, 10% v/v) at 27°C. Although Sf9 cells can also be grown as monolayers in flasks, metabolic labelling is more easily carried out when they are grown in suspension using Erlenmeyer flasks placed in an orbital shaker in the presence of pluronic acid (0.001%) to prevent cell tearing due to agitation. To permit expression of the receptor at sufficient levels in the entire cell population, infection should be carried out at a multiplicity of infection (MOI) of approximately 5 recombinant baculovirus molecules per Sf9 cell when they are in a logarithmic phase of growth at a density of 1-2 X 106 cells ml-1 (see Chapter 7 for further details). 2.1.2 Mammalian cells Although the levels of expression achieved in mammalian cells following either transient or stable transfection protocols rarely reach those obtained in Sf9 cells, both these systems have been used successfully to study palmitoylation. The major advantage of transient over stable expression systems is that one does not need to go through the tedious process of selection and cellular cloning (4-6 weeks), and one can readily use the cells 48 h after transfection. Moreover, higher levels of expression are generally obtained using transient systems. However, stable expression systems provide homogeneous populations of cells that all express the receptor at the same level and allow for repeated experiments under identical conditions. In general, we feel that stable cell lines offer a better model system to study palmitoylation of GPCRs. These cells are generally obtained by transfecting the cells with expression plasmids that encode the receptors of interest, as well as a selectable marker such as an antibiotic resistance gene. Selection of the expressing cells is then realized by treatments with appropriate concentrations of the antibiotic. A large number of cell lines can be used to generate stable expression systems. Considerations in selecting a cell line should include the ease of cultivation of large quantities of these cells, the absence of endogenous expression for the receptor to be transfected, the presence of the signalling pathway(s) to which the receptor should be functionally coupled, the presence of other 38

3: Post-translational acylation of signal transducing polypeptides signalling molecules which could be considered in the course of the study, etc. Chinese hamster fibroblasts (CHW-1102, NIGMS GM0459; and CHO, ATCC CRL 9096), and human embryonic kidney cells (HEK-293, ATCC CRL 1573), stably expressing various GPCRs, have been used successfully to detect palmitoylation. For the B2-adrenergic receptor, stable expression levels reaching 10 pmol per mg of protein were obtained in HEK-293 cells using the pCDNA3 plasmid (Invitrogen), greatly facilitating the study of palmitoylation in mammalian cells. Once the cell line of choice has been generated, the culture conditions should be maintained as usual until one day before the experiments. In our case, CHW or HEK-293 cells are grown as monolayers in Dulbecco's minimum Eagle's medium (DMEM), supplemented with 10% FBS, penicillin (100 units ml-1), streptomycin (100 mg ml-1), fungizone (0.25 mg ml-1), and glutamine (2 mM), in an atmosphere of 95% air-5% CO2 at 37°C. In the case of stably transfected cell lines, we found that maintaining the selective pressure with the selectable antibiotic (in most cases neomycin) helps to maintain the level of receptor expression at stable levels for longer period of times (between 30 and 60 passages, depending on the clones).

2.2 Metabolic labelling The approach most widely used to study receptor palmitoylation is directly monitoring the covalent attachment of radiolabelled palmitate molecules to the receptor in situ, following isotopic labelling of the cellular palmitoyl-CoA pool. The next section describes the method used to perform such metabolic labelling. 2.2.1 Optimization of labelling The substrate involved in the palmitoylation reaction in cells is the metabolic intermediate palmitoyl-CoA. However, the polar characteristic of this activated lipid prevents its direct utilization in metabolic labelling experiments, since it can not cross biological membranes. The labelling of the cellular pool of palmitoyl-CoA is therefore achieved by incubating the cells with [3H]palmitate, which penetrates into the cells, and is transformed by ATP-driven esterification with extra-mitochondrial CoA-SH to yield palmitoyl-CoA. This newly formed palmitoyl-CoA can then be used as the palmitate donor in protein acylation reactions, but is also used in the fatty acid oxidation and elongation pathways in various cellular compartments, including the mitochondria. As a consequence of the large cellular pool of palmitoyl-CoA, its rapid metabolism in distinct metabolic pathways, and its subcellular compartmentalization, it is virtually impossible to determine the specific activity of the acyl-CoA pool that is used for the palmitoylation reactions. Moreover, its rapid turnover makes it very difficult to reach isotopic equilibrium for extended period of times. It follows from the previous observations that establishing the conditions that allow optimum steady-state labelling of the receptor is of 39

Michel Bouvier et al. primary importance, and that the time of labelling is a crucial parameter. The optimum time of labelling varies with the cell type used, and therefore must be established for each system considered. For instance, maximum [3H]palmitate incorporation into the B2-adrenergic receptor was observed following 1 h labelling in Sf9 cells (31), whereas 3 h labelling is required to attain maximal incorporation in HEK-293 cells (Lynda Adam and M. B., unpublished observation). When possible, labelling longer than 4 h should be avoided, to reduce the incorporation of the label into amino acids following metabolic degradation of the fatty acid, and use of the labelled acetyl-CoA in the amino acid biosynthetic pathways. Because tritium is a relatively weak radioisotope that is required in large quantities for detection by autoradiography, selecting a cell line that expresses a large number of receptors is of primary importance. For this reason and as indicated above, Sf9 cells have often been used as the model of choice to study palmitoylation. However, mammalian cells expressing 1 pmol of receptor per mg of membrane protein or more can also be used. 2.2.2 Solubilization and purification of the receptor Even in Sf9 cells that express as much as 50 pmol of receptor per mg of membrane proteins, the presence of a fairly large number of palmitoylated proteins prevents the detection of the palmitoylated receptor in crude membrane preparations. It follows that, after metabolic labelling, receptors must be solubilized and purified. Solubilization represents an important and delicate step in the isolation of the receptors. Indeed, only a few detergents (e.g. digitonin and n-dodecyl maltoside) preserve the ligand-binding activity of most GPCRs. This is important, since this biological activity may be used in affinity purification schemes. Moreover, maintaining the ligand-binding activity allows the measurement of the number of functional receptors throughout the purification steps. Such quantification is important for comparing the palmitoylation states in different conditions. The best detergent (or detergent mixture) and optimum Solubilization conditions should be determined for each receptor in a given cell type. However, either digitonin or n-dodecyl maltoside represent good starting choices, as they have been found to solubilize many distinct receptors successfully. The various approaches that are generally used to purify GPCRs can be classified in three categories: (a) methods that take advantage of the ligandbinding properties of the receptor and use an affinity resin; (b) immunoprecipitation techniques using polyclonal or monoclonal antibodies raised against the receptor sequence; and (c) methods based on the construction of fusion recombinant proteins between the receptor and epitopes specifically designed to allow purification. The use of an affinity resin requires that the receptor maintains its binding properties after Solubilization. When this is not the case, immunoprecipitation or an epitope-based purification scheme should be used. 40

3: Post-translational acylation of signal transducing polypeptides The following protocol describes a procedure to detect the palmitoylation of the human B2-adrenergic receptor expressed in Sf9 cells, using an affinity chromatography purification scheme. Procedures using mammalian cells and alternative purification procedures are described later in this chapter.

Protocol 1. Pulse metabolic labelling of the human B2-adrenergic receptor with [3H]palmitate in Sf9 cells Equipment and Reagents • Sf-9 cells • Recombinant baculovirus expressing the human B2-adrenergic receptor • [9, 10-3H] palmitic acid • Grace's insect medium (Gibco)

.

• Autoradiographic film Fluorographic enhancer . Centriprep and Centricon cartridges (Amicon) • Alprenolol-sepharose resin

Method 1. Infect 100 to 200 ml of Sf9 cells (2 X 106 cells ml-1), grown in suspension, with a recombinant baculovirus encoding the human 32AR at a multiplicity of infection (MOD of approximately five recombinant baculovirus molecules per Sf9 cell. 2. At 30 h post-infection, transfer the cells to serum-free medium for 18 h to stop cell growth and reduce metabolic activity.a 3. Following this starvation period, incubate 25 ml of Sf9 cell suspension (50 X 106 cells) for 1 h at 27°C in the presence of Grace's insect medium (Gibco) supplemented with 1% FBS, and add [9,103 H]palmitic acid [60 Ci mmol-1] dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 0.2 mCi ml-1, for periods of time varying between 15 min and 4 h.b 4. Terminate the labelling period by centrifugation at 500 g for 5 min at 4°C. From this point the samples are always kept at 4°C unless indicated otherwise. 5. Wash the pelleted cells twice with 50 ml of ice-cold phosphatebuffered saline (PBS), and disrupt them by sonication in 5ml of icecold buffer containing 5 mM Tris-HCI, pH 7.4, and a protease inhibitor cocktail composed of 2 mM EDTA, 5 mg ml-1 leupeptin, 10 mg ml-1 benzamidine, and 5 mg ml-1 soybean trypsin inhibitor (buffer A).c 6. Centrifuge the lysate at 500 g for 5 min, and centrifuge the supernatant again at 45000 g. Wash the pelleted membrane three times in buffer A. 7. To solubilize the receptor, resuspend the pelleted membrane preparations to a final concentration of 2 mg of protein per ml, in 2-4 ml of a 41

Michel Bouvier et al. Protocol 1.

Continued

buffer containing 100 mM NaCI, 10 mM Tris-HCI, 5 mM EDTA, pH 7.4, 0.3% digitonin (or 0.3% n-dodecyl maltoside) and protease inhibitors (as described above). Stir the suspension gently, using a magnetic stirring bar, for 90 min. 8. Remove the non-solubilized material by centrifugation at 100000 g for 60 min. You can use the supernatant containing the solubilized receptor immediately for the purification procedures, or quickly freeze it and keep it at -80°C for several days if necessary. 9. To purify the receptor, equilibrate an alprenolol-Sepharose resin (32, 33), 6 ml of gel per sample, with 50 ml of buffer B (100 mM NaCI, 10 mM Tris-HCI, 2 mM EDTA, pH 7.4, 0.05% digitonin [or 0.02% ndodecyl maltoside] and protease inhibitors). Then add the solubilized receptor (2-4 ml) to the affinity resin, and shake gently for 2 h at room temperatured to allow binding of the receptor to the matrix. 10. Following this batch-wise loading procedure, transfer the resin to 15 ml columns, let the soluble preparation flow through, and place the columns at 4°C.d Once the temperature of the resin has reached 4°C, wash the columns with 15 ml of an ice-cold buffer containing 500 mM NaCI, 50 mM Tris-HCI, 2 mM EDTA, pH 7.4, 0.05% digitonin (or 0.02% n-dodecyl maltoside). After the high salt washing, restore the original ionic strength by washing with 30 ml of buffer B. 11. In preparation for the bio-specific elution, return the columns to room temperature.d Then elute with 15 ml of buffer B containing 60 mM of alprenolol at a rate of 5 ml h-1 at room temperature, and collect the eluate on ice to reduce proteolytic degradation 12. Concentrate the eluted receptor to a final volume of 50 ml by ultrafiltration, using Centriprep and Centricon cartridges (Amicon). After assessing the number of receptors isolated,e prepare the samples for SDS-PAGEf immediately, or keep them at -80°C for a few days following quick-freezing in liquid nitrogen. 13. Following electrophoresis, incubate the fixed gels with a fluorographic enhancerg for 30 min, dry, and expose the gel to Kodak XAR5 or Biomax-MS films with intensifying screens at —80°C for several days. 14. Analyse the fluorogram obtained by densitometry, using a ladder of [3H]palmitic acid blotted on a Whatman paper as an internal fluorography control.h a

Good labelling of the receptors can also be achieved without the starvation period. Once the optimum labelling time has been established, experiments can be carried out using that time. For the p2-adrenergic receptor expressed in Sf9 cells, the maximal labelling was observed following a 1 h labelling period, b

42

3: Post-translational acylation of signal transducing polypeptides c

ln some cases, adding PMSF, bestatin, and aprotinin may be necessary to prevent proteolysis completely. d To favour rapid binding on- and off-rate of the receptor to the resin, the loading and elution procedures are carried out at room temperature. However, to slow down the off-rate during the washing procedures, the resin temperature is reduced to 4°C to prevent non-specific elution of the receptor. e After desalting the purified sample on a G-50 gel filtration column to remove alprenolol, B2AR recovery can be measured by soluble binding using [125I] cyanopindolol (CYP) as the radioligand (34). f The thioester link between the receptor and the palmitate moiety is somewhat sensitive to reducing agent, and it may be preferable to carry out the SDS-PAGE under non-reducing conditions. However, many GPCRs will tend to form high molecular weight aggregates that do not penetrate the polyacrylamide gels under non-reducing conditions. Using a low concentration of reducing agents, such as 1% (B-mercaptoethanol or 5 mM DTT, is a good compromise that does not significantly affect the palmitoylation of most proteins. Boiling the samples is not recommended, as it may promote more aggregation. g Using this protocol, incorporation of tritiated palmitate can be detected after one to two weeks of exposure. A commercial fluorographic enhancer can be advantageously substituted by a solution of 1 M salicylic acid. h Fluorography is linear only over a small range of signal intensity. Blotting standardized amounts of [3H]palmitate onto a Whatman paper allows the generation of a standard curve that facilitates quantification.

Unfortunately, affinity resins that permit bio-specific purification are not available for all GPCRs and, as mentioned above, solubilization does not always permit preservation of the binding activity of the receptor. This has led to the use of alternative approaches. Although immunoprecipitation, using antibodies raised against receptors, would be the technique of choice to purify GPCRs, attempts to raise antibodies against these receptors have generally failed to produce high affinity antibodies that can be used satisfactorily in immuno-affinity purification procedures. To date, only a few such antibodies have been generated. To circumvent this problem, many investigators have constructed recombinant receptors fused in frame with sequences encoding specific epitopes that can be recognized by commercially available antibodies. Epitopes derived from parts of the c-myc and the haemagglutinin coding sequences, as well as the FLAG™ epitope, are among the most popular. Antibodies that have been used successfully to immunoprecipitate epitopetagged receptors include the anti-c-myc 9E10 antibody (Santa Cruz Biotechnology), the anti-haemagglutinin 12CA5 antibody (Roche Diagnostics), and the anti-FLAG M2 antibody (Sigma). In addition to immuno-reactive epitopes, stretches of six or more histidines have also been used. These polyhistidine epitopes interact by a chelation reaction with high affinity to nickel, and thus allow purification of proteins bearing this epitope using an agarose resin coupled to nickel (Ni-NTA-agarose, Qiagen). The poly-Histagged receptors can be eluted from the resin using neutral imidazole, which preserves the binding activity of the receptor. Theoretically, the epitopes can be introduced anywhere within the coding sequence of the receptor. However, positioning them either at the N- or C-terminus has generally been favoured over an insertion within the core of the receptors, with the idea that 43

Michel Bouvier et al. (a) it would result in a greater accessibility of the epitopes to the antibodies or nickel, and (b) it should have less detrimental effects on the 3D structure of the receptors. Successful purification schemes have been developed using both N- or C-terminally positioned epitopes. In addition to permitting immunopurification of the receptors, the presence of the epitope allows visualization of the receptor by immunoblotting throughout the purification steps. Protocol 10 will describe how the FLAG epitope positioned at the Cterminus of the human 8-opioid receptor can be used to study the palmitoylation of this receptor in HEK-293 cells. Now, we will describe how the polyHis epitope can also be used to study palmitoylation of the |32-adrenergic receptor in Sf9 cells.

Protocol 2. Purification of the B2-adrenergic receptor using nickel-NTA-chromatography Equipment and Reagents • Recombinant baculovirus expressing the human B2-adrenergic receptor containing a hexa-histidine tag

• Sf-9 cells . Nickel-NTA-agarose (Qiagen)

Method 1. Infect 100-200 ml of Sf9 cells (2 X 106 cells ml-1), grown in suspension, with a recombinant baculovirus encoding the human |32-adrenergic receptor which is tagged at its N-terminus with six histidine residues. Use a multiplicity of infection (MOD of approximately five recombinant baculovirus molecules per Sf9 cell. 2. Repeat Steps 2 to 8a of Protocol 1, replacing the Tris-HCI buffer with a 25mM Hepes buffer, pH 7.4. 3. Load the solubilized membrane preparations on columns packed with 10 ml of Ni-NTA-agarose (Qiagen), at a rate of 0.5 ml min-1 in the presence of 10 mM imidazole to reduce the non-specific binding of proteins to the resin. All the chromatographic steps are carried out at 4 °C to prevent proteolytic degradation. 4. Wash the columns with 20 volumes of a buffer containing 25 mM Hepes (pH 7.4), 500 mM NaCI, 10 mM imidazole, 0.02% n-dodecylmaltoside, and the protease inhibitor cocktail (buffer C) at a rate of 1 ml min-1. 5. Re-equilibrate the columns to a lower ionic strength with 5 volumes of buffer C in which the NaCI concentration has been reduced to 100 mM. 44

3: Post-translational acylation of signal transducing polypeptides 6. Elute the poly-His-tagged receptor at 0.5 ml min-1 with 3 volumes of a buffer containing 25 mM Hepes (pH 7.4), 100 mM NaCI, 100 mM imidazole, 0.02% n-dodecyl-maltoside, 20% glycerol (v/v), and the protease inhibitor cocktail. 7. Treat the purified receptor as in Protocol 1 (steps 12-14). a

The solubilization and all chromatographic steps should be carried out in the absence of EDTA or EGTA. These chelating agents would bind to nickel and strip it away from the resin.

Immuno-purification and nickel-based chromatography methods usually lead to overall purification yields that are higher than those attained by affinity chromatography (yields can easily reach 85% of the solubilized receptor, compared to ~50% for the alprenolol-Sepharose affinity method described in Protocol 1). Generally, however, more contaminating bands may accompany the receptor following these epitope-tag-based procedures. Identification of the palmitoylated band(s) that correspond to the receptor thus becomes particularly important. Western blot analysis of the same samples, using either an anti-tag antibody or an antibody directed against the receptor itself, can be used to identify directly the receptor band. Another approach that permits the identification of the receptor is photo-affinity labelling using radiolabelled photo-reactive ligands, or chemical cross-linking if peptide ligands are available for the receptor of interest.

2.3 Characterization of incorporated radiolabel The most direct way to identify the nature of the fatty acid incorporated into the receptor would certainly be analysis by mass spectrometry (35). However, simpler approaches, requiring considerably less purified protein, can satisfactorily confirm the identity of the [3H]palmitate incorporated. First, the covalent nature of the attachment of the labelled lipid to the receptor can be confirmed by assessing the resistance of the labelling to organic extraction. Noncovalently bound lipids would be extracted from the purified receptor.

Protocol 3. Organic extraction of non-specifically adsorbed [3H]lipids Method 1. Incubate a purified and concentrated receptor preparation, obtained as in Protocol 1 or 2 from metabolically labelled cells, in a mixture of chlorofornrmethanol (2:1, v/v), and mix vigorously.

45


Continued

2. After a 30 min incubation at room temperature, centrifuge the mixture at 4500 g to pellet the proteins. 3. Repeat the above extraction and centrifugation steps twice with the same solvents, three times with a mixture of chloroform:methanol: water (1:1:0.3, v/v/v), and finally once with methanol alone. After each extraction step, incubate the mixture for 10 min at room temperature. 4. Recover the pelleted proteins, solubilize them in SDS-PAGE sample buffer, and prepare the samples for electrophoresis and fluorography as in Protocol 1.a a The detection of tritium incorporation into the protein following the organic extraction would confirm the covalent attachment of the lipid moiety to the receptor.

The nature of the covalent link between the label and the receptor can also be deduced from the fact that, unlike the N-terminal amide linkage or the Cterminal thioether linkage of myristoylation and prenylation, respectively, thioesterification of cysteine by palmitic acid is sensitive to hydroxylamine. Sensitivity to hydroxylamine can be determined by treating either the soluble purified protein or, alternatively, the polyacrylamide gel following electrophoresis.

Protocol 4.

Hydroxylamine treatment of purified receptor

Equipment and Reagents • 1M hydroxylamine, pH7.0

• Centricon 30 cartridges (Amicon)

Method 1. Incubate the purified and concentrated receptor preparation, obtained in Protocol 1 or 2 a, with a 1 M Tris solution, with or without hydroxylamine, at a final concentration of 1 M, and pH of 7.0. 2. After an incubation for 1 h at room temperature, desalt and concentrate the samples using Centricon 30 cartridges (Amicon). 3. Prepare the concentrated samples for SDS-PAGE and fluorography as above.b • Samples that have been extracted with organic solvent, as in Protocol 3, can also be used. b Alternatively, the sensitivity of the labelling to hydroxylamine can be tested directly on fixed polyacrylamide gels following SDS-PAGE of the purified labelled receptors. For this, incubate the fixed gel with a 1 M hydroxylamine solution containing 1 M Tris, at a final pH of 7.0, for 16h, wash extensively, dry, and expose for fluorography.

46

3: Post-translational acylation of signal transducing polypeptides Third, analysis of the [3H]lipids incorporated into the receptor can also be performed using ascending chromatography. Protocol 5. Thin layer chromatography (TLC) of the[3H]lipid incorporated into the receptor Equipment and Reagents • Trypsin . Silica gel TLC plates (Aldrich)

. pH] lipid standards . 1M sodium salicylate

Method 1. Following SDS-PAGE of the purified labelled receptor, identify the receptor band using the fluorogram, and excise it from the polyacrylamide gel.a 2. Homogenize and digest the band in 5 ml of a solution containing 0.1 M (NH4)HCO3, (pH 7.7) with 0.3 mg ml-1 of trypsin. Incubate for 15 min at 37°C. 3. Acidify the digest with HCI, and extract with 1 volume of hexane. 4. Lyophilize the extract, and treat with 1 ml of 1 M KOH for 12 h at 37°C to cleave the attached lipids. 5. Concentrate the extract under nitrogen, and apply to a silica gel TLC plate (Aldrich) along with tritiated lipid standards in parallel lanes.b 6. Develop the chromatogram with hexane:ethyl acetate:acetic acid (80:20:1, v/v/v). 7. Air-dry the silica plates, immerse them in a 1 M salicylate solution for 1 h, and seal them into a hybridization bag prior to autoradiography.c a

The same protocol can be apply to the purified labelled receptor before it is resolved by SDSPAGE. b The identity of the palmitate is confirmed by directly comparing the chromatographic mobility of the labelled lipid cleaved from the receptor with that of commercial standards. Reverse-phase HPLC could be used instead of TLC. c Alternatively, the solid support of the silica gel can be cut into small slices, and directly counted by liquid scintigraphy.

3. Kinetics of palmitoylation 3.1 Pulse labelling As indicated above, studying the dynamics of palmitoylation is complicated by the fact that the cellular pool of palmitoyl-CoA is large and turns over very rapidly. It follows that the kinetics of receptor palmitoylation observed in pulse-labelling experiments is largely dependent on the rate of incorporation of the [3H]palmitate into the cells, its thioesterification with Co-A-SH, and the 47

Michel Bouvier et al. metabolic fate of [3H]palmitoyl-CoA once it is formed. Until now, it has not been possible to demonstrate that true isotopic equilibrium of the palmitoylCoA pool occurs in usual metabolic labelling experiments. Consequently, useful information can be gathered by assessing the entry of [3H]palmitate into cells, and its incorporation into cellular lipids. For this purpose, wholecell lipid labelling must be studied under the same metabolic labelling conditions that are used for assessing receptor palmitoylation. Protocol 6. Whole cell lipid labelling in Sf9 cellsa Equipment and Reagents • 1M Salicylate . [3H] lipid standards • Silica gel TLC plates (Aldrich)

• Speedvap (or similar) centrifugal vacuum evaporator

Method 1. Aliquot 1 ml of Sf9 cells that were metabolically labelled as in Protocol 1, and harvest them by centrifugation at 500 g for 5 min at 4°C.b 2. Rinse the cells twice with ice-cold PBS, and resuspend them in 200 ml of this buffer before lysing the cells by sonication.c 3. Extract total cell lipids by adding 200 ml of CHCI3:methanol:H20 (5:5:1, v/v/v) and mixing vigorously. 4. Separate the organic and the aqueous phase by centrifugation at 500 g for 5 min. Quantitatively recover the organic phase, repeat the extraction procedure twice, and pool the fractions. 5. Dry the pooled organic phases under vacuum, and resuspend the dried material in 100 ml of CHCI3:methanol:H20 (6:6:1, v/v/v). 6. Apply 20 ml of the resuspended lipid extract to a silica gel TLC plate for ascending chromatography, using n-butanol:CH3COOH:H20 (5:2:3, v/v/v) as the mobile phase. In parallel lanes load commercially available [3H]lipid standards.d 7. Immerse the silica gel plates for 1 h in a 1 M salicylate solution, and seal them into hybridization bags prior to autoradiography.a a

The same protocol can be used for mammalian cells. An aliquot of the supernatant can be collected and counted by liquid scintigraphy to determine the proportion of radiolabelled tracer that was not incorporated into the cells. c An aliquot of the cell lysate can be collected and counted by liquid scintigraphy to determine the proportion of radiolabelled tracer that was incorporated into the cells. d At least a [3H]palmitate standard should be included to identify the labelled band corresponding to cellular palmitate. In that system, fatty acids will migrate at the top of the chromatogram, while phospholipids will be found at the lower end of the TLC. e Because of the high level of radioactivity incorporated in the lipids, the use of salicylate is not required, and rapid direct detection can be achieved using the Biomax-MS films from Kodak with a low energy (LE) screen. b

48

3: Post-translational acylation of signal transducing polypeptides In the systems that we have studied, [3H]palmitate was found to enter the cells and to be rapidly metabolized. The rapid incorporation of tritiated fatty acids into cellular phospholipids and into the receptor indicates a fast turnover of both the cellular palmitoyl-CoA and receptor-bound palmitate. Comparing the labelling of the free palmitate with that of the receptor-bound palmitate over time allows determination of the time at which an apparent steady-state of labelling has been reached. A steady state is assumed when the labelling intensity of neither palmitate nor receptor changes with time. For palmitoylation of the human B2-adrenergic receptor in Sf9 cells, the steady state was reached between 40 and 80 min of labelling (31). Although determining the time required to attain the steady state is useful to select the optimal labelling conditions, it does not provide any real kinetic information about the receptor palmitoylation process. However, one such kinetic parameter, the turnover rate of the receptor-bound palmitate, can be obtained by performing pulse-chase labelling experiments.

3.2 Pulse-chase labelling Protocol 7. Determining the half-life of the receptor-bound palmitate Equipment and Reagents • [9, 10-3H] palmitic acid

• Grace's insect medium (Gibco)

Method 1. Incubate the cells with [9,10-3H]palmitic acid (60 Ci mmol-1) dissolved in DMSO to a final concentration of 0.2 mCi ml-1 (as indicated in Protocol 1) for the period of time required to attain steady-state labelling (as determined in Protocol 6). 2. At the end of the labelling period, harvest the cells by centrifugation at 500 g for 5 min, and rinse them three times with 50 ml of PBS at room temperature. 3. Initiate the chase by adding 100 ml of fresh Grace's insect medium with 1% FBS, containing 0.2 M of unlabelled palmitate. 4. Continue the incubation at 27°Ca for times varying between 5 and 60 min. 5. Stop the chase by placing the cells on ice, harvest the cells by centrifugation, prepare membranes, and purify the receptor as indicated in Protocol 1. 6. Following SDS-PAGE and fluorography, calculate the half-life of the receptor-bound palmitate by analysing the decay of the labelling 49

Michel Bouvier et al. Protocol 7. Continued intensity of the receptor band, using the one compartment metabolic turnover equation: q(t) = q(t—>oo) + q(t = 0)e(-R)t where t is the time of incubation (in minutes), R is the rate of decay, and q represents the level of labelling (as a percentage of the control). The half-life is estimated as f when q(t) = 50%. * Incubation should be carried out at 37oC with the appropriate medium if mammalian cells are used.

To determine if the decay in [3H]palmitate labelling is the consequence of receptor-bound palmitate turnover, and does not reflect the turnover of the receptor protein itself, pulse-chase labelling with [35S]methionine-cysteine should be carried out in parallel experiments. For this purpose, cells are preincubated in Grace's supplemented medium deprived of methionine and cysteine for 30 min. Tran35S-Label (ICN) is then added at a concentration of 100-200 mCi ml-1 to the medium for the time of labelling, and the chase is initiated by adding complete Grace's medium or DMEM containing 1 mM methionine and 1 mM cysteine.

3.3 Agonist-mediated regulation of receptor palmitoylation The effect of agonist stimulation on receptor palmitoylation can be assessed by various approaches. In pulse-labelling experiments, the effect of the agonist can be assessed by comparing the extent of receptor labelling observed in the presence and absence of various concentrations of agonists. The agonists can be added to the medium for the entire labelling period, or for shorter times (5-15 min) once steady-state labelling has been achieved. Alternatively, the effect of agonist stimulation on the turnover rate of the receptor-bound palmitate can be studied by adding agonists at the time of the chase in pulse-chase experiments. In any case, it is important to verify that the effects observed do not result from an agonist-promoted change in the specific activity of the palmitate donor pool. This is easily assessed by verifying that the treatment does not non-specifically influence the incorporation of [3H]palmitate into all the proteins.

Protocol 8. Incorporation of [3H]palmitate into total proteins Equipment and Reagents • 1M sodium salicylate

• Densitometer

50

3: Post-tmnslational acylation of signal transducing polypeptides Method 1. Aliquot 30 ml of Sf9 cells that have been metabolically labelled or chased in the presence and absence of agonist, and harvest them by centrifugation at 500 g for 5 min at 4°C, 2. Rinse the cells twice with ice-cold PBS, and resuspend them in 5ml of this buffer before lysing the cells by sonication. 3. Prepare membranes as indicated in Protocol 1, and directly solubilize them in SDS-PAGE sample buffer. 4. After SDS-PAGE separation, incubate the fixed polyacrylamide gel for 30 min in a solution of 1 M salicylate, and expose it to an autoradiographic film for several hours. 5. Assess the intensity of labelling of the various bands by densitometric analysis.

4. Assessing the palmitoylation state of the receptor at the cell surface Several studies have shown that agonist stimulation can selectively modulate the palmitoylation state of GPCRs (19, 36). More specifically, it has been proposed that agonists promote an increase in the turnover rate of receptorbound palmitate (31). This suggests that palmitoylation-depalmitoylation cycles can occur at the cell surface where the receptor is accessible to the agonists. To assess directly the palmitoylation occurring at the plasma membrane level, we developed a procedure that allows detection of the incorporation of [3H]palmitate into only those receptors that are expressed at the cell surface (U. P-R and M.B., manuscript in preparation). For this, we took advantage of the possibility of biotinylating cell surface proteins, using a water-soluble reagent that does not cross the plasma membrane, before carrying out metabolic labelling with [3H]palmitate. Following the metabolic labelling, cell-surface proteins can be purified through a streptavidin affinity chromatographic step, and the receptor subsequently purified from this mixture. The following protocols describe this approach, using the human 8opioid receptor expressed in mammalian HEK-293 cells. Because the 8-opioid receptor does not readily retain its binding properties following solubilization, a receptor construct harbouring a Flag epitope at its C-terminus was used to allow immunopurification.

51


Biotinylation of cell-surface receptor and metabolic labelling

Equipment and Reagents • HEK293 cells expressing a Flag™-tagged 8-opioid receptor

• Sulfo-NHS-biotin (Pierce) • [9,10-3H] palmitic acid

Method 1. Grow HEK-293 cells expressing the Flag-tagged 8-opioid receptor in 150 cm2 flasks until nearly confluent, and wash them three times with PBS containing calcium and magnesium.a 2. Place the flasks on ice, and add 0.5 mg of sulfo-NHS-biotin (Pierce) per ml to the cells into a final volume of 6 ml of PBS. Incubate for 30 min at 4°C while shaking gently.b 3. Stop the biotinylation reaction by adding Tris-HCI (1 M, pH 7.4) to a final concentration of 50 mM, and incubate for 10 min at 4°C. 4. Wash the cells three times with 10 ml of PBS containing 50 mM TrisHCI, pH 7.4; finally add 10 ml of serum-free DMEM, and let the cells warm up by placing the flask at 37°C. 5. Initiate the metabolic labelling by adding [9,10-3H]palmitic acid (60 Ci mmol-1) in serum-free DMEM (10ml per 150 cm2 flask) to a final concentration of 0.4 mCi ml-1 in the presence or absence of the agonist Leu-enkephalin, for times varying between 15 and 120 min.c 6. Stop the labelling by aspirating the medium and washing the cells three times with 10 ml of ice-cold PBS. 7. The cells can then be quickly frozen and kept at —80°C, or processed immediately for purification. aThe presence of calcium and magnesium in the washing buffer helps to maintain the attachment of the cells to the flask. bWater-soluble biotinylation reagents with several different reactive groups are available from Pierce. Biotin can thus be coupled to either lysine or cysteine residues, or even to carbohydrate groups of the receptors. Reagents should therefore be selected keeping in mind which coupling would be the least detrimental to receptor function. cln HEK-293 cells, optimal labelling is obtained following a 60 min labelling period.

Protocol 10. Solubilization and purification of cell surface palmitoylated 5-opioid receptor from HEK-293 cells Equipment and Reagents • Magnetic stirrer • Streptavadin-agarose (Pierce)

• Anti-Flag™ peptide (Sigma) • Centricon cartridges (Amicon)

52

3: Post-translational acylation of signal transducing polypeptides Method 1. Following biotinylation and metabolic labelling, lyse the cells by sonication in 3 ml of a buffer containing 25 mM Tris-HCI (pH 7.4), 2 mM EDTA, 0.5 mM PMSF, 2 mM 1,10-phenanthroline, 2 mg ml-1 aprotinin, 5 mg ml-1 leupeptin, 5 mg ml-1 soybean trypsin inhibitor, 10 mg ml-1 benzamidine (buffer D). 2. Sediment the cell membranes by centrifugation at 45000 g for 20 min at 4°C, and wash the pellet twice in buffer D. 3. Solubilize the membrane proteins in 1 ml of a buffer containing 0.5% n-dodecyl maltoside, 25 mM Tris-HCI (pH 7.4), 140 mM NaCI, 2 mM EDTA, 0.5 mM PMSF, 2 mM 1,10-phananthroline, 2 mg ml-1 aprotinin, 5 mg ml-1 leupeptin, 5 mg ml-1 soybean trypsin inhibitor, 10 mg ml-1 benzamidine (buffer E) for 60 min at 4°C on a magnetic stirrer. 4. Remove the non-solubilized material by centrifugation at 100000 g for 60 min. 5. To the solubilized fraction, add 50 ml of streptavidin-agarose resin (Pierce)a in a 1.5 ml Eppendorf tube, and incubate for 2 h at 4°C to allow batchwise binding of the biotinylated proteins to the resin. 6. Centrifuge the Eppendorf tube in a microfuge for 1 min at 1000 g to sediment the resin. Aspirate and discard the supernatant. Wash the resin twice with 0.5 ml of Buffer E, four times with 0.5 ml of buffer E in which the concentration of n-dodecyl maltoside has been reduced to 0.1% (Buffer F), and finally once with 0.5 ml of a 25 mM Tris-HCI (pH 7.4) solution. After each washing procedure, recover the resin by centrifugation. 7. Initiate the elution of the biotinylated proteins by adding 100 ml of a solution containing 25 mM Tris-HCI (pH 7.4), and 1% SDS to the resin. Incubate for 15 min at room temperature, and 5 min at 95°C. Centrifuge and recover the eluate. Wash the resin with an additional 900 ml of Buffer E, centrifuge again, and pool this last supernatant with the 100 ml eluate. 8. To purify the Flag-tagged 8-opioid receptor from this mixture of cell surface biotinylated proteins, add 25 ml of anti-Flag(M2) antibodyagarose (Sigma), and incubate overnight at 4°C while maintaining gentle agitation. 9. Following the loading of the immuno-affinity resin, centrifuge and wash twice with 0.5 ml of Buffer E, and four times with 0.5 ml of Buffer F. 10. Initiate the final elution by adding 150 ml of 175 mg of Flag-peptide (Sigma) per ml of Buffer F, and incubate for 10 min on ice. Centrifuge the resin and recover the supernatant. Repeat this elution procedure 53


Continued

two more times, and pool the three eluates. Concentrate the samples using Centricon cartridges, and prepare them for SDS-PAGE.b 11. Detection of the palmitoylated receptor is carried out by fluorography, as described in Protocol 1. aImmobilized monomeric avidin (Pierce) can also be used as an affinity matrix if it is advantageous to maintain functional activity of the receptor. Indeed, biotinylated proteins can be eluted from this resin with biotin in non-denaturing conditions. b This elution procedure allows specific elution of the Flag-tagged receptor. However faster and less expensive elution can be achieved by incubating the immuno-affinity resin directly in 100 MI of SDS-PAGE sample buffer, 15 min at room temperature and 2 min at 95°C. The sample is then centrifuged, and the supernatant loaded on the PAGE. However, elution of non-specific bands can occur, and needs to be controlled.

5. In vitro palmitoylation of synthetic peptides As indicated in the introduction, very little is known about the mechanism controlling the palmitoylation state of GPCRs. Despite considerable efforts to characterize and purify the enzyme(s) catalysing the palmitoylation of GPCRs, palmitoyl-transferase activity directed at GPCRs has remained elusive. Several investigators have even suggested that a non-enzymatic reaction may play a role. In fact, autocatalytic transfer of palmitate from palmitoyl-CoA to specific cysteines within a protein has been shown to occur in vitro for at least one GPCR, rhodopsin (14), for G a subunits (37), and for several other proteins (38, 39). Interestingly, the sites found to be autocatalytically palmitoylated in vitro were identical to those identified in whole cell palmitoylation experiments, suggesting that the molecular determinants of palmitoylation selectivity may be contained in the primary sequence of the protein, independently of the presence of an enzyme. Therefore, in an effort to identify the primary sequence required to direct palmitoylation of a specific cysteine within a protein (C. B. and M. B., manuscript in preparation), we developed an in vitro autocatalytic palmitoylation assay using synthetic peptides of various compositions with [3H]palmitoyl-CoA as the palmitate donor. Protocol 11. [3H]palmitoyl-CoA synthesisa Equipment and Reagents • [9,10-3H] palmitic acid . CoA-SH, lithium salt (Sigma) • Acyl-CoA synthetase (Sigma)

• Slilica gel TLC plates (Aldrich) • Film and screens for autoradiography

54

3: Post-translational acylation of signal transducing polypeptides Method 1. In a final volume of 1 ml of a buffer containing 0.1 M MOPS-NaOH (pH 7.4), 20 mM MgCI2, 2 mM CHAPS, 10 mM ATP, and 1 mM DTT, incubate 0.5 mmol of [9,10-3H]-palmitate (~25 mCi) with 25 mmol of CoA-SH lithium salt (Sigma), in the presence of 0.2 units of acyl-CoA synthetase (Sigma) at 28°C for 1 h. 2. To assess the efficacy of the enzymatic [3H]palmitoyl-CoA synthesis, take a 1 ml aliquot for TLC analysis. 3. Deposit the aliquot on a silica-gel TLC plate, and develop the chromatogram using n-butanol:pyridine:acetic acid:H2O (45:30:9:36, v/v/v/v) as the mobile phase.b 4. Air-dry the silica plates, and expose them directly to Biomax-MS films from Kodak with a low energy (LE) screen for autoradiography.c 5. Keep the synthesis products at -20°C.d a3

[ Hlpalmitoyl-CoA is commercially available, but is significantly more expensive than [3H]palmitate. b In this chromatographic system, the palmitate migrates with the solvent front, whereas the palmitoyl-CoA is found half-way in the chromatogram (Rf = 0.5). On a routine basis, the synthesis yield is ~80%. cAlternatively, detection can be achieved by fluorography, following immersion of the plates in 1 M salicylic acid as previously described. dThe [3H]palmitoyl-CoA produced is stable for several months.

Protocol 12. Auto-palmitoylation of synthetic peptides Equipment and Reagents • [3H] palmitoyl-CoA (Amersham)

• Silica gel TLC plates (Aldrich)

Method 1. Following synthesis,a solubilize the peptide in 100% DMSO, and dilute it in a buffer containing 0.1 M MOPS-NaOH (pH 7.4)b and 5 mM DTT, to obtain a final concentration of 10 mM peptide and 10% DMSO. Keep the stock solution of the peptide at -20 °C under nitrogen. 2. Add 5 ml of peptide to 5 ml of [3H]palmitoyl-CoA (~0.1 mCi) in a final volume of 30 ml of a buffer containing 0.1 M MOPS-NaOH (pH 7.4). Incubate at 37°C for times varying between 15 and 150 min.c 3. To visualize the products of the reaction, separate the palmitoylated peptides from palmitoyl-CoA by TLC chromatography on silica gel plates, using n-butanol:pyridine:acetic acid:water (60:30:5:15, v/v/v/v) as the mobile phase.d 55


Continued

4. Detect the palmitoylated peptide by direct autoradiography as described above, and quantify the [3H]palmitate incorporated in the peptides by liquid scintillation counting, after cutting the band of the autoradiogram corresponding to the palmitoylated peptides.e aPeptides as short as eight amino acids were found to be good auto-palmitoylation substrates. The inclusion of DTT in the peptide stock solution and the conservation under nitrogen are required to prevent the formation of intermolecular disulfide bonds. bThe pH at which the reaction is carried out greatly influences the level of palmitoylation achieved, lower pH inhibiting the reaction. cWith most peptides, maximal palmitoylation is observed after 120 min. d ln this system, palmitoyl-CoA does not migrate away from the origin, facilitating the separation of the palmitoylated peptides. However, peptides with very polar properties may not migrate well. eThe covalent attachment of the palmitate, and the nature of the thioester linkage with the peptide, can be assessed by analysing the isolated palmitoylated peptide as indicated in Protocols 3 and 4.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Omary, M. B., and Trowbridge, I. S. (1981). J. Biol. Chem., 256, 12888. Bonatti, S., Giovanni, M. S., and Simons, K. (1989). J. Biol. Chem., 264, 12590. Omary, M. B., and Trowbridge, I. S. (1981). J. Biol. Chem., 256, 4715. Alvarez, E., Girones, N., and Davis, R. J. (1990). J. Biol. Chem., 265, 16644. Hancock, J. F., Paterson, H., and Marshall, C. J. (1990). Cell, 63, 133. Skene, J. H. P., and Virag, I. (1989). J. Cell Biol., 108, 613. Robinson, L. J., Busconi, L., and Michel, T. (1995). /. Biol. Chem., 270, 995. Paige, L. A., Nadler, M. J. S., Harrison, M. L., Cassady, J. M., and Geahlen, R. L. (1993). J. Biol. Chem., 268, 8669. Shenoy-Scarcia, A. M., Dietzen, D. J., Kwong, J., Link, D. C., and Lublin, D. M. (1994). J. Cell Biol., 126, 353. Linder, M. E., Middleton, P., Hepler, J. R., Taussig, R., Oilman, A. G., and Mumby, S. M. (1993). Proc. Natl. Acad. Sci. USA, 90, 3675. Morello, J.-P., and Bouvier, M. (1996). Biochem. Cell Biol., 74, 449. Stoffel, R. H., Randall, R. R., Premont, R. T., Lefkowitz, R. J., and Inglese, J. (1994). J. Biol. Chem., 269, 27791. Premont, R. T., Macrae, A. D., Stoffel, R. H., Chung, N., Pitcher, J., Ambrose, C., Inglese, J., MacDonald, M. E., and Lefkowitz, R. J. (1996). J. Biol. Chem., 271, 6403. O'Brien, P. J., and Zatz, M. (1984). /. Biol. Chem., 259, 5054. Ovchinnikov, Y. A., Abdulaev, N. G., and Bogachuk, A. S. (1988). FEBS Lett., 230, 1. O'Dowd, B. F., Hnatowich, M., Caron, M. G., Lefkowitz, R. J., and Bouvier, M. (1989). J. Biol. Chem., 264, 7564. Kennedy, M., and Limbird, L. E. (1994). J. Biol. Chem., 269, 31915. Ng, G. Y. K., O'Dowd, B. F., Caron, M., Dennis, M., Brann, M. R., and George, S. R. (1994). J. Neurochem., 63, 1589. 56

3: Post-translational acylation of signal transducing polypeptides 19. Ng, G. Y., Mouillac, B., George, S., Caron, M., Dennis, M., Bouvier, M., and O'Dowd, B. (1994). Eur. J. Pharmacol. Mol. Pharmacol. Sec., 267, 7. 20. Ng, G. Y., George, S. R., Zastawny, R. L., Caron, M., Bouvier, M., Dennis, M., and O'Dowd, B. F. (1993). Biochemistry, 32, 11727. 21. Butkerait, P., Zheng, Y., Hallak, H., Graham, T. E., Miller, H., Burris, K., Molinoff, P. B., and Manning, D. R. (1995). J. Biol. Chem., 270, 18691. 22. Kosugi, S., and Mori, T. (1996). Biochem. Biophys. Res. Commun., 221, 636. 23. Sadeghi, H. M., Innamorati, G., Dagarag, M., and Birnbaumer, M. (1997). Mol. Pharmacol., 52, 21. 24. Hayashi, M. K., and Haga, T. (1997). Arch. Biochem. Biophys., 340, 376. 25. Kawate, N., and Menon, K. M. (1994). J. Biol. Chem., 269, 30651. 26. Horstmeyer, A., Cramer, H., Sauer, T., Muller-Esterl, W., and Schroeder, C. (1996). J. Biol. Chem., 271, 20811. 27. Okamoto, Y., Ninomiya, H., Tanioka, M., Sakamoto, A., Miwa, S., and Masaki, T. (1997). J. Biol. Chem., 272, 21589. 28. Alaluf, S., Mulvihill, E. R., and Mcllhinney, R. A. (1995). J. Neurochem., 64, 1548. 29. O'Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992). Baculovirus expression vectors, a laboratory manual, W. H. Freeman and Co., New York. 30. Vialard, J., Lalumiere, M., Vernet, M., Briedis, D., Alkhatib, G., Henning, D., Levin, D., and Richardson, C. (1990). J. Virol., 64, 37. 31. Loisel, T. P., Adam, L., Hebert, T., and Bouvier, M. (1996). Biochemistry, 35, 15923. 32. Benovic, J. L., Shorr, R G. L., Caron, M. G., and Lefkowitz, R. J. (1984). Biochemistry, 23, 4510. 33. Caron, M. G., Srinivasan, Y., Pitha, J., Kociolek, K., and Lefkowitz, R. J. (1979). J. Biol. Chem., 254, 2923. 34. Bouvier, M., Hnatowich, M., Collins, S., Kobilka, B. K., De Blasi, A., Lefkowitz, R. J., and Caron, M. G. (1988). Mol. Pharmacol., 33, 133. 35. Papac, D. I., Thornburg, K. R., Bullesbach, E. E., Crouch, R. K., and Knapp, D. R. (1992). /. Biol. Chem., 267, 16889. 36. Mouillac, B., Caron, M., Bonin, H., Dennis, M., and Bouvier, M. (1992). J. Biol. Chem., 267, 21733. 37. Duncan, J. A., and Gilman, A. G. (1996). J. Biol. Chem., 271, 23594. 38. Bizzozero. O. A., McGarry, J. F., and Lees, M. B. (1987). J. Biol. Chem., 262, 13550. 39. Mack, D., Berger, M., Schmidt, M. F. G., and Kruppa, J. (1987). J. Biol. Chem., 262, 4297.

57


4

G protein-coupled receptor phosphorylation and desensitization ANDREW B. TOBIN, ANGELA RAE, and DAVID C. BUDD

1. Introduction Phosphorylation of G protein-coupled receptors (GPCRs) has been linked with receptor desensitization, primarily through work on the B-adrenergic receptor(1-3). Extensive studies have demonstrated that agonist stimulation of the B-adrenergic receptor results in phosphorylation of the receptor by two kinases; protein kinase A (PKA), and the receptor-specific kinase B-adrenergic receptor kinase (GRK-2) (1-3). Each of these kinases phosphorylates distinct sites on the receptor. PKA sites are at RRSS motifs in the third intracellular loop and C-terminal tail (4), and GRK-2 sites are in an acid serine/threoninerich region of the C-terminal tail (5). Phosphorylation at both PKA and GRK-2 sites results in receptor desensitization (1). In the case of GRK-2mediated desensitization the mechanism has been demonstrated to involve the intermediary protein B-arrestin, which binds to the phosphorylated receptor and in doing so is able to 'displace' the Gas-protein, thereby uncoupling the receptor from adenylyl cyclase (6). Cloning studies have revealed that GRK-2 is a member of a protein kinase family called the G protein-coupled receptor kinase (GRK) family (7). There are at present six members of this family (GRK-1 to GRK-6); each is thought to be able to mediate agonist-stimulated phosphorylation of certain GPCRs (7). The process of agonist-mediated phosphorylation and desensitization of the B-adrenergic receptor is considered to represent a general regulatory phenomenon applicable to a wide range of GPCRs. There is now compelling evidence that nearly all GPCRs, including both adenylyl cyclase and phospholipase C (PLC)-coupled receptors, undergo agonist-dependent phosphorylation (8, 9). Whereas receptor phosphorylation appears to be a universal regulatory mechanism, the role that receptor phosphorylation plays in receptor function across this broad range of receptors is still unclear. However, because of the analogy with the B-adrenergic receptor system, most laboratories focus on a possible role in receptor desensitization. In the present chapter we shall describe methods we have developed for

Andrew B. Tobin et al. investigating receptor phosphorylation in intact cultured cells and broken cell preparations. We shall also describe approaches we have adopted in the investigation of desensitization of PLC-coupled receptors.

2. Techniques in the investigation of GPCR phosphorylation 2.1 Determination of GPCR phosphorylation in intact cultured cells by immunoprecipitation Early studies on the B-adrenergic receptor and m2-muscarinic receptor relied on ligand-based affinity-purification techniques to isolate phosphorylated receptors solubilized from tissues or cell lines that had been labelled with [32P]orthophosphate (e.g. 10, 11). The ability to purify these two particular receptor subtypes has allowed in vitro assays to be developed, in which purified receptors reconstituted in phospholipid vesicles were used as substrates for the receptor-specific kinases (e.g. GRK-2). Not only were these reconstitution techniques used in the early studies on the purification of GRK-2 (10), but they have also been employed recently to determine the modes of GRK-2 regulation, in particular the regulatory roles of G protein By-subunits (12, 13) and the phospholipid phosphatidylinositol 4,5-bisphosphate (14, 15). Similar affinity chromatography techniques have been used to isolate phosphorylated forms of other receptor subtypes, for example the CCK receptor (16). However, these techniques are extremely technically demanding due to the low expression levels of GPCRs, and the problems associated with solubilization of the receptor in a form that still allows ligand binding. The universal role of receptor phosphorylation in GPCR regulation has necessitated the development of less demanding techniques to monitor receptor phosphorylation that could readily be applied to a number of different receptor subtypes. Our laboratory was one of the first to use receptor-specific antibodies to immunoprecipitate a GPCR in the study of receptor phosphorylation (17). The use of receptor-specific antisera or epitope-tagged receptors is now widespread, and has been directly responsible for revealing the diversity of receptor subtypes that undergo phosphorylation (9). We have applied this technique in the investigation of phosphorylation of the PLC-coupled m3muscarinic (17-19) and ml-muscarinic receptors (20), expressed as recombinant proteins in Chinese hamster ovary cells (CHO cells), and more recently with the B2-adrenergic receptor expressed in CHO cells (Figure 7). The protocols we describe here are based on our experience with these receptor systems. Any immunoprecipitation protocol naturally centres on the quality of the primary antibody. By far the best anti-receptor antiserum we have used is that against the m3-muscarinic receptor, which was raised against a glutathione-Stransferase (GST) bacterial fusion protein containing a region of the third 60

4: G-protein coupled receptor phosphorylation

Figure 1. Time course for the agonist-mediated phosphorylation of the B2-adrenergic receptor. CHO cells expressing recombinant B2-adrenergic receptor were labelled with [32P]-orthophosphate and stimulated with the adrenergic agonist isoproterenol (1 mM). The reaction was stopped with ice-cold RIPA buffer, and the B2-adrenergic receptor immunoprecipitated using a receptor-specific antiserum. The positions of molecular weight markers (kDa) are shown.

intracellular loop of the human m3-muscarinic receptor (17). Although construction of bacterial fusion proteins can he lime-consuming and somewhat unpredictable, due to the potential problems with expressing certain receptor regions (particularly very hydrophobic regions), they do generally make excellent antigens. Alternatively, we have raised a number of receptor antisera (e.g. ml-muscarinic and metabotropic glulamate la receptors) against peptides conjugated to keyhole limpet haemocyanin. The procedures we use for antisera production are based on those described in the excellent methods book by Harlow and Lanc (21). The problem encountered with many GPCRs is that they are poor immunogens, so that raising high quality antisera for immunoprecipitation protocols has been difficult. An alternative approach to raising receptor subtype-specific antibodies for immunoprecipitalion studies is to epitope-tag the receptor, usually at the C-terminus. The solubilized receptor can then be immunoprccipitated using commercially available monoclonal antibodies raised against the epitope tag (see e.g. ref. 22). One epitope tag employed in GPCR studies is the influenza hacmagglutinin (HA) tag YPYDVPDYA, which is recognized by the 12CA5 monoclonal antibody available from BabCo, Berkeley Antibody Co, and Boehringer Mannheim. A shorter IIA tag, DVPDYA, has also been reported to be recognized by the 12CA5 antibody. Also the FLAG tag DYKDDDDK, which is recognized by the FLAGM2 monoclonal antibody available from Kodak IBI, is commonly used. 61

Andrew B. Tobin et al. It is essential to characterize the antiserum correctly, even (or especially!) when it has been obtained from a commercial source. The glycosylation of GPCRs results in unpredictable migration on SDS-PAGE, and their hydrophobic nature often results in broad 'fuzzy' bands (Figure 1). The use of transfected cell lines is probably the most reliable test for antiserum specificity. The antiserum should be tested against transfected and sham-transfected controls in both immunoprecipitation and Western blots. In testing the most appropriate receptor solubilization conditions, we have found that the RIPA buffer described here was the most efficient. The advantage of immunoprecipitation over methods that employ purification of the receptor on a ligand affinity matrix is that maintaining the ligand-binding properties is not important for immunoprecipitation. Therefore, the solubilization conditions can be harsh, ensuring total solubilization of the receptor. This is particularly important in the case of GPCRs that appear to be differentially solubilized by mild detergent solutions designed to maintain the ligand binding properties of the receptor (23). In our early studies we labelled cells with [32P]-orthophosphate in suspension (17). Recently, however, we have found it more convenient and reliable to label plated-down cells. Both methods are given here in Protocols 1 and 2 respectively. Protocol 1. [32P]-labelling of the cellular ATP pool and receptor solubilization (of cells in suspension) Equipment and Reagents • [32P]-orthophosphate (Amersham) • Cells expressing receptor of interest

• Microcentrifuge

Method 1. Harvest cells using HBS/0.5 mM EDTA (HBS: 10 mM Hepes, pH 7.4; 0.9% NaCI) and wash twice in phosphate-free Krebs/Hepes buffer (10 mM Hepes, 118 mM NaCI, 4.3 mM KCI, 1.17 mM MgS04.7H2O, 1.3 mM CaCI2.2H20, 25 mM NaHC03, 11.7 mM glucose, pH 7.4) 2. Resuspend the cells in phosphate-free Krebs/Hepes buffer at a density of 1-3 x 106 cells ml-1, and aliquot into 1ml aliquots. 3. Add [32P]-orthophosphate (50 mCi ml-1), and incubate the cells at 37°C for 60 min. Under these conditions we have calculated that the specific activity of the intracellular ATP pool is 840 ±161 c.p.m. per pmol ATP (in the case of CHO cells; ref. 17). 4. The cell suspensions can now be challenged with experimental reagents for the required time. Terminate the reaction by rapid centrifugation at ~2000 r.p.m. on a bench top Microfuge for 30 s. Aspirate the medium 62

4: G-protein coupled receptor phosphorylation and resuspend the cell pellet in 1 ml ice-cold RIPA buffer (10 mM TrisHCI, 10 mM EDTA, 500 mM NaCI, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, pH 7.4). 5. Following 30 min solubilization on ice, clear the sample by centrifugation in a Microfuge (maximum speed for 3 min). Save the supernatant for immunoprecipitation of the solubilized receptor, see below.

Protocol 2. [32P]-labelling of the cellular ATP pool and receptor solubilization (of adhered cells) Equipment and Reagents • Cells expressing receptor of interest • [32P]-orthophosphate (Amersham)

• Microcentrifuge

Method 1. Grow cells to ~70% confluence on six-well dishes. It is important not to allow cells to become too confluent, since the solubilization procedure will precipitate the genomic DNA to give a stringy mass that will result in high background if the cell density is too high. 2. Remove cell culture medium, and wash cells twice in phosphate-free Krebs/Hepes buffer. 3. Incubate the cells in 1 ml phosphate-free Krebs/Hepes buffer containing 50 mCi [32P] orthophosphate for 60-120 min at 37 °C. 4. Add stimulatory reagents for the required time. 5. Terminate the reaction by aspirating the medium and adding 1 ml RIPA buffer (4°C). 6. Following 30 min solubilization on ice, transfer the reaction to a 1.5 ml Eppendorf centrifuge tube, clear by centrifugation on a bench top microfuge for 3 min at maximum speed. Remove the supernatant and save for immunoprecipitation of the solubilized receptor.

Protocol 3. Receptor immunoprecipitation Equipment and Reagents • Appropriate antiserum or antibody • Protein-A-sepharose CL-4B (Pharmacia) • Rotating mixer

• Microcentrifuge • X-ray film

63

Andrew B. Tobin et al. Protocol 3.

Continued

Method 1. Add antiserum (0.2-1.0 mg) to the solubilized cell extract obtained from Protocols 1 or 2 above. Incubate on ice for 60-90 min. (Note: it is preferable to purify the antiserum in some way, e.g. by protein A before use) 2. Add 175mlof protein A-sepharose slurry (Protein A-sepharose CL-4B (Pharmacia) 1.5 g resuspended in 50 ml of TE buffer (2.5 mM Tris-HCI, 2.5 mM EDTA, pH 7.4)) to the sample and mix on slowly rotating rollers for 15 min at 4°C. 3. Pellet the protein A-sepharose by brief centrifugation (maximum speed in a Microfuge for 30 s), aspirate the supernatant, and wash the pellet 3-5 times with TE-buffer. (It is important to note that the wash steps can be changed to eliminate background. High-salt and low-salt washes can be included, as well as detergent washes. For example, in the immunoprecipitation of the B2 adrenergic receptor (see Figure 1) from CHO cells expressing recombinant receptor, we wash the protein A pellet twice in a Tween-20 buffer (100 mM Tris-HCI, 0.5% Tween 20, 1.5 M NaCI, pH 7.4) followed by a further wash in TE-buffer. 4. At the final wash step remove as much of the supernatant as possible using a fine pipette tip. 5. Resuspend the pellet in 20 ml of 2 X SDS-PAGE sample buffer. Heat the sample to ~85 °C for exactly 2 min, and then resolve the proteins by SDS-PAGE. Do not boil the sample since this results in aggregation of the receptors. 6. Stain the gel with 0.2% Coomassie blue in 40% methanol-10% acetic acid, and destain in 40% methanol-10% acetic acid. This procedure will stain the immunoprecipitated antibody, thereby confirming equal immunoprecipitation/loading; however, the receptor will not be visible due to the low quantity. Dry the gel, and obtain an autoradiograph.

2.2 Identification of GPCR phosphorylation in a crude membrane preparation We have been able to demonstrate that the kinase responsible for phosphorylation of the m3-muscarinic receptor expressed in CHO cells (CHO-m3 cells) is, at least in part, associated with the plasma membrane (18, 24). Using a membrane preparation from CHO-m3 cells we have reported that the m3muscarinic receptor can still be phosphorylated in an agonist-dependent manner (24). This membrane kinase assay has allowed the characterization of the endogenously expressed receptor kinase using cell-impermeable inhibitors (24). Furthermore, we have been able to reconstitute purified receptor kinases 64

4: G-protein coupled receptor phosphorylation with the membrane preparation, and investigate the ability of exogenously added kinase to increase the level of agonist-sensitive receptor phosphorylation (18, 19). Similar studies have used reconstituted membrane preparations from cell lines to investigate the phosphorylation of the B-adrenergic receptor (25), the A3-adenosine receptor (26), and the bradykinin B2 receptor (27). The method outlined here for identification of receptor phosphorylation in membrane preparations from cloned cell lines is drawn from our work on the m3-muscarinic receptor expressed in CHO-m3 cells (expression levels ~1 pmol mg-1 protein). The technique can be divided into three distinct sections; (i) preparation of the membrane fraction, (ii) stimulation of receptor phosphorylation, and (iii) solubilization and immunoprecipitation of the receptor.

Protocol 4. Preparation of a membrane fraction from cell lines Equipment and Reagents • Polytron

Method 1. Harvest cells using HBS/0.5 mM EDTA (use at least two confluent 175 cm2 flasks), and resuspend the cell pellet in ice-cold TE-buffer (15 ml) plus protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg ml-1 soybean trypsin inhibitor, 1 mg ml-1 leupeptin, 1 mg ml-1 pepstatin A, 100 mg ml-1 benzamidine, 100 mg ml-1 iodoacetamide). 2. Leave cells on ice for 10 min to swell and homogenize (15 s pulse in a Polytron). 3. Remove cell debris by centrifugation at 2000 g for 3 min. Collect the supernatant which contains the membrane fraction, and pellet the membranes by centrifugation at 15 000 g for 10 min. 4. Resuspend membrane pellet in kinase buffer (20 mM Tris-HCI, 10 mM MgCI2, 1 mM EGTA, pH 7.5) plus protease inhibitors. Adjust protein concentration to 1 mg protein per ml.

Protocol 5. Stimulation of receptor phosphorylation in crude membranes Equipment and Reagents • Microcentrifuge

65


Continued

Method 1. To 50 ml of the membrane preparation obtained using Protocol 4 (e.g. for CHO-m3 cells this is equivalent to ~0.1 pmoles of receptor) add stimulatory reagents and/or exogenous protein kinase preparation or buffer blank. Add kinase buffer to bring the final volume to 100ml,and start the reaction by adding 100 mM [y32P] ATP (1-4 c.p.m. fmol-1 ATP). 2. Continue the reaction at 32°C for the required time. It is important to remember that the membrane preparation contains ATPase activity. We have calculated that in our experiments using CHO-m3 membranes ~80% of the ATP is consumed within 10 min. 3. Stop the reaction either by adding 1 ml ice-cold RIPA buffer directly, or by first pelleting membranes by a brief 30 s spin in a Microfuge (maximum setting) followed by aspiration of the supernatant and resuspension of the pellet in 1 ml ice-cold RIPA buffer. 4. Solubilize the membranes on ice for 30 min. Clear the supernatant by centrifugation (13 000 g, 3 min), and immunoprecipitate the solubilized receptor as described in Protocol 3. Resolve the immunoprecipitated proteins by SDS-PAGE gel electrophoresis. Stain and destain the gel (as outlined in Protocol 3) before drying and obtaining an autoradiograph.

3. Identification of desensitization of phospholipase C-coupled receptors Many of the techniques that report desensitization of PLC-coupled receptors analyse the accumulation of total inositol phosphates in the presence of a lithium block of inositol monophosphatase (28). Although this technique can give a good indication of the rate of phosphatidylinositol polyphosphate hydrolysis at early time points (particularly within the first few seconds and minutes of agonist exposure), prolonged receptor stimulation results in problems associated with lipid-pool depletion and changes in the specific activity of the radiolabelled phosphatidylinositol polyphosphate pool (27). The technique we describe here measures the product of phosphatidylinositol 4,5bisphosphate hydrolysis, namely inositol 1,4,5-trisphosphate (Ins(l,4,5)P3). Using analysis of Ins(l,4,5)P3 production, we have been able to identify rapid desensitization of the peak Ins(l,4,5)P3 response that occurs after 10 seconds of agonist stimulation (29) (Figure 2). In contrast, the sustained Ins(l,4,5)P3 response appears to be resistant to desensitization. Since the sustained component contributes the majority of the inositol phosphates when measuring total inositol phosphates in the presence of lithium, any receptor desensitiza66

4:

G-protein

coupled

receptor

phosphorylation

Figure 2. Desensitization of the lns(1,4,5)P3 response in CHO-m3 cells. CHO-m3 cells expressing ~1 pmol mg-1 protein of recombinant human m3-muscarinic receptors were pre-incubated for 5 min with 1 mM muscarinic agonist carbachol (open circles) or vehicle (closed circles). Cells were then washed three times, and allowed to recover for 5 min before stimulation with 1 mM carbachol. Reactions were stopped with TCA (1 M), and lns(1,4,5)P3 extracted and assayed using a radio-receptor binding assay. Data represent the mean ± SEM (n = 3).

tion that results in a reduction in the early peak Ins(l,4,5)P3 response would not be detected if total inositol phosphate accumulation is employed. It is for this reason that we mainly measure Ins(l,4,5)P3 production using a radioreceptor assay to assess desensitization of PLC coupled receptors. This procedure can be divided into three components: (i) stimulation of the cultured cells; (ii) extraction and neutralization of Ins(l,4,5)P3; and (iii) Ins(l,4,5)P3 radio-receptor assay. (Although methods for (ii) and (iii) are given here, they have also been described in detail previously, refs 30,31)

Protocol 6. Stimulation of cultured cells in the investigation of PLC-coupled receptor desensitization Equipment and Reagents • Cells expressing at appropriate receptor e.g. M3 muscarinic acetylcholine receptor

Method

1. Seed cells on to 24-well dishes, and allow to grow overnight. 67


Continued

2. Remove medium and wash once in Krebs/Hepes buffer (10 mM Hepes, 118 mM NaCI, 4.3 mM KCI, 1.17 mM MgS04.7H20, 1.3 mM CaCI2.2H20, 25 mM NaHC03, 11.7 mM glucose, 1.17 mM KH2PO4, pH 7.4). 3. Allow cells to stabilize for 10 min at 37°C. 4. Pre-expose cells to agonist/vehicle for the appropriate time with 200 ml agonist in Krebs/Hepes buffer (e.g. 5 min with 1 mM of the muscarinic agonist carbachol). 5. Stop pre-incubation by washing cells rapidly with 200 ml of Krebs/ Hepes buffer (37°C) three times. This should take ~2-3 min per dish. 6. Allow cells to incubate for a further 2-3 min, so that the total wash and recovery phase is at least 5 min. This recovery time is the minimum employed since calcium pool refilling takes ~2-3 min, and any depletion of the calcium pool will result in a reduced lns(1,4,5)P3 response, due to disruption of calcium feedback on PLC activity (32). 7. Stimulate cells by replacing buffer with 100 ml of Krebs/Hepes buffer containing agonist for the appropriate time (general time course employed 0, 5, 10, 60, 500 s). 8. Stop reaction with equal volume of 1 M trichloroacetic acid.

Protocol 7.

Extraction and neutralization of lns(1,4,5)P3

Method 1. Remove 160 ml of the reaction mixture obtained in Protocol 6, step 8. 2. Add 40 ml of 10 mM EDTA and 200 ml of Freon/tri-n-octylamine (1:1 v/v). Vortex the mixture and separate the phases by brief centrifugation in a microfuge. 3. Remove 100 ml of the upper phase, and to this add 50 ml of 25 mM NaHCO3. 4. Prepare buffer blanks for use in making up lns(1,4,5)P3 standards (see Protocol 9). Mix 400ml of Krebs/HEPES buffer with 400 ml 1 M TCA and then add 200 ml of 10 mM EDTA. This mixture is extracted with 1 ml of Freon/tri-n-octylamine. Remove 500 ml of the upper phase and to this add 250 ml of 25 mM NaHCO3.

68

Figure 3. Typical standard curve obtained for the lns(1,4,5)P3 radio-receptor assay. Cold lns(1,4,5)P3 at concentrations ranging from 0.036-36 pmol was used to displace [3H]lns(1,4,5)P3 (7349 d.p.m. per assay) from binding proteins present in an adrenal cortex preparation. Bound lns(1,4,5)P3 was separated from free by rapid filtration through GF/B filters. (Non-specific binding is defined at 1200 prnol cold lns(1,4,5)P3).

Protocol 8. Preparation of adrenal cortex lns(1,4,5)P3 binding protein Equipment and Reagents • Bovine adrenal glands

• Polytron

Method 1. Obtain bovine adrenal glands and cut each gland longitudinally. Remove the medulla and scrape the cortex from the outer capsule. 2. Dispense the cortex into 50 ml centrifuge tubes (~5 g tissue per tube), and homogenize in 30-40 ml of ice-cold buffer A per tube (20 mM NaHCO3, 1 mM dithiothreitol, pH 8.0). 3. Centrifuge the homogenate (5000 g, 10 min, 4oC). Pool the supernatants and extract the pellet again by rehomogenization in 30-40 ml of buffer A and recentrifugation. 4. Combine the supernatants from the two extractions, and discard the twice-extracted pellet. 69


Continued

5. Obtain a P2 fraction by centrifugation of the supernatant (40000 g, 20 min, 4°C). Discard the supernatant and rehomogenize the pellet in buffer A. Recentrifuge (40 000 g, 20 min, 4 °C). Repeat this wash step of the P2 pellet three times. 6. Resuspend the washed P2 pellet in buffer A, and adjust protein concentration to 15-18 mg ml-1. 7. Aliquot the adrenal cortical preparation, and store at -20 °C. The preparation can be stored for 6-12 months.

Protocol 9. lns(1,4,5)P3 radio-receptor assay Equipment and Reagents • [3H]-inositol (1,4,5) trisphosphate (Amersham or NEN) . Inositol (1,4,5) trisphosphate K

« Adrenal cortex-binding protein (see Protocol 8) . Filtration apparatus or cell harvester (Millipore)

Method 1. To prepare the lns(1,4,5)P3 standards, dilute 40 mM stock lns(1,4,5)P3 using the buffer blank (Protocol 7) to give the following concentrations: 0, 1.2, 4, 12, 40, 120, 400, and 1200 nM. Non-specific binding is determined using 40 mM stock. Note that by using 30 ml of each standard in a final reaction volume of 120ml,the concentration of lns(1,4,5)P3 in the assay tubes ranges from 0.036-36 pmol lns(1,4,5)P3 per assay. An example of a typical standard curve is given in Figure 3. 2. Take 30 ml of neutralized extract obtained from Protocol 7, step 3, or 30 ml of lns(1,4,5)P3 standard, and add to 30 ml of a 4x concentrated assay buffer (100 mM Tris-HCI, 4 mM EDTA, pH 8.0). 3. To this mix add 30 ml of 3H-lns(1,4,5)P3 diluted in water to give 60008000 d.p.m. per assay (Stock [3H]-lns(1,4,5)P3 obtained from Amersham, 30-50 Ci mmol-1, or from NEN, 17-20 Ci mmol-1). 4. Start the reaction by adding 30 ml of adrenal cortex-binding protein. Total reaction volume is 120 ml. 5. Vortex the mixture and incubate on ice for 30 min. 6. Stop the reaction by adding 3 ml of ice-cold wash buffer (25 mM TrisHCI, 1 mM EDTA, 5 mM NaHC03, pH 8.0) and immediately filter through GF/B filter disks using rapid vacuum filtration (Millipore vacuum manifold). 70

4: G-protein coupled receptor phosphorylation 7. Wash filter disks with 2 x 3 ml of ice-cold wash buffer. Note that filtering procedure should be done as quickly as possible. 8. Transfer the filters to scintillation vials, add 4mls of scintillation fluid, and allow to extract overnight before counting.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Hausdorff, W. P., Caron, M. G., and Lefkowitz, R. J. (1990). FASEB. J., 4, 2881. Lohse, M. J. (1993). Biochim. Biophys. Acta, 1179, 171. Haga, T., Haga, K., and Kameyama, K. (1994). J. Neurochem., 63, 400. Hausdorf, W. P., Bouvier, M., O'Dowd, B. F, Irons, G. P., Caron, M. G., and Lefkowitz, R. J. (1989). J. Biol. Chem., 264, 12657. Fredericks, Z. L., Pitcher, J. A., and Lefkowitz, R. J. (1996). J. Biol. Chem., 271, 13796. Sterne-Marr, R., and Benovic, J. L. (1995). Vitamins and hormones, 51, 193. Lefkowitz, R. J. (1993). Cell, 74, 409. Tobin, A. B., and Waugh, M. G. (1996). In The phospholipase C pathway: its regulation and desensitisation (ed. A. B. Tobin) p. 65. Springer Press, New York. Tobin, A. B. (1997). Pharmacol. Ther., 75, 135. Benovic, J. F., Mayor, F., Staniszewski, C., Lefkowitz, R. J., and Caron, M. G. (1987). J. Biol. Chem., 262, 9026. Haga, K., and Haga, T. (1990). FEBS Lett., 268, 43. Haga, K., and Haga, T. (1992). J. Biol. Chem., 267, 2222. Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. K., Casey, P. L., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992). Science, 257, 1264. Pitcher, J. A., Fredericks, Z. L., Stone, C. W., Fremont, R. T., Stoffel, R. H., Kock, W. J., and Lefkowitz, R. J. (1996). J. Biol. Chem., 271, 24907. DebBurman, S. K., Ptasienski, J., Benovic, J. L., and Hosey, M. M. (1996). J. Biol. Chem., 271, 22552. Klueppelberg, U. G, Gates, L. K., Gorelick, F. S., and Miller L. J. (1991). /. Biol. Chem., 266, 2403. Tobin, A. B., and Nahorski, S. R. (1993). J. Biol. Chem., 268, 9817. Tobin, A. B., Keys, B., and Nahorski, S. R. (1996). J. Biol. Chem., 271, 3907. Tobin, A. B., Totty, N. F., Sterlin, A. E., and Nahorski, S. R. (1997). J. Biol. Chem., 272, 20844. Waugh, M. G., Burford, N. T., Nahorski, S. R., and Tobin, A. B. (1995). Brit. J. Pharmacol., 114, 143P. Harlow, E., and Lane, D. (1984). Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, NY. Ali, H., Richardson, R. M., Tomhave, E. D., Didsbury, J. R., and Snyderman, R. (1993). J. Biol. Chem., 268, 24247. Rinken, A., Kameyama, K., Haga, T., and Engstrom, L. (1994). Biochem. Pharmacol., 48, 1245. Tobin, A. B., Keys, B., and Nahorski, S. R. (1993). FEBS Lett., 335, 353.

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Andrew B. Tobin et al. 25. Pei, G., Tiberi, M., Caron, M. G., and Lefkowitz R. J. (1994). Proc. Natl. Acad. Sci. USA, 91, 3633. 26. Palmer, T. M., Benovic, J. L., and Stiles, G. L. (1995). J. Biol. Chem., 270, 29607. 27. Blaukat, A., Alla, S. A., Lohse, M. J., and Muller-Ester, W. (1996). J. Biol. Chem., 271, 32366. 28. Wojcikiewicz, R. J. H., Tobin, A. B., and Nahorski, S. R.(1993). Trends Pharmacol. Sci., 14, 279. 29. Tobin, A. B., Lambert, D. G., and Nahorski, S. R. (1992). Mol. Pharmacol., 42, 1042. 30. Challiss, R. A. J., Batty, I. H., and Nahorski, S. R. (1988). Biochem. Biophys. Res. Commun., 157, 684. 31. Challiss, R. A. J. (1995). In Signal transduction protocols. (ed. D. A. Kendall, and S. J. Hill). p. 167. Humana Press Inc, Totowa, NJ. 32. Willars, G. B., and Nahorski, S. R. (1995). Mol. Pharmacol., 47, 509.

72

5

G proteins and their identification IAN MULLANEY

1. Introduction The demonstration that G proteins mediate many intracellular signalling processes has led to the development of specific techniques that can be used to identify which of these polypeptides is involved upon receptor activation by ligand. This chapter deals with the methodology involved to produce specific immunological tools, and the use of these reagents to probe functionally the specificity of receptor-G protein interaction. Many hormones and neurotransmitters exert their actions on target cells through changes in the levels of a number of intracellular second messengers via activation of cell surface receptors. Activation of these receptors initiates a sequence of biochemical cascades, leading to certain physiological events such as contraction of smooth muscle, neurotransmitter release, etc. The discovery of the critical role that guanine nucleotides play in mediating hormonal stimulation of adenylyl cyclase, the enzyme directly responsible for modulating intracellular cyclic AMP levels, led to the isolation and purification of the first of a number of closely related guanine nucleotide-binding proteins [or G proteins], Gs (1). These polypeptides, which function through the cyclical binding and hydrolysing of GTP, have proved to be central in transducing the effects of activated heptahelical integral membrane receptors. The 'classical' G proteins exist as heterotrimers comprising non-identical a, B, and y subunits. Initial identification of G proteins involved the ability of the a subunit to act as a substrate for mono-ADP-ribosylation, catalysed by the ADP-ribosyl transferase activity of a number of bacterial exotoxins, a modification that functionally altered the involvement of the G protein in signal transduction. The use of [32P] NAD+ as a substrate allowed the visualization of ADP-ribosylated polypeptides following separation in SDS-PAGE gels and autoradiography. In this way Gs, the G protein involved in the hormonal stimulation of adenylyl cyclase, was identified as a substrate for ADPribosylation by cholera toxin (2). Similarly, Gi, the inhibitory G protein, was characterized by its ability to act as a substrate for pertussis toxin (3-5). Although the use of toxins yields little information about the molecular identity of the G proteins involved, it can be useful in the initial investigation

Ian Mullaney Table 1. The peptide sequences which have been widely used to generate a series of antipeptide antisera directed against the a subunits of various G proteins Peptide used

G protein sequence

Antiserum identifies

*RMHLRQYELL TPEPGEDPRVTRAKY *KENLKDCGLF LERIAQSDYI *KNNLKECGLY *ANNLRGCGLY GCTLSAEERAALERSK NLKEDGISAAKDVK *QLNLKEYNLV EKVSAFENPYVDAIKS EKVTTFEHQYVNAIKT *QENLKDIMLQ *HDNLKQLMLQ *QLNLREFNLV *ARYLDEINLL *QNNLKYIGLC

Gsa 372-381 Gsa 325-339 TD1a 341-350 Gi2a 160-169 Gi3a 345-354 G0a 345-354 G0a 1-16 G0a 22-35 Gqa 350-359 Gqa 119-134 G11a 119-134 G12a 370-379 G13a 368-377 G14a 346-355 G15a 365-374 Gza 346-355

14 19 T D , a , TD2a, Gi1a, Gi2a20 14 Gi2a 14,21 Gi3a 14 G0a G0a 21 22 G0a 23 Gqa,G11a (G14a?) 24 Gqa 24 G11a G12a 6 G13a 25 7 G14a, Gqa, G11a 7 G16a, G16a 26 Gza

Reference

Gsa Gsa

* denotes C-terminal sequences. Amino acids are represented using the one letter code. TD = transducin

into G protein function in a particular system. However, a number of G protein a subunits have shown themselves to be refractory to influence by these toxins. The ability of agonists to activate phospholipase CBl, and cause hydrolysis of inositol-containing phospholipids, has been shown to be unaffected by treatment with both pertussis and cholera toxin in the vast majority of cells and tissues studied, implying the involvement of toxininsensitive G proteins (6, 7). Indeed, the use of the polymerase chain reaction, based on conserved sequence domains across the G protein family, and the isolation of cDNAs has now identified at least 17 G protein a subunits, including members of two new, toxin-insensitive subfamilies (Gq and G12). The elucidation of G protein primary amino acid sequences has allowed for the production of specific antisera directed against these proteins, using specific peptide stretches known to be both unique and functionally important (see Table 1). In this chapter, methods designed to probe the identity of G protein a subunits will be outlined. These will include the use of bacterial toxins, resolution of polypeptides by SDS-PAGE, and production of specific G-protein antisera. Further, the use of these tools to probe receptor-G protein interactions will be presented, specifically, the immunoprecipitation of [35S]-labelled product, and agonist-induced cholera toxin-mediated ADP-ribosylation. Finally, functional assays that use the guanine nucleotide activation-deactivation cycle will be described. 74

5: G proteins and their identification

2. Production of crude plasma membrane fractions for analysis of G proteins Since the majority of G protein assays use cell-free systems, crude plasma membrane fractions remain the most common biological preparation in which to study the function of these polypeptides. Isolation of plasma membrane sheets by the differential centrifugation method described by Koski and Klee has become the standard technique in the field (8). Immunoblotting of supernatant fractions produced during membrane production routinely shows little immunoreactivity, reinforcing the concept that the G protein a subunits are located at membranes. Protocol 1 describes the preparation of plasma membrane from cells grown in culture, whilst Protocol 2 outlines a method for preparing these fractions from intact animal tissue. We have found it beneficial, in terms of final yield, to store tissue and cell pastes at -80°C before homogenization. This freezing procedure fractures the cells, making them more amenable to rupture by the tissue grinder. However, it is advisable to keep the pellet produced from the first centrifugation step on ice. This pellet contains mainly unbroken cells and can, if the final product yield is low, be rehomogenized and re-fractionated. Although early preparations did not include the use of protease inhibitors in the homogenization buffer, it has become clear that some elements of the signalling cascade can be sensitive to proteolytic degradation. The inclusion of common inhibitors in the buffer is recommended. Since many of the techniques involved in investigating G protein function rely on resolution by electrophoretic techniques, the method involved in preparation of samples for analysis is outlined in Protocol 3. Protocol 1. Preparation of crude plasma membrane fraction from cells grown in cell culture Equipment and reagents • Refrigerated ultracentrifuge and benchtop centrifuge • Tight fitting Teflon-on-glass homogenizer • Phosphate-buffered saline (PBS) (0.2 g KCI, 0.2 g KH2PO4, 8 g NaCI, 1.14 g Na2HP04 (anhydrous), pH 7.4, H20 to 1000 ml) . TE buffer (10 mM Tris-HCI, 0.1 mM EDTA, pH 7.5)

• Spectrophotometer . plasma membrane buffer (TE buffer containing the following protease inhibitors: 10 mM NaF, 100 MM Na3VO4, 1 mM phenylmethanesulfonyl fluoride, 3 mM benzamidine, 0.1 mM soybean trypsin inhibitor, 10 mM. Ieupeptin, 0.2 mM aprotinin, 1.5 mM antipam)

Method 1. Gently remove cells from the surface of the flasks with a Pasteur pipette or rubber policeman, collect in a 50 ml conical centrifuge tube on ice, and centrifuge at 1000 r.p.m. at 4°C for 5 min on a benchtop centrifuge.a

75

Ian Mullaney Protocol 1. Continued 2. Discard the supernatant, resuspend the cell pellet in 30 ml ice-cold PBS, and centrifuge as before. 3. Repeat this procedure twice, and store the resultant washed cell paste at -80°C until needed. 4. Thaw the frozen cell pastes, and resuspend in 2 ml of ice-cold TE buffer containing the protease inhibitor cocktail, and homogenize with 20 strokes of a Teflon-on-glass tissue grinder. 5. Centrifuge the homogenates for 10 min at 500 g in an ultracentrifuge. 6. Discard the pellet, and recentrifuge the supernatant at 48000 g for 10 min. 7. Discard the supernatant, resuspend the pellet in 5 ml of plasma membrane buffer, and recentrifuge for 10 min at 48000 g. 8. Finally, discard the supernatant, and resuspend the pellet in plasma membrane buffer. 9. Triturate the resuspended pellet with a syringe with a fine-gauge needle, and aliquot into appropriate volumes and store at -80°C until needed. 10. Determine protein concentrations. Membranes should have protein concentrations of 1-2 mg ml-1. a

The protocol is suitable for between 107 to 1012 cells per preparation.

Protocol 2. Preparation of crude plasma membrane fraction from whole animal tissue Equipment and reagents • Benchtop centrifuge and refrigerated ultracentrifuge • Polytron • Spectrophotometer

• PBS (see Protocol 1) . TE buffer (see Protocol 1) . plasma membrane buffer (see Protocol 1)

Method 1. Remove tissue (up to 10 g), and briefly wash in 10 volumes of PBS 2. Chop the tissue with scissors, rinse in two washes of PBS, and homogenize on ice for 60 s (4 X 15s bursts) in 10 volumes of ice-cold plasma membrane buffer with a polytron at setting number 4. 3. Follow steps 5-10 in Protocol 1. 76

5: G proteins and their identification Protocol 3. TCA-deoxycholate precipitation of samples for SDSPAGE Equipment and reagents • Microcentrifuge and benchtop centrifuge . 2% (w/v) 7-deoxycholic acid, sodium salt, in H2O . 24% (w/v) TCA in H20 • 1 M Tris base (do not adjust the pH)

• Laemmli sample buffer (0.605 g Tris, 30 g urea, 5 g SDS, 6 g dithiothreitol, 10 mg bromophenol blue, in 100 ml H2O, pH adjusted to 8.0 with HCI)

Method 1. Take 25-150 ug of membranes, and place on ice in a 1.5 ml Eppendorf centrifuge tube. 2. Centrifuge for 5 min at 12000 r.p.m. on a microcentrifuge, remove the supernatant, and resuspend the pellet in 20 ul of TE buffer. 3. Add 6.5 ul of 2% (w/v) 7-deoxycholic acid to each tube, followed by 750 ul of double-distilled H2O, then 250 ul 24% (w/v) TCA. a 4. Vortex mix each sample, and spin in a benchtop microcentrifuge for 10 min at 12 000 r.p.m. 5. Carefully discard the supernatant, and bring the pellet to weakly alkaline pH by addition of 20 ul 1 M Tris base.b 6. Add 20 ul Laemmli sample buffer, and load sample onto gel. a

The solutions must be added in the order 7-deoxycholate, H2O, and then TCA. Solutions can be kept indefinitely at room temperature. b If the blue Laemmli buffer solution turns brown-orange then the sample is still acidic. Add 5 ul amounts of 1 M Tris base until the sample turns blue.

3. Gel electrophoresis of G proteins 3.1 Mono-ADP-ribosylation of G proteins by bacterial exotoxins Until specific G protein antisera became available, the ability of a subgroup of G proteins to act as targets for bacterial exotoxins from Vibrio cholerae (cholera toxin) and Bordetella pertussis (pertussis toxin or islet-activating protein) was the most widely used approach to identify and probe the function of these polypeptides. These toxins, which exert their effects by catalysing the covalent transfer of an ADP-ribose moiety on to the G protein a subunit, were initially noted for their ability to disrupt receptor-mediated control of adenylyl cyclase. Whilst cholera toxin is able catalyse ADPribosylation of a variety of proteins in vitro, its key substrates are the splice variant forms of the stimulatory G protein of adenylyl cyclase, Gsa where an 77

Ian

Mullaney

Figure 1. G12 but not G9 is a substrate for in vivo pertussis toxin-catalysed ADP-ribosylation. Membranes from U937 cells which were either untreated (lanes 1, 3) or pretreated with pertussis toxin (25 ng ml-1, 16 h) as described in Protocol 4 were resolved by SDS-PAGE, and immunoblotted with antisera directed against G12 (panel A) or G9 (panel 2) (see Protocols 7, 75). (Data from Mitchell et al., 1991, ref. 23).

arginine residue acts as the substrate amino acid. In contrast, pertussis toxin modifies a number of G proteins, including the inhibitory G protein of the adenylyl cyclase cascade, which have acceptor cystcinc residues located four amino acids from the carboxyl terminus. To date, this family of pertussis toxin substrates includes G11 G12, G13, G01, G02, and both forms of transducin. Protocol 4 outlines the method for toxin-mediated ADP-ribosylation of cultured cells in vivo. Both toxins are members of the A-B type toxin family, comprising two components, the A (active) component that consists of the ADP-ribosyltransferase that catalyses the transfer of ADF-rihose to the acceptor amino acid residue, and the R (binding) component lhat binds to the ceil surface, enabling the A component to enter the cell. The use of these toxins in conjunction with specific immunological tools has proved invaluable in identifying the G protein family. It should be recognized that although many G proteins are toxin-sensitive, a sizeable number are refractory to the toxins. Figure l shows an immunoblot of untreated and pertussis toxin-pretrealed membranes prepared from the monocyte-dcrived cell line, U937. The specific G12 antiserum detected a single immunoreactive species that migrated more slowly in membranes prepared from the toxin-pre-treated cells. In contrast, the mobility of G q , which does not contain the acceptor cysteine, was unaffected by the toxin, confirming its insensitivity to pertussis toxin, 78


Protocols

Mono-ADP-ribosylation of cultured cells by bacterial toxins (in vivo)

Equipment and reagents • Pertussis toxin (0.44 mg ml-1) • Cell culture medium

• Cell culture facilities • Cholera toxin (1 mg ml-1)

Method 1. Use cells at 70-80% confluence, and replace with culture medium containing either cholera or pertussis toxin at a final concentration of 100 ng toxin per ml of medium. 2. Incubate for 16-24 h, and harvest the cells. 3. Prepare the plasma membrane fraction (see Protocol 1).

Cholera and pertussis toxins can facilitate the transfer of ADP-ribose to plasma membrane fractions in vitro (Protocol 5). The use of these cell-free

Figure 2. Cholera and pertussis toxin-catalysed ADP-ribosylation in membranes of NG108-15 cells. Membranes (25 (i.g) of control (a, c, d, f) or 6-day dibutyryl cAMP differentiated (b, e) NG108-15 cells were treated with [32P]NAD1 and cholera toxin (a, b), pertussis toxin (d, e), or without toxin (c, f) as described in Protocol 4. Samples were recovered by deoxycholate-TCA precipitation and resolved by SDS-PAGE (10% w/v acrylamide) (see Protocols 5, 76). (Data from Mullaney et al., 1988, ref. 22),

79

Ian Mullaney systems has made the need for the binding component of the toxin redundant. Preactivation of the toxins with dithiothreitol to dissociate the binding apparatus from the catalytic component is recommended. Further, the use of the radiolabelled substrate [32P]NAD+, combined with SDS-PAGE and autoradiography, allows direct visualization of the polypeptides (9). Figure 2 shows plasma membranes prepared from the neuroblastoma X glioma cell line, NG108-15, and subjected to mono-ADP-ribosylation with radiolabelled NAD+ and either pertussis or cholera toxin. Protocol 5. Mono-ADP-ribosylation of membranes by bacterial toxins (in vitro) Equipment and reagents • Benchtop centrifuge, microcentrifuge, and refrigerated ultracentrifuge • Water bath • Cholera toxin (1 mg ml-1) • Pertussis toxin (0.44 mg ml-1) . Nicotinamide adenine dinucleotide, diftriethylammonium) salt, [adenylate-32P] (product no. NEG023), specific activity 10-50 Ci mmol-1 (NEN-Dupont)

1.5 M sodium phosphate buffer ADP-ribosylation cocktail mix (for 15 samples): 75 ul 0.2 M thymidine, 30 ul 1 mM GTP, 125 ul 1.5 M sodium phosphate buffer, 10 ul 0.04 M ATP, 15 ul 1 M arginine hydrochloride, 30 uCi [32P] NAD+ to 300 ul H2O Laemmli sample buffer (see Protocol3) 100 mM dithiothreitol (DTT)

Method 1. Pre-activate toxins by addition of equal volumes of 100 mM DTT for 60 min at room temperature prior to assay. 2. Take 25-50 u,g of plasma membrane fraction (see Protocol 1), and place on ice in a 1.5 ml Eppendorf centrifuge tube. 3. Centrifuge for 5 min at 12000 r.p.m. in a microcentrifuge, remove the supernatant, and resuspend the pellet in 25 ul of TE buffer. 4. Add 20 ul of the ADP-ribosylation cocktail mix to each tube, and start the incubation by adding 5 ul of the appropriate pre-activated toxin. a 5. Incubate for up to 90 min in a 37°C water bath. 6. Place on ice and precipitate samples using the TCA-deoxycholate method (Protocol 3). 7. Add 20 ul Laemmli sample buffer, and load the sample onto the gel (Protocol 7). a

Add 5 ul of 50 mM DTT instead of toxin for negative control

Under assay conditions where no exogenous GTP is added, it is possible for pertussis toxin-sensitive G proteins to act as substrates for mono-ADPribosylation by cholera toxin in plasma membrane preparations (Protocol 6). Further, addition of selective agonist markedly enhances incorporation of 80

5: G proteins and their identification radioactivity into 40 kDa (Gi-like) polypeptides, but not Gs, in a dosedependent manner. Used in conjunction with immunoprecipitation with specific G protein antisera, this provides a method to identify direct receptor activation of G protein a subunits (10,11) Protocol 6. Agonist-mediated mono-ADP-ribosylation of membranes by cholera toxin Equipment and reagents • Benchtop centrifuge, microcentrifuge, and • 1.5 M sodium phosphate buffer refrigerated ultracentrifuge . ADP-ribosylation cocktail mix (for 15 sam• Water bath ples): 75 ul 0.2 M thymidine, 125 ul 1.5 M . Cholera toxin (1mg ml-1) sodium phosphate buffer, 10 ul 0.04 M . Nicotinamide adenine dinucleotide, di(tri- ATP, 15 ul 1M arginine hydrochloride, 30 ethylammonium) salt, [adenylate-32P] (produCi [32P] NAD+ to300ulH2O° uct no. NEG023), specific activity 10-50 • Laemmli sample buffer -1 Ci mmol (NEN-Dupont) . 100 mM dithiothreitol

Method 1. Pre-activate toxin by addition of an equal volume of 100 mM DTT for 60 min at room temperature prior to assay. 2. Take 25-50 u.g of plasma membrane fraction (see Protocol 1), and place on ice in a 1.5 ml Eppendorf centrifuge tube. 3. Centrifuge for 5 rnin at 12000 r.p.m. on a microcentrifuge, remove the supernatant, and resuspend the pellet in 20 ul of TE buffer. 4. Add 20 ul of the ADP-ribosylation cocktail mix and 5ul of an appropriate agonist to each tube, and start the incubation by adding 5 ul of the appropriate pre-activated toxin. 5. Incubate for up to 2 h in a 37°C water bath. Terminate the reaction by transfer of tubes to ice. 6. Samples can either be precipitated using the TCA-deoxycholate method (Protocol 3) and separated by SDS-PAGE, or can be immunoprecipitated with G protein antiserum (Protocol 78) prior to separation with SDS-PAGE (Protocol 7)

3.2 Gel electrophoresis of G proteins Adaptations of the discontinuous SDS-PAGE technique developed by Laemmli provide the single most important method by which to separate the various members of the G protein superfamily (11). Although the toxinmediated incorporation of radiolabel appears to consist of single bands under the gel conditions used in Figure 2 (10% (w/v) acrylamide plus 0.27% (w/v) bisacrylamide) (Protocol 7), they actually represent the total pools of substrates for each particular toxin. Indeed the majority of G protein a subunits 81

Ian Mullaney have predicted molecular masses of between 39 and 45 kDa, and gel conditions and sample treatments have been optimized to separate polypeptides within this narrow molecular mass range. The most useful of these has been to reduce the concentration of bisaerylamide in the resolving gel from 0.27 to 0.06% (w/v). Figure 3 shows membranes prepared from NG108-15 cells that have been subjected to [ 32 P]ADP-ribosylation with pertussis toxin, and the radiolabclled products resolved by SDS-PAGE on 12.5% (w/v) acrylamide/ 0.06% (w/v) bisaerylamide gel (Protocol 8). Three pertussis toxin-sensitive G proteins are clearly separated in these cells using this gel system (panel A), that were identified as G(). G12, and G13, using specific antisera (panels B-D). In addition, treatment of the sample with N-ethylmaleimide differentially alkylates the a subunits of the pertussis toxin-sensitive G proteins G11, G12, G13, and the isoforms of G0 on accessible cysteine residues (Protocol 9). This has the effect of altering the migration of these a subunits on SDS-PAGE, with the result that it is possible to obtain greater resolution of the G0 isoforms from the Gi-like G proteins. If sample alkylation is performed in conjunction with resolution on a 12.5% acrylamide/0.06% bisaerylamide gel, the separation achieved can be dramatic. This technique is particularly useful when trying to identify G proteins with antisera that cross-react, a common example being that antisera directed against the carhoxyl terminus of G n can cross-react with G13 because of the presence of an immunodominant tyrosine in the peptide sequence of these two polypeptides. To separate the isoforms of G0a or G proteins of the Gq family, the best strategy is to resolve the membranes on 12.5% acrylamide gels containing 6 M urea (Protocol 10). It is also possible to separate these proteins on SDS-PAGE gels which contain a 4-8 M urea gradient, or on two-dimensional gels. However, both of these methods arc technically move difficult and have little advantage over the 6 M

Figure 3. Resolution and identification of pertussis toxin-sensitive G proteins. Membranes from NG108-15 cells 1100 ug) were treated with thiol-activated pertussis toxin and [32P]NAD1 for 90 min at 37 o C and resolved by SDS-PAGE (12.5% w/v aeryIamide, 0,06% w/v bisacrylamide) (see Protocols 4, 70). Samples were transferred to nitrocellulose, and either autoradiographed (panel Al or immunoblotted with antisera directed against G13 (panel B), G12 (panel B), and Gn (panel B).

82

5: G proteins and their identification Table 2. SDS PAGE gel compositions a Solution Resolving gel Gel buffer 1 Acrylamide solution 1 Acrylamide solution 2 50%(v/v)glycerol 10%(w/v)APS TEMED H2O Stacking get Gel buffer 2 Acrylamide solution 1 10%(w/v)APS TEMED H2O

10% (ml)

12.5% (ml)

6.0 8.0 0.0 1.6 0.09 0.01 8.2

6.0 0.0 10 2.0 0.09 0.01 5.8

3.75 1.5 0.15 0.01 9.75

3.75 1.5 0.15 0.01 9.75

a

The volumes given are for one gel (180 mm x 160 mm with spacers of 1.5 mm) run as part of a Bio-Rad Protean I electrophoresis apparatus.

urea SDS-PAGE gel system. It should be noted that polypeptides do not run normally on SDS-urea gels. They may appear to run at different molecular masses from those seen in the absence of urea, making it more difficult to identify particular a subunits. It is suggested that purified or recombinant a subunits first be resolved on these gels and used as reference standards. Table 2 outlines the amounts of reagents needed to produce the various gels. Protocol 7. SDS-PAGE (10% (w/v) acrylamide) of G protein a subunits Equipment and reagents • Hamilton syringe • Gel buffer 2 solution (6 g Tris, 4 ml 10% . SDS-PAGE gel apparatus and power pack (w/v) SDS in 100ml H2O, pH 6.8) (e.g. Bio-Rad Protean I electrophoresis • 50% (v/v) glycerol in H2O system) . TEMED . 10% (w/v) SDS in H2O . 10% (w/v) ammonium persulfate in H2O . Acrylamide solution 1 (30 g acrylamide, 0.8 . Electrophoresis running buffer (6 g Tris, g bisacrylamide in 100 ml H2O) 28.8 g glycine, 20 ml 10% (w/v) SDS in 2000 • Gel buffer 1 solution (18.17 g Tris, 4 ml 10% ml H2O. Do not adjust pH) (w/v) SDS in 100 ml H2O, pH 8.8)

Method 1. Set up the gel apparatus according to the manufacturer's guidelines. Add all gel reagents in the order and amounts given in Table 2 into a 250 ml conical flask and mix gently. Cast the gel using a Pasteur pipette. 83

Ian Mullaney Protocol 7.

Continued

2. Carefully overlay the cast gel with approximately 1 ml of 0.1% (w/v) SDS, and allow the gel to polymerize. This should take between 1 and 2 h at room temperature. 3. After polymerization, remove SDS overlay, and wash the gel with distilled water to remove any remaining traces of SDS. Add all stacker gel reagents in the order and amounts given in Table 2 into a 100 ml conical flask and mix gently. Pour the stacker gel on top of the resolving gel, and place the well-forming comb in the top of the gel, ensuring that no air bubbles are trapped under the comb, and leave to polymerize for 1 h at room temperature. 4. After polymerization, remove the sample-well comb, and place the gel in the gel tank containing enough running buffer in the base to cover the bottom edge of the gel, and add the remaining running buffer to the top. 5. Load the prepared samples in the preformed wells using a Hamilton syringe. 6. Run the gel overnight (approximately 16 h) at 60 V and 15 mA per plate, until the dye front reaches the bottom of the gel plates.

Protocol8.

SDS-PAGE (12.5% (w/v) acrylamide) of G protein a subunits

Equipment and reagents • Hamilton syringe • Gel buffer 2 solution (6 g Tris, 4 ml 10% . SDS-PAGE gel apparatus and power pack (w/v) SDS in 100 ml H2O, pH6.8) (e.g. Bio-Rad Protean I electrophoresis • 50% (v/v) glycerol in H2O system) . TEMED . 10% (w/v) SDS in H2O . 10% (w/v) ammonium persulfate in H2O . Acrylamide solution 2 (30 g acrylamide, . Electrophoresis running buffer (6 g Tris, 0.15 g bisacrylamide in 100 ml H2O) 28.8 g glycine, 20 ml 10% (w/v) SDS in 2000 • Gel buffer 1 solution (18.17 g Tris, 4 ml 10% ml H2O. Do not adjust pH) (w/v) SDS in 100 ml H2O, pH 8.8)

Method 1. Set up the gel apparatus according to the manufacturer's guidelines. Add all gel reagents in the order and amounts given in Table 2 into a 250 ml conical flask, and mix gently. Follow steps 1-5 from Protocol 7. 2. Run the gel overnight (approximately 18-20 h) at 100 V and 15 mA per plate, until the dye front reaches the bottom of the gel plates. 84

5: G proteins and their identification Protocol 9.

NEM treatment of samples for SDS-PAGE

Equipment and reagents • Microcentrifuge • 100'C heating block

. 100 mM NEM in H2O • Laemmli sample buffer (see Protocol 3)

Method 1. Take 25-150 ug of membranes, and place on ice in a 1.5 ml Eppendorf centrifuge tube. 2. Centrifuge for 5 min at 12000 r.p.m. in a microcentrifuge, remove the supernatant, and resuspend the pellet in 20 ul of TE buffer. 3. Add 10 ul of 5% (w/v) SDS, 50 mM DTT, and incubate at 90°C for 5 min. 4. Cool the samples on ice, add 10 ul of freshly prepared 100mM NEM to each tube, and leave at room temperature for 20 min. 5. Add 20 ul Laemmli sample buffer, and load the sample onto the gel.

Protocol 10. SDS-urea PAGE (12.5% (w/v) acrylamide) of G protein a subunits Equipment and reagents 10% (w/v) SDS in H2O • 6 M urea-gel buffer 2 solution (6 g Tris, 4 ml 10% (w/v) SDS, 36.025 g urea in 100 ml H2O, pH 6.8) > 50% (v/v) glycerol in H2O i TEMED > 10% (w/v) ammonium persulfate in H2O Electrophoresis running buffer (6 g Tris, 28.8 g glycine, 20 ml 10% (w/v) SDS in 2000 ml H2O. Do not adjust pH)

• Hamilton syringe • SDS-PAGE gel apparatus and power pack (e.g. Bio-Rad Protean I electrophoresis system) • 6 M urea-acrylamide solution 2 (30 g acrylamide, 0.15 g bisacrylamide, 36.025 g urea in 100 ml H2O) • 6 M urea-gel buffer 1 solution (18.17 g Tris, 4 ml 10% (w/v) SDS, 36.025 g urea in 100 ml H2O, pH 8.8)

Method 1. Set up the gel apparatus according to the manufacturer's guidelines. Add all gel reagents in the order and amounts given in Table 2 for a 12.5% SDS-PAGE gel into a 250 ml conical flask, and mix gently.8 2. Follow steps 1-5 from Protocol 7. 3. Run the gel overnight (approximately 18-20 h) at 120 V and 50 mA per plate, until the dye front reaches the bottom of the gel plates. ' If the room temperature is on the cold side, there is the possibility that the urea will come out of solution as the gel is polymerizing. To avoid this, allow the gel to set in a warmer room, e.g. tissue culture room, 30°C hot room, etc.

85

Ian Mullaney The detection of radiolabelled polypeptides is best done by autoradiography (Protocol 11} using conventional X-ray film. However, newer technologies such as phosphorimage analysis allow greater sensitivity and easier quantification of results (Protocol 12). Protocol 11. Autoradiography Equipment and reagents • • • • •

Imaging densitometer Gel drier and vacuum pump Whatman 3 mm filter paper X-ray film developing apparatus -80°C freezer

Coomassie stain buffer (0.25% (w/v) Coomassie Brilliant Blue R-250, 45% (v/v) methanol, 10% (v/v) glacial acetic acid) Gel destaining buffer (45% (v/v) methanol, 10% (v/v) glacial acetic acid)

Method 1. Remove the SDS-PAGE gel with the resolved radiolabelled polypeptides from the electrophoresis apparatus, and soak for 1 h in Coomassie stain buffer. 2. Remove the stain buffer, add the destaining solution, and leave for 2-3 h. 3. Remove destaining buffer, and dry the gel onto Whatman 3 mm filter paper under suction from an electric vacuum pump attached to a gel drier at 70°C for 2 h. 4. Transfer the dried gel to a Kodak X-o-matic cassette with intensifying screens (or similar) containing Kodak X-omat S X-ray film, and allow to autoradiograph at -80°C for an appropriate time. 5. Develop the film (e.g. on a Kodak X-o-mat developing machine), and quantify the autoradiograph using an imaging densitometer.

Protocol 12.

Phosphorimaging

Equipment • Phosphorimager

Method 1. Follow steps 1-3 in Protocol 11. 2. Transfer the dried gel to a phosphorimager plate, and leave for an appropriate length of time. 3. Develop the image using a phosphorimager, and quantify the resultant image. 86


4. Immunological methods Initial attempts to define the specificity of cellular signalling systems made great use of the bacterial exotoxins produced by Vibrio choleras and Bordetella pertussis (2-5). These toxins were found to modulate the inhibitory and stimulatory mechanisms of cyclic AMP regulation by causing mono-ADPribosylation of those G proteins that transduced receptor interactions with adenylyl cyclase. However, the realization that multiple G protein gene products could act as substrates for these toxins within a single cell or tissue made the need for more selective, discriminatory tools. Immunological probes were first generated against purified G protein preparations, and although these proved extremely useful in the initial identification of G proteins, the problems of obtaining homogeneous polypeptide preparations meant that their use was limited. It was the isolation of cDNA species corresponding to the G protein a subunits that revolutionized our understanding of these molecules, allowing the generation of specific antisera directed against short peptide stretches unique to particular proteins (for a review, see ref. 13). To date, the primary amino acid sequences of seventeen G protein a subunits have been deduced, allowing the generation of these antisera for use as specific immunological tools. Although the G protein superfamily is highly conserved, there are regions of sequence variation, particularly in the carboxy terminal region, which contains the receptor coupling sites, that have been successfully used to produce specific polyclonal antipeptide antisera (see Table 1), Such antisera are generally produced by subcutaneous injection of peptide conjugated to carrier protein into rabbits, and can be tested either in ELISA assays against the antigen peptide, or by immunoblotting using either purified or recombinant G protein a subunits. This section outlines the procedures needed in the production and characterization of specific antipeptide antisera. In addition, methods that use these immunological tools are also described.

4.1 Immunization and serum collection Protocol 13 outlines a method for the production of antipeptide antisera directed against specific peptides corresponding to specific regions of G protein a subunits in commercially purchased New Zealand White rabbits (14). Protocol 14 describes the steps needed to harvest the serum for laboratory use. Pre-immune blood samples should be taken from each of the animals prior to injection, and the serum checked for any significant titre or immunological identification of cellular proteins. All antisera produced by this method should be tested for specificity against other C-terminal G protein sequences and non-related peptides by ELISA, to test for non-specific immunoreactivity (see Protocol 15). ELISA determinations use plates precoated with the immunogenic peptide and with peptides corresponding to the 87

Ian Mullaney

Figure 4. ELISA reactivity of antiserum raised against the carboxyl terminal peptide of Gqa/Gna against other C-terminal peptides of G proteins. ELISA assays were performed by the method described in Protocol 15 with various antiserum dilutions using 100 ng of peptides corresponding to the C-terminal decapeptides of Gq/G11 (open dotted squares), G2 (open diamonds), G11 + G12 (filled squares), G13 (open squares), and G0 (filled diamonds). (Data from Mitchell et al., 1991, ref. 23).

equivalent regions of other G protein a subunits. Half-maximal antiserum dilutions for useful antisera in immunoblots and immunoprecipitations are typically between 1:10 000 and 1:100 000, although it should be stressed that ELISA alone gives little information on the suitability of candidate antisera for these techniques. Figure 4 shows an ELISA performed on an antiserum raised against the terminal decapeptide of Gq with carboxyl terminal peptides corresponding to a variety of G protein a subunits. Immunoreactivity is specific to Gq peptide, with a half-maximal dilution value in excess of 1:100000. Alternatively, immunoblot analysis using antisera with added peptide to compete specific binding may prove useful in determining antiserum specificity. Table 1 contains peptide sequences commonly used in G protein anti-peptide antisera production. 88

5: G proteins and their identification Protocol 13. Immunization of New Zealand white rabbits Equipment and reagents • Probe sonicator . 21 mM glutaraldehyde in H2O • Keyhole limpet haemocyanin • 0.1 M Na phosphate buffer (pH 7.0) • Freund's complete and incomplete adjuvants • Peptide

Method 1. Dissolve 10 mg of keyhole limpet haemocyanin and 3 mg of peptide in 1 ml of 0.1 M Na phosphate buffer (pH 7.0). 2. Add 0.5 ml of 21 mM glutaraldehyde dropwise with stirring, and incubate at room temperature overnight. 3. Mix with an equal volume of Freund's complete adjuvant, and sonicate at full power for 20 s. 4. Immediately after sonication, inject the resultant emulsion in 0.2 ml volumes into multiple subcutaneous sites in the rabbit. Immunizations are normally performed simultaneously into two rabbits to maximize successful antibody production. 5. After two weeks, give each animal a booster immunization with material prepared identically, except that one-half as much peptide and keyhole limpet haemocyanin are injected in Freund's incomplete adjuvant.

Protocol 14. Serum collection Equipment • Benchtop centrifuge

• Glass universal for serum collection (20 ml)

Method 1. Four weeks after the booster injections, bleed the animals from ear arteries, collect the blood into glass universals, and allow to clot overnight at 4 °C. a 2. Remove the straw-coloured serum from the clot, and centrifuge at 1000 r.p.m. for 5 min on a benchtop centrifuge to remove any residual erythrocytes. 3. Aliquot the serums in appropriate volumes (100-200 ul), and store at -20°C. * Use glass universals; plasticware does not allow the clot to shrink to allow serum harvest.

89

Ian Mullaney Protocol 15. ELISA Equipment and reagents • . . . .

ELISA plate reader • ELISA secondary antibody solution (1:1000 96-well ELISA plates dilution of horseradish peroxidasePeptide solution (10ugml-1) conjugated donkey anti-rabbit IgG, diluted in ELISA PBS (see Protocol 1} antibody carrier solution) ELISA blocking solution (1 g powdered milk . Citrate-phosphate buffer (17.9 ml 0.1 M in 100 ml PBS) citric acid; 32.1 ml 0.2M Na2HPO4; 50 ml . PBS-Tween 20 (0.5 ml Tween 20 in 1000 ml H2O, pH 6.0). PBS) • H2O2 solution (10 ul of stock H2O2 in 10 ml

. ELISA antibody carrier solution (0.05 ml Tween 20, 0.1 g powdered milk in 100 ml PBS) . ELISA primary antibody solution (G protein antiserum diluted in ELISA antibody carrier solution)"

H2O) « o-phenylenediamide dihydrochloride (OPD) substrate solution (4 mg OPD, 9 ml citratephosphate buffer, 1 ml H202 solution) • 2 M H2S04

Method 1. Coat a 96-well ELISA plate with antigen peptide by adding 100 ul of 10 ug ml-1 peptide solution to each well, covering the plate with cling film, and incubating overnight at 4°C. 2. Remove the liquid, and wash each well twice with PBS. 3. Blot dry, add 100 ul of ELISA blocking solution to each well, and incubate at 37°C for 1 h. 4. Remove blocking solution, wash each well twice with PBS-Tween 20, and blot dry. 5. Add 100 ul of increasing dilutions of antiserum (from 1:10 to 1:100 000) to each well, cover the plate with cling film, and incubate overnight at 4°C. 6. Remove antiserum dilutions, wash each well twice with PBS-Tween 20, and blot dry. 7. Add 100 ul of secondary antibody solution to each well, and incubate at 37°C for 1 h. 8. Remove the secondary antibody solution, wash each well five times with PBS-Tween 20, and blot dry. 9. Add 100 ul of OPD substrate solution to each well, wrap the plate in aluminium foil, and incubate, in the dark, at room temperature, for 15-20 min. 10. Stop the reaction by addition of 50 ul 2 M H2S04 to each well, and read at 492 nm using an ELISA plate reader. 11. Plot the absorbance reading against antiserum dilution to determine specificity. a

Dilute the antiserum simply by a series of 1:1 dilutions starting from 1:10, 1:20, 1:40, 1:80, 1:160, etc.

90


4.2 Immunoblotting and immunoprecipitation Protocol 16 describes the transfer of proteins from SDS-PAGE gels onto nitrocellulose, and subsequent incubation with antisera. This method, which essentially follows the strategies reported by Towbin (15), can be used in conjunction with the variety of gels, including low bisacrylamide and SDSurea gels, that have been previously described. Although we prefer to use a fully immersed blotting procedure, semi-dry transfer can also be successfully used. Protocol 16. Electroblotting of proteins onto nitrocellulose Equipment and reagents • Electroblotting apparatus and power pack (e.g. Bio-Rad Trans-Blot Cell) • Whatman 3mm chromatography filter paper • Nitrocellulose • Blotting buffer (15 g Tris, 72 g glycine, 1000 mlmethanol, made up to 5000 ml with H20. Do not adjust pH) . Ponceau S solution (15 g trichloroacetic acid in 500 ml H20; allow to dissolve and add 0.5 g Ponceau S). Keep stock solution at room temperature and re-use . PBS-NP40 (2 ml Nonidet P40 in 1000 ml PBS)

• Blocking buffer (5 g gelatin in 100 ml PBS) . First antiserum solution (appropriate anti-G protein antiserum dilution in PBS-NP40 containing 1% (w/v) gelatin) 'Second antibody solution (1:500 dilution of commercial horseradish peroxidaseconjugated donkey anti-rabbit IgG in PBSNP40 containing 1% (w/v) gelatin) • o-dianisidine solution (10 mg o-dianisidine hydrochloride solution in 1 ml H20) • Sodium azide solution (1 g NaN3 in 100 ml H2O) . 30% (v/v) hydrogen peroxide

Method 1. Transfer proteins, separated by SDS-PAGE, onto nitrocellulose using electroblotting apparatus according to the manufacturer's instructions.3 2. Transfer the electroblotted nitrocellulose sheet into a dish, cover with 100 ml immunoblot blocking buffer, and incubate for 2 h at 30°C. 3. Remove blocker, wash the nitrocellulose using copious amounts of double-distilled water, add the first antiserum solution (normally the specific anti-G-protein antiserum), and incubate overnight at 30°C. 4. Remove primary antiserum, and wash the blots thoroughly with double-distilled water to remove all the unbound antiserum. 5. Wash the blot with PBS-NP40 for 2 X 1 0 min, then incubate at 30°C for 2 h in the second antiserum solution. 6. Thoroughly wash the blot with double-distilled water, then with PBSNP40 for 2 x 1 0 min, and finally with two washes each of 10 min with PBS. 7. Place the blot in the dish containing 40 ml PBS. Add 1 ml newly prepared o-dianisidine solution, then 10 n-l of stock hydrogen peroxide. 91

Ian Muilaney Protocol 16.

Continued

8. Remove the developer, terminate the reaction by addition of sodium azide solution, and leave for 2 mm. Pour off the sodium azide and wash with water. a

To check if transfer is complete, remove the nitrocellulose from the blotting sandwich, place in a clean container, and cover with Ponceau S solution. Gently rock until protein banding appears. Discard the staining solution, and wash the blot with blotting buffer from the electroblotting tank until the bands disappear.

Sustained exposure of cell surface receptors to agonist frequently results in downregulation of both the receptor and the activated G protein in a coordinated manner. We have used the ability of many of these anlisera to immunopreeipitate G protein a subunits to probe the mechanisms involved in the turnover of these polypeptides. Protocol 17 describes pulse-chase assay techniques, in which cells are first incubated with [ 35 S]-methionine labelled medium, then chased in the presence or absence of a receptor agonist. Subsequent immunoprecipitation of these radiolahelled polypeptides with specific antisera has revealed that the mechanism of downregulation for a variety of G protein a subunits is agonist-induced accelerated turnover of the a sutmnit (Protocol 18). As an example, we have demonstrated that activation

Figure 5. The effect of agonist on the rate of degradation of Gqa/G11a in Chinese hamster ovary cells expressing the human M1 muscarinic acetylcholine receptor. Cells were labelled with Trans[ 35 S]-label (Protocol 77) and the rate of turnover of Gqa/G11a was measured by immunoprecipitation (Protocol 181with the specific antiserum, CQ2, following various periods in which the ceils were maintained in the presence (+) or absence (-) of 1mM carbachol. Panel A depicts a fluorogram from a typical experiment. Panel B shows the resultant quantification of the fluorogram by densitometric scanning. Signals from imrnunoprecipitants from cells maintained in the absence (open square) or presence (filled circle) of carbachol. pirn = preirnmune serum, (Data from Mitchell et al., 1993, ref. 27).


of the human ml muscarinic receptor by the selective agonist carbachol results in an enhanced degradation of Gqa/G11a, the G proteins which interact with this receptor (Figure 5). Protocol 17. [35S]-trans pulse-chase Equipment and reagents i 35S labelling medium: methionine- and cysteine-free Dulbecco's modified Eagle's medium (ICN Biomedicals, Inc.) supplemented with 50 uCi ml-1 Tran35S-label (ICN) and 1% (v/v) heat-inactivated, dialysed, fetal bovine serum •

• Tissue culture facilities • Benchtop centrifuge, microcentrifuge, and refrigerated ultracentrifuge • 100°C heating block • 2 ml screw-cap Eppendorf tubes . 2% w/v SDS

Method 1. Seed cells into 75 cm3 flasks or 6-well culture dishes. 2. When cells are approximately 60% confluent, replace growth medium with 35S labelling medium, and incubate cells in 35S-label for 20-48 h (pulse). 3. Wash the cell monolayer twice with normal growth medium, and leave in fresh growth medium (chase). 4. At appropriate times, gently wash the cells off the surface of the flask with a Pasteur pipette or rubber policeman, collect in a 12.5 ml plastic centrifuge tube on ice, and centrifuge at 1000 r.p.m. at 4°C for 5 min in a benchtop centrifuge. 93


Continued

5. Take off medium, resuspend the cells in 100 ul H2O, and add 100 ul 2% (w/v) SDS.b Transfer to a 2 ml screw-cap Eppendorf tube. 6. Tighten the screw cap onto the tube, and heat to 100°C for 20 min.c 7. Transfer the tube to ice, pulse spin to collect all moisture at the bottom of the tube, and proceed to the immunoprecipitation method (see Protocol 18). a

Caution: 35S is volatile. Stocks should be opened and aliquotted in a fume hood. Put aliquots in 2 ml screw-cap Eppendorf tubes and store at -80°C. b To ensure proper solubilization of the cell pellet, always resuspend in H2O, then add the SDS. Do not resuspend the cell pellet directly in SDS. c If the sample is still viscous at this stage, pass it through a 25-gauge needle and syringe, and re-boil as before for 10 min. At this stage it is possible to freeze the sample at -20°C and store until further use.

Protocol 18.

Immunoprecipitation

Equipment and reagents • Benchtop centrifuge, microcentrifuge, and refrigerated ultracentrifuge . 100°C heating block ., . .. . u .Rotating wheel inhibitor, 10 . 1.33% (w/v) SDS . Pansorbin or protein A-sepharose . IP wash buffer (80 ml IP buffer, 20 ml 1% (w/v) SDS).

• IP buffer (1% Triton X-100, 10 mM EDTA, 100 mM NaH2PO4, 10 mM NaF, 100 ixM Na2VO4, 50 mM Hepes, 1 mM PMSF, 3 mM benzamidine, 0.1 u.M soybean trypsin uM leupetin, 0.2 uM aprotinin, 1.5uMantipain, pH 7.2, at 4°C) . IP final wash buffer (50 mM Tris, pH 6.8, at 4°C) . Laemmli sample buffer

Method 1. To each sample in a final volume of 50 ul, add 150 ul of 1.33% (w/v) SDS in 2 ml screw-cap Eppendorf tubes. 2. Heat the samples to 95°C for 5 min, then place on ice to cool and centrifuge the samples to the bottom of the tubes by briefly spinning the samples at maximum speed in a microcentrifuge. 3. Add 0.8 ml of ice cold IP buffer containing protease inhibitors to each sample, mix by inverting, and leave on ice for 1 h. 4. Spin samples in a microcentrifuge at 12000 r.p.m. for 10 min at 4°C, and transfer the supernatant to a new tube. 5. Add an appropriate amount of antibody (between 2 and 20 ul, depending on the antiserum) and incubate with rotation at 4°C overnight. 6. Add 50 ul of Pansorbin (or 20 ul protein A-sepharose) to each sample, and incubate at 4°C with rotation for a minimum of 4 h. 94

5; G proteins and their identification 7. Spin samples in a microcentrifuge at 12000 r.p.m. for 2 min at 4°C, and wash three times with 1 ml IP wash buffer, pelleting the Pansorbin complex between each wash by centrifugation for 30 s at 12000 r.p.m.. 8. After the final spin, remove the supernatant and wash the pellet with 1 ml IP final wash buffer, and repeat the centrifugation step. 9. Remove the final wash buffer, and add 50 ul Laemmli buffer. Heat samples to 100CC for 10 min, centrifuge for 10 min at 12000 r.p.m. to pellet the Pansorbin, then load the supernatant onto the gel.

5. Quantification of G protein a subunits The ability to produce high-level expression of recombinant mammalian G protein a subunits in Escherichia coli has revolutionized our ability to measure accurately the cellular levels of these polypeptides. Protocol 19 describes a method published by Wise and Milligan that uses plasmid pT7.7 into which the various Ga genes have been subcloned (16). This expression system uses the promoter for bacteriophage T7 RNA polymerase in the plasmid vector pT7.7. This expression vector contains an initiation codon ATG, and a ribosome binding site positioned downstream from the T7 promoter such that maximal expression is ensured. Expression constructs were transformed into the lysogen BL21 (DE3), which contains a single chromosomal copy of the gene for T7 RNA polymerase under control of the isopropyl-p-D-thiogalactopyranoside (IPTG)-inducible lac UV5 promoter. It should be noted that preparations of partially purified G protein a subunits are available commercially.

Protocol 19. Preparation of competent E. constrain BL21 DE3, transformation of cells with the expression vector pT7.7, and expression of mammalian G protein a subunits Equipment and reagents • Tissue culture facilities and shaking • Sterile L-broth-glucose (10 g tryptone, 5 g incubator yeast extract, 10 g NaCI, 3.6 g glucose in . Clinical autoclave (e.g. Prestige medical 1000 ml H2O, pH 7.0) series 2100) • Sterile ampicillin stock (50 mg ml-1) • Benchtop centrifuge • Sterile L-broth-agar (10 g tryptone, 5 g . Spectrophotometer yeast extract, 10 g NaCI, 15 g agar in 1000 o . 100 C heating block mlH2O,pH7.0) . SDS-PAGE gel apparatus and power pack ' 100 mM isopropyl-B-D-thiogalactopyrano. E. coli strain BL21 DE3 with G protein cDNA side (IPTG) • Laemmli sample buffer (0.605 g Tris, 30 g jnsert urea, 5 9 SDS, 6 g DTT, 10 mg bromosterile L-broth (10 gtrytone, 5g yeast

phenol blue in 100 ml h2o

extract, 10 g NaCl in 1000 ml H2,lo

95


Continued

Method 1. Take 50 (il of E. coli stock, add 5 ng of plasmid DNA, and incubate on ice for 15 min. 2. Heat the cells at 42°C for exactly 90 s, then return to ice for 2 min. 3. Add 450 ul of L-broth-glucose, and allow the cells to recover by incubation at 37°C for 1 h in a shaking incubator. 4. Spread 100 ul of the transformants onto L-broth-agar plates containing 100 ul ml-1 ampicillin, and incubate overnight at 37°C. 5. Select single colonies from the plate, and put them into 10 ml L-broth containing 100 ug ml-1 ampicillin, and incubate in a shaking incubator overnight at 37°C. 6. Take 0.5ml of the overnight culture, inoculate into 50 ml L-broth containing 100 ug ml-1 ampicillin, and incubate in a shaking incubator at 37°C until an absorbance at 550 nm of between 0.3 and 0.5 absorbance units is reached. 7. Remove 1 ml of the cell suspension as a control, add 0.5 ml of 100 mM IPTG to the remainder, and incubate in a shaking incubator at 37°C for 4h. 8. Remove 1 ml of the cell suspension, spin in a benchtop microcentrifuge for 10 min at 12000 r.p.m., and discard the supernatant. 9. Add 25 ul of Laemmli sample buffer to the pellet, heat to 90°C for 10 min, and resolve the whole cell extracts by SDS-PAGE.

Quantification is most conveniently achieved by immunoblotting various amounts (0-100ng) of either E. coli-expressed G protein or purified G protein along with known amounts of the plasma membrane fractions. Data from these immunoblots can then be obtained either by densitometric scanning (Protocol 20), or overlay techniques that use commercial radiolabelled second antibody (Protocol21). A standard curve can be constructed, and levels of G protein in the sample can be assessed and expressed in terms of membrane protein, tissue amount, or even cell number. Figure 6 shows such an experiment where the cellular levels of Gsa were assessed in NCB20 cells.

96

5; G proteins and their identification

Figure 6. Quantification of levels of E. coli-expressed Gsa, (A). Various amounts (0-25 ng) of E coli-expressed Gsa (long isoform) were resolved by SDS-PAGE, and immunoblotted with an antipeptide antiserum directed against G s a. Lane 1, 2.5 ng; lane 2, 5 ng; Iane3, 7,5 ng; lane 4: 10 ng; lane 5, 15 ng; lane 6, 20 ng; lane 7, 25 ng. Membranes (20 ug) from untreated (lane 8) and isoprenaline-treated (10 uM, 16 h) (lane 9} NCB-20 cells stably transfected to express the human B2-adrenoceptor were also immunoblotted. (B). The developed immunoblot was subjected to densitometric analysis as described in Protocol 20. and a standard curve was constructed, (Data from Mullaney et al., 1995, ref. 28).

Protocol 20.

Densitometric quantitation of immunoblots

Equipment • Imaging densitometer

Method 1. Place the developed immunoblot onto filter paper and allow to dry.

2, Scan the blot into an imaging densitometer (e.g. Bio-Rad GS-670) and quantify.


Continued

3. Plot the standard curve showing amount of recombinant or purified G protein against arbitrary densitometric values, and extrapolate the values for the unknowns from the curve.

Protocol 21. [125l]-labelled donkey anti-rabbit immunoglobulin overlay technique Equipment and reagents • Gamma counter . [126l]-labelled donkey anti-rabbit immuneglobulin, (product no. IM 134), specific activity 750-3000 Ci mmol-1 (Amersham International plc., Amersham, UK)

• [125l]-overlay solution: (50ml PBS-NP40 containing 1% (w/v) gelatin and 5 (iCi [125l]-labelled donkey anti-rabbit immunoglobulin)

Method 1. Place the developed blots in a dish containing 50 ml [125l]-overlay solution, and incubate for 1 h at 30°C. 2. Remove the overlay solution, wash the blots thoroughly with doubledistilled water to remove all the unbound label, and then wash twice for 30 min with PBS. 3. Allow the blot to air dry, excise the immunoreactive bands, and measure by liquid scintillation counting. 3. Plot the standard curve showing amount of recombinant or purified G protein against the c.p.m. obtained from the bound [125l]-overlay solution, and extrapolate the values for the unknowns from the curve.

6. Functional aspects of G protein signalling The ability to bind and hydrolyse guanine nucleotides as integral parts of their activation-deactivation cycle is perhaps the most prominent functional characteristic of signal-transducing heterotrimeric G proteins. Measurement of the effects of receptor activation on high-affinity GTPase activity provides the basis of a variety of methods used to probe G protein function. Activation of receptor by agonist initiates the release of guanosine 5'-diphosphate (GDP) from the a subunit of the heterotrimer, followed by the binding of guanosine 5'-triphosphate (GTP). This leads to the dissociation of the aBy heterotrimer into GTP-liganded a subunit and free By dimer, both of which can interact with a variety of effectors to modulate intracellular second messenger concentrations. Deactivation occurs from hydrolysis of GTP by the intrinsic GTPase activity of the a subunit, and subsequent reassociation of the heterotrimer (1,13). 98


6.1 Determination of GTP hydrolysis in membrane preparations Using a modified version of the method developed by Cassel and Selinger (17), it is possible to measure the rate of agonist-mediated GTP hydrolysis (Protocol 22). Determination of high affinity GTPase activity is achieved by measuring the agonist-mediated breakdown of substrate y[32P]-GTP to GDP and [32Pi], which is counted as an index of enzymatic activity. Although receptor stimulation of GTPase as a measurement of activity for Gsa has been reported, the degree of stimulation is often modest. On the other hand, robust GTPase activity measurements mediated through pertussis toxin-sensitive G proteins are common, a reflection of their cellular abundance and higher intrinsic rate of GTP hydrolysis.

Protocol 22. Determination of agonist-stimulated high-affinity GTPase activity in plasma membranes Equipment and reagents • Liquid scintillation counter • 2x stock reagent assay mixture (2 mM . Refrigerated benchtop centrifuge App(NH)P, 2 mM ATP, 2 mM ouabain, 20 mM . TE buffer (see Protocol 7) creatine phosphate, 5 units ml-1 .,,_. ___ , ... creatine kinase, 200 mM NaCI, 10 mM Qn ...... . ^FP] GTP (product no-1 PB 10244), specific MgCl2, 4 mM2 mM EDTA, activity >5000 Cimmol (Amersham Phar- 20 mM Tris-HCl (pH 7.5), 1 uM GTP 32 a,b macia Biotech UK Limited., Amersham, UK) containing -y[ P] GTP). • Charcoal slurry (5% (w/v) activated charcoal in 20 mM phosphoric acid, pH 2.3)

Method 1. Take an appropriate amount of plasma membranes (typically between 2 and 10 ug in 20 ul TE buffer), place in a 1.5 ml Eppendorf centrifuge tube, and make up to 20 ul with TE buffer.c 2. Add 50 ul of assay mixture and 10 ul of the appropriate agonist to each tube. Blank values are determined by replacing membranes with 20 ul TE buffer, and non-G protein-mediated low-affinity GTPase activity is assessed by incubating in parallel tubes that include 100 uM GTP. 3. Make up each sample to 100 ul final volume with water, briefly vortex mix, and initiate the assay by transferring the tubes to a 37°C water bath. After 20 min, terminate the assay by removal of the tubes to ice.d 4. Add 900 ul of charcoal slurry to each tube, mix, and centrifuge at 12000 gfor 20 min in a refrigerated benchtop centrifuge 99


Continued

5. Carefully remove 500 u1 of the clear supernatant from each sample to a scintillation vial, and count by either liquid scintillation or Cerenkov counting. a

The reaction mix is twice the final reagent concentration -y[32P] GTP is added as a trace amount to the cold GTP. Each assay tube should contain approximately 50000 c.p.m. Count an aliquot of the final reaction mix to determine the exact number of counts per assay tube C AII manipulations should be performed on ice. d Further hydrolysis of GTP is negligible on ice. b

To decrease non-specific hydrolysis of GTP, ATP, creatine phosphate, and creatine kinase are included in the assay as an ATP regenerating system to prevent the nucleoside diphosphokinase-mediated transfer of [32Pi] to endogenously present ADP, which can in turn be hydrolysed by specific ATPases. In addition, App(NH)P is added to inhibit nucleoside triphosphatases and ouabain is included as an inhibitor of Na +/ K + ATPase. Exogenous Mg2+, an important co-factor in G protein activation, is also added. Results are expressed as pmol GTP hydrolysed per minute per mg of membrane protein.

6.2 Measurement of receptor-stimulated [35S]GTPyS binding in membrane preparations The binding of radiolabelled GTP analogues which are not hydrolysed by the GTPase activity of the G protein provides a convenient tool with which to probe the initial steps of G protein activation. Measurement of receptormediated increases in 5'-0-(y/-[35S]thio)triphosphate ([35S]GTP-yS) binding is most frequently used (Protocol 23). Protocol 23. Determination of agonist-stimulated [35S] GTP-yS binding in plasma membranes Equipment and reagents • • • .

Liquid scintillation counter Refrigerated benchtop centrifuge TE buffer (see Protocol 1) [35S] GTPyS, (product no. NEG-030H)), specific activity >1250 Ci mmol-1 (NENDupont). To avoid decomposition, the stock should be diluted 100x in 10 mM Tricine, 10 mM dithiothreitol (pH 7.6), aliquotted, and stored at -80°C

• Brandel cell harvester or other binding drum apparatus . Whatman GF/C glass fibre filters • 2X reagent assay mixture (6 mM MgCI2, 200 mM NaCI, 20 uM GDP, 0.4 mM ascorbic acid, 40 mM Hepes, pH 7.4) containing [35S] GTPyS at 50 nCi per assay point a, b • Ice-cold filter wash buffer (3 mM MgCI2, 20 mM Hepes, pH 7.4)

Method 1. Take an appropriate amount of plasma membranes (typically between 10 and 50 ug in 25 ul TE buffer), and place in a 5 ml disposable glass test tube.c 100

5: G proteins and their identification 2. Add 50 ul of assay mixture and 10 ul of the appropriate agonist to each tube. Blank values are determined by replacing membranes with 25 ul TE buffer, and non-specific binding is assessed by incubating parallel tubes that include 10 uM unlabelled GTP-yS. 3. Make up each sample to 100 ul final volume with water, briefly vortex mix, initiate the assay by transferring the tubes to a 4°C ice slurry bath, and incubate for 60 min. 4. Incubation is terminated by addition of 3 ml filter wash buffer to each tube, and immediate passing of the sample through a GF/C filter. Wash the filter with 2 x 5 ml filter wash buffer. 5. Remove the filter into scintillation vials containing 10 ml scintillation cocktail, and count by liquid-scintillation counting. a

The reaction mix is 2x final reagent concentration Each assay tube should contain approximately 100 000 c.p.m of [35S] GTP-yS. Count an aliquot of the final reaction mix to determine the exact number of counts per assay tube c All manipulations should be performed on ice. b

One attraction of this method is that it can be adapted to give optimal responses for receptor stimulation of different G proteins in a variety of cells and tissues. When measuring receptor-mediated [35S] GTP-yS binding to Gi or G0 with incubations either at 4°C or 25-37°C, addition of GDP (0.1-10 uM) and Nad (100-150 mM) is recommended. In contrast, measurement of receptor-mediated [35S]GTP-yS binding to Gs should be performed at 4°C in the absence of both GDP and NaCl, since these reagents act to decrease binding to this G protein (18). Results are expressed as pmol [35S]GTP-yS bound per mg of membrane protein.

Acknowledgements I would like to thank Professor Graeme Milligan for providing laboratory space and helpful discussion in the preparation of this manuscript. I would also like to thank Drs F. R. McKenzie, F. M. Mitchell and A. Wise for their contributions towards the information contained in this chapter.

References 1. 2. 3. 4. 5. 6.

Gilman, A. G. (1987). Annu. Rev. Biochem., 56,615. Gill, D. M., and Meren, R. (1978). Proc. Natl. Acad. Sci. USA, 75,3050. Katada, T., and Ui, M. (1982). J. Biol. Chem., 257,7210. Katada, T., and Ui, M. (1982). Proc. Natl. Acad. Sci. USA, 79,3129. Kurose, H., Katada, T. Amano, T., and Ui, M. (1983). J. Biol. Chem., 258,4870. Strathmann, M., and Simon, M. I. (1990). Proc. Natl. Acad. Sci. USA, 87,9113. 101

Ian Mullaney 1. Wilkie, T. M., Scherle, P. A., Strathmann, M. P., Slepak, V. Z., and Simon, M. I. (1991). Proc. Natl. Acad. Sci. USA, 88,10049. 8. Koski, G., and Klee, W. A. (1981). Proc. Natl. Acad. Sci. USA, 78,4181. 9. Hudson, T. H., and Johnson, G. L. (1980). J. Biol. Chem., 255,7480. 10. Milligan, G., Carr, C., Gould, G. W., Mullaney, L, and Lavan, B. E. (1991). J. Biol. Chem., 266, 6447. 11. Georgoussi, Z., Merkouris, M., Mullaney, L, Megaritis, G., Carr, C., Zioudrou, C., and Milligan, G. (1997). Biochim. Biophys. Acta, 1359,263. 12. Laemmli, U. K. (1970). Nature, 227,680. 13. Milligan, G. (1988). Biochem. J., 255,1. 14. Goldsmith, P., Gierschik, P., Milligan, G., Unson, C. G., Vinitsky, R., Malech, H. L., and Spiegel, A. (1987). J. Biol. Chem., 262,14683. 15. Towbin, H., Staehelin, T., and Gordon, J. (1979). Proc. Natl. Acad. Sci. USA, 76, 4350. 16. Wise, A., and Milligan, G. (1994). Biochem. Soc. Trans., 22,12S. 17. Cassel, D., and Selinger, Z. (1976). Biochim. Biophys. Acta, 452,538. 18. Wieland, T., and Jakobs, K. H. (1994). In Methods in enzymology (ed. lyengar, R.) Vol. 237, p. 3. Academic Press, London 19. Milligan, G., Unson, C. G., and Wakelam, M. J. O. (1989). Biochem. J., 262,643. 20. Falloon, J., Malech, H., Milligan, G., Unson, C., Kahn, R., Goldsmith, P., and Spiegel, A. (1986). FEBS Lett., 209,352. 21. Mullaney, L, and Milligan, G. (1990). /. Neurochem., 55,1890. 22. Mullaney, L, Magee, A. L, Unson, C. G., and Milligan, G. (1988). Biochem. J., 256, 649. 23. Mitchell, F. M., Mullaney, L, Godfrey, P. P., Arkinstall, S. J., Wakelam, M. J. O., and Milligan, G. (1991). FEBS Lett., 287,171. 24. Milligan, G. (1994). In Methods in enzymology (ed. Iyengar, R.). Vol. 237, p. 268. Academic Press, London. 25. Milligan, G., Mullaney, L, and Mitchell, F. M. (1992). FEBS Lett., 297,186. 26. Casey, P. J., Fong, H. K. W., Simon, M. L, and Gilman, A. G. (1990). J. Biol. Chem., 265,2383. 27. Mitchell, F. M., Buckley, N. J., and Milligan, G. (1993). Biochem. J., 293,495. 28. Mullaney, I., Shah, B. H., Wise, A., and Milligan, G. (1995). J. Neurochem., 65, 545.

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6

Construction and analysis of receptor—G protein fusion proteins ALAN WISE

1. Introduction It is now well established that a multitude of diverse extracellular signals such as neurotransmitters, hormones, odorants, and light elicit intracellular responses via vectorial signal transduction mechanisms across the lipid bilayer, following binding to specific cell surface receptors possessing seven transmembrane-spanning domains. Agonist-occupation of these receptors leads to activation of effector proteins, and the concomitant mobilization or production of second messengers that initiate the desired physiological response within the cell. In all eukaryotic organisms the family of heterotrimeric G proteins plays a pivotal role, acting as intermediates between such cell surface receptors and cytoplasmic or membrane-bound effector molecules, leading to the regulation of humoral, neural, metabolic, and developmental functions. Examples of intracellular effectors which are regulated by G protein activation include adenylate cyclase, phosphoinositidase C, cyclic GMP phosphodiesterases, phospholipase A2, and a number of ion channels (1,2). Structurally, the heterotrimeric G proteins are composed of distinct a, (3, and -y subunits of molecular masses 39-52, 35-36, and 7-8 kDa, respectively. The a subunit binds GTP, possesses intrinsic GTPase activity, and determines the specificity of the holoprotein for its receptor(s) and effector(s), whilst the By complex exists as a tightly associated complex which can itself activate effector molecules. All heterotrimeric G proteins studied so far function in a similar manner: agonist-occupation of G protein-coupled receptors (GPCRs) increases the rate of exchange of GTP for GDP in the nucleotide-binding pocket of the G protein a subunit. The GTP-liganded a subunit can then dissociate both from the receptor and from the G protein By complex, allowing effector regulation. Deactivation of the system is achieved by the intrinsic capacity of the a subunit to serve as a GTPase, resulting once again in the presence of GDP in the binding site (1). Molecular cloning techniques have allowed identification of at least sixteen genes encoding a subunits, and six and twelve encoding B and y subunits,

Alan Wise

Figure 1. Shows homology of the G protein a subunits at the amino acid level. The four major subclasses, Gs, Gi, Gq, and G12 are grouped.

respectively. The a subunits can be divided into four major subclasses, termed Gs, Gi; Gq and G12, based on structural homology at the amino acid sequence level (Figure 1). In addition, the number of cDNAs encoding GPCRs so far identified exceeds 400 and is still growing rapidly (2). Given that many cells express multiple G proteins and GPCRs, the potential signalling circuitry is immense. The fact that multiple receptors can couple to a single G protein, that single receptor subtypes can couple to more than one G protein, that individual ligands can bind to multiple functionally distinct receptor subtypes, and that G protein species can stimulate more than one effector, demonstrates that G protein-mediated signalling pathways form 104

6: Construction and analysis of receptor-G protein fusion proteins highly complicated networks. The advent of heterologous expression systems has proved invaluable in the characterization of signalling components and in elucidating receptor-G protein-effector coupling specificities. The techniques and methodologies developed to study and delineate the activation, function, and receptor-effector coupling specificities of individual G protein a subunits have proved extremely useful in unravelling the complexities of signalling across the plasma membrane (see Chapter 5). This chapter describes a recent approach taken by us and others (3-11) which allows the interaction between a receptor and its cognate G protein to be studied in isolation, following heterologous expression of both entities as constrained fusion proteins. In particular, GPCR coupling to members of the pertussis toxin-sensitive Gi family will be examined.

2. Methods to study G protein function 2.1 Second messenger production Over the last two decades a number of assay systems have been developed which measure changes in levels of second messenger molecules, including cAMP, IP3 and Ca2+, following exposure of whole cells to agonists. Hence they provide compelling evidence for the identification of effector proteins involved in particular G protein-mediated signalling cascades. Such technologies are now widely used to identify and unravel G protein and effectorcoupling specificities of receptors. More recently, reporter gene technologies have been identified and used as alternative ways of studying G protein function, and will be discussed elsewhere in this book (Chapter 8).

2.2 G protein activation One of the earliest points in the signalling cascade at which G protein function can be measured is at the level of G protein activation by receptor. This may be achieved using assays that measure the exchange of GDP for GTP, and/or the subsequent hydrolysis of GTP induced by agonist ligands. Such assays are, however, limited to members of the Gi family (with the exception of Gz), since they possess significantly higher rates of guanine nucleotide exchange (turnover numbers in the region of 4 min-1) than other G protein family members (1).

2.3 Use of pertussis toxin-resistant G protein mutants The a subunits of many individual G proteins are substrates for bacterial toxins elucidated by Vibrio cholerae and Bordetella pertussis. Each toxin mediates the transfer of an ADP-ribose moiety from NAD+ to specific amino acid residues (Figure 2). The site of ADP-ribose incorporation catalysed by cholera toxin has been identified as a single Arg residue, corresponding to 105

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Figure 2. Cholera and pertussis toxins catalyse the transfer of an AOP-ribose group from NAD1 to specific amino acid residues on G5 and G1 family G protein a subunits, respectively. In the case of the Gi G proteins, this modification occurs at a conserved cysteine residue four amino acids from the C-terminus, and leads to receptor-G protein uncoupling. Mutation of this cysteine residue to glycine renders the G protein insensitive to pertussis toxin modification.

position 188 of Gsa, and such modification leads to inhibition of the intrinsic GTPasc activity of the a suhunit and constitutive activation of the signalling pathway (12). Pertussis toxin-mediated ADP-ribosylation occurs at a cysteine residue at the fourth position from the C-terminus on members of the Gi-family (13, 14). This abrogates functional contact between receptor and Gi, thus maintaining the G protein in its inactive aBy heterotrimeric conformation (15). Since other G protein a subunits do not serve as substrates for pertussis toxin-catalysed ADP-ribosylalion, it is possible to determine whether a particular agonist at a GPCR invokes activation of members of the Gi family of G proteins by measuring attenuation of function following pertussis toxin treatment of cells or tissue (16). The pertussis toxin-sensitive G proteins Gl1a, G12a, and G13a are often co-expressed in cells, hence definition of the specificity of interactions of a receptor with individual G proteins from this family cannot be attempted using such a limited approach. To circumvent this problem, we (17) and others (18. 19) have mutationally altered these proteins, such that the cysleine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue, thus rendering these proteins refractory to pertussis toxin-catalysed ADP-ribosylation (Figure 2). These pertussis toxin-insensitive C y s > G l y mutants of G11a, G12a, and G13a can then be introduced into cells with a receptor of interest. Specificity of interaction between expressed receptor and Cys—»Gly G1a can be studied in isolation, following exposure of cells to pertussis toxin to eliminate any potential coupling to endogenous G 1 -family G proteins. We have previouslyused such technology to investigate interactions of the porcine a2 A -adrenoceptor with individual members of the G1 G protein family following transient expression in COS-7 cells (17). 106

6: Construction and analysis of rcceptor-G protein fusion proteins

3. Receptor—G protein fusions 3.1 Background The direct receptor-stimulated GTPase activity of G proteins has historically been difficult to measure, due to a lack of information on the absolute levels of expression of the receptor and its cognate G protein, and their localization in relation to one another in cells and at the plasma membrane. Indeed, such measurements have often resulted in estimates too low to account for the rapid kinetics of ligand-indueed activation and deactivation of signal transduction cascades (1,20.21). These problems can he overcome by constraining receptor and G protein within a single fusion protein, thus defining the stoichiomelry of expression of the two entities as 1:1 and ensuring their colocalization following expression (Figure 3). We and others (3-11) have used this approach to study thc mechanisms and specificities governing receptor-G protein interaction. In particular, we have generated fusion proteins between the a 2A -adrenoccptor and G11a, and between the A] adenosine receptor and Cys—>Gly pertussis toxin-insensitive variants of G11a, G12a, and G13a. These constructs have proved useful tools to study the enzymic capacity of G1 G proteins, to measure ligand efficacy, and to evaluate receptor coupling specificities of related G proteins. Strategies employed in the construction, expression, and measurement of function of such fusions will be described herein.

Figure 3. Receptor-G protein fusion protein. Construction involves fusion of the Nteminus of the G protein a subunit to the C-terminus of the receptor. 107

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Figure 4. Construction of receptor-G protein fusion cDNA. In this case the cDNAs encoding the a2 A -adrenoceptor and C y s - G i y pertussis toxin-insensitive variant of G11a were fused together, using a PCR-based approach as described in Protocol 7. This procedure results in production of an in-frame construct, whereby the 3' end of the a2Aadrenoceptor ORF is exactly adjacent to the 5' end of the Cys >Gly G11a ORF. Finally- the entire construct is subcloned into an expression vector such as pCDNA3. The letters E, N, and K denote restriction enzyme sites for EcoRI, A/col and Kpnl, respectively.

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3.2 Construction of receptor-G protein fusion proteins The following protocol details the construction of a fusion protein made between the a2A-adrenoceptor and a Cys—»Gly pertussis toxin-insensitive variant of G11a (4), which is depicted in Figure 4. The protocol takes advantage of the use of the polymerase chain reaction (PCR), which significantly expedites the manufacture of such constructs. Pfu DNA polymerase (Stratagene) from Pyrococcus furiosus is also employed, which exhibits a 12-fold higher fidelity of DNA synthesis than the more traditionally used Taq DNA polymerase (22). It also utilizes the existence of an Ncol site, which is found straddling the ATG initiation codon of G, family G proteins. Hence a similar strategy can be applied to the manufacture of other fusions that harbour GPCRs linked to other members of the Gj G protein family. Basically, the strategy involves the PCR-mediated addition of an Ncol restriction site at the 3' end of the ORF of the cDNA encoding the a2Aadrenoceptor, such that receptor and G protein cDNAs can be ligated together. Introduction of the Ncol site at the 3' end of the ORF results in the C-terminal amino acid of the receptor being altered from valine to alanine, and to removal of the stop codon. Rat Cys—»Gly G11a cDNA contains two Ncol sites, one straddling the ATG start codon and the other 268 bp downstream from this. Therefore, this 268 bp fragment must be excised from G11a to permit ligation to the 3' end of the receptor ORF. Once this ligation has been achieved, the 268 bp fragment can then be reinserted to yield the full fusion construct. A suitable host vector is pBluescript (Stratagene), since it does not possess an Ncol restriction site. This procedure results in production of an in-frame construct, whereby the 3' end of the at2A-adrenoceptor ORF is exactly adjacent to the 5' end of the Cys—>Gly G11a ORF. The full fusion construct can then be excised and ligated into a suitable eukaryotic expression vector. Protocol 1. Construction of receptor-G protein fusion cDNA The porcine a2A-adrenoceptor (23) was obtained from Dr. L. E. Limbird, Vanderbilt University, Nashville, TN, USA. The Cys-»Gly pertussis toxininsensitive variant of G11a was made using site-directed mutagenesis, which is detailed in ref. 17. Equipment • PCR machine

• 37°C shaking incubator

Method 1. PCR-amplify the ORF of the a2A-adrenoceptor DNA using the oligonucleotides: sense, 5'-TTGGTACCATGTATCCTTACGACGTrC-3'; anti109

Alan Wise Protocol 1. Continued

2.

3.

4.

5.

6.

sense, 5'-AAGAATTCCATGGCGATCCGTTTCCTGTCCCCACGGC-3'. The restriction sites for Kpnl, EcoRI and Ncol are underlined. PCR is performed in a reaction volume of 50 ul containing 20 mM Tris-HCI, pH 8.2; 10 mM KCI; 6 mM (NH4)2S04; 2 mM MgCI2; 1% (v/v) Triton X-100; 10 ug ml-1 BSA; 25 pmol of each oligonucleotide; 250 uM dNTPs; 2.5 ng of supercoiled a2A-adrenoceptor miniprep plasmid DNA in the vector pCDNAS (Invitrogen); and 2.5 units of native Pfu DNA polymerase. DMSO at a final concentration of 5% (v/v) is also included to assist primer annealing. The temperature cycling conditions (30 cycles) are: 94°C for 42 s (denaturation), 60°C for 1 min (annealing), and 72°C for 5 min (polymerization). Restriction digest a 1% (w/v) agarose gel-purified PCR-amplified fragment of approx. 1.5 Kb with Kpnl and EcoRI, and ligate into pBluescript through these restriction sites. Restriction digest rat Cys—»Gly G11a in pBluescript with Ncol, and remove the 268 bp fragment from the 5' end of the ORF by 1% (w/v) agarose gel electrophoresis. Religate the shortened Cys-»Gly G11a cDNA, and excise from pBluescript by digestion with EcoRI, and clone into the EcoRI site of the PCRamplified a2A-adrenoceptor in pBluescript, adjacent to the 3' end of the receptor ORF. Subclone the Ncol excised 268 bp fragment of rat Cys-»Gly G11a between the Ncol sites at the 3' end of the a2A-adrenoceptor ORF and at the 5' end of the Cys->Gly G11a ORF. The full fusion construct is then excised from pBluescript with Kpn\ and EcoRI, and ligated into the eukaryotic expression vector pCDNA3.

4. Expression of receptor-G protein fusions in cultured cells 4.1 Choice of recipient cell line Most of the functional studies that we have performed so far on receptor-G protein fusions have been carried out following transient transfection into two recombinant cell lines stably harbouring the SV40 large T antigen: human embryonic kidney cells (HEK293T), and the simian line COS-7. Both cell lines have been widely used to study receptor-G protein function following transient expression of foreign receptor cDNA; they are particularly appropriate for such studies as they do not express many GPCRs endogenously. They can also be cultured in monolayers for ease of manipulation, and possess rapid doubling times (Gly G11a fusion protein. Section 6 documents how these assay technologies have been used and adapted to provide insights into the mechanisms and specificities of receptor-G protein coupling, and how a fusion approach can be employed as a means of measuring agonist efficacy.

5.2 Receptor-promoted binding of guanosine-5'-[y-35thio]triphosphate ([35S]GTPyS) Binding of [35S]GTP-yS to membranes can be performed using two techniques, which differ only in the means by which bound nucleotide is separated from free: (1) using wheat germ agglutinin scintillation proximity assay (SPA) bead technology, and (2) using traditional separation of bound nucleotide from free by filtration. The assay using SPA technology is amenable to a 96well format, is more convenient than the filtration assay, and is detailed below in Protocol 4. The footnote to Protocol 4 briefly outlines the alternative filtration assay. Protocol 4. High affinity [35S]GTP-yS binding - SPA format Equipment and reagents • Assay buffer (20 mM Hepes, 100 mM NaCI, 10 mM MgCI2, pH 7.4) . [35S]GTP-yS (1170 Ci mmor') (Nycomed Amersham) . Wheatgerm agglutinin SPA beads (Nycomed Amersham) . 96-well clear-bottomed polystyrene plates (Wallac)

• Scintillation counter capable of reading in 96-well format (e.g. 1450 Microbeta Trilux, Wallac) • If using the filtration assay, additional equipment includes: vacuum filtration apparatus capable of filtering greater than 12 samples individually; filters of 2.5 cm diameter.

Method All membrane manipulations should be carried out at 4°C. Assays are performed in a 96-well format, using a method modified from that described in ref. 30. 1. Dilute membrane proteins (Protocol 3)(5 ug per point) to 0.083 mg ml-1 in assay buffer supplemented with saponin (10 mg l-1). 2. Preincubate diluted membranes with 40 uM GDP for 2-5 min.

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6: Construction and analysis of receptor-G protein fusion proteins 3. Apply 60 ul of diluted membrane mix (5 ug) to each well of a clearbottomed 96-well plate. 4. Add 20 ul of agonist or vehicle. 5. Determine non-specific values by the addition of 20 ul of 3 mM GTP (0.6 mM final concentration). 6. Start the assay following the addition of 20 ul of [35S]GTPyS (1170 Ci mmol-1) at 0.3 nM final concentration (100 ul final assay volume), and allow to proceed at room temperature for 30 min. 7. Add wheatgerm agglutinin SPA beads (Nycomed Amersham) (0.5 mg ml-1) in 25 ul assay buffer, and incubate the whole at room temperature for 30 min with agitation.a 8. Centrifuge the plates at 1500 g for 5 min, and determine bound [35S]GTP-yS by scintillation counting. a

The binding assay using filtration to separate bound nucleotide from free is essentially the same from steps 1-5. However, the reaction is terminated by rapid filtration through Whatman GF/C glass-fibre filters under vacuum. Filters are then washed three times with 5 ml of assay buffer. Filters are placed in a scintillation vial with 5-10 ml scintillation fluid, and radioactivity is quantified by scintillation counting.

5.3 Measurement of GTPase activity Protocol 5. Agonist stimulation of GTPase activity Equipment • 37°C incubator or water bath

• Scintillation counter

Method All manipulations are performed at 4°C. 1. Membrane proteins (Protocol 3) are diluted in 10 mM Tris HCI, 0.1 mM EDTA, pH 7.4 (TE buffer) to a concentration of 0.25 mg ml-1 (5 ug per assay). 2. An assay mixture is made with the following reagents: Reagent

Concentration in mixture

Final concentration in assay

App(NH)p ATP Ouabain Creatine phosphate Creatine phosphokinase Sodium chloride

2 mM 2 mM 2 mM 20 mM 5 units ml-1 200 mM

1 mM 1 mM 1 mM 10 mM 2.5 units ml-1 100 mM

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3.

4. 5. 6. 7.

8.

9. 10.

Magnesium chloride 10 mM 5 mM Dithiothreitol 4 mM 2mM EDTA 0.2 mM 0.1 mM Tris-HCI 20 mM 10mM y[32P]GTP 1uM 0.5 uMa Final pH of the assay mix is 7.5. An aliquot of this is retained for radioactive counting, so that the exact number of counts in each tube is known. Add aliquots (50 ul) of the above reaction mixture to Eppendorf tubes, together with 20 ul of diluted membrane protein and 10 ul of appropriate agonist. Final assay volume is 100 ul, which may be made up with water. Assess low affinity hydrolysis of y[32P]GTP by incubating parallel tubes in the presence of 100 uM GTP. Initiate the reaction by transferring the tubes to 37°C. Terminate the assay after 20 min by removal of the tubes to ice (hydrolysis of y[32P]GTP on ice is negligible). To separate free [32Pi] from the unhydrolysed y[32P]GTP, add 900 ul of a 5% (w/v) activated charcoal slurry in 20 mM phosphoric acid (pH 2.3) to each tube, giving a total volume of 1000 ul. Centrifuge tubes at 12000 g for 20 min at 4°C to pellet the charcoal along with unhydrolysed -y[32P]GTP. The free [32Pi] is present in the supernatant. Remove 500 ul aliquots of supernatant, and add to scintillation vials for radioactive counting. Radioactivity may be assessed by either liquid scintillation or Cerenkov counting.

a

Since the radioactive GTP will be present in trace amounts, it is necessary to add cold GTP up to the required concentration.

5.3.1 Calculation of GTPase assay results Each assay tube contained 50 pmol of GTP, in addition to approx. 50000 c.p.m. of y[32P]GTP. The amount of y[32P]GTP should be calculated each time by counting 50 u1 aliquots of the assay mixture. Hence the specific activity of the GTP is approx. 1000 c.p.m. per pmol. The rate of hydrolysis is calculated by: (C/S.A.) X 2 X (1000/P) X (1/T) where: C = counts in 500 ul sample S.A. = specific activity of GTP P = amount of protein in ug T = duration of assay 116

6: Construction and analysis of receptor-G protein fusion proteins This will give the rate of hydrolysis of GTP in pmol per min per mg of membrane protein.

5.4 Receptor binding studies Levels of expression of the a2A-adrenoceptor-Cys—»Gly G11a fusion protein can be assessed using the a2-adrenoceptor-selective antagonist [3H]RS-79948197 (Nycomed Amersham). The following protocol should be easily adaptable to permit measurement of expression levels of other GPCR-G protein tandems for which a radiolabelled antagonist ligand is commercially available.

Protocol 6. Measurement of a2A-adrenoceptor-Cys—>Gly G11a fusion protein levels Equipment • 30°C incubator or water bath • Scintillation counter

• Brandel cell harvester

Method All manipulations are to be performed at 4°C. 1. Dilute plasma membrane proteins (Protocol 3) to 0.04 mg ml-1 in 10 mM Tris HCI, 50 mM sucrose, 20 mM MgCI2, pH 7.4 (assay buffer). 2. Add 50 ul (2 u,g) of diluted membrane mix to tubes on ice. 3. Determine non-specific binding by adding 50 ul of 500 uM idazoxan (final 100 uM) to parallel tubes. 4. Start the reaction by adding 50 ul of [3H]RS-79948-197 (final concentration 1 nM) to diluted membrane mix. The assay volume of 250 ul is made up with assay buffer. 5. Incubate for 45 min-1 h at 30 °C, and then filter under vacuum through Whatman GF/C filters to separate bound from free. 6. Wash filters with 3 x 4 ml of cold assay buffer. 7. Determine radioactivity in each filter by liquid scintillation counting. Specific binding is the difference between total and non-specific binding.

6. Receptor-G protein fusions as research tools 6.1 Measurement of agonist-induced guanine nucleotide turnover by G11a Quantification of the agonist-induced stimulation of GTPase activity of G proteins, whether performed in complex membrane preparations or in recon117

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6: Construction and analysis of receptor-G protein fusion proteins Figure5. Expression of an a2A-adrenoceptor-Cys—»Gly G11a fusion protein. Membrane fractions of pertussis toxin-treated COS-7 cells transfected with the a2A-adrenoceptorCys—»Gly G11a were subjected to saturation binding studies using [3H]RS-79948-197 (upper panel) as described in Protocol 6. Specific binding is shown. The data were then converted to a Scatchard plot (lower panel). From ref. 4, with permission.

stituted systems, has been problematic, since absolute levels of receptor and/or G protein are difficult to measure and it is unclear whether agonistoccupied receptor has the capacity to activate the full complement of its cognate G protein, due to differences in cellular localization. Indeed, measurements of the GTPase activity of isolated G proteins have routinely resulted in estimates which are too low to account for the rapid kinetics of ligand-induced activation and deactivation of signal transduction cascades (20,21, and reviewed in ref. 1). Use of the a2A-adrenoceptor-Cys—»Gly G11a fusion protein as a tool to measure agonist-induced catalytic-centre activity of G11a constrained therein (4) is described as follows. Basically, transient transfection of the a2A-adrenoceptor-Cys-»Gly G11a fusion protein cDNA in COS-7 cells resulted in high levels of expression of this construct (6-15 pmol mg-1 of membrane protein in separate transfections) when measured by saturation analysis of the specific binding of the a2Aadrenoceptor selective antagonist [3H]RS-79948-197 (Figure 5) as described in Protocol 6. The Kd for this interaction was 0.35 nM, similar to that obtained following individual coexpression of the a2A-adrenoceptor and Cys—»Gly protein was then measured by performing a GTPase assay, as outlined in Protocol 5, with various concentrations of cold GTP in the absence and presence of the a2A-adrenoceptor agonist UK14304 (10 uM). By varying the concentration of substrate (i.e. GTP), it is possible to measure Vmax and Km values for G11a in the absence and presence of agonist, using MichaelisMenten kinetics. The results of such an experiment are shown in Figure 6, with data being presented as an Eadie-Hofstee transformation with the y axis detailing Vmax measurements. In the experiment shown, the increase in Vmax produced by UK14304 was 18.8 pmol per min per mg of membrane protein. Since receptor and G protein within the fusion are by definition present in a 1:1 molar ratio, and the fusion protein was expressed at 6.2 pmol mg-1 in this experiment, then agonist-induced turnover number was calculated to be 3.0 min-1. The measured Km for GTP of the fusion protein was 0.37 uM, which is in agreement with previous estimates of 0.1-0.5 uM (1). In all experiments, cells were exposed to pertussis toxin (50 ng ml-1) for 24 h to eliminate any potential interactions between the receptor element of the fusion protein and endogenously expressed Gi family G proteins. To conclude, Figure 6 shows the utility of using such a receptor-G protein fusion construct to measure G protein enzymic capacity. Such an approach 119

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Figure 6. Agonist stimulation of the high affinity GTPase activity of the a2A-adrenoceptorCys-»Gly G11a fusion protein. Membrane fractions from pertussis toxin-treated COS-7 cells transfected to express the c^A-adrenoceptor-Cys—>Gly G11a fusion protein were used to measure high affinity GTPase activity (Protocol 5). Upper panel: high affinity GTPase activity was measured over a range of concentrations in the absence (filled circles) or presence (open circles) of UK14304 (10 uM). Lower panel: the data are presented as an Eadie-Hofstee transformation. From ref. 4, with permission.

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6: Construction and analysis of receptor-G protein fusion proteins may offer novel means of studying the ability of GTPase-activating proteins such as the RGS family members (31, 32) to modify the kinetics of G protein signalling mechanisms.

6.2 Measurement of agonist efficacy Agonist efficacy is a measure of the capacity of a ligand to induce a functional response (33, 34). Adequate measurement of agonist efficacy at GPCRs has proven troublesome, because estimates of efficacy can vary with tissue, with expression levels of receptor, and also with the point within a signalling cascade at which the response is measured (35, 36). One of the earliest, and therefore most direct points at which efficacy can be measured is at the level of G protein activated by a GPCR. Hence receptor-G protein fusions, particularly those harbouring Gs family G proteins, offer an alternative means by which efficacy can be measured. The use is described of the a2A-adrenoceptor-Cys—»Gly G11a fusion protein to evaluate efficacy of a number of agonists at this GPCR, by measuring the ligand-induced rate of GTP turnover by the physically associated G11a, following expression in COS-7 cells (4). GTPase measurements were performed on membranes from pertussis toxin-treated cells expressing the a2Aadrenoceptor-Cys—>Gly G11a fusion protein, essentially as described in Protocol 5, in the presence of varying concentrations of a range of a2Aadrenoceptor agonists, as shown in Figure 7. Of those examined, only adrenaline and noradrenaline were found to function as full agonists, with UK14304, xylazine, clonidine, and BHT933 acting as partial agonists. For comparison, individual cDNAs encoding the a2A-adrenoceptor and Cys—»Gly G11a were coexpressed in COS-7 cells, and the efficacy of the same agonists assessed following pertussis toxin treatment of the cells. Table 1 shows that all

Table 1. Capacity of known agonists at a2-adrenoceptors to stimulate high affinity GTPase activity of a2A-adrenoceptor-Cys—»Gly G11a fusion protein or of the a2A-adrenoceptor plus Cys->Gly G11a was assessed following transient expression in COS-7 cells Efficacy to activatea

Ligand

a2A-adrenoceptor-Cys-»Gly G11a a2A-adrenoceptor + Cys-»Gly G11a (% of adrenaline) (% of adrenaline) Adrenaline Noradrenaline a-methylnoradrenaline UK14304 BHT933 Xylazine Clonidine

100 97 105 56 20 16 15

100 99 93 85 44 28 26

'Efficacy was determined relative to the function of adrenaline (1 x 10-4 M) in parallel assays.

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Alan Wise

Figure 7. Efficacy measurements of ligands at the a2A-adrenoceptor-Cys—>Gly G11a fusion protein. The capacity of varying concentrations of adrenaline (filled triangles), noradrenaline (open squares), UK14304 (filled squares), xylazine (open triangles), BHT933 (open circles), and clonidine (filled circles) to stimulate the high affinity GTPase activity of the a2A-adrenoceptor-Cys-»Gly G11a fusion protein was assessed as described in Protocol 5. Data are presented as the % of the stimulation produced by 1 x 10-4 M adrenaline. From ref. 5, with permission.

of the ligands found to be partial agonists at the a2A-adrenoceptor-Cys—»Gly G11a fusion protein were also partial agonists for separated receptor and G protein in comparison to adrenaline: the rank order of efficacy was also similar in the two systems. Similar enzymic capacity measurements were made, as described in Section 6.1, to elucidate whether the differences in efficacy of the agonists were not just a reflection of distinct agonist-induced variations in the Km of the fusion construct for GTP. Figure 8 shows that this indeed was not the case. The data shown provide compelling evidence that a receptor-G protein fusion can provide a novel means to measure agonist efficacy at a GPCR. Two sources of variation which have until now proved problematic to the measurement of efficacy - the accurate assessment of receptor/G protein levels and localization within the cell - have been circumvented by fixing the receptor and G protein stoichiometry to 1:1, and by defining their co-localization by physical linkage. 122


FigureS. Partial agonism at the a2A-adrenoceptor-Cys—>Gly G11a fusion protein is manifest in varying values for the Vmax of the GTPase activity without alteration in Km for GTP. High-affinity GTPase activity was measured at varying concentrations of GTP (upper panel) in membranes of pertussis toxin-treated COS-7 cells transfected to express the a2A-adrenoceptor-Cys^>Gly G11a fusion protein in the absence of ligand (filled circles), or in the presence of 1 x 10-4 M adrenaline (filled triangles), a-methylnoradrenaline (open circles), UK14304 (filled squares), or xylazine (open triangles), as detailed in Protocol 5. Data were then transformed as an Eadie-Hofstee plot (lower panel) to allow direct estimation of V max , Km for GTP, and the efficacy of the ligands compared to adrenaline. From ref. 5, with permission.

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6.3 Elucidating the role of N-terminal acylation of G11a All known G protein a subunits are known to be modified at their N-terminus by co-translational myristoylation and/or post-translational palmitoylation (37-39). These acylations are considered to play key roles in targeting G protein a subunits to the plasma membrane, and may also contribute to proteinprotein interactions between the G protein a subunit and both receptors and the 37 complex (40, 41). Addition of the C14 fatty acid myristate is restricted to a subunits of the Gi family, because they contain the consensus sequence (MGXXXS) for the enzyme W-myristoyl-CoA-transferase. Glycine at position 2 serves as the acceptor residue for the covalent attachment of this fatty acid group, following removal of the initiating methionine. Addition of the C16 fatty acid palmitate occurs on either one or two cysteine residues within the first ten amino acids of the a subunits of all the widely expressed G proteins (37-39). Prevention of myristoylation by site-directed mutagenesis of the acceptor glycine reduces the affinity of interaction of Gi G proteins and the By complex, and renders them cytosolic (37,42). Prevention of palmitoylation by mutation of the N-terminal cysteine acceptor residues also limits membrane association, and has also been reported to abrogate receptor interaction (40, 41, 43, 44). Figure 9 shows the lack of interaction of the various acylationdeficient forms of Cys—»Gly G11a with the a2A-adrenoceptor, following coexpression in COS-7 cells and exposure to pertussis toxin. In this case a GTPase assay, as described in Protocol 5, is employed to measure receptor-G protein coupling (45). However, it has been unclear whether poor receptor regulation of acylation-defective G protein a subunits is simply due to lack of appropriate targeting and thus proximity to a receptor, or is inherently due to the acylation status of the G protein. To address this question use was again made of the a2A-adrenoceptorCys-»Gly G11a fusion protein (4). However, fusion chimaeras were also constructed between the a2A-adrenoceptor and mutant forms of Cys-^Gly G11a in which the sites that are normally palmitoylated and myristoylated, Cys3 and Gly2, were mutated to Ser and Ala, respectively. In addition, the a2Aadrenoceptor is also known to be a target for post-translational palmitoylation, at Cys442 within the C-terminal tail (46). Because this acylation is proposed to create a 'fourth intracellular loop' in the receptor structure, and because this receptor has a relatively short C-terminal tail, fusion proteins were made between a C442A mutant of the a2A-adrenoceptor and the various forms of G11a detailed above. All fusion constructs were made using Protocol 1 and are depicted in Figure 10. Expression of each of these a2A-adrenoceptor-Cys—»Gly G11a fusion proteins in COS-7 cells resulted in similar, high levels of membrane expression of the receptor binding site (15-25 pmol mg-1) as measured by specific binding of the selective and high affinity a2-antagonist [3H]RS-79948-197 as described in Protocol 6. To assess coupling between the a2A-adrenoceptor and its fused 124


Figures. Lack of receptor interactions of acylation-deficient mutants of GJ,(I. High affinity GTPase measurements (Protocol 5) were performed in the presence (hatched barsl or absence (open bars) of UK14304 (10 uM) on membranes from pertussis toxin-treated COS-7 cells transfected with vector alone (1) or with the a2A-adrenoceptor in combination with C y s - G l y G11a (2), C3SCys >Gly G11a. (3), G2ACys-*Gly G11a W or G2A/C3SCys-^GIy G11a (5). From ref. 45, with permission.

Figure 10. Construction of a2A-adrenoceptor/acylation-deficient Cys^Gly G11a fusion proteins. Fusion of the N-terminus of Cys-Gly G11a to the C-terminus of the a 2A adrenoceptor resulted in the receptor C-terminal amino acid (valine) being converted to alanine, as described in Protocol 1. Arrows represent the amino acids altered from the wild-type sequence in the various fusion proteins generated. Marked cysteine residues are known targets for post-translational palmitoylation in both receptor and G protein. Glycine 2 in G11a undergoes cotranslational myristoylation.

125

Alan Wise acylation-deficient pertussis toxin-resistant G11a partners, GTPase assays, as described in Protocol 5, were performed on membranes of transfected COS-7 cells which had been exposed to pertussis toxin (50 ng ml-1) for 24 h prior to harvest. Figure 11 shows the results of such an assay. All of the expressed chimaeric fusion proteins were able to stimulate pertussis toxin-insensitive high affinity GTPase activity in membranes of transfected COS-7 cells upon addition of the a2A-adrenoceptor agonist UK14304. The fusion protein approach guarantees the physical proximity of receptor and cognate G protein. Hence such data demonstrate that the lack of functional activation of acylation-negative mutants of G proteins by co-expressed receptors is related to deficiencies in cellular targeting and location, rather than an inherent inability to produce appropriate protein-protein interactions for signal transmission. Figure 11 also shows that all of the fusion chimaeras required similar concentrations of UK14304 to cause half-maximal effects. Similar data were also generated using fusion proteins containing the C442A mutant version of the a2A-adrenoceptor. These findings also support the argument that acylation

Figure 11. UK14304 stimulates high affinity GTPase activity of a2A-adrenoceptor/acylationdeficient Cys-»Gly G11a fusion proteins. Effects of varying concentrations of UK14304 are shown for a2A-adrenoceptor-Cys^>Gly G11a (open circles), a2A-adrenoceptor-C3SCys^> Gly Gna (open diamonds), a2A-adrenoceptor-G2ACys—»Gly G11a (closed diamonds), or a2A-adrenoceptor-G2AC3SCys^»Gly G11a (filled squares) fusion proteins. GTPase measurements were performed as described in Protocol 5. From ref. 6, with permission.

126

6: Construction and analysis of receptor-G protein fusion proteins is not required to produce effective protein-protein contacts between receptor and a subunit, but to position the G protein appropriately. In addition, co-expression of each of the fusion proteins with the 37 complex, pl-yZ, resulted in greater maximal UK14304 stimulation of GTPase activity, as shown in Figure 12. This was not simply due to enhanced levels of

Figure 12. Expression of B1y2 increases UK14304 stimulation of fusion protein GTPase activity. a2A-adrenoceptor-Cys—»Gly G11a (1 and 2), a2A-adrenoceptor-C3SCys—»Gly G11a (3 and 4), a2A-adrenoceptor-G2ACys-»Gly G11a (5 and 6), or a2A-adrenoceptorG2AC3SCys^»Gly G11a (7 and 8) fusion proteins were expressed with (2, 4, 6, and 8) or without (1, 3, 5, and 7) B1y2. GTPase activity (open bars, basal; filled bars, UK14304, 10 uM) (A) or fusion protein levels (B) were measured on membranes from transfected pertussis toxin-treated COS-7 cells, as described in Protocols 5 and 6, respectively. From ref. 6, with permission.

127

Alan Wise fusion protein expression in the presence of B1y2, as [3H]antagonist binding studies showed no such alterations in expression levels (Figure 12). These results are interesting given that the N-terminus of the G protein a subunit is known to play a central role in By interaction (47, 48), that the By complex may play a key role in receptor interactions with the a subunit (49), and that a subunit acylation is important in defining the strength of interaction with the By complex (37, 42). Such data demonstrate interaction between receptor-G protein fusions and the By complex, and that formation of a receptor-aBy complex is required to permit the most efficient signal transduction between receptor and G protein. Overall, the data presented further document the utility of receptor-G protein fusions as tools to study receptor-G protein interactions, and provide compelling evidence for the role of G protein acylation in directing G protein cellular targeting, but not in the transmission of information between receptors and G proteins.

6.4 Study of interactions between the A1 adenosine receptor and multiple Gi-family G proteins Agonist stimulation of the A1 adenosine receptor leads to modulation of numerous intracellular signalling events, such as inhibition of adenylate cyclase, stimulation of phosphoinositidase C, activation of inwardly rectifying K+-channels, and inhibition of neuronal calcium channels as a consequence of coupling to multiple pertussis toxin-sensitive Gia-famity G proteins (50-52). To study further the interactions between the Al adenosine receptor and individual members of the Gi- family of G proteins, fusion proteins were generated between the A1 adenosine receptor and pertussis toxin-resistant Cys—*Gly variants G11a, G12a and G13a, using essentially the same strategy as described in Protocol 1 for the construction of the a2A-adrenoceptorCys—»Gly G11a fusion protein (4). Minimal disruption was caused to each polypeptide following manufacture of the fusions, with only the C-terminal amino acid of the receptor being altered from aspartic acid to alanine, and the initiator methionine of the G protein (which would normally be removed) remaining in the new protein. All studies were conducted in HEK293T cells, following transient expression of each of the A1 adenosine receptor-Gja fusion proteins as described in Protocol 2, and exposure of the cells to pertussis toxin (50 ng ml-1) for 24 h prior to harvest. Parallel experiments were also performed in which the A1 adenosine receptor was co-expressed along with each of the Cys—»Gly variants G11a, G12a and G13a, to compare agonist-mediated receptor-G protein interactions when expressed as constrained proteins within a fusion and as separate signalling polypeptides. Expression levels of introduced fusion proteins were measured by [3H]antagonist binding studies, essentially as described in Protocol 6, but with the use of the A1 adenosine receptor selective antagonist 128


Figure 13. Agonist-mediated stimulation of [35S]GTP-yS binding to A, adenosine receptor-Cys->GlyGiix fusion proteins and to coexpressed A, adenosine receptor and Cys—»GlyGia G proteins. HEK293T cells were transfected with A1 adenosine receptor alone, together with Cys—»Gly variants of G11a, G12a, and G13a, or as fusion proteins with each of these Gi-family G proteins. Cells were treated with pertussis toxin prior to harvest, and [35S]GTP-yS binding was measured on membrane fractions without (open bars) or with (hatched bars) exposure to NECA (10 uM), as described in Protocol 4. * denotes Cys-»Gly variant of Gia. From ref. 11, with permission.

[3H]DPCPX (Dupont-New England Nuclear, 120 Ci mmor1) as radiolabel, and inclusion of NECA (10 uM) rather than idazoxan to assess non-specific binding. Receptor-G protein coupling was measured by [35S]GTPyS binding in the absence and presence of the adenosine receptor agonist NECA, as described in Protocol 4. Figure 13 shows that introduction of each fusion protein into HEK293T cells led to robust agonist-mediated stimulation of [35S]GTP-/S binding activity, which was of significantly greater magnitude than observed when receptor and G protein were co-expressed. This is probably due to more efficient coupling between receptor and G protein when constrained within a fusion protein, rather than being over-expressed in a cell as separate entities. Certainly, such enhanced G protein activation cannot be attributed to altered levels of receptor expression, since saturation [3H] antagonist binding studies 129

Alan Wise Table 2. Expression levels of transiently transfected A, adenosine receptor-Cys-»Gly G,a fusion proteins Transfections A,AR + A1AR + A1R + A1RGiI a A1ARG12a a

Gi1a Gi2a Gi3a

Antagonist binding (pmol 5.8 ±0.3 6.3 ±0.7 6.1 ±0.3 9.6 ±3.7 7.9 ±2.8 7.9 ±1.2

mg-1)

Dissociation constant, Kd, of antagonist binding (nM) 0.68 ±0.07 0.78 ±0.18 0.64 ±0.10 1.40 ±0.55 0.90 ±0.53 0.96 ±0.26

denotes pertussis toxin-resistant Cys-»Gly variant of da.

revealed similar quantities of A1 adenosine receptor in membranes from cells transfected with cDNAs encoding receptor-G protein fusions, and also in cells transfected with receptor, together with the individual Gi-family G proteins (Table 2). The affinity of interaction between the A1 adenosine receptor and the Cys—»Gly forms of G11a, G12a, and G13a, when the receptor-G protein interaction is constrained within a fusion protein, were then measured by [35S]GTP-yS binding following exposure of membranes to increasing concentrations of NECA. Figure 14 shows that similar EC50 values were attained for all three fusion proteins. Recently, evidence has arisen implying that particular agonists can interact with GPCRs in a defined manner to harness activation of specific G proteins (reviewed in refs 53, 54). The above fusion proteins were employed to study this phenomenon of 'agonist trafficking', since the A1 adenosine receptor causes activation of a variety of intracellular signalling pathways via interaction with Gi-family G proteins. Hence a range of A1 adenosine receptor agonists were characterized for their ability to promote activation of a particular Gi family G protein following expression of the individual fusions in HEK293T cells. No preferential activation of any expressed Gi G protein via the A1 adenosine receptor was observed with any of the compounds tested (representative data for ten compounds out of 40 studied are shown in Table 3). Potency of the same series of ligands was also measured following coexpression of the A1 adenosine receptor with each Cys—>Gly Gia (Table 4). Hence, no agonist-induced 'channelling' of the human A1 adenosine receptor to cause activation of distinct members of the Gi-family of G proteins was observed. To investigate 'agonist trafficking' more rigorously, it may be more pertinent to select GPCRs which are known to couple to less closely related G proteins such as the a2A-adrenoceptor, which has been shown to interact with both Gi and Gs subtypes (55). 130

6: Construction and analysis of receptor-C protein fusion proteins

Figure 14. Dose-dependent agonist-mediated stimulation of [35S]GTP-yS binding to A, adenosine receptor-Cys->GlyG,a fusion proteins and to coexpressed A, adenosine receptor and Cys->GlyGja G proteins. The ability of varying concentrations of NECA to stimulate the binding of [35S]GTP-yS was measured as described in Protocol 4, in membranes of pertussis toxin-treated HEK293T cells transfected to express (A) A1 adenosine receptor-Cys—>GlyGiOi fusion proteins containing G11a (filled triangles), G12a (open circles), and G13a (filled squares), and (B) coexpressed A, adenosine receptor and Cys-»Gly variants of G11a (filled triangles), G12a (open circles) and G13a (filled squares). Modified from ref. 11, with permission.

131

Alan Wise TableS. Efficacy measurements of ligands at the A, adenosine receptor when coexpressed with Cys—>Gly variants of Gia or as fusion proteins with these G proteins Ligand

Potency (EC50 in nM) A,ARGi1 •

NECA 56.4 ±17 CPA 5.3 ±1.2 GR79236X 19.0 ±4.5 CCI4019 46.6 ±3.6 GR56071 7.7 + 0.9 GR56072 3.5 ±1.1 33.2 ± 5.4 GR66683 S-PIA 300 ± 39 R-PIA 8.5+1.2 CHA 11.9 + 3.1 a

42.0 ± 6

49.2 ± 9

14.5

7.8 + 2.8 21.0 + 6.2 66 ± 7.5

±5.5

22.0 ± 6.3 63.7

±6.1

11.8±2.7 3.5 ±1.6 52.1 + 13.4 357 + 80 9.3 ±2.3

46.0

23.4

10.3+1.8

±3.1

10.8

128±

11.7

31.6 + 1.6 96.6 ± 5.6

317 + 38 46 + 2.8

±1.3

4.5 ±2.5

A,ARG,2a 145.8 ±10.1 41. 3 ±3.5 129 ±10.0 340+18 56+1.6

20.7 ±2.8

25.2

320 ±41

214±18.6 1850 ±300

10.9

53.6

247 + 13.8 1330 + 167 64.3 ± 3.3 90.2 ± 8.6

±6.4 ±1.7

±4.1

64.2 ± 6.0

±1.2

159.8+15.3 39.7 + 2.0 125 ±9.6 312 + 10.9 65 ± 2.8 25.5 ± 4.0 241 ± 15.0 1470+147 73.3 + 3.7 98 ± 6.7

denotes pertussis toxin-resistant Cys-»Gly variant of G^

Table 4. Efficacy measurements of ligands at the A1 adenosine receptor when coexpressed with Cys—>Gly variants of Gia or as fusion proteins with these G proteins Ligand

Efficacy to activate (% of NECA) A,ARGi3a

CPA GR79236X CCI4019 GR56071 GR56072 GR66683 S-PIA R-PIA CHA a

90 ±29 121±7 114 + 2 115±4 125±10 111±5 136 ±2 118±4 110±8

113±10 119±8 119 + 3 125 + 7 126±15 116±3 141 ±7 156 ±6 129 ±4

96 ±10 125 ±8 112±3 107 + 4 105 ±19 107 + 4 117 + 4 123 ±6 129 ±5

98 ±1 100 ±2 96 ±3 101 ±2 103 ±4 103 ±2 102 ±9 95 ±2 96 + 3

102 ±2 105 ±2 100 + 2 104 ±1 106 ±1 101 ±2 85 + 4 103 ±1 111±3

103 ±1 97 ±2 95 ±1 103 ±1 99 ±3 96±2 103+1 103 ±1 104 ±2

denotes pertussis toxin-resistant Cys-»Gly variant of G,a.

In conclusion, these results provide strong evidence that a fusion protein approach can be adopted as a reliable means of studying interactions between particular receptor-G protein tandems.

6.5 Receptor-G protein fusion regulation of effectors The work described in the previous sections of this chapter has focused entirely on the use of receptor-G protein fusions as an alternative means to study the complexities of receptor-G protein interaction. However, a number of recently published articles have looked at the downstream regulation of effector molecules by receptor-G protein fusion proteins (3, 7, 8). Indeed, the first work detailing the use of fusions, by Bertin et al. (3), involved the construction of a B2-adrenoceptor-Gs chimaera. Addition of agonist was able 132

6: Construction and analysis of receptor-G protein fusion proteins to cause activation of adenylate cyclase following expression of this fusion protein in S49 lymphoma eye" cells, which lack endogenous Gsa (3). More recently, Seifert et al. (8) used fusions between the B2-adrenoceptor and the long and short splice variants of Gsa to demonstrate subtle differences in their coupling mechanisms. In this study, regulation of adenylate cyclase by these fusion proteins was used as an important method of measuring G protein activation. Furthermore, a fusion protein between the yeast a-factor receptor, Ste2, and the G protein, Gpal was found to transduce signal efficiently in yeast cells devoid of endogenous STE2 and GPA1 genes (7). However, the fusion constructs between the a2A-adrenoceptor and Cys—»Gly G11a, and between the A1 adenosine receptor and Cys—»Gly forms of G11a, G12a,and G13a do not appear to be able to transduce signals to effector proteins via their attached G proteins (9, 11). For example, stable expression of the a2A-adrenoceptor-Cys—»Gly G11a fusion protein in Ratl fibroblasts results in an a2A-adrenoceptor agonist (UK14304)-mediated stimulation of GTPase activity in membranes from these cells. This stimulation is, however, only partially sensitive to pretreatment of cells with pertussis toxin, demonstrating that the receptor in the fusion protein can couple to both fused pertussis toxin-resistant G protein partner and endogenous pertussis toxin-sensitive Gi G proteins (9). a2A-adrenoceptor agonists can also cause inhibition of forskolin-amplified adenylate cyclase in these cells; however, this response is abolished following exposure to pertussis toxin (Figure 15), suggesting that effector regulation is purely via fused receptor and the endogenous Gi G protein population. These data demonstrate that receptors constrained within fusion proteins have the capacity to regulate both their fused G protein partner and also those endogenously expressed. This is not entirely surprising, since GPCRs that have had other proteins, such as green fluorescent protein, linked to their C-termini still appear to be capable of interacting with and activating cellular G proteins (56). However, this is also an extremely important point to consider when adopting such an approach to measure coupling between specific receptors and G proteins. The inability of the a2A-adrenoceptor-Cys—»Gly G11a fusion protein to mediate inhibition of forskolin-amplified adenylate cyclase activity via the physically linked Gi may simply reflect limited spatial opportunities of the fusion protein-linked G protein, or the relative G protein activation ratios, which in the fusion must be limited to mol per mol of agonist-occupied receptor. Indeed, both the a2A-adrenoceptor and A1 adenosine receptor possess relatively short C-terminal tails compared with the B2-adrenoceptor, and at present little is known the about the cellular distribution of signalling proteins relative to one another (57). The inability of Gi-containing receptor-G protein fusions to regulate effectors may also be due to an unknown property of the fused Gi G protein. This possibility will hopefully be addressed by construction of further Gi-based receptor-G protein tandems. 133

Alan Wise

Figure 15. Agonist occupation of a2A-adrenoceptor-Cys-»Gly G11a results in inhibition of adenylate cyclase via endogenous Gi G proteins, but not via fused Cys—»Gly G11a. Adenylate cyclase measurements were performed on Rat1 fibroblasts stably expressing the a2A-adrenoceptor-Cys-»Gly G11a fusion protein. Basal activity (1 and 5), stimulation by forskolin (50 uM) (2 and 6), and the capacity of UK14304 (100 uM ) (3 and 7), or adrenaline (100 uM) (4 and 8) to inhibit forskolin-amplified activity were then measured in untreated (1-4) and pertussis toxin-treated (5-8) cells. Significant inhibition by agonists was only observed in untreated cells. From ref. 9, with permission.

In conclusion, receptor-G protein fusion proteins can regulate effector pathways; however, careful data analysis is necessary to dissect coupling between receptors and their fused G protein partners, and those endogenously represented. From the data published so far, it would seem that the G protein partner contained within a particular fusion is a crucial determinate in denning effector interaction capability. In addition, the capacity of receptor-G protein fusions harbouring Gq and G12 family G protein members to regulate effectors is also eagerly awaited.

7. Summary and future perspectives The generation and use of fusion proteins comprising GPCRs and G protein a subunits is beginning to provide an alternative means to explore and delineate 134

6: Construction and analysis of receptor-G protein fusion proteins the complexities of mechanisms governing receptor-G protein interaction. This technology provides a direct means of studying the catalytic activation of G proteins by their cognate receptors, and will hopefully provide novel means to examine specificity in the pharmacology of cell signalling systems. The use of pertussis toxin-resistant G, G proteins also provides a means of studying interactions between particular receptor-G; G protein tandems in isolation. The generation of receptor-G protein fusions containing members of the Gq and G12 families is restricted due to endogenous expression of these G proteins, and hence the inability to demonstrate unequivocally whether a particular response results from activation of the G protein partner of the fusion protein or from activation of endogenous G proteins. This may be overcome by the use of chimaeric G, G proteins with altered receptor-coupling specificities, in which the C-terminus is exchanged with that of other G protein a subunits. Other future work will involve the generation of receptor G; G protein fusions which can efficiently regulate effector proteins. Such work should permit a more widespread use of this technology to study G proteinmediated signalling pathways.

Acknowledgements I would like to thank Professor Graeme Milligan for providing the facilities to perform most of the experiments described herein. I would also like to thank members of the Molecular Pharmacology Group at the University of Glasgow, especially Craig Carr, for excellent technical assistance. In addition, I would like to thank Stephen Rees and members of the Receptor Systems Unit at GlaxoWellcome Research and Development for their help and support to continue this work.

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6: Construction and analysis of receptor-G protein fusion proteins 41. Edgerton, M. D., Chabert, C., Chollett, A., and Arkinstall, S. (1994). FEBS Lett., 354,195. 42. Mumby, S. M., Heuckeroth, R. O., Gordon, J. I., and Gilman, A. G. (1990). Proc. Natl. Acad. Sci. USA, 87,728. 43. Parenti, M., Vigano, M. A., Newman, C. M. H., Milligan, G., and Magee, A. I. (1993). Biochem. J., 291,349. 44. Galbiati, F., Guzzi, F., Magee, A. I., Milligan, G., and Parenti, M. (1994). Biochem. J., 303, 697. 45. Wise, A., Grassie, M. A., Parenti, M., Lee, M. G., Rees, S., and Milligan, G. (1997). Biochemistry, 36,10620. 46. Milligan, G., Parenti, M., and Magee, A. I. (1995). Trends. Biochem. Sci., 20,181. 47. Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995). Cell, 83,1047. 48. Lambright, D. G., Sondek, J., Bohm, A., Skiba, N.P., Hamm, H. E., and Sigler, P. B. (1996). Nature, 379,311. 49. Taylor, J. M., Jacob-Mosier, G. G., Lawton, R. G., Remmers, A. E., and Neubig, R. R. (1994). J. Biol. Chem., 269,27618. 50. Stiles, G. L. (1992). J. Biol. Chem., 267, 6451. 51. Fredholm, B., and Dunwiddie, T. V. (1998). Trends. Pharmacol. Sci., 9,130. 52. Garwins, P., and Fredholm, B. (1992). J. Biol. Chem., 267,16081. 53. Kenakin, T. (1995). Trends Pharmacol. Sci., 16, 232. 54. Kenakin, T. (1995). Trends Pharmacol. Sci., 16,188. 55. Eason, M. G., Jacinto, M. T., and Liggett, S. B. (1994). Mol. Pharmacol, 45,696. 56. Barak, L. S., Ferguson, S. S. G., Zhang, J., Martenson, C., Meyer, T., and Caron, M. G. (1997). Mol. Pharmacol., 51,177. 57. Neubig, R. R. (1994). FASEB J., 8,939.

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7

Application of the baculoviral expression system to signal transduction ANDREW PATERSON

1. Introduction The Baculovirus Expression Vector System (BEVS) is an extremely versatile and powerful laboratory tool (1). In simplest terms, the BEVS allows the forced heterologous expression of recombinant protein in susceptible host strains of insect cells. Gene products from the simplest prokaryotes through to the higher eukaryotes have been expressed successfully in this system. Recombinant proteins are recovered in a folded and active form, and posttranslation modifications, such as phosphorylation, lipid acylation, and simple glycosylation are often preserved. Oligomerization is preserved also, as is the ability to form holomeric complexes. This has been demonstrated clearly with expression of the ~ 40 kDa G proteins in their native «p-y heterotrimeric complexes (2), and extended to the activation of the kinase, c-Raf, upon coexpression with Ras and Src tyrosine kinase (3). Since the host insect cells maintain the ability to process eukaryotic pre-mRNA, heterologous expression from genomic DNA is possible. However, the most attractive facet of BEVS must be the high level of heterologous protein expression with values of between 0.1 and 30% of total cell protein being reported, and often with gene products that fail to express with activity or reasonable yield in other systems. The rationale of the BEVS is fairly simple. The baculoviral genome is engineered to incorporate the coding sequence of a foreign protein. Incorporation is directed to a position downstream, and under the control, of a strong viral promoter. Activation of the promoter during the normal progression of baculoviral infection results in expression of the foreign protein. This is seen as an accumulation of foreign protein, which is available for harvest before completion of the viral life cycle and lytic death of the cell. It is now a fairly simple matter to introduce our chosen coding sequence into the genome of the most commonly used baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). The ability of AcMNPV to

Andrew Paterson Table 1. Signalling proteins expressed in the BEVS Reference Human B2-adrenoceptor Human M1 and M2 muscarinic acetylcholine receptor EGF receptor extracellular domain EGF receptor tyrosine kinase domain Gaq family Gai1,Gai2, Gai3, GaD, Gas Ga12, Gaz Free By-subunits Adenylyl cyclase (type I) PhospholipaseC-p Phospholipase C-y cAMP phosphodiesterase Protein kinase C isoenzymes

4 5 6 7 2 8 9 10 11 12 13 14 15,16

p60 src PI 3-kinase (p85/p110 complex) B2-adrenoceptor kinase (GRK2) Protein kinase B isoenzymes

17 18 19 20

replicate, package progeny virions, and express virally-encoded proteins in cells derived from Spodoptera frugiperda, the fall army worm, has provided the technology we require both to engineer recombinant baculovirus and to express the encoded protein. Over the last decade proteins expressed in the BEVS have been applied to many aspects of signal transduction research (see Table 1). In most of these examples the knowledge gained has been critical and, in may cases, where other expression systems fail to provide suitable material. Several excellent dedicated laboratory manuals describing the molecular biology of the baculoviridae and their use in the laboratory are available currently (21, 22). They are recommended as further reading, as are the laboratory manuals provided with baculoviral products purchased from Life Technologies, Pharmingen, and Invitrogen.

2. Sf9 cell culture The clonal Spodoptera frugiperda line, Sf9 (ATCC # CRL 1711), is most often first choice for use in the BEVS. Its maintained culture is well documented, and information regarding its growth characteristics and requirements are available readily (21). The following description of methodology for establishing novel recombinant baculoviral vectors and protein expression will focus on their use. Alternative cell lines will be discussed briefly at the conclusion of this chapter. In culture, Sf9 cells grow either as monolayers or as stirred suspensions. In 140

7: Baculoviral expression both cases, they grow as monodisperse cells exhibiting logarithmic growth kinetics, and doubling times of less than 30 h. Slow-growing cultures that aggregate to clusters should be discarded. Use of Sf9 cells with sub-optimal health is a common source of problems. Rigid discipline must be adopted with continuous cultures of Sf9 cells. Maintaining Sf9 cells in logarithmic, suspension growth and preventing cell clustering is best achieved over what is an extremely narrow range of cell density (1.0-2.5 X 106 cells mH). Healthy Sf9 cultures may double in density in less than 27 h, and this demands subculturing every 36 h or so.

2.1 Media Choice of basal medium is a matter of personal preference. Many formulations have evolved, and these include TNM-FH, IPL-41, Ex Cell 400, and TC 100. These have been reviewed extensively elsewhere (21, 22). Much of the methodology following relies heavily on complete TNM-FH. The suggested formulation for complete TNM-FH is Grace's Antherean Culture medium (Vaughn's modification) supplemented with 3.3 g l-1 lactalbumin hydrolysate, 3.3 g l-1 Tissue Culture Yeastolate, 10% (v/v) fetal bovine serum, 100 ug ml-1 streptomycin, 100 IU ml-1 penicillin, and 0.25 ug ml-1 amphotericin B. It should be possible to substitute complete TNM-FH with other proprietary serum-containing media in most of the following methodology.

2.2 Reviving Sf9 cells from frozen Thaw a vial of frozen cells in a 27°C water bath. This must be done rapidly and care taken to prevent the thawed culture warming much above freezing point. Dilute the thawed cells in 10 ml ice-cold complete TNM-FH and transfer to a 75 cm2 TC flask. Allow the cells to attach for 15-30 min at 27°C. Replace the medium and unattached cells with 12 ml complete TNM-FH, and incubate at 27°C until the monolayer reaches confluence.

2.3 Maintaining monolayer cultures of Sf9 Cells Monolayers should be passaged 12-24 h after confluence. Detach the monolayers into their growth medium with a sterile cell scraper. Dilute the suspension fourfold with warmed complete TNM-FH, and replate 12 ml in a 75 cm2 TC flask. A cell density of approximately 6 X 104 Sf9 cells cm2 is desired. Incubate the monolayer at 27°C overnight, replace the medium with fresh complete TNM-FH, and further incubate the cells at 27°C until confluence (approximately 2-3 days). As the culture establishes it is possible to increase the dilution at each passage. After 3-4 passages it will be possible to split the monolayer 8- to 10-fold without loss of cell viability. The culture can be maintained in 75 cm2 TC flasks until scale-up is required. For instance, it will be desirable to seed 141

Andrew Paterson at least two 150 cm2 TC flasks in preparation for adaptation to suspension growth.

2.4 Adaptation to suspension culture Growing Sf9 cells as monolayers is convenient. It is relatively simple, inexpensive, and does not require capital investment in specialized equipment. Provided that a temperature of 25-27°C can be established, Sf9 cells will grow. Many low-cost incubators are available, and there is no requirement to supply gassed atmospheres as in mammalian cell culture. Disused 37°C, CO2 incubators are adapted readily for use with Sf9 cells. These are convenient approaches but, in truth, most laboratories have a draught-free area that will maintain a temperature between 25 and 27°C and can be used. Enterprising laboratories have been known to grow these cells in disused cupboards or cabinets fitted with a heat lamp. Splitting Sf9 monolayers by scraping or other means results in loss of significant numbers of cells. The resulting dead cells are removed easily by washing the monolayer with fresh medium on the day following plating. However, their presence does prevent use of monolayer-derived cells for transfections (see Protocol 1), plaque assays (see Protocol 2), etc. It is recommended strongly that monolayers are adapted to and maintained in suspension as soon as possible. 2.4.1 Equipment Suspension cultures of Sf9 cells should be maintained in microcarrier spinner flasks fitted with an impeller blade. Those manufactured by the Bellco Glass Co are recommended. These are available in a range of sizes. Continued culture of Sf9 cells from frozen aliquots to the production of milligram amounts of protein will entail the manipulation of cell suspensions in volumes ranging from 40 to 400 ml. In each case an adequate surface area to volume ratio is required, and the cell suspension should never exceed half the spinner flask's capacity. For example, in the routine culture of Sf9 cells it is recommended that 100 ml of culture be grown in a 250 ml flask (Bellco #1965-00250). A range of spinner flasks should therefore be available to accommodate this. To facilitate stirring, and to help inhibit formation of cell clusters or clumps, the spinner flask impeller blades should have only 5 mm clearance from the vessel wall. Impeller blades marketed for larger spinner flasks (Bellco #A523-199) can be cut to size and fitted in 250 ml spinner flasks. Microcarrier spinner flasks are operated in conjunction with a magnetic stirring plate. Those intended solely for use with microcarrier spinner flasks are recommended. The stirring plate should be non-heating, and capable of maintaining a range of speeds from 50 to 150 r.p.m. These are marketed in a range of models (Bellco ranges #7760-, 7761-, and 7765- ). Although not essential, models featuring tachometers remove the chore of calibrating impeller speeds. 142

7: Baculoviral expression 2.4.2 Suspension growth of Sf9 cells Detach the confluent monolayers from at least two 175 cm2 TC flasks, and collect by centrifugation at 500 g for 5 min at room temperature. Aspirate the medium and cell debris, and resuspend the cell pellet to a density of 1 X 106 cells per ml with complete TNM-FH. Seed 50 ml of this suspension in a 100 ml microcarrier spinner flask (Bellco #1965-00100) and maintain the suspension at 70 r.p.m. in a 27°C incubator. Spinner flask side-arm caps may be loosened to allow gaseous exchange, provided the incubator is humidified. As the cell density reaches 2.5 X 106 per ml (approximately 48 h) the culture should be diluted to 1.0 X 106 per ml. Care should always be taken that the cell density does not exceed 3 X 106 per ml. A cell suspension may be maintained in this manner for several months. The simplest procedure is to maintain a given volume of culture, and to discard the surplus suspension generated with each dilution. The surplus cells grown in the first week after adaptation to suspension culture should not be employed for baculoviral work. Superior results will be obtained with cells that have grown in suspension for at least 7 days. Adapted cells should grow without clumping, and exhibit greater than 90% trypan blue exclusion, with doubling times of 24-30 h. Satisfied with the health of the suspension, the cells may then be used for generating novel virus, amplification of viral stocks, or for expression of recombinant protein.

2.5 Freezing Sf9 cells For practical reasons it is suspension cultures that are frozen for storage. The culture to be frozen must be in optimal health. Collect the cells from at least 50 ml of culture by centrifugation at 500 g for 2 min at room temperature. Aspirate the medium and cell debris, resuspend the cells to 1 x 107 cells per ml in DMSO:fetal bovine serum (1:9), and place 1 ml aliquots in sterile screwtop cryovials. Prepare 5-10 vials for storage. Wrap the filled cryovial in cotton wool, place in a polystyrene box, and freeze slowly to -70°C overnight. Remove the frozen vials, and place in a liquid nitrogen cell vat. Confirm viability of the frozen stock by reviving a vial (see Section 2.2) after at least 2 days in storage.

2.6 Adaptation to serum-free medium Serum-free medium is employed widely for culture of Sf9 cells. Apart from the obvious advantage of cost, the absence of serum-derived protein, and inclusion of detergent to prevent cell shearing in stirred culture is beneficial. However, reduced cell viability and decreased levels of recombinant protein expression are experienced with cells maintained for extended periods in serum-free medium. A compromise is to maintain cells in serum-containing medium, and to adapt a portion to serum-free medium at least a week before required. 143

Andrew Paterson Formulation of serum-free medium is a matter of choice (21). Several excellent proprietary serum-free media are available. These include Ex-Cell 401 and Ex-Cell 420 from JRH Biosciences. Before use these should be completed with 100 IU ml-1 penicillin, 100 ug ml-1 streptomycin, and 0.25 ug ml-1 amphotericin B. Cells grown in complete TNM-FH are weaned gradually onto serum-free medium over a period of 10 to 20 days. Cells should be diluted to 0.5 X 106 per ml in a 1:1 mixture of complete TNM-FH and serum-free medium, and incubated at 27°C until a density of 2.5 X 106 per ml is reached. Cells are then diluted to 0.5 X 106 per ml in a 1:4 mixture of complete TNM-FH and serumfree medium, and allowed to grow as before. Finally, the cells are diluted in a 1:10 mixture, and grown for a further 3 days until they reach 2.5 X 106 per ml. At this stage the cells can be assumed to have adapted to serum-free medium, and can be grown in the absence of TNM-FH. An alternative and less involved approach is to adapt the cells in a single step. Collect cells grown in complete TNM-FH by centrifugation at 500 g, and resuspend them to 1.0 X 106 per ml in ExCell 401 completed with antibiotic and antimycotic. Stir the cells at 70 r.p.m. for two days at 27 °C until the cell density reaches 2.5 X 106 per ml. Dilute the cells to 1 X 106 per ml in fresh complete ExCell 401 and grow as before. The cells tend to clump as they adapt to serum-free medium. Increasing the rate of stirring from 70 to 120 r.p.m. reduces this.

3. The baculoviral life cycle, and constructing recombinant baculoviral vectors 3.1 Time course of viral infection and the polh locus Baculoviral infection leads to lytic death of an infected insect cell. This will take approximately 4 days in AcMNPV-infected Sf9 cells, and results in release of many competent progeny virions. During infections baculoviralencoded genes are transcribed and expressed in a cascade of co-ordinated events. These can be separated and classified into the three phases of viral gene activity: early, late, and very late. These are described further below. Early: Transcriptionally active in the absence of any viral gene expression, and is thus dependent on the host cell's transcriptional machinery. Early genes are active in the first 10 h of infection. Late: Dependent on early viral gene expression and DNA replication. Late genes are active approximately 8-18 h post-infection. For instance, the p6.9 basic core-associated protein promoter (pcor) is active at this time. Very late: Dependent on early viral gene expression and viral DNA replication. Transcription of very late genes will initiate approximately 18 h postinfection, but will probably be at their highest levels 27-48 h post144

7: Baculoviral expression infection. The polyhedrin protein promoter (ppolh) is active during this phase of viral infection. A recombinant baculoviral expression vector is constructed by modifying the AcMNPV genome to encode a foreign protein. In the vast majority of cases the coding sequence is inserted at the viral polyhedrin (polh) locus and its translation controlled by the polh promoter (ppolh). This promoter is active in the very late phase of viral infection and drives polyhedrin expression until it predominates in the infected cell. This protein is not essential for viral function in cultured Sf9 cells, and the coding sequence can be substituted without affecting viral viability. AcMNPV is a double-stranded DNA virus. The circular genome is large (approximately 120 kb), and foreign cDNA cannot easily be introduced at the viral polh locus with techniques as convenient as restriction digestion followed by ligation. Rather, insertion of cDNA is achieved either by recombination between a transfer plasmid harbouring the sequence and AcMNPV, or by Tn7-mediated transposition into a baculoviral shuttle vector.

3.2 Construction of a recombinant transfer plasmid Some transfer plasmids which are available commercially are given in Table 2. Their application to virus construction either by cotransfection or through baculoviral shuttle vectors is indicated also. They offer several possibilities. The recombinant protein can be expressed either with its full-length, native sequence, or fused to a tag which facilitates rapid purification. In addition, all transfer plasmids encode polyadenylation and transcription termination sequences downstream of the multiple cloning site (mcs). When constructing the recombinant transfer plasmid, coding sequence should be introduced with the minimum of 5'-untranslated DNA. The 5'untranslated mRNA should not form significant secondary structure nor possess any transcription termination sequences. Any sequence to be inserted 3' to the translation termination site should be minimized, and should not contain a transcription termination sequence.

3.3 Construction of recombinant baculo virus by cotransfection Possibly the most common method for constructing recombinant baculovirus is to cotransfect insect cells with a transfer plasmid construct and AcMNPV genomic DNA. The mcs of the transfer plasmid is flanked by considerable tracts of sequence derived from the AcMNPV polh locus. This facilitates recombination and integration of the mcs at the viral polh locus when cotransfected with AcMNPV genomic DNA (Figure 1A). The double recombination required for successful integration occurs with low, but finite efficiency. The efficiency of recombination can be increased with linearized 145

Table 2. Transfer plasmids Name

Promoter

Pre-existing tag

Compatible with cotransfection"

Compatible with baculoviral shuttle vectors6

Supplier

pVL1392/pVL1393 pBacPAK8/9 pBac-1 pAcG1/2T/3X pAcHLT-A/B/C pAcGHLT-A/B/C pBacPAK-His1/2/3 pBac-2cp pAcMP2/3 pFastBAC 1 pFastBAC HTa/b/c

polh polh polh

none none none N-terminal GST A/-terminal His6 /V-terminal GST/His6 A/-terminal His6 A/-terminal His6 none none A/-terminal His6

yes yes yes yes yes yes yes yes yes no no

no no no no no no no no no yes yes

Invitrogen/Pharmingen Clontech Novagen Pharmingen Pharmingen Pharmingen Clontech Novagen Pharmingen Life Technologies Life Technologies

a b

see Section 3.3. see Section 3.4.

polh polh polh polh polh cor polh polh

Figure 1. Recombinant baculovirus construction. (A) Homologous recombination between AcMNPV-derived sequence in the transfer plasmid and the polh locus of AcMNPVwt genomic DNA. A double recombination event involving ORF 603 and ORF 1629 is given as an example. (B) AcMNPV DNA linearized at a novel Bsu36l site within ORF 1629 is nonviable. The virus is rescued by recombination with transfer plasmid. (C) Transposition in E. coli. The transfer plasmid mini-Tn7 element transposes to the baculoviral shuttle vector affTn7 site. Transposon function is supplied by a third helper plasmid. Transposition results in disruption of lacZa, and E. coli, which fail to stain blue when grown in the presence of IPTG and X-gal.

147

Table 3. AcMNPV viral preparations Name

Compatible with cotransfection3

Compatible with baculoviral shuttle vectorsb

Supplier

Linearized AcMNPV viral DNA

Linearize AcRP23.lacZ

yes

no

Pharmingen

Bsu361 -cut viral DNA

Baculogold BacPakB BacVector-1000 BacVector-2000 BacVector-3000

yes yes yes yes yes

no no no no no

Pharmingen Clontech Novagen Novagen Novagen

Baculoviral shuttle vector

Bacmid (bMON14272c)

no

yes

Life Technologies

' See Section 3.3. b See Section 3.4. c Supplied cotransformed with transposition helper plasmid, pMON7124, in the competent E. coli strain DH10Bac.

7: Baculoviml expression preparations of AcMNPV genomic DNA (see Table 3); however, less than 30% of the virus recovered from a cotransfection will be recombinant. The background of non-recombinant virus can be removed. Non-viable, linearized viral DNA preparations are now available. The AcMNPV genome has been modified by introduction of a novel Bsu36I site within ORF1629. This open reading frame is essential for viral viability, and maps close to the viral polh. Exhaustive digestion with Bsu36l both linearizes the viral DNA and truncates ORF1629. The resulting deletion destroys viral viability, and must be rescued by recombination with polh-based transfer plasmid (Figure 1B). Protocol 1 describes the cotransfection of Sf9 cells with Bsu36I-digested AcMNPV. Bsu36I-cut viral DNA preparations are available commercially form Pharmingen (BaculoGold), Clontech (BacPak6), and Novagen (BacVector-1000, -2000, and -3000). Although the high background of non-recombinant virus can be reduced significantly with Bsu36I-digests of AcMNPV, but the resulting progeny virus is still not clonal. Kitts and Posee (23) report that up to 5% of the virus constructed by cotransfection with Bsu361-digested AcMNPV did not express the protein encoded by the transfer plasmid. This can be eliminated and clonality ensured by purifying recombinants expressing virus to homogeneity. Purification by plaque assay is described in Protocol 2, and this should be performed with the supernatant recovered from cotransfected Sf9 cells (Protocol 1, Step 11). Plaques formed by virus expressing recombinant protein will be identified after SDS-PAGE analysis. Protocol 1. Cotransfection of Sf9 cells with Bsu36l-digested AcMNPV DNA Equipment and reagents • 25 cm2 TC flask (Corning # 430639) • Sf9 cells adapted to suspension growth in . Sterile microfuge tubes complete TNM-FH. The culture should be in -,,.,., . . ., . ,.- , exponential growth and at a density of #2059) 0.1). C. Isolation of specific antibodies 1. Apply pooled IgG fractions to the column (column 1) containing the non-phosphorylated peptide affinity matrix, and allow the unbound material to flow directly on to a tandemly-linked second column (column 2) containing the phosphorylated peptide affinity matrix. 2. Detach column 1. 3. Wash column 2 extensively with Column buffer. 4. Elute the affinity purified antibody as in B., Step 4. 5. Pool the fractions containing protein, and concentrate using a Centricon filter (Amicon).

6. Measurement of MAP kinase enzyme activity Whilst in many cases it may be sufficient to use one of the procedures outlined in Section 5 to assess relative activation of MAP kinases, often it is desirable to obtain a more precise measurement of the activity of the enzyme. Direct enzyme assays are also appropriate when attempting to chromatograph several MAP kinase activities from a single extract. MAP kinase assays are generally performed either directly in partially purified cell or tissue extracts, or following immunoprecipitation with an antibody which preserves the enzyme in its activated state. Although each class of MAP kinase possesses its own set of known physiological substrates, there is considerable overlap, and in vitro most of the commonly used protein or peptide substrates are phosphorylated by all types of MAP kinase. The presence of a prolyl residue on the carboxy terminal side of the target serine or threonine often appears to be sufficient to render the peptide a MAP kinase substrate in vitro. For assays of MAP kinase following immunoprecipitation, MBP is relatively inexpensive and easily obtained. On the other hand, for assaying MAP kinase in an 349

Neil G. Anderson unpurifled or partially purified extract, it would be preferable to use an appropriate peptide substrate to reduce phosphorylation of a protein substrate by other protein kinases.

6.1 Assay of MAP kinase in partially purified cell extracts The number of protein kinases present in a typical cell extract makes it inappropriate to assay MAP kinase directly in unpurified extracts. A simple procedure is available for partial purification of ERK MAP kinases from cell or tissue extracts by batch adsorption to phenyl-Sepharose (9). This is outlined in Protocol 4. In early studies on MAP kinases, it was realized that ERKs bind tightly to this matrix, only eluting with relatively high concentrations of ethylene glycol. Consequently, phenyl-Sepharose chromatography represents a simple but powerful method for rapid removal of > 90% of cellular protein from an extract in a single step. The final eluate contains ERK MAP kinases free from the majority of other contaminating kinases. The hydrophobic nature of the interaction between MAP kinase and the phenyl group means that detergent-solubilized cell or tissue extracts cannot be used, however. Protocol 4. Partial purification of extracts by batch adsorption to phenyl-Sepharose Equipment and reagents • Cell lysis buffer: 25 mM Tris-HCI, pH 7.5 (4°C); 40 mM 4-nitrophenyl phosphate; 25 mM NaCI; 10 uM dithiothreitol. Store at -20°C in the dark. Prior to use add the cocktail of protease inhibitors and sodium orthovanadate, exactly as described in Protocol 1.

• Phenyl-Sepharose (Pharmacia) . Ethylene glycol .Refrigeratedmicrocentrifuge

Method 1. Dispense 0.2 ml aliquots (packed volume) of phenyl-Sepharose into the required number of microcentrifuge tubes on ice. Wash twice with lysis buffer (0.5 ml each wash). 2. Lyse tissue or cells by sonication or homogenization in lysis buffer 3. Centrifuge homogenate at 20000 g max (4°C) to remove unbroken cells. 4. Measure the protein concentration of the supernatant, and apply 0.2-0.5 mg of protein to 0.2 ml (packed volume) of phenyl-Sepharose in a microcentrifuge tube. 5. Gently invert the tube to allow the cell extract to mix with the matrix. 6. After 5 min spin tubes at 10 000 g for 10 s.

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14: Detection, isolation and quantitative assay of mitogen activated 7. Using a glass Pasteur pipette linked to an aspirator, carefully remove the unbound material. 8. Replace with 0.5 ml of lysis buffer, mix gently, and re-centrifuge. 9. Repeat Step 7, then wash twice with lysis buffer containing 35% ethylene glycol. 10. Add 0.2 ml lysis buffer containing 60% ethylene glycol. 11. Gently mix, and place on ice for 5 min. 12. Centrifuge as in Step 5. 13. Remove the 60% eluate, and retain for assay.

To obtain information on which MAP kinases are activated in a particular cell type in response to a novel ligand or other stimulus, a useful initial experiment is to conduct a broad chromatographic analysis of the cell extract to separate the different MAP kinase activities. Anion exchange chromatography using diethylaminoethyl (DEAE)-cellulose or DEAE-Sepharose can be used. However, the greater resolution and speed offered by chromatography on matrices such as Mono-Q using an FPLC system (Pharmacia) make such methods preferable. Comparing the profiles of enzyme activities from control and treated cells gives an overall picture of the MAP kinases activated (Figure 5). To determine which of the peaks of activity contain known MAP kinases, the fractions can be immunoblotted in parallel with the appropriate antibodies (see Protocol 5). The initial analysis over a wide salt gradient can subsequently be refined to detect 'peaks within peaks', using shallower NaCl gradients in the desired range. The assays are conducted in the presence of a range of protein kinase inhibitors (see Protocol 6), to minimize background phosphorylation of substrate. This is especially important if myelin basic protein is used as substrate. Protocol 5. Anion exchange chromatography of cell extracts using Mono-Q columns Equipment and reagents • FPLC system (Pharmacia) • Mono Q HR 5/5 FPLC column (Pharmacia) • Cell lysis buffer (see Protocol 7)

• 100% trichloroacetic acid • 0.25% sodium deoxycholate

A. Fractionation of extract 1. Prepare lysates from untreated and stimulated cells (1-5 x 107) according to Protocol 1. 2. Apply the cell lysate to a Mono Q column previously equilibrated in equilibration buffer (lysis buffer without Triton X-100 and protease inhibitors).

351

Neil G. Anderson Protocol 5. Continued 3. Wash the column with equilibration buffer until A280 is zero. 4. Elute the bound material with a 0-0.8 M NaCI linear gradient, collecting forty 1 ml fractions on ice. 5. Assay each fraction immediately according to Protocol 6. B. Precipitation of fractions for immunoblotting 1. Precipitate the remaining protein in each fraction by adding the appropriate volume of trichloracetic acid and sodium deoxycholate (to give final concentrations of 5% and 0.025% respectively). 2. Leave on ice for 10 min. 3. Centrifuge at 12000 g for 10 min. 4. Remove the supernatant, and resuspend the pellet in SDS-sample buffer. 5. Boil for 5 min, and then store the samples at -20°C.

Figure 5. Use of anion exchange chromatography to separate MAP kinase activities from cell extracts. Lysates were prepared, according to Protocol 4, from untreated (open circles) and growth hormone-treated (filled circles) 3T3-F442A fibroblasts. The lysates were then applied to a 1 ml EconoPac Q column (BioRad laboratories). After washing, the column was chromatographed using a linear sodium chloride gradient (0-0.4 M), indicated by the dashed line. Fractions were collected and assayed for MAP kinase activity, using myelin basic protein as substrate.

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14: Detection, isolation and quantitative assay of mitogen activated Protocol 6. MAP kinase assay Reagents • MBP peptide [APRTPGGRR] (Upstate Biotech.): prepare stock solution at 0.8 mg ml-1 in 50% glycerol, store at -20oC • [y-32P]-ATP (specific activity > 3000 Ci mmor-1) • P81 phosphocellulose paper (Whatman), cut into 2 x 2 cm squares . Phosphoric acid (180 mM) • ATP stock solution (10 mM)a

• MgCI2 stock solution (1 M) . Kinase assay buffer: 10 mM MOPS, pH 7.2 (30°C); 10 mM p-glycerophosphate; 2 mM EGTA; 25 uM dithiothreitol; 0.1 mM sodium orthovanadate; 2 uM protein kinase inhibitor (PKI) peptide (Sigma); 10 uM calmidazolium (Calbiochem); 2 uM PKC inhibitor peptide 19-31 (Calbiochem)

Method 1. Prepare 8x concentration of ATP-Mg mix containing 400 uM ATP, 80 mM MgCI2, and [y-32P]ATP (0.5 uCi ul-1). 2. On ice add 5 ul sample to 5 ul substrate peptide and 25 ul kinase buffer. 3. Pre-incubate tubes for 2 min at 30°C, then add 5 ul ATP mix. Stagger the starts at 20 s intervals. 4. Briefly vortex, then return to the 30°C water bath. 5. Assay for the appropriate time (see Section 6.3). 6. Remove 30 ul of the reaction mix, and spot onto a P81 paper square numbered in pencil. 7. After about 10 s, drop the paper into a beaker containing 180 mM phosphoric acid (use —20 ml per assay). 8. Once all assay papers are in phosphoric acid, stir for 5 min. 9. Decant the phosphoric acid, and do four further washes of the papers (5 min each wash). 10. Finally, rinse the papers in ethanol, and place on absorbent paper to dry. 11. Place P81 papers in scintillation fluid, and count the radioactivity in a scintillation counter. * Dissolve ATP (disodium salt) in water to give approximately 10 mM. Adjust pH to 7. Measure absorbance at 260 nm. Calculate the exact concentration of ATP using e260 = 15 400.

6.2 Assay of MAP kinases following immunoprecipitation A number of antibodies are now available commercially which allow immunoprecipitation of several of the MAP kinases in an active form which can be assayed on the immunocomplex. The major advantage of this method is its ability to purify, rapidly and selectively, the MAP kinase of interest from 353

Neil G. Anderson a crude cell extract, to enable kinase activity to be determined in the absence of other protein kinases. However, it is important to consider potential limitations of this method, which could result in underestimation of the amount of kinase activity present in the original cell extract. Firstly, when complexed with an antibody, the kinase may lose intrinsic activity. This possibility can be examined by measuring the activity of a known amount of the purified enzyme in the presence or absence of an immunoprecipitating antibody. Secondly, co-immunoprecipitation of other kinases cannot usually be ruled out. The use of narrow specificity MAP kinase substrates for the assay will minimize the contribution of contaminating kinases to the activity measured. Finally, for quantitative measurements, it is necessary to determine empirically the quantity of antibody required to precipitate the entire MAP kinase cellular protein from a given cell extract. The efficiency of the antibody can be determined by immunoblotting the cell extract for the relevant MAP kinase before and after immunoprecipitation. Protocol 7. Assay of ERK MAP kinase by immune complex kinase assay Equipment and reagents • • . •

Rotating wheel for microcentrifuge tubes Microcentrifuge Aspirator Protein A-agarose or protein G-agarose (depending on the class of immunoprecipitating antibody used)

• Cell lysis buffer (see Protocol 7) • High salt buffer (10 mM Tris-HCI, pH 8.0; 0.5 M LiCI) • Kinase buffer (see Protocol 6); the inhibitors PKI, calmidazolium, and PKC inhibitor peptide may be omitted

Method 1. Prepare cell or tissue lysates (see Protocol 1). 2. Pre-clear lysates by adding 20 ul (packed volume) protein A- or protein G-agarose. 3. Rotate on the wheel for 1 h. Meanwhile conduct a protein assay on an aliquot of each sample. 4. Microcentrifuge for 10 s, and transfer the supernatant to a fresh tube. 5. To equal quantities of cellular protein, add an appropriate amount (previously determined) of precipitating antibody. 6. Incubate from 2 h to overnight at 4°C. 7. Transfer samples to fresh tubes containing 20 ul (packed volume) of protein A- or protein G-agarose. 8. Rotate on the wheel for 1 h. 9. Wash immunoprecipitates by centrifugation and careful aspiration of the resulting supernatant with 0.5 ml lysis buffer (twice), then with 0.5 ml high salt buffer. 354

14: Detection, isolation and quantitative assay of mitogen activated 10. Finally wash immunoprecipitates with 0.5 ml kinase buffer. 11. Remove all remaining buffer from the immunoprecipitates, using a fine pipette. 12. Resuspend beads in 25 ul kinase buffer plus 5 ul MBP peptide or protein.a 13. Preincubate at 30°C for 2 min. 14. Add 5 ul [32P]ATP-Mg mix as in Protocol 6. 15. Incubate for an appropriate time, then centrifuge reaction tubes. 16. Carefully remove 25 ul of reaction mixture, and spot onto P81 paper. 17. Proceed as in Protocol 6. a

If MBP protein is to be used as substrate for the kinase assay, terminate the reaction by adding 10 ul of 4X concentrated Laemmli buffer. Boil the samples for 2 min, then centrifuge the tubes for 5 min in a microcentrifuge. Apply 20 ul of the supernatant to a 13% mini-gel. After running the gel, stain with Coomassie Blue to locate the MBP band. Excise the bands and count the associated radioactivity.

6.3 Assay conditions Whichever method is used to assay MAP kinase activity, it is essential to establish optimal standardized assay conditions to allow valid comparisons to be made between experiments. MAP kinase phosphotransferase activities are usually quoted as picomoles of phosphate incorporated into substrate per minute under standard assay conditions. It should first be established that the measured incorporation of 32P into substrate is linear with respect to the assay time. Non-linearity will result in under- or overestimation of true phosphotransferase activity. Pre-incubation of the reaction mixture at 30°C, prior to addition of ATP, is essential to eliminate the lag in reaction rate as the mixture reaches 30°C. Depletion of substrate (peptide or ATP) during the course of the assay can lead to underestimation of enzyme activity. This is more likely to be a problem when conducting assays in unpurified extracts. The concentration of ATP should be at least 50 uM, and that of substrate at least 25 uM. To detect enzyme activity with reasonable sensitivity, the use of a specific radioactivity of at least 2500 c.p.m. per picomole of ATP is suggested. This entails the use of 2.25 uCi of radioactivity per assay under the conditions quoted in Protocol 6. When performing several assays under these conditions, the accompanying radiation hazard is a serious consideration. Assays of purified or immunoprecipitated kinases could, almost certainly, be conducted satisfactorily at lower specific activities. Finally, when conducting assays in unpurified cell extracts, the potential influence of endogenous diffusible inhibitors should be investigated. This is easily accomplished by checking the linearity of the relationship between amount of sample and phosphotransferase activity measured. If inhibitors are present, this relationship breaks down at high concentrations of sample. 355

Neil G. Anderson

6.4 Other MAP kinase assays 6.4.1 Assay of JNK and p38 MAP kinases For assay of specific MAP kinase family members, several immunoprecipitating antibodies are now available. Following the general procedure given in Protocol 7 allows determination of the activities of these kinases in cell or tissue extracts. As stated previously, all MAP kinases will phosphorylate myelin basic protein or peptides encompassing the relevant phosphorylated sites in vitro. Obviously there is a much greater degree of specificity in vivo, and this can be used for the more selective assay of individual family members. For example, although ERK MAP kinases can phosphorylate c-jun in vitro, it is now well established that JNKs are primarily responsible for c-jun phosphorylation in intact cells. Based on this, an assay for JNKs has been established, involving the use of recombinant c-jun fragments fused to glutathione S-transferase to precipitate JNKs from cell extracts (10). A solid state assay is then conducted on glutathione-Sepharose beads, and the phosphorylated c-jun resolved by electrophoresis. Immune-complex assays for p38 MAP kinases have employed a fragment of the transcription factor ATF-2 as a relatively selective substrate for this class (11). 6.4.2 Non-radioactive MAP kinase assay procedures A number of novel non-radioactive methods for detection of MAP kinase activities have recently been developed. These procedures generally rely on the specific detection of a phosphorylated peptide assay product, either immunochemically or colorimetrically. For example, antibodies which recognize the phosphorylated residue in myelin basic protein or c-jun have been developed (now available from Upstate Biotech.) which can be used to detect the reaction product by Western blotting or ELISA. In this way, the MAP kinase of interest can be immunoprecipitated and assayed using the method given in Protocol 7. The reaction mixture can then be analysed by gel electrophoresis, and Western blotting using the phospho-specific antibody. As discussed above, quantification of the immunoreactive phospho-protein is the potential limitation of such a procedure. The high cost of such a procedure is also likely to be a factor for most investigators. However, the linkage of this principle to an ELISA detection method is clearly worth considering for high throughput analysis of multiple samples.

7. Determination of MAP kinase subcellular localization and activation by immunocytochemistry Downstream biological consequences of MAP kinase activation are often a result of translocation of the activated protein to another part of the cell 356

14: Detection, isolation and quantitative assay of mitogen activated where the relevant substrate resides. For example, it has been demonstrated that prolonged activation of ERK MAP kinases by some mitogens or differentiating agents can lead to their translocation to the nucleus. This is hardly surprising, given the fact that many of the true physiological substrates of MAP kinases are transcription factors, e.g. c-jun, p62TCF, and ATF2 (Table 2). However, nuclear translocation does not always occur, and determining the movement of a particular MAP kinase in response to a novel ligand may be of fundamental importance in describing the biological consequences of MAP kinase activation. Many of the antibodies now available are sufficiently specific to allow immunostaining of fixed cells or frozen tissue sections. In addition, the recent development of phospho-specific MAP kinase antibodies should allow the selective localization of the active forms of the protein. A generalized procedure for immunocytochemistry of ERK MAP kinases in fibroblasts is given in Protocol 8. However, it is likely that these procedures would require some optimization for other cell types and for frozen sections. Protocol 8. Detection of ERK MAP kinase by immunocytochemistry Equipment and reagents • Glass coverslips • Fluorescence microscope • 3.7% formaldehyde solution (prepared in PBS containing 0.2% Triton X-100)

• Blocking buffer (1% BSA in PBS) • Mounting medium (50% glycerol, 0.1% pphenylenediamine dihydrochloride, in PBS) « Transparent nail polish

Method 1. Grow cells on glass coverslips in 12-well cell-culture plates. 2. Treat cells with growth factor or other agent. 3. Rinse cells twice with ice-cold PBS. 4. Drain off PBS, and add 0.5 ml formaldehyde solution. 5. After 10 min, drain off formaldehyde solution, and air-dry coverslips. 6. Place coverslips on filter paper in small Petri dishes. 7. Apply sufficient blocking buffer (~50ul) to cover cells, and leave for 30 min. 8. Drain off blocking buffer onto filter paper. 9. Apply anti-ERK antibody (1:50 dilution), and leave for 1h. 10. Remove antibody solution, and rinse coverslips three times with PBS. 11. Apply an appropriate dilution of FITC-conjugated secondary antibody for 1 h. 12. Remove the antibody solution, and rinse three times with PBS. 357

Neil G. Anderson Protocol 8.

Continued

13. Invert the coverslip onto a microscope slide bearing 10 ul mounting medium. 14. Seal the edges of the coverslip with nail polish. 15. View under a fluorescence microscope.

8. Assay of upstream activators of MAP kinases All MAP kinases are activated by at least one class of MAP kinase kinases (MAPKK), which in turn are activated by MAP kinase kinase kinases (MAPKKKs). For example, activation of ERKs involves the activation of Raf kinase (the MAPKKK), which then activates MEK (the MAPKK), which then activates ERK. However, for each of the three major classes of MAP kinase, several kinases are known to exist at each level of the three-member signalling module. Thus the two ERKs can be activated by two different MAPKKs (MEK 1 and MEK 2), which in turn can be activated by three different Raf kinases (A-Raf, B-Raf, and Raf-1). The upstream regulation of the SAPKs is even more complex. For this reason, it is beyond the scope of this chapter to describe individual methods for assaying all of these enzymes. Most of these kinases can be assayed routinely using immunoprecipitating antibodies and procedures based on the ERK immunocomplex assay given in Protocol 7. The choice of substrate is critical, since the MAPKKKs and MAPKKs generally have a much narrower substrate specificity than do MAP kinases. For example, ERKs are the only known physiological substrate of MEK1 or MEK2. Consequently, to measure MEK catalytic activity, it is necessary to use ERK protein as substrate. Similarly, Raf kinase activity should be measured using MEK as substrate. It is now possible to generate recombinant versions of both ERK and MEK, and their use in these assays has been described in detail previously (12, 13).

9. Chemical inhibitors of MAP kinase pathways Analysis of the role of MAP kinases in various biological processes has been aided with the recent identification of two classes of chemical inhibitor. The Parke-Davis compound PD098059 shows a high degree of selectivity towards MEK1 and MEK2 (14). This compound inhibits the activation of MEK by Raf, and therefore the activation of ERKs and responses downstream of ERKs are abrogated. However, the non-competitive nature of the inhibition means that previously activated MEK is not affected by the inhibitor. In addition, the effectiveness of PD098059 in blocking ERK activation appears to be dependent on the level of Raf activation. In Swiss 3T3 fibroblasts, the 358

14: Detection, isolation and quantitative assay of mitogen activated inhibitor almost completely blocks ERK2 activation by insulin, which is a weak activator of Raf-1, but is only partially effective in blocking ERK2 activated by epidermal growth factor, which is a strong activator of Raf-1 (14). PD098059 generally shows maximal effectiveness in cultured cells at concentrations of 30-100 uM. The SmithKline-Beecham compounds SB202190 and SB203580 potently inhibit p38 MAP kinases without affecting ERKs, JNKs, or a wide range of other kinases. However, it has been shown that chronic inhibition of the p38 MAP kinases can lead to the activation of ERK kinases by unknown mechanisms (15). Such observations demonstrate that caution should be exercised in the use of MAP kinase inhibitors, especially when measuring long-term cell responses, such as proliferation or apoptosis. In addition, a recently identified novel isoform of p38, termed p38-8, was shown to be insensitive to SB203580 (16).

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Ray, L. B., and Sturgill, T. W. (1987). Proc. Natl. Acad. Sci. USA., 84, 1502. Minden, A., and Karin, M. (1997). Biochim. Biophys. Acta, 1333, F85 Ferrell, J. E., Jr. (1996). Curr. Top. Dev. Biol., 33, 1. Anderson, N. G., Mailer, J. L., Tonks, N. K., and Sturgill, T. W. (1990). Nature, 343, 651. Nakielny, S., Cohen, P., Wu, J., and Sturgill, T. W. (1992). EMBO J., 11, 2123. Erickson, A. K., Payne, D. M., Martino, P. A., Rossomando, A. J., Shabanowitz, J., Weber, M.J., Hunt, D. F., and Sturgill, T. W. (1990). J. Biol. Chem., 265, 19728. Gordon, J. A. (1991). In Methods in enzymology (ed Hunter, T. and Sefton, B. M.). Vol. 201, p. 477. Academic Press, London. Anderson, N. G., Wolf, B. B., and Sturgill, T. W. (1991). Adv. Prot. Phosphatases, 6, 119. Anderson, N. G., Kilgour, E., and Sturgill, T. W. (1991). J. Biol. Chem., 266, 10131. Westwick, J. K., and Brenner, D. A. (1995). In Methods in enzymology (ed Balch, W. E., Der, C. J. and Hall, A.). Vol. 255, p. 342. Academic Press, London. Kummer, J. L., Rao, P. K., and Heidenreich, K. A. (1997). J. Biol. Chem., 272, 20490. Reuter, C. W. M., Catling, A. D., and Weber, M. J. (1995). In Methods in enzymology (ed Balch, W. E., Der, C. J. and Hall, A.). Vol. 255, p. 245. Academic Press, London. Alessi, D. R., Cohen, P., Ashworth, A., Cowley, S., Leevers, S. J., and Marshall, C. J. (1995). In Methods in enzymology (ed Balch, W. E., Der, C. J. and Hall, A.). Vol. 255, p. 279. Academic Press, London. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995). /. Biol. Chem., 270, 27489. Jarvis, W. D., Fornari, Jr., F. A., Auer, K. L., Freemerman, A. J., Szabo, E., Birrer, M. J., Johnson, C. R., Barbour, S. E., Dent, P., and Grant, S. (1997). Mol. Pharmacol., 52, 935. Jiang, Y., Gram, H., Zhao, M., New, L., Gu, J., Feng, L., Di, P-F., Ulevitch, R. J., and Han, J. (1997). J. Biol. Chem., 272, 30122. 359


15 Measuring inositol 1,4,5-trisphosphate-evoked 45 Ca2+ release from intracellular Ca2+ stores COLIN W. TAYLOR and JONATHAN S. MARCHANT

1. Introduction The receptors for inositol 1,4,5-trisphosphate (Ins(l,4,5)P3) are intracellular Ca2+ channels which open in response to the concerted effects of cytosolic Ca2+ and Ins(l,4,5)P3, and thereby allow Ca2+ stored within the lumen of the endoplasmic reticulum to leak into the cytosol. The resulting increase in cytosolic [Ca2+] regulates many aspects of cellular behaviour. In the twentyfive years since Ins(l,4,5)P3 was shown to provide the link between receptors in the plasma membrane that stimulate polyphosphoinositide hydrolysis and the release of Ca2+ from intracellular stores (1), novel experimental approaches have often provided the impetus for the steps towards our current understanding. Simple methods to separate [3H]-inositol phosphates, and the use of Li+ to block the final step in their degradation (2), allowed the effects of numerous stimuli on polyphosphoinositide hydrolysis to be established in many cells. Methods that allowed selective permeabilization of the plasma membrane, without destroying the integrity of intracellular organelles (3), were instrumental in allowing the effects of Ins(l,4,5)P3 on intracellular Ca2+ stores to be demonstrated (1). The introduction of fluorescent Ca2+ indicators (4), and their subsequent use with caged compounds (5, 6) and confocal microscopy methods (7), revealed the complex spatial organization of intracellular Ca2+ signals, and established that the signals evoked by Ins(l,4,5)P3 are often regenerative. Small, very localized Ca2+ release events, probably reflecting the opening of the channels of relatively few Ins(l,4,5)P3 receptors, can, if the stimulus exceeds a critical threshold, lead to recruitment of additional Ca2+ release events and thereby to the propagation of a regenerative Ca2+ wave across the cell (8). The luminescent Ca2+ indicator protein, aequorin, has recently enjoyed a revival of interest because it can be targeted to specific

Colin W. Taylor and Jonathan S. Marchant intracellular compartments, and so provide further opportunities to resolve the spatial organization of Ca2+ signals (9). The families of Ca2+ indicators derived from green fluorescent protein look set to provide even more versatile means of visualizing the subcellular organization of intracellular Ca2+ signals (10). Preparation of [32P]-Ins(l,4,5)P3 (11), and then the commercial availability of [3H]-Ins(l,4,5)P3 with high specific activity, allowed Ins(l,4,5)P3 receptors to be identified using radioligand binding methods, and paved the way for their purification by conventional affinity purification methods (12), and subsequent functional reconstitution. Molecular cloning then provided the first structural information, allowed receptor subtypes and splice variants to be identified, and has begun to establish the relationships between Ins(l,4,5)P3 receptor structure and function (13). Throughout these developments, both synthetic chemistry (14) and natural sources (15, 16) have provided ligands with which to address structure-activity relationships and to manipulate Ins(l,4,5)P3 receptor behaviour. Because Ins(l,4,5)P3 receptors can respond very rapidly to Ins(l,4,5)P3, it is important that assays of their function have the appropriate sub-second temporal resolution, and that the methods used minimize the likelihood of the Ca2+ released exerting feedback effects on the behaviour of the receptor (17). From a practical standpoint, the latter is a very significant problem. The biphasic effects of cytosolic Ca2+ on Ins(l,4,5)P3 receptor behaviour are kinetically complex (17), and while fluorescent Ca2+ indicators used with conventional stopped-flow methods (18, 19) provide the simplest and least timeconsuming means of examining the rapid kinetics of Ins(l,4,5)P3-evoked Ca2+ release, they are inherently prone to the difficulty of disentangling direct effects of Ins(l,4,5)P3 from feedback regulation by Ca2+. This chapter describes an alternative method, rapid superfusion of permeabilized cells, and while it focuses specifically on analysis of the responses of rat hepatocytes to Ins(l,4,5)P3, superfusion methods are also readily applicable to analyses of other ion channels. Two other volumes in this series describe methods specifically related to Ca2+ signalling (20), and to inositol phosphate actions and metabolism (21), and additional volumes include relevant chapters on bilayer recording (22) and radioligand binding (23). Detailed descriptions of various methods used in Ca2+ signalling research are also provided in other books (24-26).

2. Preparation, permeabilization, and 45Ca2+ loading of hepatocytes 2.1 Isolation of rat hepatocytes In the thirty years or so since suspensions of viable hepatocytes were first isolated in high yield from intact liver, the methods used to isolate hepatocytes have evolved in many laboratories. The essential principles underlying the 362

15: Measuring Ca2+ release method, and many of the technical details, are comprehensively reviewed in an excellent book (27). Protocol 1 describes the method currently used in our laboratory. Protocol 1. Isolation of viable rat hepatocytes Equipment and reagents • Fine and coarse scissors and forceps • 50 ml plastic syringe • Thin-walled (1 mm inside diameter, 2 mm outside diameter) PVC cannulation tubing (Portex) • • • • • •

.

• 50 ml of MEM, supplemented with additional Ca2+ (final concentration 5 mM) and collagenase (20 mg per 50 ml type 1 from Clostridium histolyticum, ~250 units mg-1 Worthington) • 100ml of MEM supplemented with bovine Silk sutures serum albumin (BSA, Sigma, type V; 2 g Gauze per 100 ml) Petri dish . Eagle's medium (Sigma) supplemented 25 ml conical centrifuge tubes with NaHCO3 (26 mM) and BSA (2 g per 100 Liver perfusion chamber (27) ml) at 4°C Ca2+-free minimal essential medium, MEM • Trypan Blue solution (0.4 mg Trypan Blue (500ml: 116 mM NaCI, 5.4 mM KCI, 0.8 mM in 100 ml of medium containing 150 mM MgSO4, 1 mM NaH2P04, 25 mM NaHCO3, Nacl, 10 mM Hepes, pH 7.4) 11 mM glucose, 500 uM EGTA, gassed with • Gyratory waterbath (New Brunswick 95% O2-5% C02, pH 7.4 at 37°C) Scientific, model G76) MEM (500 ml: as above, but with the EGTA • Bench-top centrifuge omitted and replaced by 1.8 mM CaCI2) • Haemocytometer and microscope

Method 1. Rapidly open the abdomen of an adult male rat (-150 g), killed by cervical dislocation, expose the hepatic portal vein, and tie a loose silk ligature around the vein. 2. Cannulate the vein, using tubing attached to a 50 ml syringe containing Ca2+-free MEM (gassed with 95% O2-5% C02, pH 7.4 at 37°C), secure the cannula with the silk ligature, sever the vena cava, and then slowly manually perfuse the liver with the contents of the syringe. The entire liver should be uniformly cleared of blood. 3. Remove the liver, and secure the cannula to a gravity-fed perfusion system (—10 ml min-1) within a chamber held at 37°C. Continue the perfusion with Ca2+-free MEM for 10 min. Perfuse for 1 min with MEM, and then with MEM supplemented with collagenase (recirculate this medium if necessary). The medium must be gassed with 95% 02-5% C02 throughout. 4. After 10-15 min, the liver will be substantially digested, and it can be removed for gentle mechanical disruption in a Petri dish. If necessary, transfer the dispersed cells to the MEM containing collagenase, and incubate at 37°C in a gyratory waterbath (80 r.p.m.) for 10 min to complete the collagenase digestion. 5. Filter the cells through gauze into 25 ml centrifuge tubes, and recover the cells by centrifugation (40 g, 2 min). 363

Colin W. Taylor and Jonathan S. Marchant Protocol 1. Continued 6. Wash the cells by aspirating the supernatant, gently resuspending the cells in MEM containing 2% BSA, and re-centrifuging. Repeat. 7. Finally, resuspend the cell pellets in 100 ml of the cold Eagle's medium, and leave for about 15 min before assessing cell viability. The viability of the cells, assessed by their ability to exclude Trypan Blue, should exceed 95%. Count the cells using a haemocytometer; a single liver typically provides >3 x 108 cells. 8. The cells can be stored in Eagle's medium at 4°C for 1-2 days without their viability falling below 90%.

2.2 Cell permeabilization, loading of intracellular stores with45Ca2+, and the effects of Ins(l,4,5)P3 In order that Ins(l,4,5)P3, a charged and therefore membrane-impermeant messenger, can gain access to its receptors in the membranes of the intracellular Ca2+ stores, the plasma membrane must be selectively permeabilized while minimizing damage to intracellular organelles. A variety of approaches (3) have been used to permeabilize the plasma membrane, including electroporation, bacterial toxins (streptolysin O and a-toxin), activation of P2X7 receptors by ATP to open large membrane pores, and even removal of bivalent cations from the extracellular medium. The most widely used and simplest means of achieving selective permeabilization of the plasma membrane is provided by detergents (notably saponin and digitonin) that preferentially interact with cholesterol. Careful use of these detergents introduces pores in the plasma membrane that allow the passage of molecules with relative molecular masses < 200 000 (28), while sparing the cholesterol-deficient membranes of intracellular organelles. Protocol 2 describes a typical procedure. It is, however, important to stress that while it is possible with meticulous attention to the permeabilization conditions to minimize disruption of the intracellular morphology (29), under less stringent conditions the intracellular stores retain their ability to sequester and then release Ca2+, but the intracellular architecture may be severely perturbed. The continuity of the endoplasmic reticulum may be disrupted (30). Even very large proteins may be lost from the cytosol: under the conditions (Protocol 2) we use to permeabilize hepatocytes; for example, most lactate dehydrogenase (Mr 130 000) is lost within 3 min (31). Finally, when permeabilized hepatocytes are pelleted by centrifugation, a fraction of the original Ins(l,4,5)P3-sensitive stores are recovered from the supernatant, indicating severe disruption of intracellular architecture (18, 31). Despite these limitations, permeabilized cells afford a simple means of studying Ins(l,4,5)P3 receptor behaviour under precisely controlled conditions; indeed, permeabilized hepatocytes have recently been shown to be capable of generating Ca2+ oscillations (32). 364

15: Measuring Ca2+ release With the plasma membrane no longer protecting the cell interior from the incubation medium, it is important to ensure that the composition of the latter approximates to that of the cytosol, and that the ATP required for active Ca2+ uptake into intracellular stores is maintained at an appropriate level throughout the experiment. The ATP that is hydrolysed to ADP during the incubation can be enzymatically regenerated by adding an ATP-regenerating system, typically creatine phosphate and creatine phosphokinase, to the incubation medium. Unless it is important to be able to remove ATP during the experiment, such an ATP-regenerating system is therefore usually added to the cytosol-like medium. 45 Ca2+ provides the most common and straightforward means of observing ATP-dependent Ca2+ uptake into the intracellular stores of permeabilized cells (Protocol 2). Addition of a fluorescent indicator to a cytosol-like medium with no other Ca2+ buffers provides an alternative approach; as the stores actively sequester Ca2+, the free [Ca2+] in the medium falls, and the fall can be readily recorded using a conventional fluorescence spectrofluorimeter. For experiments using 45Ca2+, either filtration through glass fibre filters or centrifugation can be used to separate the 45Ca2+ trapped within intracellular stores from the much larger amount of 45Ca2+ present in the incubation medium; the former is simpler and most easily adapted to large numbers of samples. In a typical experiment, permeabilized cells would be allowed to load to steady state with 45Ca2+ in a cytosol-like medium (Figure 1), the appropriate additions would be made to the cells (e.g. Ins(l,4,5)P3), and soon afterwards the effects of the additions on the 45Ca2+ content of the stores would be assessed by stopping the incubations, and then rapidly separating the 45Ca2+ trapped within the stores from that in the incubation medium. Because some 45Ca2+ binds non-specifically to both the cells and filters, appropriate controls are required to identify the active ATP-dependent 45Ca2+ uptake into the intracellular stores (i.e. the 45Ca2+ content determined before addition of ATP, or after addition of the ionophore ionomycin to prevent active Ca2+ uptake). Protocol 2 describes a typical method for loading the intracellular stores of permeabilized hepatocytes with 45Ca2+, and then assessing the effects of Ins(l,4,5)P3. Protocol 2.

Loading intracellular Ca2+ stores with 45Ca2+

Equipment and reagents • Rat hepatocytes, isolated according to • ATP-regenerating system containing a 3:5:5 Protocol 1 mixture of ATP (500 mM), phosphocreatine • Cytosol-like medium (CLM: 140 mM KCI; 20 (1 M), and creatine phosphokinase (1 mM NaCI; 1 mM MgCI2; 1 mM EGTA; 20 unit ul-1); each of the reagents is from mM Pipes; CaCI2, 300 uM, to give a free Boehringer [Ca2+] of 200 nM; pH 7.0 at 37°C) • Trypan Blue solution (Protocol 1) 2+ • Ca -free CLM, in which the CaCI2 is • Carbonyl cyanide p-trifluoromethoxyomitted from CLM phenylhydrazone (FCCP: 10 mM in ethanol) • Saponin solution (Sigma, 1 mg 20ul-1 in • lns(1,4,5)P3 (American Radiolabeled Ca2+-free CLM) Chemicals)

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Colin W. Taylor and Jonathan S. Marchant Protocol 2. 45

Continued 1

• CaCI2 (~8 Ci g- , Dupont) . Glass fibre filters (Whatman, GF/C 25 mm • For some variations of the protocol, the diameter) following may also be required: thapsigar• Ten-place vacuum filtration manifold gin (Alamone Laboratories), BAPTA or (Hoeffer Scientific Instruments) EGTA (Sigma), and a solution of apyrase . Scintillation vials (5 ml) and hexokinase in glucose (Sigma; final • Ecoscint A scintillation fluid (National concentrations, 50 units ml-1 for each Diagnostics) enzyme, and 10 mM for glucose) •Gyratory waterbath • Wash medium (310 mM sucrose; 1 mM trisodium citrate, PH 7) • Bench-top centrifuge • Disposable plastic tubes (75 x 12 mm)

Method 1. Confirm the viability of the hepatocytes using Trypan Blue. Use only preparations with >95% plasma membrane integrity. 2. Remove a volume of Eagle's medium containing ~107 cells, pellet them by centrifugation (40 g, 2 min), and gently resuspend the cells in 25 ml of Ca2+-free CLM supplemented with saponin (5 ul, final saponin concentration 10 ug ml-1) at 37°C. 3. Incubate the cells in a conical flask in a gyratory waterbath (80 r.p.m.) at 37 °C, and periodically sample the cells to monitor the integrity of the plasma membrane using Trypan Blue. Permeabilization of >90% of the cells typically takes 5-10 min, but add more saponin if it takes longer. 4. When >90% of the cells are permeabilized, remove the cells from the medium by centrifugation (650 g, 2 min), resuspend them in 6 ml of CLM at 37°C, and add FCCP (6 ul, final concentration 10 uM) and 45 Ca2+ (final concentration ~2 uCi ml-1). 5. Incubate the cells in a flat-bottomed tube in a gyratory waterbath at 37°C, to keep the cells in suspension. 6. Remove duplicate 200 ul samples to disposable plastic tubes containing ice-cold wash medium (5 ml), rapidly filter the contents through Whatman GF/C filters, and then rinse the tube with a further 5 ml of wash medium. These tubes provide a measurement of the ATPindependent (i.e. non-specific) binding of 45Ca2+, which will be subtracted from all subsequent measurements. 7. Add the ATP-regenerating system (13 ul ml-1, giving final concentrations of 1.5 mM ATP, 5 mM creatine phosphate, and 5 units ml-1 creatine phosphokinase) to the cells to initiate active 45Ca2+ uptake. For hepatocytes, the steady-state 45Ca2+ content is attained within 5 min (Figure 1), but the timing needs to be independently determined for each cell type and incubation condition. The ATP concentration should be increased (to 7.5 mM) and the creatine phosphokinase omitted from experiments in which the ATP is to be enzymatically removed during the experiment. 366

15: Measuring Ca2+ release 8. At appropriate times, remove samples of cells (200 ul) to add to appropriate stimuli (e.g. lns(1,4,5)P3, 4 ul at fifty times its final concentration). lns(1,4,5)P3 typically causes a half-maximal response (EC50) when its concentration is about 200 nM. 9. Stop the reactions after a suitable interval (typically 30s- 5 min), using the method described under Step 6. 10. Add each filter to a scintillation vial, add Ecoscint A scintillation fluid (5 ml), leave overnight for the filters to dissolve, and then determine the 45Ca2+ activity using a liquid scintillation counter. Determine the 45 Ca2+ activity of the CLM by counting a 50 ul aliquot, and from that and its total Ca2+ concentration (300 uM), determine the specific activity of the 45Ca2+ in the incubation medium. Calculate the ATP-dependent 45Ca2+ content of each of the samples of permeabilized cells, and from the cell density and specific activity of the 45Ca2+ compute the steady-state ATP-dependent Ca2+ content of the stores (typically 1-2 nmol per 106 cells for hepatocytes; Figure 1). The effects of the experimental manipulations can then be expressed relative to the steady state Ca2+ content.

Methods similar to those described in Protocol 2 have been successfully applied, with appropriate modifications, to a variety of cell types and

Figure 1. ATP-dependent accumulation of 45Ca2+ into intracellular stores. Permeabilized rat hepatocytes (107 cells ml-1) were incubated in CLM (free [Ca2+] = 200 nM) supplemented with 45 Ca 2+ , ATP, and FCCP for the indicated periods. Cells were then filtered through Whatman GF/C filters using a Brandel receptor-binding harvester, and the amount of radioactivity trapped on the filters was determined by liquid scintillation counting. The results are typical of five independent experiments.

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Colin W. Taylor and Jonathan S. Marchant experimental requirements. Some of the more useful variations are described below: • The method can be easily adapted to larger numbers of samples by using a Brandel 24-place receptor binding harvester (SEMAT) in place of the filter manifold to allow simultaneous filtration of 24 samples. • Unidirectional 45Ca2+ efflux measurements, rather than the steady-state measurements described in Protocol 2, are straightforward if further active 45 Ca2+ uptake is inhibited before addition of the experimental stimulus. Thapsigargin (to selectively inhibit the Ca2+ pump of the endoplasmic reticulum), removal of ATP (using apyrase, glucose, and hexokinase), or rapid chelation of all free Ca2+ by addition of excess BAPTA or EGTA, alone or in combination (33), provide effective means of rapidly inhibiting further 45Ca2+ uptake. • The composition or temperature of the medium in which the cells are stimulated with Ins(l,4,5)P3 may be varied independently of that used to load the intracellular stores (typically CLM because it most closely mimics the cytoplasmic conditions within an unstimulated cell). This allows the effects of modulators (e.g. Ca2+ or other bivalent cations) (34) on Ins(l,4,5)P3 receptor behaviour to be characterized. The computer programs and fluorescent indicator methods described in a previous volume (20) provide the means of estimating and then reliably determining the free [Ca2+] of incubation media.

3. Rapid kinetic measurements of 45Ca2+ release from intracellular stores 3.1 Rapid superfusion methods In cell populations, initial rates of Ins(l,4,5)P3-stimulated Ca2+ release provide the most direct measure of the activity of the Ca2+ channels, but such rates can be determined only if Ca2+ release can be measured with sufficient temporal resolution. As the temperature is reduced, rates of Ca2+ mobilization are substantially slowed (18, 35), but they are still too fast for initial rates to be resolved with conventional methods. There is also the additional problem that changes in temperature may themselves significantly influence important characteristics of the response. To overcome these problems, rapid filtration (36-38) and rapid superfusion techniques (31, 39-41) have been used to resolve the kinetics of responses to Ins(l,4,5)P3. Both techniques are based upon the pressure-driven movement of medium (typically a cytosol-like medium) around a biological sample immobilized on a porous support. Rapid filtration systems, such as that manufactured by Bio-Logic (36, 37, 42), are designed to allow measurement of the amount of 45Ca2+ left within the cells after a period of superfusion with medium containing the appropriate stimulus 368

15: Measuring Ca2+ release (e.g. Ins(l,4,5)P3). Each immobilized sample of cells therefore provides only a single determination of 45Ca2+; the effects of stimuli must then be assessed by comparison with parallel experiments. Rapid supervision techniques aim to record 45Ca2+ release continuously from a sample of immobilized cells, and are thereby able to detect effects of stimuli within a single run, avoiding the variability introduced by comparisons that depend wholly on parallel runs. Furthermore, the amount of 45Ca2+ released (the signal in superfusion experiments) is typically small relative to that remaining within the cells (the signal recorded in rapid filtration experiments); rapid superfusion therefore offers a much improved signal-to-noise ratio. Both approaches, however, suffer from the drawback that the high temporal resolution of the techniques requires that media are superfused at high flow rates (typically several ml s-1; Protocol 3) throughout the experiment, and that can prove prohibitively expensive for some reagents. Several groups have used rapid superfusion to study the kinetics of intracellular Ca2+ release (31,39-41,43), and the remainder of this chapter will describe the methods in detail. It is, however, worthwhile to consider first the advantages offered by rapid superfusion, because it is undoubtedly more costly, more technically demanding, and more time-consuming than alternative methods. The first major advantage is the ability to measure unidirectional 45Ca2+ release from the intracellular stores, under conditions where local cytosolic Ca2+ gradients (44) are effectively minimized. To resolve the Ins(l,4,5)P3 concentration-dependence of the opening of the Ins(l,4,5)P3 receptor, for example, initial rates of Ca2+ release must be measured under conditions that prevent the feedback regulation by Ca2+ that would otherwise contribute to the response (45). Unless such feedback is eliminated, it becomes very difficult to resolve whether a steep Ins(l,4,5)P3 concentration-effect relationship results from positive feedback by cytosolic Ca2+, or from a genuine need for several subunits of the receptor to bind Ins(l,4,5)P3 before the channel opens (31). The combination of a high concentration of a Ca2+ buffer (EGTA or BAPTA) in the superfusion medium, together with the very rapid flow of medium through the sample, provides the best means of dispersing microdomains of high [Ca2+] in the vicinity of active receptors (36). Even this method may not, of course, prevent the mouth of an open channel from being exposed to a substantial increase in [Ca2+]. When fluorescent indicators are used to record Ca2+ mobilization, the medium [Ca2+] must increase (there is otherwise no signal to detect) (45). Furthermore, with high affinity indicators like Fura-2 and Fluo-3, both the speed and amplitude of local cytoplasmic Ca2+ changes can be severely underestimated, especially during the early stages of Ca2+ mobilization, when spatial gradients of Ca2+ are high (46). Secondly, rapid superfusion can effect rapid changes in ligand concentration on a millisecond timescale (see below). Unless Ins(l,4,5)P3 receptors are exposed to such rapid changes in Ins(l,4,5)P3 concentration, channel activity will be desynchronized by the slowly rising concentration of agonist: 369

Colin W. Taylor and Jonathan S. Marchant some channels will be opening, some closing, and some still waiting to bind agonist. It then becomes very difficult to resolve the kinetics of the various aspects of receptor behaviour. Thirdly, rapid superfusion affords the opportunity to add and remove components of the superfusion medium rapidly: no other method is at present capable of allowing such sequential changes in the composition of the medium, while retaining the temporal resolution required to record the activity of Ins(l,4,5)P3 receptors. Ultimately, of course, single channel methods may provide an alternative means of achieving these aims (47), but the electrophysiological methods so far used to examine Ins(l,4,5)P3 receptor behaviour are incompatible with very rapid changes of medium (48, 49).

3.2 Rapid superfusion apparatus 3.2.1 Overview In this section, we describe the construction of a rapid superfusion apparatus which we have used to investigate the behaviour of Ins(l,4,5)P3 receptors in permeabilized rat hepatocytes (31, 41). The equipment could, however, easily be adapted to other applications, because similar methods have previously been used to examine neurotransmitter release from synaptosomes (50-54), the kinetics of cGMP-stimulated cation efflux in bovine retinal rods (55), the activation of neuromuscular nicotinic acetylcholine receptors (31), and the enzymology of the Na+ pump (56). Various designs of superfusion system have been reported (43, 53, 54, 57); that described here has evolved from its original description (56) and through two subsequent versions (55, 58). Figure 2 shows both a schematic representation and a photograph of our rapid superfusion apparatus. The components needed for its assembly are all available commercially, and are referred to in each of the subsequent sections by a component number (C1-C33, Table 1). The total cost of constructing the apparatus is about GBP4000. Briefly, immobilized cells are mounted in a chamber linked by four solenoid valves to pressurized stainless steel vessels containing the superfusion media. Fluid flows continuously from one of the pressurized reservoirs over the sample, and is partitioned into discrete samples during a single rotation of a fraction collector. Each fraction contains the 45Ca2+ released within a particular collection interval, and therefore represents the average rate of Ca2+ mobilization during that period. More detailed descriptions of the main components of the superfusion apparatus follow in the next four sections. 3.2.2 The superfusion chamber The biological sample is housed in the exit port of a stainless-steel manifold (Cl), fixed in position by an x-y adjustable support arm attached to the turntable casing. Four solenoid valves (C2) gate independent inputs to the 370

15: Measuring Ca2- release

Figure 2. The rapid superfusion apparatus. (a) Four fluid reservoirs are independently connected to a common superfusion chamber housed within the solenoid manifold. The superfusion chamber is accessed by four coaxial ports, each gated by a solenoid-driven valve. Fluid flows continuously from one of the pressure reservoirs over the sample, and is captured by the fraction collector during a single revolution, (b) Photograph of the rapid superfusion apparatus.

371

Colin W. Taylor and Jonathan S. Marchant Table 1. Components of rapid superfusion apparatus

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 C31 C32 C33

Component

Supplier

Part number

solenoid manifold assembly solenoid (series-9) Teflon poppet armature Kalrez O-ring buffer spring armature spring Teflon bulkhead connector stainless steel washer 1/4-28" UNF outlet fitting PTFE tubing (1/8" by 1/16") pressure filtration vessels stainless steel support screen 1/8" NPT-1/4-28" UNF fitting 3-way valves tube end fittings gripper fittings PTFE cones air thermostat Treff 1.5 ml microfuge tubes optical tachometer stepper motor pulley belt motor indexer X-ware I/O board 8-channel valve driver Reed switch master switch cellulose nitrate SC filter (8 um) pre-filter post-filter Teflon tube-to-tube connector

General Valves General Valves General Valves General Valves General Valves General Valves General Valves General Valves Anachem General Valves Omnifit Gelman Gelman Custom built Omnifit Omnifit Omnifit Omnifit Merck Anachem RS MicroDrives Contitech Parker Hannifin Parker Hannifin Amplicon General Valves RS RS Schleicher & Schuell Schleicher & Schuell Schleicher & Schuell General Valves

9396901+ 93130502 3230502 91810312 9701001 720011 7206401 1310622 P407X 511252 3011 4280,4287 4235 1109 2210 2312 1524 ST60 968160902 CT6-205665 HY2003450350A8 140XL037 Digiplan, PDX15

PC36AT 9031100 338169 333669 AE99 GF51 GF52 1320622

superfusion chamber. Each solenoid is constructed from non-passivated stainless steel, and only inert materials line the path of the superfusion media. Valve (C2) leakage has been estimated to be less than 1 X 10-8 He per second per atmosphere, such that pressures up to 85 atm can be withstood with response times as low as 2 ms (General Valves Corporation, product literature). However, we find that erosion of a Teflon poppet (C3) within the solenoid valve occurs with time, resulting in valve leakage. This component and others within the valves assembly (C4—C7) are therefore routinely replaced. The volume of the exit port of the solenoid manifold is reduced with an adapted Teflon connector (C8) to accommodate the filter array (see later and Figure 4). A stainless steel washer (C9) is immobilized immediately below the common outlet port. The filter array (Figure 4) is secured within the superfusion 372

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release

chamber using a modified fitting (C10), with the internal bore narrowed by PTFE tubing (Cll) to maintain laminar flow. The superfusate leaves through this outlet as a continuous uninterrupted stream. When the outlet fitting is not narrowed, the data points show substantial scatter (not shown). Superfusion media are held under nitrogen pressure in 200 ml stainless steel filtration vessels (C12). A narrow-gauge filter support (C13) is inserted at the base of each pressure vessel, to avoid solid contaminants reaching the solenoid valves. Connecting valves and tubing were selected to present negligible flow resistance relative to the filter. The inlet and outlet ports of each reservoir are connected to PTFE tubing (Cll) by way of machined stainless steel connectors (C14) and 3-way valves to allow pressure release (C15). Connection to the solenoid entry ports is made with tube end fittings (C16) and stainless steel gripper fittings (C17) to provide flangeless, inert, leak-free connections. All valved connections are made with pressure-resistant, inert PTFE cones (C18). The temperature of the entire apparatus can be regulated by housing it within a heated Perspex chamber (C19), or in a refrigerator. Rapid changes in the composition of the superfusion medium are effected by virtue of rapid solution flow through minimal dead volume. Exchange kinetics are routinely followed in every experiment, by supplementing one of the media with a biologically inert radiotracer ([3H]-inulin), which serves the additional role of an internal time marker, permitting changes in the rate of 45 Ca2+ efflux from the intracellular stores to be directly referenced to the time of solution exchange. The half-time for solution exchange under experimental conditions with the filter array present is measured at different superfusion pressures, by introducing a variety of radiolabels ([3H]inulin, [3H]glucose, and 45 Ca2+) into one of two superfusion media, and measuring the rate of appearance and disappearance of radioactivity in the superfusion effluent. Figure 3 shows two switching events between CLM, and CLM supplemented with trace amounts of [3H]-inulin. The kinetics of both solution influx and efflux are described by mono-exponential functions, with a half-time of 30 ± 4 ms at 30 p.s.i. (n = 10). This corresponds to a 5-95% rise time of -130 ms. The halftimes for solution exchange at 20 p.s.i. and 60 p.s.i. are 46 ± 6 ms (n = 10) and 20 ± 1 ms (n = 6) respectively. Each vial on the fraction collector collects 97 ± 3% of the activity expected, calculated from the known dilution of radiolabel, and a measured flow rate of 2 ml s-1. 3.2.3 Variable speed fraction collection The effluent from the superfusion chamber is collected into 100 fractions by cuvettes rotating at the perimeter of a turntable disc (diameter ~42 cm) mounted on a variable-speed fraction collector. The system provides a continuously variable sample interval of between 9 ms and 3000 ms. The turntable platter is made from lightweight material (total mass ~300 g) to minimize torque and inertia, and thereby maximize rotational velocity. Because the length of an experiment is limited by the time taken for a single 373

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Figure3. Kinetics of solution exchange in the super-fusion chamber. The rate of appearance and disappearance of [3H]inulin in the superfusate was measured during two switching events. Each point denotes the amount of [3H]inulin detected in a 20 ms sample collected during superfusion of the chamber with CLM at 2 ml s-1 and 30 p.s.i. Data are expressed as percentages of the expected value, calculated from the dilution of radiolabel and a calculated collection volume of 40 ul (20 ms at 2 ml s-1). The kinetics of solution exchange were fitted with mono-exponential functions (solid line).

rotation of the turntable, a variable-speed fraction collector is essential: it allows the samples that are important for the kinetic analysis to be collected at high temporal resolution, while the remaining samples, which are essential to establish the size of the total 45Ca2+ pool (Protocol 3), can be collected with much lesser temporal resolution into the minimal number of vials. The versatility provided by the variable-speed fraction collection is also advantageous in experiments involving long preincubation periods, where the effluent from the preincubation can be collected into very few vials at a slow collection speed, leaving most of the 100 cuvettes available for collection of the most important samples, with maximal temporal resolution. The turntable is driven from a stepper motor (C22), indirectly geared (3:1, C23) to the turntable platter to generate a wide range of collection intervals. All connections are tightly welded to prevent slippage. The stepper motor is driven by a motor indexer (C24), programmed independently to the superfusion protocol using X-Ware software (C25). The stepper motor (0.03° increments) can accelerate extremely rapidly (±999 rev s-2) over the practicable range of velocities (5-0.01 rev s-1), and can generate a broad range of collection intervals (continuously variable between 9 ms and 3000 ms). The time taken to change velocity within a revolution is verified by sampling the volume of fluid collected by successive vials during a range of velocity steps. In practice, acceleration and deceleration are essentially instantaneous over 374

15: Measuring Ca2+ release the experimental range of collection intervals (9-3000 ms), although to avoid any possible problems, large steps in velocity are effected as gradual ramps and completed before switchings between superfusion media. Rotational speed is verified with an optical tachometer (C21). Cuvettes are closely juxtaposed on a single strip of masking tape, and screwed into the turntable platter. The lumina are bridged using microfuge tubes (C20) to minimize splatter. 3.2.4 Electronic control system Superfusion is controlled from a 24-line programmable digital I/O board (C26) inside a Viglen III/LS microcomputer. The I/O board communicates with both the valve driver (C27) and motor indexer (C24). The sequence and duration of the superfusion protocol is specified using a custom-written GWBASIC program. Motion protocols for the turntable are programmed in XWare (C25) via the RS232 interface. Two sequential signals are required to initiate superfusion: first, a signal from the operator via the computer, to indicate that the superfusion chamber is ready; and second, an automated input from the motor indexer, triggered by closure of the magnetic reed switch (C28), to indicate correct alignment of the turntable. A switch (C29) triggers indefinite turntable rotation until the motor indexer receives this home-position input. The valve driver then energizes the solenoids in the desired sequence, and this is registered on status LEDs and by logic output signals reported through the program interface to confirm independently the switching protocol. The motor driver will terminate superfusion if a programmable end-of-travel limit is reached. These two sequential signals protect the system against current fluctuations, and permit turntable rotation independent of superfusion. This arrangement also permits 'signal averaging', the collection of sequential identical experiments into the same sample holders. 3.2.5 The filter array Selection of the filter system for immobilization of the biological sample is a primary concern, as the overall temporal resolution of the apparatus is very much determined by it. Ideally, the filter array should retain the maximal amount of the biological sample without clogging, provide mechanical stability to prevent damage from shear forces and turbulence at high flow rates, yet occupy minimal volume and provide minimal resistance to flow. Although high flow rates increase the temporal resolution, an increased pressure gradient across the sample decreases the amount of intact material retained, and also leads to artefactual release of radiolabel from the biological sample when the fluid flow is perturbed on switching. Temporal resolution is inevitably therefore a compromise with the need to maintain mechanical stability of the sample: the exact compromise will depend upon the nature of the biological specimen. We have found that the optimal filter composition for stable immobilization of permeabilized rat hepatocytes consists of a cellulose nitrate filter triplet 375

Colin W. Taylor and Jonathan S. Marchant (8 uM, C30), protectcd by a pre-filter to dampen pressure surges on switching (C31), and a post-filter (C32) to trap dislodged material (Figure 4). The filter array is supported by a 5 mm diameter stainless steel mesh (C33) and a stainless steel washer (C9), The use of permeabili/cd cells has obviated the need for low porosity filters, thereby permitting a high flow rate, and hence high temporal resolution without sacrificing the stability of the sample. During an experimental run (Protocol 3), the filter array is assembled externally to the supcrfusion apparatus in one end of a connector fitting (C33). Permeabilized cells loaded to steady state with 45Ca2+ (Protocol 2) are immobilized onto the filter triplet by filtration, and the filter array is then rapidly transferred to the superfusion apparatus. The steps between transferring the cells to the filter and initiating the experimental protocol are completed within 20 s. Since the half-time for passive 45Ca2 efflux from the intracellular stores is 162 ± 13 s at 20°C (n = 3), the Ca2+ contents of the intraeellular stores at the onset of the experiment are no less than -92% of the steady-state level. Using this filter array, the rate of 45 Ca 2+ release from permeabilized hepatocytes is unaffected by switchings between control media, confirming that the apparatus allows rapid changes in superfusion medium without dislodging or damaging the intraeellular stores (Figure 5). The only artefactual release of

Figure 4. Filter array used for immobilization of permeabilized rat hepatocytes. The intraeellular stores of permeabilized rat hepatocytes were loaded with 45Ca?' and immobilized on a cellulose ester filter triplet, sandwiched between a pre-filter and post-filter baffle and supported by stainless steel gauze and washers. An outlet fitting (C10), narrowed with PTFE tubing, secured the assembly into the superfusion chamber shown on the left. 376

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Figure 5. Stable immobilization of permeabilized rat hepatocytes on the filter array. The intracellular stores of permeabilized hepatocytes were loaded to steady state with 45Ca2+, and immobilized on the filter array (Protocol 3). After two 5 s prewashes, cells were superfused with CLM for 8.5 s, interrupted by control switchings at 3.25 s and 5.875 s. The timecourse of the switching event is reported with the inert volume marker [3H]inulin (dotted line, right axis). After 8.5 s, cells were superfused with CLM supplemented with Triton X-100 (T, 50 mg per 100 ml). 46Ca2+ release (black circles) collected during each 125 ms interval is expressed as a percentage of the total amount of 45Ca2+ within the stores trapped on the filters (left axis). The trace has been corrected for the unstimulated rate of 46 Ca2+ efflux.

45

Ca2+ occurs when the fluid flow is first initiated, and this probably reflects the washout of 45Ca2+ from cells damaged during setting-up. However, subsequent superfusion of the detergent, Triton X-100 (50 mg per 100 ml), causes a rapid increase in the rate of release of 45Ca2+, such that -90% of the 45Ca2+ remaining on the filter is released within 250 ms. Similar results, although with a slower time course, are obtained with the Ca2+ ionophore ionomycin (not shown). Integration of the area beneath the data points can be used to estimate the total amount of 45Ca2+ initially immobilized on the filter. Because the total amount of sequestered 45Ca2+ immobilized on the filters varies between independent runs, cells are routinely superfused with CLM 377

Colin W. Taylor and Jonathan S. Marchant containing Triton X-100 (50 mg per 100 ml) at the end of every experiment, to quantify the amount of stored 45Ca2+ immobilized on the filters.

3.3 Rapid responses to Ins(l,4,5)P3 Protocol 3 describes a typical method using the rapid superfusion apparatus to examine the rapid kinetics of 45Ca2+ evoked by Ins(l,4,5)P3. Protocol 3.

Rapid kinetics of lns(1,4,5)P3-stimulated 45Ca2+ efflux

Equipment and reagents • Rapid superfusion apparatus • PVC tubing (Portex, 1 mm inside diameter, 2 mm outside diameter) • Permeabilized rat hepatocytes loaded to steady state with 45Ca2+ in CLM (Protocol 2} • lns(1,4,5)P3 • Triton X-100 (50 mg per 100 ml)

CLM [3H)-inulin (as an inert volume marker) Treff 1.5 ml microfuge tubes (Scotlab) Scintillation vials Ecoscint A scintillation fluid (National Diagnostics)

Method 1. Connect three cylinders to the superfusion chamber, one containing CLM (Protocol 2), the second containing CLM supplemented with lns(1,4,5)P3 (10 uM) and trace amounts of [3H]-inulin, and the third containing CLM with Triton X-100 (50 mg per 100 ml). Block all unused solenoid valve ports with an occluded screw (C16), and pressurize the cylinders with nitrogen. 2. Load the intracellular Ca2+ stores of permeabilized rat hepatocytes (107 cells ml-1 to steady state with 45Ca2+ by incubation for 5 min at 37°C (Protocol 2). During this time, prime all the tubing leading to the superfusion chamber by sequentially energizing the connected valves for 500 ms. Wash out residual traces of medium from the superfusion chamber by perfusing for 5 s with CLM. 3. Immobilize the cells on the filter array by first transferring them to PVC tubing connected to a 1 ml syringe, and then gently forcing the suspension into the filter triplet, prerinsed with CLM, and previously assembled in one end of a Teflon connector (C33). Rapidly transfer the filter array into the superfusion chamber and secure it by adding the stainless steel mesh and washer (C9) and then tighten with a Teflon screw (C10). 4. Adjust the flow rate of the superfusate through the sample to 2 ml s-1 during two prewashes (5 s), by tightening or loosening the Teflon screw (C10). 5. Secure the solenoid manifold in position above the fraction collector, and initiate a superfusion protocol. A typical sequence would com378

15: Measuring Ca2+ release prise 2 s exposure to CLM, followed by a 5 s exposure to lns(1,4,5)P3, and finally a 4 s exposure to Triton X-100 (50 mg per 100 ml) to release all 45Ca2+ remaining within the intracellular stores. The exact timings are modified to meet the specific aims of the experiment. 6. Determine the radioactivity released into each sample (3H and 45Ca2+) by dual-label liquid scintillation counting in EcoScint-A scintillation cocktail. 7. Most experiments are likely to be analysed by expressing the amount of 45Ca2+ released by lns(1,4,5)P3 as a fraction of the entire 45Ca2+ content of the immobilized cells (i.e. that released during the entire experiment, together with that released by Triton X-100 at the end (Figures 6-8). Alternative forms of analysis may be more appropriate for some experiments (59) (e.g. 'fractional release rate', i.e. rates expressed as fractions of the Ca2+ content of the stores at the beginning of each interval during which the 45Ca2+ release is recorded). Whichever form of analysis is adopted, simple spreadsheets (e.g. Microsoft Excel) provide the most convenient means of processing the large amounts of data generated.

Results from a typical rapid superfusion experiment are shown in Figure 6a. A maximal concentration of Ins(l,4,5)P3 (10 uM) caused a transient release of 45Ca2+ from the intracellular stores: the rate of 45Ca2+ release rapidly accelerated towards a peak, which was abruptly followed by a protracted decline in the rate of 45Ca2+ mobilization (59). A saturating concentration of Ins(l,4,5)P3 released -30% of the intracellular Ca2+ stores (Figure 6b), after which no further 45Ca2+ release could be evoked by a further addition of Ins(l,4,5)P3. The size of the Ins(l,4,5)P3-sensitive Ca2+ stores detected by the superfusion protocol is similar to that observed with conventional methods (35), and substantially larger than that found in brain synaptosomes (—6%) (39) or hepatic microsomes (~3%) (40). The fraction of the intracellular Ca2+ stores released by prolonged superfusion with Ins(l,4,5)P3 increased with Ins(l,4,5)P3 concentration, indicating that the unusual 'quantal' pattern of Ins(l,4,5)P3-evoked Ca2+ release was retained (60). The concentrationdependence of the ultimate extent of Ins(l,4,5)P3-stimulated 45Ca2+ release (half-maximally effective concentration, EC50 477 ± 21 nM), and of the maximal rate of 45Ca2+ mobilization differed by only twofold (EC50 941 ± 21 nM) (31, 41). This observation, that rates of Ca2+ release are more sensitive than their eventual extents to Ins(l,4,5)P3 (59), further underscores the necessity for rapid measurements, if the characteristics of the initial states of the Ins(l,4,5)P3 receptor are to be examined. The rank order of potency of several different agonists (Ins(l,4,5)P3 > 3-deoxy-Ins(l,4,5)P3 > 2,3-dideoxyIns(l,4,5)P3 > Ins(2,4,5)P3) was the same as previously reported with conventional methods (31, 61) (Figure 6c). In summary, therefore, the key 379


Figure 6. Rapid superfusion with lns (1,4,5)P3 evokes responses with similar characteristics to those recorded using conventional methods. (A) The time course of 45Ca2 mobilization in response to two sequential superfusions with 10 uM lns(1,4,5)P3 in CLM (solid bar) is shown. The shaded area indicates the kinetics of delivery of lns(1,4,5)P3 to the superfusion chamber, detected using [3H]inulin. Each point represents the average rate of 45Ca21 efflux during each 80 ms collection interval. (B) The cumulative amount of 45 Ca 2 1 released with time, obtained by integration of the results shown in (a), and expressed relative to the size of the intracellular Caz store, (C) Concentration-dependence of the eventual extent of 45Ca21 release in response to lns(1,4,5)P3 (black circles), 3-deoxylnsl1,4,5)P3 (open squares), 2,3-dideoxy-lns(1,4r5)P3 (black squares), and lns(2,4,5)P3 (open circles). Results, which are means ± SEM of at (east three independent experiments, are expressed as percentages of the maximal response.

380

15: Measuring Ca2+ release characteristics of Ins(l,4,5)P3-evoked Ca2+ release are preserved under the conditions required for their analysis by rapid superfusion methods. An additional advantage of these rapid superfusion methods is shown by the results in Figure 7, in which the kinetics of the initial activation of the Ins(l,4,5)P3 receptor are resolved (31). Our previous detailed analysis of the delay between exposure of Ins(l,4,5)P3 receptors to Ins(l,4,5)P3 and channel opening led us to propose a model in which binding of Ins(l,4,5)P3 causes a Ca2+-binding site to become exposed, and only when all four subunits of the tetrameric receptor have bound both Ins(l,4,5)P3 and Ca2+ does the Ca2+ channel open (31). A unique advantage of the superfusion system is evident from Figure 8, in which the kinetics of the termination of 45Ca2+ release after brief pulses of Ins(l,4,5)P3 are examined (59). By rapidly removing Ins(l,4,5)P3 at different phases of the response, the superfusion apparatus allows the rate of termination of 45Ca2+ release, reflecting the rate of dissociation of Ins(l,4,5)P3 from its receptor, to be measured. The kinetics of the decay in the rate of 45Ca2+ release are faster when Ins(l,4,5)P3 is removed after 380 ms (the half-time for recovery is 195 ± 20 ms, n = 10) than when it was removed after 640 ms (when the half-time for recovery is 360 ± 35 ms, n = 4). The results are consistent with Ins(l,4,5)P3 first binding to a lowaffinity active receptor conformation, after which the receptor rapidly (with a

Figure 7. Delayed Ca2+ release after maximal stimulation of lns(1,4,5)P3 receptors with lns(1,4,5)P3. Permeabilized hepatocytes loaded with 45Ca2+ were stimulated with a supramaximal concentration of lns(1,4,5)P3 (300 uM), and its arrival in the effluent was monitored by inclusion of [3H]-inulin (black circle). 45Ca2+ release during each 9 ms interval of the superfusion was recorded (open circle). Even though the concentration of lns(1,4,5)P3 to which the cells are exposed exceeds that required for maximal stimulation within 9 ms, there is no detectable release of 45Ca2+ for 31 ± 2 ms. These results, together with those from additional rapid superfusion experiments, provide the evidence from which we suggest that both lns(1,4,5)P3 and Ca2+ must sequentially bind to the lns(1,4,5)P3 receptor before its channel can open. Reproduced with permission from ref. 31.

381


Figure 8. Kinetics of channel closure after removal of lns(1,4,5)P3. Cells were exposed to lns(1,4,5)P3 for the periods indicated, and the rate of decay in the rate of 45Ca2+ release was measured in response to a 380 ms pulse (black triangle), a 640 ms pulse (open triangle), or a 4 s challenge with 10 uM lns(1,4,5)P3 (open circle). For clarity, only the latter parts of the traces that employed short pulses of lns(1,4,5)P3 are shown. Results are typical of at least four independent experiments. The inset shows normalized monophasic curve fits to the kinetics of the decay in the rate of 45Ca2+ release following the removal of lns(1,4,5)P3, after 380 ms (solid curve), and 640 ms (dashed curve). Reproduced with permission from ref. 59.

half-time of 250 ms) switches to a higher affinity, less active conformation (59). In summary, rapid superfusion methods are beginning to reveal some of the complex changes in Ins(l,4,5)P3 receptors that follow their initial activation (31, 59), and it is particularly satisfying that the results obtained with these methods, which allow rigorous control of experimental conditions, are in close agreement with those obtained using flash photolysis of caged Ins(l,4,5)P3 in intact cells (62).

Acknowledgements Supported by The Wellcome Trust and BBSRC.

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384

A1 List of suppliers Alamone Labs Ltd., Shatner Center 3, PO Box 4287, Jerusalem, 91042, Israel. American Radiolabeled Chemicals Inc., 11624 Bowling Green Drive, St Louis, MO 63146, USA. Amersham Pharmacia Biotech, Amersham Place, Little Chalfont, HP7 9NA, UK. Amicon Amicon, Inc., 72 Cherry Hill Drive, Beverly, MA 01915, USA. Amicon Ltd., Upper Mill, Stonehouse, Gloucestershire, GL10 2BJ, UK. Amplicon, Centenary Industrial Estate, Hollingdean Road, Brighton, BN2 4AW, UK. Anachem, Anachem House, 20 Charles Street, Luton, Bedfordshire, LU2 OEB, UK. Anatrace, Inc., 434 West Dussel Drive, Maumee, OH 43537, USA. Bellco Bellco Glass Inc, PO Box B, 340 Edrudo Road, Vineland, NJ 08360, USA. Philip Harris Scientific, Lynn Lane, Shenstone, Lichfleld, Staffordshire, WS14 OEE, UK. Bio-Rad Laboratories Ltd, Bio-Rad House, Maylands Avenue, Hemel Hempstead, Hertfordshire, HP2 7TD. Calbiochem-Novabiochem Calbiochem-Novabiochem Corporation, 10394 Pacific Center Court, San Diego, CA 92121, USA. Calbiochem-Novabiochem (UK) Ltd., Boulevard Industrial Park, Padge Road, Beeston, Nottingham, NG9 2JR, UK. Clontech Laboratories Clontech UK, Unit 2, Intec 2, Wade Rd., Basingstoke, RG24 8NE, UK. Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA 94303, USA. Contitech Power Transmissions Systems Ltd., PO Box 26, Wigan, WN2 4WZ, UK. Dynatech Laboratories Inc., 14340 Sullyfield Circle, Chantilly, VA 22021, USA. Gelman Sciences Ltd., Brackmills Business Park, Caswell Road, Northampton, NN4 0EZ, UK.

List of suppliers Genera] Valves, 19 Gloria Lane, PO Box 1333, Fairfield, NJ 07004, USA. Gibco BRL, 3175 Staley Road, Grand Island, NY 14072, USA. Hoeffer Scientific Instruments, Newcastle, Staffs, ST5 0TW, UK. ICN Biomedicals Ltd., Lincoln Road, Cressex Industrial Estate, High Wycombe, Bucks, HP12 3XJ. Invitrogen BV, De Schelp 12, 9351 NV Leek, The Netherlands. Jencons Scientific Ltd., Cherrycourt Way Industrial Estate, Stanbridge Road, Leighton Buzzard, LU7 8UA. JRH Biosciences JRH Biosciences Inc., 13804 W. 107th Street, Lenexa, KS 66215, USA. AMS Biotechnology, 12 Thorney Leys Park, Witney, Oxon, OX8 7GE, UK. Life Technologies Ltd, 3 Fountain Drive, Inchinnan Business Park, Paisley, PA2 9RF, UK. Macherey-Nagel, Postfach 101352 D-52313, Diiren, Germany. MicroDrives, Aercon House, Alfred Road, Gravesend, Kent, DA11 7QF, UK. Molecular Devices Molecular Devices Ltd., Unit 6, Raleigh Court, Rutherford Way, Crawley, West Sussex, RH10 2PD. Molecular Devices Corporation, 1311 Orleans Drive, Sunnyvale, CA 94089. Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, OR, USA. National Diagnostics, Unit 3, Chamberlain Road, Aylesbury, Bucks, HP19 3DY, UK. New Brunswick Scientific Co. Ltd., Edison, NJ, USA. Novagen Novagen Inc., 601 Science Drive, Madison, WI 53711, USA. Cambridge Bioscience, 24-25 Signet Court, Newmarket Road, Cambridge, CB5 8LA, UK. NUNC, distributed by Life Technologies. Omnifit, 2 College Park, Coldhams Lane, Cambridge, CB1 3HD, UK. Packard Bioscience Packard Instrument Company, 800 Research Parkway, Meriden, CT 06450, USA. Canberra Packard Ltd., Brook House, 14 Station Road, Pangbourne, Berkshire RG8 7AN, England. Parker Hannifin, 21 Balena Close, Poole, Dorset, BH17 7DX, UK. Parr Parr Instrument Company, 211 Fifty-third Street, Moline, Illinois 61265-1770, USA. Scientific & Medical Products Ltd., Shirley Institute, 856 Wilmslow Road, Didsbury, Manchester, M20 2SA, UK. Pharmacia, 800 Central Avenue, Piscataway, NJ 08854, USA. Pharmingen Pharmingen, 10975 Torreyana Road, San Diego, CA 92121, USA. 386

List of suppliers Becton Dickinson UK Ltd., Between Towns Road, Cowley, Oxford, OX4 3LY, UK. Pierce-Warriner Pierce Chemical Co., 3747 N. Meridian Rd., PO Box 117, Rockford, IL 61105, USA. Pierce & Warriner (UK) Ltd., 44 Upper Northgate Street, Chester CHI 4EF, UK. Portex Ltd., Hythe, Kent, CT21 6JL, UK. Qiagen Ltd, Boundary Court, Gatwick, Road, Crawley, RH10 2AX, UK. Research Biochemicals International, 1 Strathmore Road, Natick, Massachusetts 01760-2447, USA. RS, Venture Close, Lammas Road, Corby, Northants, NN17 1UB, UK. Santa Cruz Biotechnology Santa Cruz Biotechnology Inc., 2161 Delaware Avenue, Santa Cruz, California 95060. Calbiochem-Novabiochem (UK) Ltd, Boulevard Industrial Park, Padge Road, Beeston, Nottingham, NG9 2JR. Insight Biotechnology Ltd., PO Box 520, Wembley, Middlesex, HA9 7YN, UK. SEMAT Technical (UK) Ltd, 1 Executive Park, Hatfield Road, St Albans, Herts, AL1 4TA, UK. Sigma-Aldrich. PO Box 14508, St. Louis, MO 63178, USA. Transduction Laboratories Transduction Laboratories, 133 Venture Court, Lexington, KY 40511-2624, USA. Affiniti Research Products Ltd., Mamhead Castle, Mamhead, Exeter, EX6 8HD, UK. Tropix, 47 Wiggins Ave., Bedford, Massachusetts 01730, USA. Upstate Biotechnology Upstate Biotechnology, 199 Saranac Avenue, Lake Placid, NY 12946, USA. TCS Biologicals Ltd., Botulph Claydon, Buckingham, MK18 2LR, UK. Wallac Wallac Inc., 9238 Gaither Road, Gaithersburg, Maryland 20877, USA. EG&G Ltd., Milton Keynes, 20 Vincent Avenue, Crownhill Business Centre, Crownhill, Milton Keynes, MK8 0AB, UK. Whatman International Ltd., St Leonard's Road, 20/20 Maidstone, Kent, ME16 0LS. UK. Worthington Biochemical Corporation, Halls Mill Road, Freehold, NJ 07728, USA. Zymed Zymed Laboratories Inc., 458 Carlton Court, South San Francisco, CA 940802012, USA. Cambridge Bioscience, 24-25 Signet Court, Newmarket Road, Cambridge CBS SLA, UK. 387


Index AcMNPV see baculovirus A1 adenosine receptor, interactions with G proteins 128-32 adenovirus culture 228-30 replication-defective 228-9 transfection into mammalian cells 228—32 adenylyl cyclase assay 233–1,240,244-8 see also cyclic AMP, assay and hormonal stimulation 73 purification 224–5 recombinant 226 regulation 223–4,240-1 structure 223, 225 transfection into mammalian cells 226–33 ADP-ribosylation by bacterial toxins 77-81 G proteins 73, 77-81,325 «2 adrenergic receptor assay 117-19 specific antagonist binding 117-19 a2A adrenergic receptor, agonists 121-3 (padrenergic receptor, agonist stimulation and phosphorylation 59 (p2 adrenergic receptor (p2AR) agonist binding and conformational change 8–9,32-3 assay 6 expression in baculovirus system 156 expression in mammalian cells 39 fluorescence labelling 2, 6–16 palmitoylation 41-3 purification 2–6,44-5 site-selective labelling 11-16 adrenoceptor see adrenergic receptor aequorin, measurement of intracellular calcium 173, 177-8,182-4,214-15, 361-2 affinity labelling, identification of binding sites 20 affinity purification, G protein-coupled receptors (GPCRs) 40–3 agonists activation of phospholipase D 303–1 for a2A adrenergic receptor 121-3 incubation with cells 303—4 inverse, assays 200-1 measurement of efficacy 121-3 reporter-gene assays 198–201 agonist trafficking 130 alcohols, substrates for phospholipase D 302 alkaline phosphatase see secreted placental alkaline phosphatase (SEAP)

antagonists, reporter-gene assays 199 antibodies G-protein a subunits 87-90 mitogen-activated protein (MAP) kinases 346 phospho-specific 348-9 phosphotyrosine 343 production 87-90, 348-9 specificity 87-8,90,343 antigenicity bacterial fusion proteins 60–1 G protein-coupled receptors (GPCRs) 61 apoptosis, involvement of ceramide 321 ATP, metabolic labelling for cyclic AMP assay 234–5 Autographa californica multiple nuclear polyhedrosis virus see baculovirus autoradiography, SDS-PAGE gels 86 baculovirus amplification 155–6 cell lines for culture 140–4,166 construction of recombinant 145, 147-55 expression of G protein-coupled receptors (GPCRs) 37-8 infection of Sf9 cells 4, 157-60 life cycle 144–5 promoters for recombinant protein expression 145–6,167 protease-null strain 158 recombinant protein expression levels 156–8 baculovirus expression vector system (BEVS) 139–69 see also baculovirus binding sites, identification affinity labelling 20 site-directed mutagenesis 20, 24 substituted-cysteine accessibility method (SCAM) 20–4, 30–1 biotinylation, cell-surface receptors 52 blue fluorescent protein (BFP) 216 Ca2+ channels, inositol 1,4,5-triphosphate (Ins(l,4,5)P3) receptors 361-2 calcium ions fluorescent indicators 214-15, 361-2, 365, 369 intracellular measurement 173, 177-8, 182-4,205,214-17,361-2 loading intracellular stores 365-8 mobilization by sphingosine kinase 324

Index calcium ions (continued) regulation of cellular behaviour 361-2 release from intracellular stores 368-70, 378-82

response to inositol 1,4,5-triphosphate (Ins(l,4,5)P3) 361-2, 365, 367-8, 378-82 second messenger 105, 361-2 cAMP see cyclic AMP cell culture oxygen air-lift fermenters 167 serum-free media 143–1, 168 Sf9 cells 4, 38, 140-4,155-6,158-60,167-8 see also culture media cell membranes see plasma membranes cells, viability testing 366 cell-surface receptors biotinylation 52 palmitoylation 51–t and phosphoinositide 3-kinases (PI3Ks) 283 ceramide and apoptosis 321 signal transduction 321-2 chloramphenicol acetyltransferase (CAT) assay 207-10 reporter gene 195 cholera toxin, action on G proteins 73-4, 77-81, 87, 105-6 chromatography inositol glycerophospholipids 258-9 separation of mitogen-activated protein (MAP) kinases 351-2 see also HPLC; thin-layer chromatography (TLC) c-jun kinases (JNKs) antibodies 346 assay 356 properties 337-8 see also mitogen-activated protein (MAP) kinase conformational change, and receptor activation 32-3 constitutively active mutants, and receptor activation 32-3 constitutive receptor signalling, reporter-gene assays 200–1 cotransfection, construction of recombinant baculovirus 145, 147-52 CRE (cAMP response element), component of promoters 172, 174-80, 240-1 CRE-binding element (CREB), transcription factor 174-5, 178-9,240 culture media mammalian cells 39 for reporter-gene assays 192-5 Saccharomyces cerevisiae 259-61 serum-free 143–4, 168

Sf9 cells 38, 141,143-4,168 cyclic adenosine monophosphate see cyclic AMP cyclic AMP (cAMP) assay 233–4 fluorescent analogues 249 isolation of tritiated 236–9 second messenger 105 stimulation of protein kinase A 248-9 cysteine fluorescent labelling 6-8,12-15 prenylation 35 reaction with methane thiosulfonate (MTS) derivatives 25-7 substitution in receptors 21-2 cytosol, preparation 311-12 deacylation, inositol glycerolipids 271–1 dopamine D2 receptor, mapping by substituted-cysteine accessibility method (SCAM) 20, 30-2 ecdysone-inducible expression system 195 effectors, regulation by receptor-G protein fusion proteins 132–4 electroblotting see immunoblotting electroporation permeabilization of membranes 364 transfection method 233 ELISA chloramphenicol acetyltransferase (CAT) assay 207-10 mitogen-activated protein (MAP) kinase assay 356 testing antiserum specificity 87-8, 90 epitope tagging, G protein-coupled receptors (GPCRs) 60-1 extracellular signal-regulated kinases (ERKs) activation 339-40,342 antibodies 346, 348-9 assay of upstream activators 358 chemical inhibition of signalling pathways 358-9 immunocytochemical localization 357-8 immunoprecipitation 354-5 phosphorylation 339 properties 337-8 purification with phenyl-Sepharose 350–1 substrates 339–40 see also mitogen-activated protein (MAP) kinases 'FLAG' epitope purification of P2 adrenergic receptor (p32AR) 3 390

Index purification of G protein-coupled receptors (GPCRs) 43-5, 61 fluorescence resonance energy transfer (FRET) measurement of calcium ions 216-17 study of protease activity 216-17 using green fluorescent protein (GFP) 173, 216-17 Fluorometric Imaging Plate Reader (FLIPR) calcium-ion measurement 214 fluorimetric assay 205 fusion proteins construction 108-10 expression 110-11,119-21 functional assay 113-17 interactions with A1 adenosine receptor 128-32 measurement of agonist efficacy 121-3 measurement of GTPase activity 117, 119-21 purification 163-4 receptor-G protein 103-37 regulation of effectors 132-4 study of role of N-terminal acylation 124-8 G0 cells, extracellular signal-regulated kinase (ERKs) activity 340 p-galactosidase adenylyl cyclase assay 246 assay 210-12 reporter gene 173,195 sensitivity 210-11 gel electrophoresis see SDS-PAGE p-glucuronidase, adenylyl cyclase assay 246 glutathione S-transferase (GST) fusion proteins, purification 163-4 glycine, myristoylation 35 GPCR phosphorylation determination by immunoprecipitation 60-4 in membranes 64-6 GPCR signal transduction, use of reporter genes 178-89 G-protein a subunits antisera 87-90 classification 103—4 downregulation 92-3 fusion proteins 103-37 immunoprecipitation 92-5 N-terminal acylation 124-8 quantification 95-8 see also G proteins G protein-coupled receptor kinases (GRKs) 59 G protein-coupled receptors (GPCRs) activation 1-2,32,321-3 activation of MAP kinases 184-9

activation of sphingosine kinase 324 affinity purification 40-3 antigenicity 61 desensitization 59 EDG family 322-3 effects of agonist binding 1,32,59 epitope tagging 60-1 fusion proteins 103-37 immunoprecipitation 40, 43-5,60-4 palmitoylation 35-56 and phosphoinositide 3-kinases (PI3Ks) 283, 290 phosphorylation 59-72 purification 2-6, 40-5, 60-4 regulation of adenylyl cyclase 241 solubilization 62-3 structure 1 study of signal transduction 171-221 see also GPCR phosphorylation; GPCR signal transduction G proteins activation 100-1, 103, 105 activation of phosphoinositide 3-kinases (PI3Ks) 284 ADP-ribosylation 73-4, 77-81, 87,105-6, 325 expression in baculovirus-infected cells 166-7 extraction from cells 75-7 fusion proteins 103-37 GTPase activity 98-100, 103, 113-14, 117, 119-21 identification 73-101 interactions with A1 adenosine receptor 128-32 mode of action 103 myristoylation 124-5 receptor-stimulated binding of GTP 100-1, 103 regulation of adenylyl cyclase 223–1,241 SDS-PAGE 81-6 structure 73-4, 103 substrate for toxins 73–1, 87, 99, 105-6, 124-6 toxin-insensitive 74, 78, 105-6, 124-6 see also G-protein a subunits green fluorescent protein (GFP) applications 215-17 assay 215-17 localization of intracellular calcium 362 long half-life 216 see also blue fluorescent protein (BFP) GTP, receptor-stimulated binding to G proteins 100–1 GTPase activity of G proteins 98-100, 103, 113-14, 117,119-21 measurement of activity 115-17 391

Index guanine nucleotide-binding proteins see G proteins

isoproterenol, binding to (32 adrenergic receptor (P2AR) 8-9, 32

hepatocytes, isolation from rat 362-4 hexahistidine see polyhistidine high-pressure liquid chromatography see HPLC hormone response elements see transcriptionfactor binding sites HPLC analysis of phosphoinositides 291, 295-8 anion-exchange columns 275, 277 calibration 277-9 inositol phospholipids 271-80 sample injection 277-8

p-lactamase, assay 212-13 lipids, extraction from Saccharomyces cerevisiae 263-5 Lipofectamine, transfection of cells 111-12, 191-4 lipofection, methods 111-12, 191-4, 233 luciferase applications 233-48 assay 201-4 dual assays 203-5, 243 firefly 201, 239-43 LuFLIPRase dual assay 205 Renilla 203, 246 reporter gene 175-7, 182, 186-8 ,195, 197, 239-3 lysine, fluorescent labelling 15-16 lysosphingolipids, activation of G proteincoupled receptors (GPCRs) 322

immunoblotting G proteins 91-2, 96-8 mitogen-activated protein (MAP) kinases 343-4, 351-2 immunoprecipitation determination of GPCR phosphorylation 60-4 extracellular signal-regulated kinases (ERKs) 354-5 G-protein a subunits 92-5 G protein-coupled receptors (GPCRs) 40, 43-5, 60-4 mitogen-activated protein (MAP) kinases 353-6 phosphoinositide 3-kinases (PI3Ks) 287-90 inositol 1,4,5-triphosphate (Ins(l,4,5)P3) and desensitization of PLC-coupled receptors 66-71 effects on intracellular calcium 361-2, 365, 367-8, 378-82 extraction from cells 68 preparation of binding protein 69-70 radio-receptor assay 70-1 receptor activation 381-2 receptors 361-2 second messenger 105 inositol glycerolipids, deacylation 271-4 inositol glycerophospholipids analytical methods 257-9 biosynthesis 256-7 nomenclature 256 radioactive labelling 259-63 structures 256, 258-9 inositol lipids extraction from yeasts 263-5 standards for HPLC 278-9 inositol phospholipids, identification 266-71 ion-exchange chromatography isolation of tritiated cyclic AMP 236-9 purification of recombinant proteins 162-3

mammalian cells, expression of G proteincoupled receptors (GPCRs) 38-9 mammalian tissue, phospholipase D assay 317 MAP kinase kinase (MEK), activation of MAP kinase 339 MEK see MAP kinase kinase membranes see plasma membranes methane thiosulfonate (MTS) derivatives, use in substituted-cysteine accessibility method (SCAM) 24-33 methylamine, deacylation of inositol glycerolipids 272-4, 292 microtubule-associated protein 2 (MAP-2) kinase see mitogen-activated protein (MAP) kinase mitogen-activated protein (MAP) kinases activation 184-9, 339-43 antibodies 346 assay 343-56 assay of upstream activators 358 chemical inhibition of signalling pathways 358-9 chromatographic separation 351-2 extraction from cells 340-2 extraction from tissues 342-3 gel electrophoresis 356 immunoblotting 351-2 immunocytochemical localization 356-8 immunoprecipitation 353-6 phosphorylation 339 phosphorylation of transcription factors 185-6 phospho-specific antibodies 348-9

392

Index properties 337-8 substrates 339-40 mitogens, activation of extracellular signalregulated kinases (ERKs) 340 mobility-shift assay, activation of mitogenactivated protein (MAP) kinases 344-7 myelin basic protein (MBP), substrate for mitogen-activated protein (MAP) kinases 339-10, 349, 355 myristoylation of glycine residues 35 G proteins 124-5 nickel-agarose, binding of polyhistidine 43-5, 164-6 nitrobenzdioxazol (IANBD) cysteine specificity 6-8,12-15 fluorescence and solvent polarity 6-8 labelling p2 adrenergic receptor (p2AR) 6-15 nitrogen cavitation, lysis of cells 161-2 S-opioid receptor, palmitoylation 51-4 p38 MAP kinases activation 339 antibodies 346 assay 356 chemical inhibition of signalling pathways 358-9 phosphorylation 339 properties 337-8 substrates 339-40 see also mitogen-activated protein (MAP) kinase palmitoylation P2 adrenergic receptor (p2AR) 41-3 autocatalytic 54-6 cell-surface receptors 51-4 chromatographic analysis 47 detection 36-47 determination of half-life 49-50 G protein-coupled receptors (GPCRs) 35-56 kinetics 47-51 mechanism 54 nature of chemical linkage 45-7 8-opioid receptor 51-4 regulation by agonists 50-1 rhodopsin 35-6, 54 synthetic peptides 54-6 palmitoyl-CoA, synthesis 54-5 pertussis toxin (PTX) ADP-ribosylation of G proteins 73—4, 77-81, 87, 105-6, 325

disruption of signalling pathways 325 inhibition of sphingosine-1-phosphate (SPP) 325 use in reporter-gene assays 196-7 phosphoinositide 3-kinases (PI3Ks) activation 283-4, 287-98 activation of protein kinase B 284 analysis of lipid products 290-8 catalytic site inhibitors 285 and cell-surface receptors 283 constitutively active alleles 286-7 dominant negative alleles 285-6 functions 283-1 and G protein-coupled receptors (GPCRs) 283, 290 identifying a role in signalling pathways 285-7 immunoprecipitation 287-90 inhibition 285 receptor mutants 286 phosphoinositides [3H]inositol labelling 293-4 32 P-labelling 291-3 analysis 291, 294-8 deacylation 292 mass analysis 297-8 phospholipase C-coupled receptors, desensitization 66-71 phospholipase D activation 301, 303-4 assay 302-17 fluorescent substrate 311-13 mammalian 301, 309-12, 315, 317 molecular structure 301 plants 301, 315, 317 and signal transduction 301 substrates 301-2 transphosphatidylation 301-2 yeast 301, 315, 317 phospholipids extraction from cells 305 labelling with tritiated fatty acids 302-3 thin-layer chromatography 306-7 phosphorimaging, of SDS-PAGE gels 86 phosphorylation and agonist stimulation of p-adrenergic receptor 59 desensitization of receptors 59 G protein-coupled receptors (GPCRs) 59-72 mitogen-activated protein (MAP) kinases 339 phosphotyrosine, antibodies 343 plants, phospholipase D 301, 315, 317 plasma membranes GPCR phosphorylation 64-6 permeabilization 364 preparation 65, 112-13, 311-12

393

Index plasma membranes (continued) source of G proteins 75-7 stimulation of receptor phosphorylation 65-6 plasmids, transfer 145-6 polyhistidine binding to nickel-coupled resins 43-5 purification of G protein-coupled receptors (GPCRs) 43-5 purification of recombinant proteins 164-7 proline-directed protein kinases see mitogenactivated protein (MAP) kinases promoters CRE 172, 174-80 natural 174 reporter genes 173-7 synthetic 174-7 protease activity, study using fluorescence resonance energy transfer 216-17 protein kinase A assay 243-4 stimulation by cyclic AMP 248-9 protein kinase B, activation by phosphoinositide 3-kinases 284 protein kinase C, activation by agonist binding to GPCR 182 protein tyrosine kinases, activation of phosphoinositide 3-kinases 283-4 quantification densitometric 97-8 G-protein a subunits 95-8 immunoblotting 96-8, 346 mitogen-activated protein (MAP) kinases 346-7, 356 phospholipase D activity 307-9, 314-16 radioactive labelling 98 receptor activation conformational change 32-3 constitutively active mutants 32-3 receptor-binding sites, probing with substituted-cysteine accessibility method (SCAM) 19-33 receptors, regulation of palmitoylation 50-1 recombinant proteins purification 162-6 release from cells 160-2 reporter-enzyme assays chloramphenicol acetyltransferase (CAT) 207-10, 244-6 dual luciferase 203-4, 243 firefly luciferase 201-2, 240-3 p-galactosidase 210-12 p-lactamase 212-13 LuFLIPRase 205

Renilla luciferase 203-4 secreted placental alkaline phosphatase (SEAP) 205-7 reporter-gene assays agonists 198-201 antagonists 199 cell preparation 197-201 choice of cell line 189-90 choice of reporter enzyme 195 constitutive receptor signalling 200-1 culture media 192-5 drug exposure 196 dual 243 dynamic range 202, 243 expression protocols 190-5 optimization 195-7 sensitivity 202, 211, 243 signal-to-noise ratio 196 use of inhibitors 196-7 reporter genes activation of MAP kinase 185-9 aequorin 173, 182-1 alkaline phosphatase (SEAP) 173, 180-1, 195, 197, 205-7, 246 assay of cyclic AMP 239-13 chloramphenicol acetyltransferase (CAT) 195, 244-6 choice of 195 construction 173-7 definition 171-3 (p-galactosidase 173, 195 (p-glucuronidase 246 green fluorescent protein (GFP) 173 p-lactamase 195, 197 luciferase 175-7, 182, 186-8, 195, 197, 239-43 promoters 173-7 study of signal transduction 171-221 transcription-factor binding sites 174-6 use for screening 173 reporter proteins 177-8 aequorin 173, 177-8, 182-1, 214-15, 361-2 green fluorescent protein (GFP) 173, 178, 215-17, 362 rhodopsin palmitoylation 35-6, 54 photoactivation 2 Saccharomyces cerevisiae advantages in phosphoinositide research 255-6 culture media 259-61 extraction of lipids 263-5 saponin, permeabilization of membranes 364-7 Schizosaccharomyces pombe, extraction of inositol lipids 264-5

394

Index scintillation proximity assay (SPA) 114-15 SDS-PAGE autoradiography of gels 86 gel composition 83 of G proteins 80-6 phosphorimaging of gels 86 phosphorylation of MAP kinases 344-7 see also gel electrophoresis SEAP see secreted placental alkaline phosphatase (SEAP) secondary structure, and substituted-cysteine accessibility method (SCAM) 31-2 second messengers 105, 361-2 secreted placental alkaline phosphatase (SEAP) colorimetric assay 205-7 heat stability 206 reporter gene 173, 180-1, 195, 197 Sf9 cells cell culture 4,38, 140-4, 155-6, 158-60, 167-8 culture media 38, 141, 143-4, 168 expression of G protein-coupled receptors (GPCRs) 37-8 expression of multimeric proteins 166-7 freezing 143 infection with baculovirus 4, 157-60 lysis 160-2 thawing 141 shuttle vector, construction of recombinant baculovirus 152-5 signalling pathways disruption by pertussis toxin 325 sphingosine kinase 325-7, 335 signal transduction ceramide 321-2 G protein-coupled receptors (GPCRs) 171-221 phospholipase D 301 sphingosine kinase 321-36 sphingosine-1-phosphate (SPP) 321, 323, 335 study using reporter genes 171-221 site-directed mutagenesis, identification of binding sites 20, 24 sphingosine kinase assay 328-35 calcium ion mobilization 324 inhibition 326-7 production of sphingosine-1-phosphate (SPP) 322-4 regulation 327-8 signal transduction 321-36 stimulation by G protein-coupled receptors (GPCRs) 324 sphingosine-1-phosphate (SPP) activation of G protein-coupled receptors (GPCRs) 321-3

caged 325 GPCR-independent action inhibition by pertussis toxin 325 inhibition of degradation 330 intracellular activity 323—4 metabolism 321-2 microinjection into cells 325 receptors 321-3, 325 signal transduction 321, 323, 335 stimulation by G protein-coupled receptors (GPCRs) 324 thin-layer chromatography (TLC) 329-30 sphingosine-1-phosphate lyase, inhibition 330 stress-activated protein kinases (SAPKs) see mitogen-activated protein (MAP) kinases substituted-cysteine accessibility method (SCAM), probing receptor-binding sites 19-33 superfusion, rapid advantages 369-70 apparatus 370-8 calcium release from intracellular stores 368-70, 378-82 cellular response to inositol 1,4,5triphosphate (Ins(l,4,5)P3) 368-70, 378-S2 kinetics 373-1, 377-82 signal-to-noise ratio 369 thin-layer chromatography (TLC) chloramphenicol acetyltransferase (CAT) assay 207-10 inositol phospholipids 266-71 lipids bound to receptors 47 phosphoinositides 291, 294-5 phospholipase D assay 312-14 phospholipids 306-7 sphingosine-1-phosphate (SPP) 329-30 transcription-factor binding sites, reporter genes 174-6 transcription factors CREB (CRE-binding protein) 174-5, 178-9 phosphorylation by MAP kinases 185-6 transfection adenovirus-mediated 228-32 calcium phosphate-mediated 233 choice of vector 111 DEAE-dextran/chloroquine mediated 226-8 efficiency 228 electroporation 233 using Lipofectamine 111-12, 191-4 liposome-mediated 233 methods 22-3, 111-12, 191-3, 226-33

395

Index transphosphatidylation, by phospholipase 301-2 Trypan blue, testing viability of cells 366 viability of cells, testing 366 viral gene activity, time course 144—5

D

wortmannin, use in reporter-gene assays 196-7 yeast phospholipase D 301, 315, 317 see also Saccharomyces cerevisiae; Schizosaccharomyces pombe

Western blotting, mitogen-activated protein (MAP) kinases 343-4, 356

396

Signal Transduction: A Practical Approach 2nd ed

Protein Phosphorylation: A Practical Approach (Practical Approach Series) 2nd ed

Signal Transduction