B3 Adrenoreceptor (Taylor & Francis Series in Pharmaceutical Sciences)

The ␤3-Adrenoreceptor

New and Forthcoming Titles in the Pharmaceutical Sciences Nuclear Medicine in Pharmaceutical Research Perkins and Frier (Eds) 1999 0 7484 0688 3 Hbk Electrically Assisted Transdermal and Topical Drug Delivery Ajay K.Banga 1998 0 7484 0687 5 Hbk Physiological Pharmaceutics Barriers to Drug Absorption 2nd Edition Washington & Wilson 1999 0 7484 0562 3 Hbk 0 7484 0610 7 Pbk Pharmaceutical Aspects of Ogligonucleotides Couveur & Malvy (Eds) 2000 0 7484 0841 X Hbk Pharmaceutical Formulation Development of Peptides & Proteins Frokjaer & Hovgaard (Eds) 2000 0 7484 0745 6 Hbk Handbook of Microbiological Quality Control Pharmaceuticals and Medical Devices Baird, Hodges & Denyer 2000 0 7484 0614 X Hbk Microbial Quality Assurance in Cosmetics, Toiletries and Non-Sterile Pharmaceuticals 2nd Edition Baird with Bloomfield (Eds) 1996 0 7484 0437 6 Hbk Immunoassay A Practical Guide Law (Ed) 1996 0 7484 0560 7 Hbk Cytochromes P450 Structure, Function and Mechanism Lewis 1996 0 7484 0443 0 Hbk Autonomic Pharmacology Broadley 1996 0 7484 0556 9 Hbk Pharmaceutical Experimental Design and Interpretation 2nd Edition Armstrong & James 1996 0 7484 0436 8 Hbk Pharmaceutical Production Facilities 2nd Edition Cole 1998 0 7484 0438 4 Hbk

full pharmaceutical science catalogue available or visit our new website on: http://www.tandf.co.uk 29 West 35th Street, New York, NY 10001, USA Tel: (212) 216 7800 Fax: (212) 564 7854

11 New Fetter Lane London, EC4P 4EE Tel: +44 (0) 20–7583 9855 Fax: +44 (0) 20–7842 2293

The ␤3-Adrenoreceptor

Edited by A.DONNY STROSBERG Cochin Institute for Molecular Genetics CNRS Unit of Molecular Immunopharmacology Paris, France

First published 2000 by Taylor & Francis 11 New Fetter Lane, London EC4P 4EE Simultaneously published in the USA and Canada by Taylor & Francis Inc, 29 West 35th Street, New York, NY 10001 Taylor & Francis is an imprint of the Taylor & Francis Group This edition published in the Taylor & Francis e-Library, 2003. © Taylor & Francis All rights reserved. No part of this book may be reprinted or reproduced or utilised in any form or by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying and recording, or in any information storage or retrieval system, without permission in writing from the publishers. Every effort has been made to ensure that the advice and information in this book is true and accurate at the time of going to press. However, neither the publisher nor the authors can accept any legal responsibility or liability for any errors or omissions that may be made. In the case of drug administration, any medical procedure or the use of technical equipment mentioned within this book, you are strongly advised to consult the manufacturer’s guidelines. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging in Publication Data The ß3 adrenoreceptor/edited by A.Donny Strosberg. p. cm.—(Taylor & Francis series in pharmaceutical sciences) Includes bibliographical references and index. 1. Beta adrenoreceptors. 2. Adrenergic beta agonists. I. Strosberg, A.D. II. Series. QP364.7.B13 2000 612'.01575–dc21 00–023999 ISBN 0-203-48444-4 Master e-book ISBN

ISBN 0-203-79268-8 (Adobe eReader Format) ISBN 0-748-40804-5 (Print Edition)

Contents

List of figures List of tables Contributors Preface Abbreviations used in this book 1

x xii xiii xv xvii

Structure and Function of the ß3-Adrenoreceptor

1

A.Donny Strosberg and Cindy C.Gerhardt 1.1 1.2

1.3

1.4

1.5

1.6

1.7

Introduction The ß3AR gene 1.2.1 Cloning of the gene 1.2.2 Structure of the coding region 1.2.3 Structure of the promoter The ß3AR protein 1.3.1 Comparison with ß1- and ß2AR 1.3.2 Comparison of ß3AR from various species 1.3.3 Polymorphism Structure-function relationships in the ß3AR 1.4.1 The ligand binding site 1.4.2 The regions of interaction with the G proteins Signal transduction and biological functions 1.5.1 ß3AR-mediated activation of adenylyl cyclase 1.5.2 ß3AR-mediated activation of other signalling pathways 1.5.3 A role for the ß3AR in adipocyte differentiation Distribution of the ß3AR 1.6.1 Functional detection of the ß3AR 1.6.2 Immunodetection of the ß3AR protein 1.6.3 Detection of the ß3AR mRNA Concluding remarks Acknowledgements v

1 2 2 3 3 4 4 7 9 10 10 11 12 12 14 15 15 16 17 18 19 19

Contents

vi

2

Regulation of the ß3-Adrenoreceptor Signalling Efficacy

20

Michel Bouvier 2.1 2.2

2.3

2.4 2.5 2.6 2.7 2.8 3

Introduction 2.1.1 The ß-adrenoreceptors and their signalling pathways General concepts of G protein-coupled signalling regulation 2.2.1 Agonist-promoted receptor phosphorylation and receptor uncoupling 2.2.2 Receptor sequestration; a process involved in desensitization and resensitization 2.2.3 Agonist-promoted down-regulation The ß3-adrenoreceptor, a prototypic system to study subtype-specific regulation 2.3.1 Comparative analysis of the structural determinants of receptor regulation The ß3-adrenoreceptor is resistant to rapid agonist-promoted uncoupling and sequestration Cell type-specific down-regulation of the ß3-adrenoreceptor Use of chimeric ß3/ß2-adrenoreceptors to delineate regulatory motifs Potential physiological consequences of the relative resistance of the ß3-adrenoreceptor to desensitization Conclusions

Using Transgenic and Gene Knockout Techniques to Assess ß3Adrenoreceptor Function

20 21 22 22 24 26 27 27 29 30 33 34 35

36

Bradford B.Lowell, Vedrana S.Susulic, Danica Grujic and Moriko Ito 3.1 3.2

3.3

3.4

Introduction Mice lacking ß3AR 3.2.1 Phenotype of mice lacking ß3AR 3.2.2 Effects of ß3AR deficiency on catecholamine-mediated stimulation of adenylyl cyclase and lipolysis in adipocytes 3.2.3 Effects of ß3AR deficiency on in vivo effects of CL-316,243 Role of ß3AR on white versus brown adipocytes in mediating effects of ß3-selective agonists on energy expenditure, insulin secretion and food intake 3.3.1 Role of white versus brown adipocyte ß3AR in mediating effects of CL-316,243 on energy expenditure 3.3.2 Role of white versus brown adipocyte ß3AR in mediating effects of CL-316,243 on insulin secretion 3.3.3 Role of white versus brown adipocyte ß3AR in mediating effects of CL-316,243 on food intake Creation of mice which express human, but not murine, ß3AR

36 36 36 38 40

40 41 42 42 43

Contents

3.5

3.6

4

vii

Using ß3AR gene knockout mice to study ‘ß4AR’ activity 3.5.1 Evidence for the existence of ‘ß4AR’ 3.5.2 Assessment of ‘ß4AR’ activity in ß3AR gene knockout mice Conclusions Acknowledgements

ß3-Adrenoreceptor Ligands and the Pharmacology of the ß3-Adrenoreceptor

46 46 47 47 47

48

Jonathan R.S.Arch 4.1 4.2

4.3

4.4

4.5 5

Introduction Antagonists 4.2.1 ß1/2AR-selective antagonists 4.2.2 Non-selective and ß3AR-selective antagonists Agonists 4.3.1 ß1/2AR-selective agonists 4.3.2 Arylethanolamine ß3AR-selective agonists 4.3.3 Aryloxypropanolamine ß3AR agonists 4.3.4 Trimetoquinol and analogues 4.3.5 Metabolism and pharmacokinetics Idiosyncrasies of ß3AR pharmacology 4.4.1 Relative agonist potencies vary with the nature of the assay 4.4.2 Prediction of human tissue pharmacology from cloned receptor pharmacology Conclusions

The Native Human ß3-Adrenoreceptor

48 49 49 52 55 55 55 66 72 73 74 74 75 76 77

Peter Arner and Fredrik Lönnqvist 5.1 5.2 5.3

5.4 5.5 5.6 5.7

Introduction The human ß3AR gene Tissue expression of mRNA for ß3AR 5.3.1 White adipose tissue 5.3.2 Brown adipose tissue 5.3.3 Heart 5.3.4 Colon 5.3.5 Brain 5.3.6 Urinary bladder 5.3.7 Other tissues Is there a fourth ßAR? Structural variations in the native human ß3AR ß3AR as a therapeutic target Conclusions

77 77 78 78 80 81 81 81 81 82 82 83 84 85

Contents

viii

6

ß3-Adrenoreceptor-Mediated Responses in Heart and Vessels

87

Max Lafontan, Dominique Langin, Jean Galitzky, Michel Berlan, Chantal Gauthier and Geneviève Tavernier 6.1 6.2

6.3 6.4

6.5 6.6 7

Introduction ßAR subtypes in the heart 6.2.1 Functional ß3AR in the human heart 6.2.2 The putative ß4AR in cardiac tissue ß3AR-mediated vasodilatation ß3AR regulation of blood flow 6.4.1 Regulation of pancreatic islet blood flow 6.4.2 Regulation of gastric mucosal blood flow ß3AR-mediated cardiovascular effects Conclusions and future trends

ß3-Adrenoreceptors in Brown and White Adipocytes: Roles in Thermogenesis and Energy Balance

87 87 88 90 91 93 93 95 95 95

97

Jean Himms-Hagen 7.1 7.2

7.3

7.4

Introduction 97 Brown and white adipocytes and tissues 98 7.2.1 Functions of brown and white adipocytes 98 7.2.2 Definition and tissue distribution of brown and white adipocytes 99 7.2.3 Definition of BAT and WAT 101 7.2.4 Multiple UCPs in BAT 101 7.2.5 Adrenoreceptors in brown adipocytes 102 7.2.6 Control of brown adipocytes by the sympathetic nervous system and the hypothalamus 103 7.2.7 Central and peripheral effects of insulin in maintenance of thermogenic capacity of BAT 104 7.2.8 Control of blood flow in BAT by the sympathetic nervous system and the hypothalamus 105 Origin of brown adipocytes 105 7.3.1 Differentiation of brown adipocytes and mitochondriogenesis 105 7.3.2 Induction of hyperplasia of brown adipocytes in BAT 108 7.3.3 Expression of multiple UCPs in brown adipocytes: function and regulation 109 7.3.4 Induction of emergence of ‘ectopic’ brown adipocytes in WAT 109 7.3.5 Is there more than one type of brown adipocyte? 110 Physiological functions of brown adipose tissue 112 7.4.1 Role in thermoregulation in a cold environment 112 7.4.2 Role in food intake 113 7.4.3 Role in defence against obesity 113

Contents

7.5

7.6 7.7 8

ix

Secretions from brown adipocytes 7.5.1 Satiety factors 7.5.2 Other factors ß3ARs in brown and white adipocytes as a target for anti-obesity and anti-diabetes drugs Perspective

The Putative ‘ß4’-Adrenoreceptor and Other Atypical ß-Adrenoreceptors

115 115 116 117 119

120

A.Donny Strosberg and Jonathan R.S.Arch 8.1 8.2 8.3 8.4

Introduction Pharmacology of the ß4AR and other atypical ßARs A summary of ‘ß4’AR pharmacology The search for molecular evidence for atypical ßARs 8.4.1 The discovery of the ß3AR gene and other putative homologues 8.4.2 The discovery of a receptor which binds iodocyanopindolol and other ß3AR ligands 8.4.3 Non-ß3AR-like pharmacological properties of ß3AR proteins

References Index

120 120 121 123 123 123 123 125 167

Figures

1.1 1.2 1.3 1.4 1.5 2.1 3.1 3.2 3.3 3.4 3.5 3.6 3.7 4.1 4.2 4.3 4.4 4.5 6.1 6.2

Primary structure of the human ß3AR Comparison of the amino acid sequences of human, monkey, bovine, dog, rat, mouse, hamster, and guinea-pig ß3ARs A composite image of the ß3AR ligand binding region Signal transduction pathways activated by the human ß3AR Immunofluorescent staining of ß3AR in infected Sf9 cells Primary sequences of the ß2AR and ß3AR and schematic representation of their proposed membrane topology The ß3AR gene, targeting vectors and recombinant allele Adenylyl cyclase activity and lipolysis in response to CL-316,243 and isoproterenol Regulation of lipolysis in mouse adipocytes (a working model) Transgenic tissue-specific re-expression of murine ß3AR in knockout mice Transgenic expression of human ß3AR in knockout mice RNase protection analysis of human (HU) ß3AR gene expression in multiple tissues In vivo effects of CGP 12,177 on O2 consumption Chemical structures of selective antagonists of ß1- and ß2ARs Chemical structures of non-selective and ß3AR-selective antagonists Chemical structures of phenylethanolamine agonists Chemical structures of aryloxypropanolamines Chemical structures of trimetoquinols Effects of BRL-37,344 and SR-58,611 on the twitch tension of human endomyocardial biopsies Effect of the intravenous (i.v.) infusion (from 0 to 10min) of CL-316,243 (0.4 nmol/kg/min) on cutaneous blood flow in normal dogs, and effect of the i.v. infusion of BRL-37,344 (0.4 nmol/kg/min) on cutaneous blood flow in normal dogs before and after bupranolol administration x

5 8 11 13 18 28 37 39 40 41 44 44 45 50 53 60 66 73 89

92

6.3

7.1 7.2

Plasma insulin concentrations and islet blood flow, a percentage of pancreatic blood flow, in rats under basal conditions, after perfusion of CL-316,243 alone or in combination with an injection of bupranolol or nadolol Diagrammatic representation of the principal features that distinguish a brown adipocyte from a white adipocyte Use of ß3AR agonists to promote oxidation of excess fat stores

xi

94 99 118

Tables

1.1 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.1 5.2 5.3 5.4 8.1

Properties of the three human ß-adrenoreceptors Affinities (pA2 or pKB) of ß1/2AR-selective antagonists for ßARs determined from functional studies using rodent tissues Affinities of ß1/2AR-selective antagonists for human and rodent cloned ßARs Affinities of non-selective and ß3AR-selective antagonists for human and rodent ßARs Potencies (pD2, i.e. pEC50 values) of ßAR agonists in rodent tissues Binding affinities and functional potencies of ßAR agonists in human cloned ßARs ß3AR selectivities of esters or amides and related acids in animal tissue and human cloned receptors Comparison of the effects of BRL-26830/28410 and Ro-16–8714 on rat right atrial rate and in vivo heart rate Clinical experience with first-generation ß3AR agonists Relative potencies of agonists at the human cloned ß3AR vary with assay Human tissues expressing ß3AR Modulation of ß3AR lipolytic function in white human fat cells Phenotypes influenced by the Trp64Arg polymorphism in human ß3AR Possible clinical use of ß3AR agents Comparison of ß3AR and ‘ß4’AR pharmacology

xii

6 51 51 54 56 57 64 65 71 74 78 79 83 85 121

Contributors

Jonathan R.S.Arch is Director of Vascular Biology, SmithKline Beecham Pharmaceuticals, Coldharbour Road, Harlow, Essex CM19 5AD, United Kingdom Peter Arner is at the Department of Medicine Karolinska Institutet Sjukhus, Huddinge University Hospital, S-141 Huddinge, Sweden Michel Berlan is at INSERM U-317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangeuil, 31403 Toulouse Cedex 4, France Michel Bouvier is at the University of Montreal, Faculty of Medicine, Department of Biochemistry, Montreal, Quebec H3C 3J7, Canada Jean Galitzky is at INSERM U-317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangeuil, 31403 Toulouse Cedex 4, France Chantal Gauthier is at the Laboratoire de Physiopathologie et Pharmacologie Cellulaires et Moléculaires, INSERM CJF 96–01 Hôpital Hôtel-Dieu and Faculté des Sciences et Techniques, Université de Nantes, 44093 Nantes Cedex, France Cindy G.Gerhardt is at Unilever Research Vlaardingen, Olivier van Noortlaan 3133AT, Vlaardingen, The Netherlands Danica Grujic is at Transkaryotic Therapeutics, 195 Albany St., Cambridge, MA 02139, USA Jean Himms-Hagen is at the Department of Biochemistry, University of Ottawa, Health Science Center, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada Moriko Ito is at Novartis, Basel, Switzerland Max Lafontan is at INSERM U-317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangeuil, 31403 Toulouse Cedex 4, France Dominique Langin is at INSERM U-317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangeuil, 31403 Toulouse Cedex 4, France Frederik Lönnqvist is at the Department of Medicine Karolinska Institutet Sjukhus, Huddinge University Hospital, S-141 Huddinge, Sweden xiii

xiv

Contributors

Bradford B.Lowell is at the Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA A.Donny Strosberg is at the Institut Cochin de Génétique Moleculaire, Laboratoire d’ImmunoPharmacologie Moléculaire, CNRS UPR 0415 and Université de Paris VII, 22, rue Méchain, 75014 Paris, France Vedrana S.Susulic is at the Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA Geneviève Tavernier is at INSERM U-317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangeuil, 31403 Toulouse Cedex 4, France

Preface

Fifteen years have now passed since the first pharmacological description of an ‘atypical’ ß-adrenoreceptor, and nearly ten since the corresponding gene, renamed ‘ß3’ adrenoreceptor (ß3AR) was actually cloned. Hundreds of peer-reviewed publications and tens of patents have appeared describing various properties of the ß3AR or its ligands. Thanks to the lipolytic activity of ß3AR ligands, several of these compounds have now entered clinical trials. Moreover, genotyping for the presence of a polymorphism in the ß3AR gene, possibly associated with increased risk of developing diabetes and/or obesity, has become one of the possible targets for pharmacogenomic approaches to treat these growing health problems. The present monograph is centred entirely on the ß3AR, and has been assembled by several of the leading investigators in the field. The first chapter, which I wrote with Cindy Gerhardt, provides a general overview on the structure, function and expression of the ß3AR. The regulation of the receptor is discussed by Michel Bouvier, whereas the biological consequences of knocking-out the ß3AR gene are reviewed by the group of Brad Lowell. Jon Arch, the leading author of one of the very first articles outlining the properties of an ‘atypical’ ßAR, gives a detailed description of the pharmacology of the ß3AR. Peter Arner and Fredrik Lönnqvist present the functional role of ß3AR in humans, while Max Lafontan and colleagues venture into the less known role of the ß3AR in vasodilatation. Finally, Jean Himms-Hagen, a specialist on brown adipose tissue, discusses the biology of this tissue, in which the ß3AR plays a crucial functional role. Although all authors place the emphasis on the ß3AR, they do it in the context of the ß1AR and ß2AR homologues, as well as the more recently described, albeit still putative, fourth ßAR, which is also discussed separately in a last chapter by Jon Arch and myself. This book attempts to present a clear and complete description of the ß3AR, prepared by the original researchers that have been involved in the work since the beginning of the ß3AR history. The reader will certainly enjoy sharing how much has been learned about this receptor during the past 15 years. A.Donny Strosberg

xv

Abbreviations

AC ADA ADD AR BASH BAT cAMP C/EBPß CHO CHW CRE CREBP1 CYP GPCR CPT EDRF ERK FABP FFA FGF GLUT G protein GPCR GRE GRK HEK-293 ISO LH LPL

adenylyl cyclase adenosine deaminase adipocyte determination differentiation factor adrenoreceptor (subtype indicated by Greek letter) brown adipocyte satiety hormone brown adipose tissue cyclic adenosine 3’5'-monophosphate CCAAT enhancer binding protein Chinese hamster ovary Chinese hamster fibroblasts cyclic AMP response element CRE binding protein 1 Cyanopindolol G protein-coupled receptor I carnitine palmitoyltransferase I endothelium-derived relaxing factor extracellular regulated kinase fatty acid binding protein (M, muscle type; A, adipose type, also known as aP2) free fatty acids fibroblast growth factor glucose transporter guanine-nucleotide binding protein G protein-coupled receptor glucocorticoid response element G protein-coupled receptor kinase human embryonic kidney cells isoproterenol lateral hypothalamus, luteinizing hormone lipoprotein lipase xvii

xviii

mRNA NA NGF PIA PGC-1 PCR PKA PKB PI3K PPARE PPAR? PTX PVN RAR RARE RT-PCR RXR TR TNFa TRE UCP UCP-DTA VMH WAT

Abbreviations

messenger ribonucleic acid noradrenaline nerve growth factor phenylisopropyladenosine PPAR gamma coactivator-1 polymerase chain reaction protein kinase A protein kinase B phosphatidylinositol-3 kinase PPARß response element peroxisome proliferator activated receptor gamma pertussis toxin paraventricular nucleus of the hypothalamus retinoic acid receptor retinoic acid response element reverse transcriptase-polymerase chain reaction retinoid X receptor thyroid hormone receptor tumour necrosis factor a thyroid response element uncoupling protein (isoform indicated by number) term used to describe transgenic mice expressing diphtheria toxin A chain under the control of the UCP-1 promoter ventromedial nucleus of the hypothalamus white adipose tissue

1

Structure and Function of the ß3-Adrenoreceptor A.DONNY STROSBERG 1 AND CINDY C.GERHARDT2 Institut Cochin de Génétique Moléculaire—Laboratoire d’Immuno-Pharmacologie Moléculaire, CNRS UPR 0415 and Université de Paris VII, 22, rue Méchain, 75014 Paris, France 2 Unilever Research Vlaardingen, Biotechnologie A1260, Oliver van Noortlaan 120–3133AT, Vlaardingen, The Netherlands 1

1.1

Introduction

The neuroendocrine, metabolic and other effects of catecholamines such as adrenaline and noradrenaline are mediated by a family of membrane-bound intrinsic proteins known as the ‘adrenergic’ receptors, more recently renamed as ‘adrenoreceptors’ or ‘adrenoceptors’. The natural biogenic amines modulate various cellular signal transduction mechanisms by binding to these receptors and thus activating GTP-binding regulatory G proteins which, in turn, stimulate or inhibit effectors such as adenylate cyclase, phospholipase C, other enzymes or even ion channels. The adrenoreceptors share general structural features with members of a very large family of single polypeptide chain membrane receptors all known to display seven transmembrane hydrophobic regions, and all presumed to be coupled to G proteins. This family includes receptors for other biogenic amines such as dopamine, serotonin, acetylcholine, other small ligands, peptides and even sensory stimuli such as light, olfactory or gustatory substances. It is one of the receptors for light, rhodopsin, which was the first one to be characterized in great detail both in terms of structure, and in terms of coupling to transducin and, through this G protein, to the effector: phosphodiesterase (for reviews, see Strosberg, 1993; Trends in Pharmacological Science, Annual Supplement 1998, 9th edition, S.P.H.Alexander and J.A.Peters, Eds). From the outset, adrenoreceptors (AR) were subdivided into
AR and ßAR subclasses, on the basis of relative potency for catecholamines and isoproterenol, which has no effect on
AR. Later, these receptors were further subdivided into
1AR,
2AR subclasses, whereas ßAR were subdivided into ß1AR and ß2AR (Lands et al., 1967a,b). Today, nine subtypes have been recognized:
1A,B,C,
2A,B,C, ß1, ß2, ß3 subdivisions of ßAR. Subdivision of ßAR into ß1AR and ß2AR was made on the following basis: ß1AR is equally sensitive to noradrenaline and adrenaline, and ß2AR is more sensitive to adrenaline (Lands et al., 1967a,b). Convincing pharmacological evidence for the existence of additional, or atypical, ßARs stems from the early 1980s (Arch et al., 1984a; Wilson et al., 1984; see Chapter 4). Five years later, a gene encoding what was designated as the ß3AR was cloned 1

Strosberg and Gerhardt

2

(Emorine et al., 1989) by low stringency-screening of a human genomic library with a ß2AR probe. Homologous ß3AR were cloned subsequently from rodents and other mammals. Pharmacological characterization of these ß3AR have however not completely reconciled all the data on atypical ßAR, and periodically new studies describe atypical non-ß3AR or ß4AR. For the sake of clarity, we discuss these ß4AR and possibly other atypical ßAR in Chapter 8. The availability of the sequence of the ß3AR for several different species, has considerably increased our knowledge about this receptor. Heterologous expression of the ß3AR in cell lines that do not express ß1AR or ß2ARs, has facilitated pharmacological studies and revealed interesting species-related differences. Studies using mutated or chimeric ß3ARs have pinpointed the regions involved in ligand recognition and signal transduction. In addition, molecular biological tools were used to localize transcripts encoding ß3ARs in different tissues. In this chapter, we provide an introductory overview on the ß3AR that is strongly orientated on molecular biological studies. We describe the structure of the gene and its promoter, and discuss interesting features in the ß 3 AR protein. ß 3 AR-specific characteristics, either in comparison with the ß1AR and the ß2AR, or with species homologues, are also outlined. We then summarize the current knowledge on structurefunction relationships of the ß3AR. The signals that are activated in response to ß3AR activation, and the biological role that these may play are subsequently described. We finish this chapter with a description of the distribution of the ß 3AR. Wherever appropriate, we refer to further discussions in the other chapters of this volume.

1.2

The ß3AR gene

The development of alprenolol-agarose based affinity chromatography, used first for the purification of the turkey ßAR (Vauquelin et al., 1977, 1979), led to the isolation of the hamster ß2AR and the partial sequencing of several cyanogen bromide-fragmented peptides by Benovic et al. (1984). Screening of a hamster genomic library with oligonucleotides corresponding to these peptide sequences led to the isolation of a single continous open reading frame encoding the hamster ß2AR (Dixon et al., 1986). Analysis of the corresponding predicted protein sequence revealed a significant similarity with the previously described bovine rhodopsin, as well as with bacterio-rhodopsin, especially in terms of the presence of seven hydrophobic stretches each of about 25 amino acid residues. The homology with the rhodopsins, that are coupled to the G protein transducin, prompted the hypothesis that other G protein-coupled receptors might have similar structural properties. Indeed, soon after the cloning of hamster ß2AR (Dixon et al., 1986), the ß1AR-like ßAR from turkey (Yarden et al., 1986) and the human ß1AR (Frielle et al., 1987) were cloned, and confirmed a general topology of G protein-coupled receptors.

1.2.1 Cloning of the gene The human ß3AR was isolated by screening a human genomic library with the entire coding sequences of the genes encoding the turkey ß 1AR and human ß2AR used as probes (Emorine et al., 1989). Upon expression of the open reading frame in CHO

Structure and function of the ß 3-adrenoreceptor

3

cells, it transpired that this third human ßAR subtype, designated as ‘ß 3 AR’, exhibited a number of the pharmacological properties previously described for ‘atypical’ ßARs mainly expressed in adipose tissues of rodents (Arch et al., 1984a; see Chapter 4). From that moment on, this adipocyte-specific atypical ßAR became gradually but widely accepted as the ß3AR. Brad Lowell in Chapter 3, and Strosberg and Arch in Chapter 8 discuss the possible existence of yet more atypical receptors, including the ß 4AR.

1.2.2

Structure of the coding region

The coding region of the human ß3AR gene contains 1224 nucleotides which putatively encode a 408-amino acid residues protein (Emorine et al., 1989; van Spronsen et al., 1993). Comparison of the amino acid sequences predicted from the genomic nucleotide sequences and from the corresponding cDNA sequences revealed however unexpected differences in the length and sequence of the carboxy-terminal regions of the ß3 receptors. Indeed, in contrast to the homologous ß1- and ß2ARs, the ß3AR-coding region was found to be present on two different exons, both in man as in mouse and rat (Bensaid et al., 1993; Granneman et al., 1993; van Spronsen et al., 1993). A first exon of 1.4kb contains the coding sequence for the first 402 and 388 amino acid residues in man and mouse or rat, respectively. In the human ß3AR gene, a second exon of 700 bp contains the sequence coding for the six carboxy-terminal residues of the receptor and the entire mRNA 3' untranslated region. In mouse and rat, a second exon of 68 bp codes for the 12 carboxy-terminal residues of the receptor, while a third exon contains the 3' untranslated region of the ß3AR mRNA. Until today, no good evidence existed for distinct functional roles for differential splice forms generating different protein variants. However, different mRNA lengths exist due to the use of widely separated promoters (Granneman and Lahners, 1994) and/ or the use of alternate acceptor splice sites in exon 3 in rodents (van Spronsen et al., 1993). In addition, human ß3AR transcripts with different 3' untranslated regions are produced by continuation of transcription beyond termination signals (van Spronsen et al., 1993) or result from the use of alternative polyadenylation signals (Granneman and Lahners, 1994).

1.2.3

Structure of the promoter

The study of the promoters of the human and murine ß3AR genes led to the identification of several potentially important sequences. Transcription of the rat ß3AR starts mainly at position -161 relative to translation initiation ATG, whereas minor transcription begins around -123 and -109. Interestingly, almost all of the (few) ß3AR messenger molecules present in the rat gastric fundus were found to start at the -123 and -109 sites (Granneman and Lahners, 1994). In human brown fat and neuroepithelioma cells, the vast majority (80%) of ß3AR transcripts begin at multiple sites around -130 (Granneman and Lahners, 1994). Motifs potentially implicated in heterologous regulation of the murine ß3AR expression by glucocorticoids (Fève et al., 1992), butyrate (Krief et al., 1993), phorbol esters (Fève et al., 1995; El Hadri et al., 1998) and insulin (Fève et al., 1994; El Hadri et al., 1998) were identified upstream of the cap sites. Four potential cyclic AMP response elements (CREs) exist in the 5' flanking region

Strosberg and Gerhardt

4

of the human ß3AR gene. Three of these CREs were reported to contribute to the agonistinduced up-regulation of the human ß 3 gene expression (Thomas et al., 1992). Interestingly, no CRE was identified in the rodent ß3AR gene, which, in contrast to the human gene, was shown to be down-regulated by agonist treatment (Granneman and Lahners, 1992, 1994; Granneman, 1995).

1.3

The ß3AR protein

The human ß3AR, as well as the ß1- and ß2ARs, belongs to the now well-established superfamily of G protein-coupled receptors. As such, it is composed of a single, 408 amino acids long peptide chain which is thought to traverse the membrane seven times. Each of those transmembrane (TM) regions is constituted of a hydrophobic stretch of about 22 to 28 residues (Figure 1.1). The extracellular, amino-terminal region of G protein-coupled receptors is of variable length and contains almost always glycosylation sites. Amino acid sequencing of the amino-terminal region of the ß3AR, immunopurified from solubilized membranes of CHO cells overexpressing the ß3AR, confirmed the sequence predicted on the basis of the DNA sequence (Guillaume et al., 1994). The Asn residue at position 7, a putative target for Asn-linked glycosylation (Asn-Ser-Ser), could not be identified in the amino acid sequence, suggesting that it is indeed glycosylated. The intracellular domains of G protein-coupled receptors often contain consensus phosphorylation sites. Receptor phosphorylation by protein kinase A or C (PKA/PKC) or G protein-coupled receptor kinase (GRK; formerly named ß-adrenergic receptor kinase, ßARK), underlies receptor desensitization by regulating for example G protein coupling or receptor internalization. Interestingly, the intracellular domains of the ß3AR do not contain any potential sites for phosphorylation by either PKA or PKC, and contain only one putative phosphorylation site for GRK (compared with eight in the ß2AR). Consequently, the ß3AR (in contrast to the ß2AR) is resistant to rapid agonist-promoted desensitization, both in transfected cells (Liggett et al., 1993; Nantel et al., 1993) and in rat adipocytes (Granneman et al., 1992) that express ß 3ARs in an endogenous environment. The regulation of ß3AR-mediated signalling is discussed in more detail in Chapter 2.

1.3.1

Comparison with ß1- and ß2AR

Although the three ßAR subtypes share a certain functional homology (e.g. their stimulation leads to the activation of adenylyl cyclase), they all play their own specific physiological role, as summarized in Table 1.1. Indeed, the structural homology between the ß1AR, ß2AR and ß3AR is restricted; the subtypes differ in length both at the N-terminus and at the C-terminus, and the number of residues conserved between the three receptors is limited. As within all members of the superfamily of G protein-coupled receptors, the conserved residues are almost exclusively restricted to the seven TM segments and the membrane-proximal regions of the intracellular loops (Figure 1.1). Site-directed mutagenesis (Guan et al., 1995; Gros et al., 1998) and studies using chimeric ß2/ß3ARs (Nantel et al., 1993, 1994; Jockers et al., 1996) support the conclusion that the conserved parts are involved in ligand binding and G

Figure 1.1 Primary structurte of the human ß3AR. The sequences are represented in the one-letter code for amino acids. The single polypeeptide chain is arranged according to the model for rhodopsin. The disulphide bond essential for Cys110 and Cys189 activity is represented by -S-S-. The two N-glycosylation sites in the amino-terminal portion of the protien are indicated by Y. palmitoylated Cys360 residue in the N-terminus of the i4 loop is indicated by a. Residues in black cicles are common to the three ßAR subtypes. The underlined sequence RSSPAQPRLCQRLDG corresponds to the synthetic peptyde which was used to obtain the anti C-terminal antibodies reacting with the whole ß3AR.

6

Strosberg and Gerhardt

Table 1.1 Properties of the three human ß-adrenoreceptors

CRE, cyclic AMP response element; GCE, glucocorticoid response element. *CL-316,243 is most selective for rodent ß3AR. Merck L-755,507 is most selective for human ß3AR (Parmee et al., 1998) **Bupranolol is not selective, but is the strongest known ß3AR antagonist. SR-59,230A is not selective but has been recognized as a good antagonist for human ß3AR expressed in several tissues, but not in model systems (De Ponti et al., 1996; Nisoli et al., 1996a)

protein interaction, respectively. The ß3AR possesses a number of pharmacological properties that distinguish it from the ß1AR and ß2AR. It has a higher affinity for noradrenaline than for adrenaline, while the ß2AR has a higher affinity for adrenaline, and the ß1AR binds both ligands equally well. In addition, several ligands that act as antagonists on ß1ARs and ß2ARs, act as agonists (e.g. bucindolol, ICI-201651, carazolol, CL-316,243) or partial agonists (e.g. CGP-12177, (cyano-)pindolol) on the ß3AR. For a detailed description of the pharmacological profile of the ß3AR, the reader is referred to Chapter 4. Recent studies by Granneman et al. (1998) using chimeric and mutant rat ß1and ß3ARs have indicated the presence of (at least partially) distinct binding sites for ß3AR-selective phenylethanolamines and catecholamines in the seventh TM domain. The findings on structure-function relationships of the ß3AR are discussed in more detail in Section 1.4. G protein-coupled receptors often contain cysteine residues in their second and third extracellular loops that can form disulphide bonds to stabilize the receptor conformation. All three ßAR subtypes contain one cysteine residue in their second extracellular loop, and three cysteine residues in their third extracellular loop. As was shown for the ß1- and the ß2AR, the cysteine residues in the human ß3AR are likely to

Structure and function of the ß 3-adrenoreceptor

7

form a functional disulphide bond. Reduction of the receptor by dithiothreitol inactivates 45% of the [125I] ICYP binding sites, while preincubation with certain ligands protects the receptor from dithiothreitol-induced inactivation (Méjean-Galzi et al., 1995). Interestingly, this protection was not induced by the agonists adrenaline, isoproterenol or BRL-37,344, while it was induced by the agonist carazolol as well as by the antagonists CGP-20712A and ICI-118551. In this respect, the human ß3AR differs from the human ß2AR, which was (in the same study) found to be protected by all ligands. Whereas the ß2AR contains two PKA target sites in its third intracellular domain and its C-terminus, none is observed in the ß3AR. Similarly, whereas the ß2AR has eight potential GRK sites in its C-terminus, the ß3AR has only one. Furthermore, the ß 2AR possesses two PKC sites and tyrosine residues involved in heterologous desensitization (Bouvier et al., 1991) and down-regulation (Valiquette et al., 1990, 1993), respectively, that are absent in the ß3AR. As described above, and discussed in more detail in Chapter 2, this has clear consequences for the desensitization properties of the ß3AR. A very low degree of homology is found in the distal C-terminal region, either within ßAR subtypes or within ß3AR species variants, and it remains to be determined whether this domain exerts a specific function. No functional differences have been found upon expression of ß3AR receptors with or without the last six (in man) or twelve (in rodent) C-terminal residues (Granneman et al., 1993; Nantel et al., 1993, 1994). Interestingly, it was reported recently that the last few residues of the C-terminal domain of the ß2AR interact directly, independently of G proteins, with a Na+/H+ exchanger regulatory factor (Hall et al., 1998). However, since this sequence is very different in the ß3AR, a similar interaction seems unlikely.

1.3.2

Comparison of ß3AR from various species

Today, the sequence of the ß3AR has been determined in human (Emorine et al., 1989), mouse (Nahmias et al., 1991), rat (Granneman et al., 1991), bovine (Piétri-Rouxel et al., 1995), guinea-pig (Atgié et al., 1996), monkey (Walston et al., 1997), dog (Lenzen et al., 1998) and hamster (S.Baude et al., unpublished results). The degree of amino acid sequence homology (Figure 1.2) between the ß3ARs from these species is considerably higher (80% to 90%) than that calculated between different subtypes (40% to 50%), a feature generally observed for receptor subtype homologies across species. Several ‘ß3-specific’ residues are shared between all partial or complete ß3 sequences analysed so far, and are not found in ß1 or ß2AR sequences (Figure 1.2). Furthermore, the human, monkey, bovine and dog ß3AR are closer to each other than to any of the rodent (rat, mouse, hamster) sequences, in particular in TM1, where a (ValLeu-Ala) deletion is observed in the rat, mouse and hamster, but not in the larger mammals. When the human ß3AR is compared with its animal counterparts, several residues are found to be unique to man (Figure 1.2). For instance, in front of TM4 and TM6 one finds a cysteine residue in the human ß3AR, while in bovine, dog, mouse, rat, guineapig and hamster and monkey, an arginine residue is present. It remains to be elucidated whether these cysteine residues play a specific role in the human ß3AR. Considerable differences in ligand binding and adenylyl cyclase activation constants

8

Strosberg and Gerhardt

Figure 1.2 Comparison of the amino acid sequences of human, monkey, bovine, dog, rat, mouse, hamster, and guinea-pig ß3ARs. To maximize homologies, gaps represented by empty spaces have been introduced in four sequences. The seven presumed transmembrane domains (TM1–TM7) are boxed and are separated by extracellular (E1 to E4) and intracellular (I1 to I4) loops. In the aminoterminal region, consensus sequences for asparagine-linked glycosylation site (N-X-S) are highlighted by bars. The presumed palmitoylated Cys361 is noted with a grey box. Residues marked with a dot are common to human ß1- and ß2-adrenoreceptors and to all ß3ARs analysed so far.

Structure and function of the ß 3-adrenoreceptor

9

have been reported when a variety of ß3AR agonists were evaluated on different species variants of the ß3AR expressed in CHO cells. For example, BRL-37,344 and CL-316,243 have much higher efficacies on rodent ß3AR than on human ß3AR. Also bupranolol, a non-selective ßAR antagonist on human and rodent ß3ARs, was found to be a partial agonist on the bovine ß3AR (Piétri-Rouxel et al., 1995). Similarly, propranolol acts as an antagonist on the mouse ß3AR, while it has partial agonist activity on the human receptor (Blin et al., 1993). Carazolol has a ten-fold higher affinity for the human ß3AR compared with the mouse ß3AR; however it has the same efficacy and potency to stimulate adenylyl cyclase, maybe because the mouse ß3AR couples to Gs (G-protein) more efficiently (Blin et al., 1994). Indeed, many reports have shown that stimulation of the human ß3AR in adipocytes stimulates adenylyl cyclase activity less efficiently than does the rodent ß3AR. It was shown recently that stimulation of the dog ß3AR hardly increases intracellular cAMP concentrations upon expression in hamster CHO/K1 cells, while it does efficiently activate adenylyl cyclase in human HEK 293 cells. Apparently, the coupling of the hamster Gs to the canine ß3AR is highly sensitive to the cellular background.

1.3.3 Polymorphism In the human ß3AR, a tryptophan residue is present at position 64 (just outside TM1), whereas in all other species an arginine (or a cysteine in guinea-pig) is found (Atgié et al., 1996). Interestingly, a polymorphism has been described (Clément et al., 1995; Walston et al., 1995; Widen et al., 1995; Strosberg and Piétri-Rouxel, 1997) that is present in about 10% to 50% of the population, and that ‘restores’ the arginine residue present in most animals. In some groups of patients, as in the initially described Pima Indians (Walston et al., 1995) but also in Japanese, Swedish, Finnish or French obese individuals, this polymorphism was found to be associated with morbid obesity and insulin resistance. However, in other cohorts no correlation could be found (for reviews, see Strosberg, 1997; Allison et al., 1998; Fujisawa et al., 1998). An increasingly likely explanation for the apparent discrepancies between the various studies could be found in the association between various alleles if different genes are implicated in the genetic control of metabolism (Strosberg, 1997). It was thus observed that the human population displays at least two alleles of UCP 1 gene, one in 65%, the other in 35% of individuals. Homozygotes for the less abundant allele seem to have an increased tendency to accumulate weight (Ricquier and Bouillaud, 1997; Gagnon, J. et al., 1998b). Individuals that bear both the Arg64 allele and the A-3826G region allele of UCP 1 have an even higher tendency towards obesity. Proenza et al. (2000) have now started to investigate various sets of alleles, to uncover genotypes predisposing humans to obesity. The recent discovery of polymorphisms in gene for MC4 or PPARg (Ristow et al., 1998) suggests that more genotypes will be uncovered in the future. The massive search for Single Nucleotide Polymorphism or ‘SNIPs’ by the pharmaceutical and biotechnology industry will soon replace the gene-by-gene approach followed so far by academic laboratories, and should therefore help define obesity-associated genotypes. Today, it is still unknown how the Arg64 mutation exactly affects adipocyte biology. To evaluate the effect of the tryptophan?arginine (W64R) substitution on receptor function in vitro, the ß3AR-Arg64 was expressed at various levels in model cells. Compared with the ß3AR-Arg64, no difference was observed in ligand binding or

Strosberg and Gerhardt

10

adenylyl cyclase activation constants (Candelore et al., 1996; Piétri-Rouxel et al., 1997). However, a consistent reduction was observed in the maximal agonist-stimulated cAMP concentration, both in CHO and in HEK293 cells (Piétri-Rouxel et al., 1997). The effect of the mutation on ERK kinase activity (Gerhardt et al., 1999) is currently being investigated. Lipolytic activity of the ß3AR in isolated visceral fat cells of heterozygote ß3AR-Arg64 carriers was not found to be significantly different from that of ß3AR-Trp64 controls in a first study by Li et al. (1996). A more recent similar study performed by the same group (Hoffstedt et al., 1997) on a larger number of the same Swedish population has however correlated the occurrence of the Arg64 allele with a ten-fold reduction of lipolysis in omental adipose tissue. In contrast, in homozygous individuals of the Pima Indian population, no differences were found in terms of in vivo lipolysis of subcutaneous adipose tissue (Snitker et al., 1997).

1.4

1.4.1

Structure-function relationships in the ß3AR

The ligand binding site

The putative ligand binding site of the ß3AR has been described on the basis of computer modelling combined with data obtained by site-directed mutagenesis and photoaffinity labelling of the ß3AR, the ß2AR or other related receptors (Figure 1.3). The following residues are considered to be most important for ligand binding: 1 Asp117 in TM3 is highly conserved in all G protein-coupled receptors for biogenic amines, and it was found to be essential for binding of these ligands. The acidic sidechain most likely forms a salt bridge with the basic group of the ligand. Indeed, substitution of this residue by a leucine residue in the human ß3AR completely suppresses agonist binding (Gros et al., 1998). 2 Ser169 in TM4 is thought by certain authors to form a hydrogen bond with the hydroxyl group of the ethanolamine side-chain (Strader et al., 1989), whereas others contest a role for this residue. 3 Ser209 and Ser212 in TM5 are also found (or substituted by Thr) in many biogenic amine receptors, and are thought to form hydrogen bonds with the hydroxyl groups of the catechol moiety. 4 Phe309 in TM6 is thought to be involved in a hydrophobic interaction with the aromatic ring of the catecholamine. Beside these residues, many other residues may play more subtle roles in the interactions between the ß3AR and (certain of) its ligands. Recent results produced by Granneman et al. (1998) have thus pinpointed two residues in TM7 to be critical for receptor activation by ß 3 AR agonists such as BRL-37,344 and CL-316,243. Replacement of Phe350 and Phe351 in TM7 of the ß1AR for Ala and Leu residues, normally present in the ß3AR, is sufficient to allow activation of the mutated ß1AR by ß3AR agonists. These residues seem to be specifically involved in binding of the ß3AR agonists, since neither the activity of catecholamines, nor the antagonism by propranolol, were affected by the substitutions. It is still not understood why several ß1/ß2AR antagonists (e.g. ICI-201651, carazolol,

Structure and function of the ß 3-adrenoreceptor

11

Figure 1.3 A composite image of the ß3AR ligand binding region. Proposed interactions in the ligand binding region of the ßAR viewed from the outside of the cell. The ligand noradrenaline is shown surrounded by several of the amino acids involved in agonist binding. These are Asp117 in TM3, Ser209 and Ser212 in TM5, and Tyr336 in TM7. The essential disulphide bond (-SS-) linking Cys110 (E2) and Cys189 (E3) is also represented. Asp83 (TM2), not represented here, is likely to be more important for signal transmission to Gs than for actual ligand binding, in which it is nevertheless involved.

CL-316,243, CGP-12177) behave as ß3AR agonists. Based on computer modelling studies, the ligand binding site of the ß3AR contains less bulky amino acid residues than that of the ß1/ß2ARs, and could therefore could more easily accommodate the larger ß1/ ß2AR antagonists (Blin et al., 1993, 1994, 1995; Strosberg et al., 1993). However, substitution of the small Gly53 for a bulky Phe (present at this position in the human ß2AR) in the human ß3AR turned out not to be sufficient to convert the ß3 agonists into antagonists (Gros et al., 1998). In addition, the molecular basis for the pharmacological differences between human and rodent ß3ARs is not yet known. The Val-Leu-Ala tripeptide, present in rodent ß3ARs but absent in the human or other large mammalian ß3AR, does not seem to be involved in this species-specific difference (Gros et al., 1998).

1.4.2

The regions of interaction with the G proteins

As is now known from a number of mutagenesis studies on different G protein-coupled receptors, the site of receptor-G protein coupling is situated in the membrane-proximal regions of the second and third intracellular loops, as well as in the C-terminal domain of the receptor. Deletion mutagenesis of the third intracellular loop of the ß3AR confirmed that the ß3AR constitutes no exception: deletion of eight amino-terminal and 13 carboxy-terminal

Strosberg and Gerhardt

12

residues from the
s subunit of the Gs protein led to functional uncoupling of the human receptor ß3AR from adenylyl cyclase (Guan et al., 1995). The site of interaction with the G protein seems to be dictated by its position rather than by its primary amino acid sequence. For example, the G protein-binding site of the ß2AR is located at the same position as in the ß3AR, but the sequence homology in these regions is only 63% at the amino-terminal and 31% at the carboxy-terminal end of the third intracellular loop. The different primary sequences may allow the receptor to couple preferentially to different subtypes of Gs protein subunits, although today no data are available on the ß 3AR specificity concerning certain combinations of Ga /ß/ g subunits. In addition to a direct, physical interaction between the receptor and a G protein, an independent process of G protein activation is necessary. It has been suggested that Asn312 in TM6 of the human ß3AR plays a role in activation of adenylyl cyclase by certain agonists (Gros et al., 1998). Thus, whereas no differences were observed in intrinsic activities of noradrenaline or CGP-12177, nadolol and tertatolol (ß3AR agonists and ß1/2AR antagonists) displayed a decreased intrinsic activity for the mutant receptor, without any change in binding affinity. With the increasing evidence of functional coupling between the ß 3AR and Gi or Go proteins (Gerhardt et al., 1999) it now becomes necessary also to investigate sites of interaction of the receptor with these other G proteins. This has not yet been attempted.

1.5

Signal transduction and biological functions

Like the ß1- and ß2ARs, the ß3AR preferentially couples to Gs in order to stimulate adenylyl cyclase, PKA and subsequent target proteins such as the hormone-sensitive lipase in adipocytes. Treatment with ß3AR agonists leads to increased concentrations of intracellular cAMP, both in transfected cell lines as well as in tissues that endogenously express ß3ARs. Below, we present a short overview on the Gs-mediated signalling by the ß3AR. For a more detailed description of this subject, the reader is referred to Chapter 2. More recently, it has become clear that the ß3AR may also modulate the activity of other signalling pathways. For example, it can lead to changes in intracellular calcium concentration, and it can modulate the activity of MAPK and PI3K (Figure 1.4). We discuss these findings in more detail, and place them in the context of the role that the ß3AR may play in adipocyte differentiation.

1.5.1 ß3AR-mediated activation of adenylyl cyclase In rodent adipocytes, ß3AR agonists potently activate adenylyl cyclase, PKA and hormone-sensitive lipase, resulting in lipolysis (e.g. Shih and Taberner, 1995). cAMP-activated lipolysis via the ß 3AR has also been well characterized in the murine 3T3-F442A pre-adipocyte cell line (Fève et al., 1991). In addition to the lipolytic activity of ß3AR agonists, these agents inhibit insulin-stimulated glucose transport. This inhibition results from a cAMP-dependent phosphorylation and subsequent loss of intrinsic acitivity of GLUT4 transporters (Carpéné et al., 1993a,b).

Structure and function of the ß 3-adrenoreceptor

13

Figure 1.4 Signal transduction pathways activated by the human ß3AR. Schematic model for intracellular signalling pathways activated upon stimulation of the ß3AR. The model is based upon studies with CHO cells, stably expressing human ß3AR, in which a coupling of the ß3AR to both Gs and Gi was established. Biological effectors and consequences that play an important role in adipocyte biology (lipolysis, thermogenesis, adipogenesis) are tentatively included into the scheme.

Stimulation with ß3AR agonist can also lead to changes in gene expression via the PKA-mediated phosphorylation of CREB. In addition to the well-documented transcriptional activation of UCP-1 expression (e.g. Champigny and Ricquier, 1996) the expression of other genes such as LPL (Kuusela et al., 1997a,b), members of the C/EBP family (Rehnmark et al., 1993) or a1ARs is up-regulated by ß3AR agonists. Stimulation of the ß3AR can furthermore lead to inhibition of gene expression, as for example the satiety hormone, leptin (Trayhurn et al., 1996). In contrast to the efficient coupling of the ß3AR to adenylyl cyclase in rodents, diverging results have been obtained on functional effects of ß3AR agonists in human tissues. Even when ß3AR is present (as determined by RT-PCR and binding studies), the increase in cAMP and the stimulation of lipolysis may be quite small (Zilberfarb et al., 1997). As outlined below, increasing evidence reveals the coupling of the ß3AR to other G proteins. It remains to be determined whether the human ß3AR, in contrast to its rodent homologue, couples preferentially to these ‘new pathways’. Interestingly, upon heterologous expression in either rodent or human cells, the human ß3AR efficiently couples to adenylyl cyclase. This may be due to different expression levels of the receptor, increased presence of G protein (subtypes), or adenylyl cyclase (subtypes) in fibroblasts as compared with adipocytes. Expression of the ß3AR in transfected model cells has enabled detailed functional pharmacological analyses, without interference from ß 1AR or ß 2AR. The ß 3ARmediated increase in cAMP concentration can be blocked by non-selective ßAR antagonists such as bupranolol, or by high concentrations of ß1- or ß 2-selective antagonists such as ICI-118,551 or CGP-20712A. SR-59230A was introduced as a selective ß3AR antagonist, but although this agent efficiently blocks endogenously

14

Strosberg and Gerhardt

expressed ß3AR (Nisoli et al., 1996a,b), it was found to act as a partial agonist in transfected cell lines (Strosberg and Piétri-Rouxel, 1997). Clearly, care should be taken when transfected cell systems are used to determine agonist/antagonist properties. Not only the number of receptors expressed, but also the assay methodology may change the ligand potency and intrinsic activity. Indeed, it has been shown that with increasing numbers of ß3AR expressed in CHO cells, the potency and intrinsic activity of a range of agonists also increased (Wilson et al., 1996). Moreover, the potency for example of noradrenaline was found to depend strongly on how the activity of adenylyl cyclase was determined.

1.5.2 ß3AR-mediated activation of other signalling pathways Several reports have described ß3AR-mediated responses that are sensitive to pertussis toxin (PTX), suggesting that this receptor can also couple to G proteins of the Gi/o class. Chaudry et al. (1992) thus described the simultaneous coupling of the ß3AR (but not the ß1AR) to either Gs or Gi in rat adipocytes. In intact adipocytes, PTX treatment significantly increased BRL-37,344-stimulated cAMP accumulation, indicating that Gi activation limits ß3AR-stimulated cAMP accumulation. Also in isolated membranes, concentrations of GTP that have been shown to activate Gi proteins, inhibited the ß3AR(but not the ß1AR-) mediated stimulation of adenylyl cyclase, again suggesting that ß3AR can interact with Gi proteins. In addition, Gauthier et al. (1996) described that treatment of isolated preparations of human ventricle with ß3AR agonists results in PTX-sensitive negative inotropic effects and reduction of the amplitude and duration of action potentials. In a followup of this study, it was shown that the negative inotropic effect was mediated via the increase in the production of nitric oxide and cGMP, indicating a ß3AR-mediated (and PTX-sensitive) activation of nitric oxide synthase in the human ventricle (Gauthier et al., 1998). It has also been reported (Seydoux et al., 1996) that isoproterenol induces an increase in intracellular calcium concentration via stimulation of both ß1-, ß2- and ß3ARs in human white adipocytes. However, this work did not address the question of the underlying mechanism of the coupling of the ß3AR to Gs, Gi/o or Gq. The pharmacological properties of the human ß3AR have been found to vary with the coupled G protein. The order of potency and intrinsic activities of both natural ligands, noradrenaline and adrenaline, is inverted between the G s -mediated activation of adenylyl cyclase, and the G i/o-mediated activation of extracellular regulated kinase (ERK)1/2 (Gerhardt et al., 1999; see also below). In addition, BRL37,344 and propranolol act as agonists in the stimulation of adenylyl cyclase, but as antagonists in the activation of ERK1/2. As was already mentioned above, these results highlight the fact that pharmacological profiles may be highly dependent not only on cellular background, but also on the signal that follows receptor activation. Although PTX similarly inhibits G
i- and G
o-subunits, and thus cannot be used to discriminate between these two G proteins, indirect evidence exists that the ß3AR couples to G o rather than to G i. In fact, preincubation with PTX of the ß 3ARexpressing CHO cells which display a functional PTX-sensitive coupling (Gerhardt et al., 1999) does not influence the positive coupling to adenylyl cyclase (PiétriRouxel et al., 1997).

Structure and function of the ß 3-adrenoreceptor

1.5.3

15

A role for the ß3AR in adipocyte differentiation

In addition to the above-mentioned cases, in which the ß 3 AR is expressed endogenously, the human ß3AR expressed in CHO cells can also couple to both Gs and Gi/o (Gerhardt et al., 1999). In these cells, the coupling of the ß3AR to Gi/o leads to the activation of PI3K, and, subsequently, to the activation of protein kinase B (PKB) and ERK1/2 (Figure 1.4). The ß3AR-mediated activation of ERK 1/2 and PKB has also been observed in cells that express the ß3AR endogenously, albeit by different mechanisms. Shimizu et al. (1997) reported that stimulation of the ß3AR (with BRL-37,344) expressed in rat adipocytes, leads to activation of ERK 1/2, but in a way mediated by the increase in cAMP, and independent of PI3K. In addition, it has been shown that ß3AR agonists can activate PKB in rat epididymal fat cells. The mechanism underlying these effects remains to be elucidated, but it seems to be independent of PI3K and cAMP accumulation (Moule et al., 1997). Since the effect of PTX was not investigated, no conclusions can be drawn about the involvement of Gi/o proteins. The activities of ERK1/2 (Sale et al., 1995), as well as of PI3K (Christoffersen et al., 1998) and PKB (Magun et al., 1996), play a crucial role in adipogenesis. Therefore, the ß3AR-mediated activation of ERK 1/2 and PKB may well form the basis for the frequent observations that treatment with ß3AR agonists, in vitro as well as in vivo, results in considerable expansion of brown adipocyte populations. For example, chronic treatment of rats with ß3 agonists leads to the appearance of brown adipocytes in white fat depots, leading to an increase in resting metabolic rate in these cells (Arbeeny et al., 1995; de Souza et al., 1997; Ghorbani et al., 1997a). Similarly, in man, brown fat reappears around catecholamine-secreting phaeochromocytoma tumours. Also in primary cultures of mouse brown pre-adipocytes, differentiation is stimulated by activation of the ß3AR (Bronnikov et al., 1992). Interestingly, the biological consequences of ß3AR stimulation are quite opposite in BAT and in WAT. For example, the positive interaction between the ß3AR and the adipocyte differentiation machinery seems to be restricted to brown adipose tissue (BAT), since in white adipose tissue (WAT) stimulation of the ß3AR actually leads to a decrease in the number of adipocytes (e.g. Cousin et al., 1993; de Souza et al., 1997; Ghorbany et al., 1997a). Although the molecular mechanism for this difference remains to be elucidated, it is remarkable that, also in contrast to BAT, ß3AR stimulation in WAT has been shown to inhibit the activity of PI3K (Ohsaka et al., 1997) and ERK 1/2 (Sevetson et al., 1993), both in a cAMP-dependent way. Effects of adrenergic stimulation on adipocyte proliferation and differentiation are discussed further in Chapter 7.

1.6 Distribution of the ß3AR In contrast to the ß1- and ß2ARs which are distributed all over the body, the ß3ARs appears to have a rather restricted pattern of expression (see also Chapter 5). Most studies have concentrated on the functional presence of atypical (non-ß1ARs, non-ß2AR) ßARs in (brown and white) adipose tissue, where they regulate lipolysis and thermogenesis, and in the gastrointestinal tract, where they regulate gut motility via smooth muscle relaxation. Since the cloning of the murine ß3AR (Nahmias et al., 1991), it has become possible to identify the atypical ßARs by molecular means, and it is now

16

Strosberg and Gerhardt

widely accepted that at least some of these receptors in adipocytes and gut are indeed ß3AR. However, the presence of atypical ßARs does not always correlate with the presence of ß3AR messenger RNA (see also Chapter 8). Moreover, their pharmacological profiles do not always overlap. Indeed, pharmacological evidence now exists for the presence of a fourth ßAR. Although molecular proof for the existence of such ß4AR is still missing, functional data are accumulating. The pharmacological characteristics of ß4ARs are described in detail in Chapter 3 and 8. Here, we describe in brief the regional distribution of the ß3AR, as assessed by means of pharmacological and functional responses, immunolocalization of the protein, and detection of mRNA.

1.6.1 Functional detection of the ß3AR A large number of reports have characterized the presence of atypical ßARs by functional effects, mainly on metabolism, smooth muscle relaxation and vasodilatation. These atypical receptors are activated by selective ß3AR agonists such as CGP-12177 and BRL-37,344, and cannot be blocked by ßAR antagonists used in concentrations which completely inhibit the ß1- and the ß2AR. Many reports describe atypical ßARs present in murine (brown as well as white) adipocytes, where they activate adenylyl cyclase and couple functionally to lipolysis via the activation of hormone-sensitive lipase. In general, the pharmacological profile of the atypical adipocyte ßAR correlates well with that of the ß3AR, and ß3AR messenger RNAs can be easily detected (see below). In man, the lipolytic activity of the ß3AR is less evident. On one hand, this may be due to the fact that strong and selective human- (in contrast to rodent) ß3AR agonists are still not widely available (e.g. Hoffstedt et al., 1996a,b), despite now being described by pharmaceutical companies (see Chapter 4). In addition, many reports have now indicated clear differences in ß3AR responses in different human fat depots. In man, ß3ARs are expressed at lower levels in subcutaneous fat than in omental and mesenteric fat, and their lipolytic activity is limited in subcutaneous fat (e.g. Arner, 1995; Hoffstedt et al., 1995; Barbe et al., 1996; Tavernier et al., 1996; van Harmelen et al., 1997) (see Chapter 5). Atypical ßARs have furthermore been localized to the gastrointestinal tract, namely in the colon and ileum, where they induce smooth muscle relaxation both in rodents (De Ponti et al., 1995; Molenaar et al., 1997a,b) and in man (De Ponti et al., 1996; Roberts et al., 1997; Bardou et al., 1998). As described below, the identification of these receptors as ß 3 ARs in the gastrointestinal tract has been confirmed by immunohistochemical and RT-PCR experiments. In addition to the atypical ßARs present in adipocytes and gut, several reports discuss ‘ß3’ AR-mediated effects on the ventricle of the heart (Gauthier et al., 1996), pulmonary vasodilatation (Dumas et al., 1998; Tamaoki et al., 1998), relaxation of oesophageal sphincter (de Boer et al., 1995; Oriowo et al., 1998), urinary bladder (Oshita et al., 1997) and bile duct (de Ponti et al., 1995). Furthermore, effects of atypical ßARs on insulin secretion (Atef et al., 1996), gastrin-and somatostatin secretion in the antrum (Levasseur et al., 1997), and glucose metabolism in skeletal muscle (Challis et al., 1988; Liu et al., 1996a,b) have been reported. However, the molecular nature of these receptors remains controversial. BRL-37,344

Structure and function of the ß 3-adrenoreceptor

17

does not stimulate adenylyl cyclase in rat soleus muscle (Roberts and Summers, 1998), and only very low levels of ß3AR messengers were detected in skeletal muscle, where adipsin transcripts were also found, suggesting the co-existence of adipocytes (Evans et al., 1996). In RT-PCR experiments on heart, no ß3AR transcripts could be detected at all (Evans et al., 1996), or only together with UCP-1 messengers (Krief et al., 1993), again suggesting a ‘contamination’ with adipocytes. On the other hand, Gauthier et al. (1996) have provided not only pharmacological, but also molecular evidence, for the presence of a ß3AR (but the absence of adipocyte-specific hormone-sensitive lipase) in human heart ventricle cells. For a more complete description of the atypical ßARs present in heart and vessels, the reader is referred to Chapter 8. Only a limited number of reports exist that have used autoradiographic techniques in order to localize atypical/ß3ARs. Binding sites have been localized in the rat ileum (Roberts et al., 1995) and colon (Sugasawa et al., 1997). However, these sites may not represent ß3ARs, since they are resistant to blockade by propranolol. Although ß3AR agonists induce smooth muscle contraction in these tissues, and ß3AR messenger RNA has been detected, different cyanopindolol binding sites may co-exist in ileum (Hoey et al., 1996a; Sugasawa et al., 1997).

1.6.2

Immunodetection of the ß3AR protein

Raising and characterization of antibodies Sixteen different synthetic peptides, corresponding to the (presumed) extra-or intracellular parts of the receptor have been used to generate antibodies in rabbits (Guillaume et al., 1994). Only seven of these peptides have yielded antibodies that reacted with the whole ß3AR protein, expressed in CHO cells. Of these seven, only one peptide (‘p12’; corresponding to the C-terminal region of the ß 3AR), generated antibody titres consistently sufficient for further experimentaion. On immunoblots of membranes of cells transfected with the ß3AR gene, p12 recognized a single dominant protein of about 70 kDa, which could be displaced by preincubation with the peptide used for immunization. No cross-reactivity was seen with ß1AR or ß2AR. It was furthermore shown that p12 could be used to immunopurify the ß3AR heterologously expressed in CHO cells, E.coli or baculovirus-infected Sf9 insect cells (Guillaume et al., 1994).

Immunohistochemistry using the p12 antibody, raised against the C-terminal reeion of the ß3AR The p12 antibody was used to detect expression of the ß3AR in CHO cells and in baculovirus-infected Sf9 insect cells (Figure 1.5). When p12 was used to detect endogenously expressed ß3ARs, positive staining was achieved in subaxillary brown adipose tissue of a 34-year-old woman (S.Cinti et al., personal communication). Immunoreactivity was furthermore detected in the gallbladder of an obese patient (Guillaume et al., 1994). Recently, p12 was successfully used to immunolocalize the ß3AR to vascular and non-vascular smooth muscle in the human gastrointestinal tract. No staining was found in epithelia, vascular endothelial cells or ganglia (Anthony et al., 1998).

18

Strosberg and Gerhardt

Figure 1.5 Immunofluorescent staining of ß3AR in infected Sf9 cells. Labelling by anti-p12 of methanol-fixed infected Sf9 cells (ß3) infected by a baculovirus recombinant for the huß3AR; (WT) non-infected cells.

Western blot analysis of ileum and sigmoid colon membrane proteins revealed a band of 55 kDa, a size which could be consistent with a partially glycosylated ß3AR. Parallel Western blot experiments on murine ileum/colon did not show any immunoreactive bands, suggesting that the antibody does not cross-react with any murine proteins expressed in these tissues. Cross-reacting murine proteins have however been suggested to exist (S.Cinti, unpublished observations). When p12 was used to characterize the expression pattern of the human ß3AR in transgenic mice, knocked-out for murine ß3AR but expressing the human homologue, various tissues stained positive (see Chapter 3). However, this staining turned out to aspecific and due to a cross-reaction with at least two murine proteins containing a sequence that was partially homologous to the C-terminal region of the human ß 3 AR (J.L.Guillaume, unpublished observations).

1.6.3 Detection of the ß3AR mRNA Due to the generally low expression level of G protein-coupled receptors, PCR-based detection of ß3AR cDNA has been a method of choice to perform expression studies. In rat tissues, ß3AR mRNA was detected in WAT and BAT, smooth muscle of colon and ileum and, to a lesser extent, in stomach fundus and skeletal muscle (Evans et al., 1996). However, the last two tissues also expressed adipsin, suggesting the presence of adipocytes in these tissues. No expression was found in heart, lung and liver. Divergent results have been obtained concerning the expression of the ß3AR in human tissues (see also Chapter 5). Some studies, for example those of Krief et al. (1993), have indicated the abundant presence of ß3AR transcripts in infant perirenal BAT. In adults, in whom (almost) all fat tissue is composed of white adipocytes, ß3AR mRNA levels were high in deep fat such as perirenal and omental fat, but low in subcutaneous fat. Furthermore, ß3AR mRNA (but not UCP-1 mRNA) was highly expressed in gallbladder and weakly in colon. In contrast to these results, it has been reported (e.g. Deng et al., 1996) that human omental white adipocytes do not express

Structure and function of the ß 3-adrenoreceptor

19

ß3AR, and that in newborn BAT, they represent a minority (9%) of adrenoreceptors compared with the ß1- (28%) and ß2ARs (63%). Although these studies were made using Northern blot analysis, which is less sensitive than RT-PCR, binding studies using [3H] SB206606, done in parallel, confirmed the absence or low presence of ß3 ARs in WAT and BAT, respectively. Berkowitz et al. (1995) used the method of RNase protection without previous PCR amplification in order to detect expression of ß3AR in a variety of human tissues. ß3AR mRNA was detected in white fat, gallbladder and small intestine, and also in the stomach and prostate gland. A few reports have shown the presence of ß3AR mRNA. in brain (Rodriguez et al., 1995; Summers et al., 1995). Expression levels were higher in children than in adults (Rodriguez et al., 1995), and highest in the hippocampus, cerebral cortex and striatum, but lower in hypothalamus, brainstem and cerebellum (Summers et al., 1995). Interestingly, expression of the ß3AR in fat tissue is restricted to mature, fully differentiated adipocytes. For the murine pre-adipocyte cell line 3T3-F442A, it has been well documented that the ß3AR can be considered as a late differentiation marker (Fève et al., 1991; El Hadri et al., 1998). Likewise, pre-adipocytes of the human brown cell line PAZ-6 do not express detectable ß3AR before differentiation into adipocytes (Zilberfarb et al., 1997). In conclusion, these studies suggest that the ß3AR is most widely found to be expressed in white and brown adipose tissue, where its expression is restricted temporally (in mature adipocytes rather than in preadipocytes) and spatially (in omental rather than subcutaneous fat). Moreover, the evidence for the expression of the ß3AR in the gastrointestinal tract, gallbladder and pancreas is growing. The functional presence of ß3ARs in the brain and heart remains a matter of controversy (see Chapter 8).

1.7 Concluding remarks Even though the ß3AR has now been well studied at the gene and protein level, considerable work remains to be done to understand fully its mechanism of action. We still do not know why a number of ß1/ß2 antagonists behave as ß3AR agonists, nor do we understand species-specific differences. The molecular basis for the ability to activate different G proteins and hence different effector systems remains unexplained. In fact, we continue to avoid analysing the exact G ß combinations involved in the coupling of the three different ßAR subtypes or activation of any of the eight adenylyl cyclase subtypes or of the PI3 kinase. Finally, no effort has been made so far to identify other proteins capable of interacting with the ß3AR such as GRK, arrestin or the equivalents of proteins found to associate to receptors and discovered by the two-hybrid system in yeast technology. We hope to answer a few of these questions in the next decade of research on the ß3AR. a

g

Acknowledgements We wish to thank all the collaborators cited in our laboratory reference list who participated in completing the investigations discussed above.

2

Regulation of the ß3-Adrenoreceptor Signalling Efficacy MICHEL BOUVIER Université de Montréal, Faculté de Médecine, Département de Biochimie, Montréal (Québec), Canada H3C 3J7

2.1 Introduction One of the most fascinating features of G protein-coupled receptor signalling systems is their high degree of plasticity. This permits the cell to adapt to its environment, and may play a major role in the sorting and integration of the information detected at the receptor levels. ß-Adrenergic signalling has been shown to be the target of various regulatory processes. In particular, negative regulation and desensitization in response to sustained activation of its own or of other signalling pathways have been well documented. Alterations in such regulatory processes probably underlie certain pathological conditions related to hyper-or hyposensitivity. In particular, reduction in ß-adrenoreceptor (ßAR) density and responsiveness which accompany conditions such as hypertension, heart failure and obesity have often been attributed to processes similar to those involved in agonist-promoted desensitization. Changes in the responsiveness of the ßAR are also believed to contribute to the phenomenon of habituation and withdrawal syndrome associated with the use of ß-adrenergic agonists and antagonists in clinical settings. Since ßARs represent important pharmacological targets in the treatment of asthma, hypertension, angina pectoris, arrhythmia, heart failure and obesity, analysis of the mechanisms leading to alterations of the ßAR responsiveness may lead to the identification of new means to modulate receptor function more efficiently for therapeutic purpose. The original characterization of the mechanisms underlying regulation of the ßAR signalling efficacy was carried out mainly with the ß2AR, with the implicit assumption that similar—if not identical—processes would also be involved in the regulation of ß1and ß3AR responsiveness. Data gathered during the past five years or so invalidated this assumption. Indeed, subtype-specific regulatory processes have been delineated for the three receptors. In particular, the regulatory profile of the ß3AR was found to be dramatically different from that of the ß2AR, and the molecular basis of the differences have begun to be unravelled. In the following sections, the general concepts of G protein-coupled signalling and regulation—as they were found to apply to the ß-adrenoreceptors—will be reviewed, with special attention being given to the specificities of the ß3AR. 20

Regulation of the ß 3-adrenoreceptor signalling efficacy

21

2.1.1 The ß-adrenoreceptors and their signalling pathways ßARs play roles of primary importance in mediating responses through the central nervous system, the sympathetic branch of autonomic nervous system, and the neuroendocrine system. Their specific interaction with the endogenous catecholamines, adrenaline and noradrenaline, or with drugs represents a key step in regulating processes as diverse as neurotransmission, cellular metabolism, secretion, cellular differentiation and growth. The three subypes (ß1, ß2 and ß3) all couple via Gs to the stimulation of the adenylyl cyclase that promotes the formation of the second messenger cAMP. Whether or not the three subtypes display the same selectivity toward the four isoforms of G
s is, however, unknown. Similarly, the possibility that different Gßg-subunit complexes may selectively interact with a given receptor subtype, as has been suggested for other receptors (Kleuss et al., 1992, 1993), remains unexplored. This could have important consequences for the signalling efficacy of the ßAR subtypes, since the six known adenylyl cyclase isoforms are differentially regulated by distinct ßg subunit complexes (Federman et al., 1992; Clapham and Neer, 1993). Stimulation of adenylyl cyclase activity is believed to mediate most of the biological actions of the three ßAR through the activation of the cAMP-dependent protein kinase (PKA). However, it has also been proposed that Ga s, upon ßAR stimulation, directly activates L-type calcium channels (Yatani and Brown, 1989). It was also suggested that the Na+/H+ exchanger type-1 is under the regulatory influence of the ß2AR via its interaction with Ga13 (Barber et al., 1989, 1992; Barber, 1991; Voyno-Yasenetskaya et al., 1994). More recently, it was shown that a direct interaction of the ß2AR with the Na+/H+-exchanger regulatory factor (NHERF) has a modulatory influence on the activity of the NA+/H+ exchanger type-3 (Hall et al., 1998). Coupling of the ß2AR (Crespo et al., 1995; Bogoyevitch et al., 1996; Yamamoto, J. et al., 1997b) ß1AR (Williams et al., 1990) and ß3AR (Gerhardt et al., 1999; Soeder et al., 1999) to the ERKl/ERK2/p38 MAP kinase signalling pathways through the ßg subunit has also been demonstrated. This activation of the MAP kinase systems has been shown to be pertussis toxin-sensitive and to involve Gai (Daaka et al., 1997). Whether the three subtypes will show selective efficacies toward these effectors, remains to be investigated. This point may have important physiological implications since the distinct physiological end-points of the three ßAR isoforms could result from subtypeselective coupling to various pathways. Even with regard to the relative signalling efficacy of the three ßAR via the more classical adenylyl cyclase stimulatory pathway, relatively little data is available. The only systematic studies comparing the capacity of equivalent receptor numbers to stimulate cAMP production in the same cellular background were carried out for the ß2- and ß1AR. In three independent studies using different expression systems, the human ß2AR was found to stimulate the adenylyl cyclase activity more efficiently than the ß1AR, suggesting a better coupling of the former to G
s (Green et al., 1992; Levy et al., 1993; Rousseau et al., 1996). Unfortunately, no study systematically compared the efficacy of the ß3AR to that of the two other subtypes. However, results obtained in several studies would indicate that the coupling efficacy of the ß3AR to the adenylyl cyclase system is very similar to that of the ß2AR and thus greater than that of the ß1AR (Nantel et al., 1993, 1994). The preceding discussion thus raises the possibility that the three ßAR may be linked to various signalling pathways with subtype-selective efficacy. Until now, however, studies on the ß3AR mostly considered the Gs/adenylyl

22

Bouvier

cyclase signalling cascade. It follows that all that we know concerning the regulation of the signalling efficacy of this receptor relates to its coupling with the production of cAMP. This review will therefore focus on the mechanisms that are known to modulate cAMP-mediated signalling.

2.2 General concepts of G protein-coupled signalling regulation Constraints on signalling networks undoubtedly exist, and provide a high degree of plasticity to transduction pathways. In fact, the concerted action of many regulatory processes assures the plasticity required to adapt to various stimulatory conditions. As the initial point of interaction with the transmitters, receptors are uniquely positioned to regulate the responsiveness of a given signalling pathway. As mentioned in the general introduction, these regulatory processes play an important role in the development of altered ßAR responsiveness related to cardiovascular and metabolic diseases. Therefore they have been the object of intense research both under normal and pathological conditions. Most efforts have concentrated on the best-characterized ß2AR, and the molecular processes regulating the signalling efficacy of this receptor have begun to be unravelled. Because much is known about the regulation of ß2AR signalling efficacy, it will be used in this section as a model system to which the ß3AR will then be compared. An important regulatory mechanism of receptor function is known as agonistinduced desensitization. This is characterized by the fact that the intensity of a response mediated by the receptor wanes over time, despite the continuous presence of the stimulus. The appearance of agonist-induced desensitization of ß 2 ARstimulated adenylyl cyclase was at first thought to be related solely to the decrease in ß2AR number (down-regulation) which follows prolonged stimulation (Lefkowitz, 1979). However, it was quickly realized that the regulatory processes are far more complex, and it is now generally accepted that at least three distinct processes are involved in the desensitization phenomenon. These are known as functional uncoupling, sequestration and down-regulation (Benovic et al., 1988; Hausdorff et al., 1990). Uncoupling corresponds to a decreased receptor-mediated activation of the adenylyl cyclase with no change in receptor number or distribution. Sequestration consists of a cellular redistribution of the ß2AR from the cell surface to an intracellular vesicular fraction (Lefkowitz et al., 1980; Staehelin and Simons, 1982; Harden, 1983). Sequestration is therefore observed as a decreased receptor density at the cell surface with no change in total cellular receptor number, whereas down-regulation refers to a loss of receptor sites and results in a decrease in total ß2AR number expressed in a given cell.

2.2.1 Agonist-promoted receptor phosphorylation and receptor uncoupling Functional receptor uncoupling represents the fastest regulatory process regulating signalling efficacy. Reduction in the ß-adrenergic-stimulated adenylyl cyclase activity can be observed as rapidly as one minute following the beginning of the stimulation, and is believed to reflect a decreased capacity of the receptor to interact productively with Gs. Phosphorylation of the ß2AR has been shown to play an important role in the expression

Regulation of the ß 3-adrenoreceptor signalling efficacy

23

of such agonist-induced desensitization. At least two distinct protein kinases, PKA and the ß-adrenoreceptor kinase (ßARK) have been implicated. Whereas PKA has broad substrate specificity, ßARK demonstrates a much more restricted specificity and phosphorylates only agonist-occupied receptor (Benovic et al., 1986). Using sitedirected mutagenesis, we and others have identified several phosphorylation sites involved in desensitization (see Figure 2.1) (Bouvier et al., 1988; Clark et al., 1989; Hausdorff et al., 1989). ßARK-mediated phosphorylation of serine and threonine residues in the distal portion of the carboxyl terminus (Ct) of the receptor has been shown to promote the association of the protein ß-arrestin with the ß2AR thus inhibiting functional coupling of the receptor to Gs (Lohse et al., 1990b; Pitcher et al., 1992b). In contrast, phosphorylation of ß2AR by PKA does not favour the interaction of the receptor with ß-arrestin (Pitcher et al., 1992b), and the mechanisms by which it promotes functional uncoupling remain largely unknown. Two phosphorylation sites for PKA have been identified within the ß2AR structure (Bouvier et al., 1989). One is located in the third intracellular loop of the receptor (Ser261,262), while the other is in the proximal portion of the carboxyl tail (Ser345,346). Upon direct activation of PKA, and in the absence of agonist occupancy of the receptor, only the third loop site is phosphorylated and contributes to the desensitization (Bouvier et al., 1989; Clark et al., 1989). However, following agonist stimulation of the receptor, the site in the carboxyl tail also becomes available to the kinase and contributes to the desensitization (Moffett et al., 1996). As discussed further below, agonist-dependent regulation of the palmitoylation state of a cysteine located four amino acids upstream of this second phosphorylation site is believed to control its accessibility to the kinase (Loisel et al., 1996; Morello and Bouvier, 1996). Because of the important role of the third cytoplasmic loop in the functional interaction of the receptor with Gs, it has been proposed that the change in charge distribution imposed to this domain by the PKA-mediated phosphorylation could be responsible for the functional uncoupling. Arguing against this hypothesis, however, is the observation that substituting acidic residues for Ser261,262, to mimic the addition of the phosphate negative charges, failed to promote receptor uncoupling and desensitization (Yuan et al., 1994). More recently, however, it was proposed that phosphorylation of the third cytoplasmic loop by PKA promotes the interaction of the receptor with G
i to the expense of G
s, thus favouring the activation of the MAP kinase and reducing the activation level of the adenylyl cyclase (Daaka et al., 1997). This would suggest that phosphorylation of the third loop sites results in a change in G protein coupling specificity rather than a loss of overall signalling efficacy. For the PKA site located in the carboxyl tail, it has recently been proposed that its phosphorylation increases the rate of phosphorylation of the more distal ßARK sites in the carboxyl tail. Although the precise mechanisms by which PKA contributes to the rapid desensitization of the ß2AR remain to be clarified, it is clear that both PKA and ßARK play important roles in receptor-specific or homologous desensitization (Hausdorff et al., 1989; Lohse et al., 1990a). In addition to its role in homologous desensitization, PKA—and in some instances PKC, but not ßARK—is involved in the heterologous desensitization of the ß2AR that occurs upon activation of other receptor systems (Bouvier et al., 1991; Yuan et al., 1994). Unlike PKA and PKC, ßARK is not activated by a second messenger and its mode of activation makes it an enzyme of choice for a role in homologous desensitization. Indeed, upon agonist binding of the receptor and G protein activation, ßARK is translocated from the cytosol to the membrane and phosphorylates only the agonist-

24

Bouvier

occupied receptors (Benovic et al., 1986; Chen et al., 1993). Interaction between dissociated Gßg complexes and ßARK is responsible for the translocation of the enzyme to the membrane and has been shown directly to increase its activity (Pitcher et al., 1992a). Prenylation of the g subunit is believed to play a crucial role in this translocation and activation mechanism (Inglese et al., 1992). ßARK is only one member of a growing family of G protein-coupled receptor kinases. To date, six kinases named GRK1 to GRK6 (ßARK1 corresponding to GRK2) have been cloned and characterized. Although absolute specificity of a given kinase for a subset of receptors has not been demonstrated, every kinase is believed to have distinct selectivity patterns towards the various G protein-coupled receptors. Selectivity towards activating Gß g has also been recently reported (Daaka et al., 1997). Interestingly, two members of the family, GRK4 and GRK6, do not require any interaction with Gßg subunits to be translocated to the plasma membrane and to phosphorylate receptors. Indeed, these two kinases are palmitoylated at their carboxyl terminus, and the lipid modification was found to be responsible for membrane association and activity (Stoffel et al., 1994; Premont et al., 1996). From the previous discussion, it is clear that phosphorylation is a post-translational modification that plays a central role in modulating ß2AR signalling efficacy by regulating its functional interaction with Gs. However, during the past few years, several studies suggested that another post-translational modification of the receptor, palmitoylation of its Cys341, may also play an important role in dictating the ability of the ß2AR to couple to Gs and to stimulate adenylyl cyclase. Like phosphorylation, palmitoylation was found to be a reversible modification that is regulated by agonist stimulation. Indeed, ß2AR stimulation leads to an increased turnover rate of the receptor-bound palmitate that ultimately favours the unpalmitoylated form of the receptor (Mouillac et al., 1992; Loisel et al., 1996). This agonist-promoted depalmitoylation of the receptor is believed to increase the accessibility of its carboxyl tail to regulatory kinases, and thus to regulate the extent of its phosphorylation and desensitization. This idea is supported by the observation that mutation of the palmitoylation site greatly increases the basal phosphorylation level of the receptor that promotes its desensitization (Mouillac et al., 1992; Moffett et al., 1993, 1996). In contrast to the wealth of information concerning the enzymes catalysing the phsophorylation of the ß2AR, nothing is known about the enzymes that control its palmitoylation state.

2.2.2

Receptor sequestration; a process involved in desensitization and resensitization

Although less rapid than functional uncoupling, sequestration of receptors away from the cell surface occurs within a few minutes after the beginning of receptor stimulation. It is intuitively sensible to propose that such a mechanism which leads to the removal of receptor sites from the cell surface would greatly contribute to the reduction of responsiveness. However, over the past few years, it has become increasingly evident that the contribution of sequestration to the overall rapid desensitization is relatively modest (Lohse et al., 1990a). This is probably due to the fact that the receptors that are removed from the cell surface have already been functionally uncoupled from Gs via phosphorylation. More recently, it was in fact found that sequestration plays a more important role in the resensitization than in the

Regulation of the ß 3-adrenoreceptor signalling efficacy

25

desensitization of the system. Indeed, sequestered receptors have been shown to be dephosphorylated and returned to the cell surface in a functional conformation (Sibley et al., 1986; Yu et al., 1993; Krueger et al., 1997). It follows that upon arrest of the stimulation, sequestration plays a crucial role in rapidly restoring a control level of responsiveness to the system. Although sequestration clearly involves internalization of the receptor in an intracellular vesicular compartment, both the cellular compartments and the molecular mechanisms involved remain poorly characterized. Sequestration of the ß2AR can readily be measured using hydrophobic and hydrophilic ligands that allow a distinction to be made between cell surface (accessible to both hydrophilic and hydrophobic ligands) and the sequestered (accessible only to the hydrophobic ligands) receptors. The sequestered receptors can also be measured following subcellular fractionation, as they are associated with a light membrane vesicular fraction. However, the nature of the vesicles involved remains a matter of controversy. Using immunofluorescence, it was shown that at least in some cells, internalization of the ß2AR occurs through classical endocytosis involving clathrin-coated vesicles (Von Zastrow and Kobilka, 1992, 1994). In contrast, electron microscopic studies demonstrated that the ß 2AR is internalized through smooth vesicles that were identified as caveolae (Raposo et al., 1989; Dupree et al., 1993). Whether the differences between the studies reflect the existence of cell type-specific pathways, or else suggest that distinct internalization pathways (for example, one being involved in recycling the other in lysosomal degradation) can co-exist in the same cell remains to be clarified. With regard to the clathrin-coated vesicle pathway, recent studies (Ferguson et al., 1996) have shown that binding of ß-arrestin to the receptor plays a central role in internalization. It was demonstrated that, by binding to both the ß2AR and clathrin, ß-arrestin serves as a clathrin-adapter protein (Goodman et al., 1996). More recently, it was also shown that ß-arrestin binds to the AP1 clathrin adapter protein that can in turn attract the ß-arrestin-bound receptor to the clathrin-coated pits (Laporte et al., 1999). However, the identity of the receptor residues involved in the interaction with ß-arrestin and in the promotion of agonist-induced sequestration remains elusive. Indeed, several studies have shown that mutation of the ßARK and PKA phosphorylation sites do not abolish sequestration, demonstrating that phosphorylation is not the main determinant of sequestration (Strader et al., 1987; Bouvier et al., 1988; Hausdorff et al., 1989; Pippig et al., 1995). Since phosphorylation of the receptor is believed to be required for ß-arrestin binding, some unanswered questions concerning the role of ß-arrestin in sequestration are left open. Interestingly, some G protein-coupled receptors were found to undergo agonistpromoted sequestration in an arrestin-independent manner (Lee et al., 1998), thus suggesting that both arrestin-dependent and independent pathways may exist. The fact that the clathrin-coated vesicle pathway has been associated with the binding of ßarrestin raises the interesting possibility that the ß-arrestin-independent pathway may involve the caveolae-like vesicular system. The NPXXY motif located at the bottom of the seventh transmembrane domain of the ß2AR has been proposed as a potential determinant for sequestration. However, mutation of this motif in the gastrin-releasing peptide (Slice et al., 1994) and the type 1 angiotensin II (Hunyady et al., 1995) receptors did not affect their agonist-promoted sequestration, arguing against a general role for this sequence. More recent studies have shown that mutation of the NPXXY motif of the ß2AR have detrimental effects on many receptor functions, suggesting that the effect of this mutation on sequestration may be

26

Bouvier

rather non-specific (Gabilondo et al., 1996). Thus, the specific receptor sequence(s) or structural motif(s) involved in ß2AR sequestration remain to be determined.

2.2.3 Agonist-promoted down-regulation Following longer-term (hours) stimulation of the receptor, another phenomenon known as down-regulation also contributes to the development of agonist-promoted desensitization. This process, which is defined as a reduction of the total number of ß2AR present in the cells, involves mechanisms acting both at the level of gene expression and of intracellular receptor processing following internalization. Many lines of evidence (reviewed in Nantel and Bouvier, 1993b) support the notion that an agonist-promoted increase in the rate of receptor degradation is involved in down-regulation. Classical models suggest that this increased rate of degradation results from the targeting of the internalized receptors toward the lysosomal degradation pathway. In a recent study, Gagnon et al. (1998a) suggested a role for the clathrinmediated endocytotic pathway in such agonist-induced down-regulation. However, this dependence of the down-regulation on the clathrin-mediated endocytosis process appears to be cell type-dependent, and different degradation pathways—including a plasma membrane restricted receptor degradation—could be involved (Jockers et al., 1999). Similarly to the case of sequestration, very little is known about the molecular motifs directing the receptors towards the degradative pathways. Only one potential molecular determinant of receptor processing has been proposed. It consists of two tyrosine residues located within a predicted ß-turn in the carboxyl tail of the receptor. Mutation of these two tyrosines (Tyr 350 and Tyr354) was found to greatly impair the agonist-induced down-regulation of the ß2AR (Valiquette et al., 1990, 1993). Similar motifs involving tyrosine residues have also been shown to play important roles in agonist-promoted down-regulation of other receptors. These include the G protein-coupled M2-muscarinic receptor (Goldman and Nathanson, 1994), as well as receptors belonging to different families such as those for low density lipoprotein, poly-immunoglobulin and mannose-6-phosphate (Vega and Strominger, 1989), epidermal growth factor (EGF; Helin and Beguinot, 1991) and transferrin (Girones et al., 1991). The internalization of these receptors involves endocytosis by means of clathrin-coated vesicles, and it is believed that the tyrosine motifs favour the interaction of receptors with proteins of the clathrin-coated protein complex, termed adaptins (Collawn et al., 1990). In addition to increasing its rate of degradation, sustained stimulation of the ß2AR has been found to regulate its synthetic pathway. First, a cAMP regulatory element located in the promoter region of its gene (Collins et al., 1990), is responsible for a rapid but transient increase in the rate of transcription of the gene. However, in the cell types studied, this transiently increased transcriptional activity was not accompanied by an elevation in receptor number (Collins et al., 1989). This may be due to the fact that, shortly following this transcriptional activation, the mRNA steady-state level begins to decline and reaches levels that are significantly lower than those observed under basal conditions. This reduction in mRNA level has since been shown to result from a cAMP-dependent destabilization of ß2AR mRNA that contributes to the agonist-induced down-regulation of the receptor (Hadcock and Malbon 1988; Bouvier et al., 1989; Hadcock et al., 1989). Although the precise

Regulation of the ß 3-adrenoreceptor signalling efficacy

27

mechanisms still need to be investigated, the cAMP-dependent binding of a ‘destabilizing’ protein to the 3' untranslated domain of the ß2AR transcript has been shown to be involved.

2.3 The ß3-adrenoreceptor; a prototypic system to study subtypespecific regulation Over the years, a number of studies carried out on many G protein-coupled receptors have confirmed the importance of the mechanism described above for the regulation of their signalling efficacy. At the molecular level it was shown in several instances that, as for the ß 2 AR, phosphorylation by GRKs and second messenger-activated kinases is responsible for the functional uncoupling that leads to rapid agonist-promoted desensitization. For down-regulation, the role of tyrosine residues in the carboxyl tail of the receptors for the degradation path, and of the mRNA level regulation for the synthetic arm, have also been confirmed for several receptors. From these studies, it was often assumed that the mechanisms described for the ß 2 AR would apply to all G protein-coupled receptors. However, primary sequence analysis of receptors that are otherwise closely related to the ß 2 AR (for example, ß 1 - and ß 3 AR) reveals that many of the sites and motifs known to play important roles in ß 2 AR regulation are not strictly conserved. This could indicate either that different sites are involved, or that some of the processes do not apply universally to all G protein-coupled receptors. As discussed below, the ß 3AR represent an excellent model to study this question, as many of the regulatory sites identified for the ß 2 AR are lacking from its primary structure.

2.3.1 Comparative analysis of the structural determinants of receptor regulation As shown in Figure 2.1, in contrast with the strong sequence identity observed between the transmembrane domains of the two receptors, the sequences of the cytosolic domains are greatly divergent between the ß 3- and the ß 2AR (see also Chapter 1). This lack of conservation within the intracellular regions of the receptors also applies to the motifs that were shown to play regulatory functions for the ß2AR. Indeed, a comparison of the ß3- and ß2AR sequences reveals that neither of the two PKA phosphorylation sites found in the ß 2AR is conserved within the ß 3 AR structure. Similarly, whereas the ß2AR has six serine and five threonine residues within the distal portion of its carboxyl tail that have been proposed as potential GRK phosphorylation sites, only five serines are found within the corresponding domain of the ß3AR. Moreover, only one of these serines is located in a context that is believed to be favourable for GRK phosphorylation. Indeed, the presence of acidic residues near serines or threonines has been proposed as a requirement for GRK phosphorylation (Onorato et al., 1991) and only one serine is found within three amino acids from an aspartate or a glutamate residue in the ß3AR carboxyl tail. This contrasts with the three threonines and five serines that are found in such an acidic context within the ß 2AR tail. Also, the two consensus sequences for phosphorylation by PKC that were proposed to play a role in the heterologous desensitization of the

Figure 2.1 Primary sequences of the ß2AR and ß3AR and schematic representation of their proposed membrane topology. The one-letter amino acid code is used. The confirmed phosphorylation sites for PKA and PKC are indicated by the closed diamonds, whereas the putative ßARK phosphorylation sites (defined as S or T located close to an acidic residue within the distal portion of the carboxyl tail) are indicated by open diamonds. Arrows identify a putative down-regulation signal. The boxes numbered 1 to 4 delimit important regulatory domains for the ß2AR and their corresponding regions within the ß3AR.

Regulation of the ß 3-adrenoreceptor signalling efficacy

29

ß2AR, upon stimulation of the phospholipase C signalling pathway (Bouvier et al., 1991), are absent from the ft sequence. Finally, the two tyrosine residues of the carboxyl tail that were found to be important in the down-regulation of the ß2AR (Valiquette et al., 1990, 1993) are absent from the sequence of the ß3AR. This lack of conservation of many of the regulatory motifs that were identified for the ß2AR has led us and others to propose that these sequence differences may underlie subtype-specific regulation profiles.

2.4 The ß3-adrenoreceptor is resistant to rapid agonist-promoted uncoupling and sequestration Based on the importance that was attributed to the phosphorylation of the ß2AR for the occurrence of functional uncoupling and rapid desensitization that follows agonist stimulation, and also given the lack of putative phosphorylation site for PKA and ßARK in the ß3AR sequence, it was reasonable to propose that the latter may be more resistant to rapid agonist-promoted desensitization than the ß2AR. This hypothesis was assessed directly using heterologous expression systems that allow comparison of the regulatory profiles of the two receptors in the same cellular background. When expressed in Chinese hamster (CHW) or murine (Ltk-) fibroblasts, the human ß3AR was found to be completely resistant to rapid desensitization. Indeed, stimulation of the ß 3AR for up to 1 h did not affect receptor responsiveness, whereas similar treatment of the ß2AR led to a reduction of 25–50% of the ß-adrenergic-stimulated adenylyl cyclase activity, depending on the cell type considered (Liggett et al., 1993; Nantel et al., 1993a). A similar resistance to rapid desensitization was also observed in cells naturally expressing the ß3AR. Indeed, exposure of isolated rat adipocytes to the non-selective ß-adrenergic agonist isoproterenol did not promote any desensitization of the ß3-adrenergic-stimulated adenylyl cyclase activity, whereas the ß1-stimulated response was significantly blunted by pretreatment with the agonist (Granneman et al., 1992). A comparable refractoriness to desensitization was also observed in hamster fat cells (Carpéné et al., 1993b). In the non-adipocytic human SKN-MC neurotumour cells, sustained treatment with isoproterenol also failed to cause desensitization of the endogenously expressed ß 3 AR, while promoting a rapid desensitization of the ß1AR also expressed in these cells (Curran and Fishman, 1996). In contrast to this general observation that ß3AR-stimulated adenylyl cyclase activity does not undergo rapid desensitization, Chaudry and Granneman observed agonistpromoted desensitization of this response both in HEK-293 cells heterologously expressing the human ß3AR and in SK-N-MC cells (Chaudhry and Granneman, 1994). However, an agonist stimulation of 1 h was the shortest assessed in this study. It could therefore be proposed that the desensitization observed did not result from receptor uncoupling, but rather from processes occurring downstream of the receptor. Consistent with this hypothesis, longer-term treatment with agonists has been shown to lead to desensitization in several cell types. It has been proposed that such desensitization results from a down-regulation of G
s (Chambers et al., 1994) or from an increased activity of phosphodiesterase activity (Unelius et al., 1993). An additional technical point needs to be raised when addressing the desensitization of the ß3AR in tissues or cells that expressed other ßAR subtypes. Indeed, in contrast to the studies using heterologous cell systems that lack endogenous ßAR (for example, CHW, CHO, Ltk-), investigations carried out using

Bouvier

30

tissues or cells such as SK-N-MC or HEK-293 that endogenously express ß1 or ß2AR cannot use the non-selective agonist isoproterenol to assess ß 3AR responsiveness. Therefore, CGP-12177A was used, in many instances, as a selective partial ß 3 agonist (being a ß 2AR antagonist) to evaluate the ß3AR response. However, caution should be used when employing CGP-12177A as a selective ß3AR agonist, as it was shown also to have partial agonistic properties towards the ß1AR (Pak and Fishman, 1996). Overall, the studies carried out during the past 5 years show clearly that, in contrast to the ß2 and ß 1AR, the ß3AR does not undergo rapid functional uncoupling as a result of short-term agonist stimulation. This of course is entirely consistent with the fact that the ß3AR lacks most of the phosphorylation sites that are believed to promote the rapid functional uncoupling and that it does not become phosphorylated upon agonist stimulation (Liggett et al., 1993). The blunted ß3AR response observed in some cell types following longer-term stimulation therefore most likely results either from a down-regulation of the ß3AR number (see below) or else from processes affecting the signalling efficacy of elements located beyond the receptor itself. As discussed in Section 2.2, shortly following functional uncoupling, the ß2AR becomes sequestered in intracellular vesicles where it is no longer accessible to the natural hydrophilic ligands, but can be dephosphorylated and recycled back to the cell surface (Sibley et al., 1986; Pitcher et al., 1995). This process is believed to contribute largely to the rapid resensitization of the system that follows interruption of the stimuli (Yu et al., 1991). Given that the ß 3AR is neither phosphorylated nor rapidly desensitized, one could predict that there is no need for sequestration of this receptor. As predicted, no agonist-promoted sequestration of the ß3AR could be observed in any of the cell systems examined (Liggett et al., 1993; Nantel et al., 1993a; Curran and Fishman, 1996; Jockers et al., 1996). Indeed, sustained stimulation of cells expressing the ß3AR does not promote translocation of the receptor from the cell surface to intracellular compartments, as assessed by either the accessibility to hydrophilic ligands or subcellular fractionation. Although one can establish a teleological link between the lack of phosphorylation and the absence of agonist-promoted sequestration (there is no need to dephosphorylate a receptor that does not become phosphorylated upon activation), the causal relationship between the lack of phosphorylation and the absence of sequestration has not yet been fully established (see Section 2.6). It should be noted that the ß3AR harbours within its transmembrane domain VII the NPXXY sequence that has been proposed as a sequestration signal for the ß2AR. The lack of agonist-promoted sequestration of the ß3AR therefore suggests that this signal is either not sufficient to permit sequestration or that, as argued in Section 2.2, it does not represent a bona fide internalization signal.

2.5

Cell type-specific down-regulation of the ß3-adrenoreceptor

In contrast to the generally observed resistance of the ß 3AR to rapid agonistpromoted desensitization and sequestration, no general consensus can be easily drawn from the existing literature on the effect of longer-term stimulation on ß3AR responsiveness. In hamster, long-term stimulation did not lead to a reduction of the ß3-adrenergic-

Regulation of the ß 3-adrenoreceptor signalling efficacy

31

mediated lipolytic response even after 6 days of continuous infusion with noradrenaline (Carpéné et al., 1993b; Langin et al., 1995), suggesting that the ß3AR was not downregulated. Similarly, no desensitization nor down-regulation of the ß3AR number could be observed in 3T3-F442A adipocytes upon exposure to the ß-adrenergic agonist isoproterenol for up to 30 h (Thomas et al., 1992). In fact, a significant increase in both ß3AR mRNA and receptor number was observed in that study, while ß1AR expression declined by approximately 70% in response to the same stimuli. This increase in ß3AR number and message was attributed to a positive-feedback mediated by transcriptional activation through three cAMP responsive elements located in the receptor gene promoter region. This resistance of the ß 3AR to long-term down-regulation is, however, not universally observed. Using the same 3T3-F442A adipocyte cell line, Granneman and Lahners (1995) observed an important reduction in ß3AR mRNA levels upon sustained stimulation with isoproterenol. A reduction in ß3AR transcript was also observed in cultured hamster brown adipocytes following treatment with ß3-adrenergic agonists and dibutyryl cAMP (Klaus et al., 1995a,b) and in white adipose tissue (WAT) of mice treated in vivo with the ß3-adrenergic agonist BRL-26830. Treatment of rats with the same agonist or with isoproterenol for 8 h also led to a significant reduction of the ß3AR mRNA levels in both WAT and brown adipose tissue (BAT) (Granneman and Lahners, 1992). In that study, the agonist-promoted down-regulation of the ß3AR mRNA was shown to be accompanied by a reduction of the ß3-adrenergic-stimulated adenylyl cyclase activity in WAT, suggesting the occurrence of a functional downregulation. However, in none of the studies mentioned above was the actual number of ß3AR shown to be down-regulated as a result of the decrease in ß3AR mRNA content. In one study, Revelli and colleagues showed, using Zucker rats, that such a decrease in ß3AR mRNA levels indeed preceded the down-regulation of ß3AR number observed in interscapular BAT following a 72-h treatment with the ß-adrenergic agonist RO 16– 8714 (Revelli et al., 1992). The possibility that agonist-mediated down-regulation of the ß 3AR mRNA may play a physiologically relevant regulatory role is further supported by the fact that sympathectomy promotes an increase in the ß3AR mRNA level, whereas exposure of rats to 4°C (which increases sympathetic activity) reduces this level (Granneman and Lahners, 1992; Onai et al., 1995). This suggests that expression of the ß 3 AR is physiologically under the dynamic control of the sympathetic nervous system activity. The reasons for the difference in the phenotype of resistance to down-regulation observed in different studies, and with the different models used, are not completely understood. However, studies carried out in heterologous expression systems may have shed some light on the apparent inconsistencies and have helped better appreciate the relative resistance of the ß3AR to agonist-promoted down-regulation. Indeed, the expression of both the ß 2- and ß 3AR subtypes in the same cellular background showed clearly that although the ß3AR can undergo agonist-promoted down-regulation, it does so less efficiently and following a pattern that is distinct from that of the ß2AR. When expressed in either hamster CHW or murine Ltk- fibroblasts, the human ß2AR was found to be down-regulated more rapidly and to a greater extent than the human ß3AR expressed at comparable density. In fact, almost no down-regulation of ß3AR number could be observed in CHW cells, even following a 24-h exposure to an agonist (Liggett et al., 1993; Nantel et al., 1994), whereas a significant loss of sites could be observed only after 6 h of agonist treatment in Ltk- cells (Nantel et al., 1994). This

32

Bouvier

contrasts with the down-regulation of 20–40% of the ß2AR sites observed as early as 1 h after the beginning of the stimulation in the two cell lines. These data indicate two things: (i) that the ß3AR is less prone to down-regulation than the ß2AR when expressed in the same cellular background; and (ii) that the extent of down-regulation of the ß3AR varies significantly from one cell type to the next. Analysis of the mechanisms leading to the agonist-promoted loss of ß3AR sites in Ltk- and CHW cells helped in our understanding the distinct down-regulation patterns of the two receptor subtypes. As indicated in Section 2.2, at least two processes contribute to the down-regulation of the ß2AR number: first, an increased rate of receptor degradation; and second, a decreased rate of receptor synthesis due to a reduction in the receptor mRNA steady-state level. The increase in receptor degradation rate can be easily appreciated for the ß2AR by the agonist-promoted reduction of the receptor half-life observed in the presence of the protein synthesis inhibitor cycloheximide (Nantel et al., 1994). In contrast, agonist treatment did not affect the half-life of the ß3AR in either CHW or Ltk- cells, thus excluding the contribution of accelerated receptor degradation to the ß 3AR down-regulation. Interestingly, the two tyrosine residues that were shown to play an important role in the down-regulation of the ß 2AR are not conserved in the ß 3 AR (see Figure 2.1), suggesting that this structural difference may account at least in part for the distinct down-regulation patterns. In contrast, sustained agonist treatment of CHW or Ltk- cells expressing the ß3AR led to a reduction of the receptor mRNA concentrations that reached statistical significance after 6 h of a maximal level of stimulation (Nantel et al., 1994). This reduction which was modest (25% at 6h) and transient (mRNA levels returning to control values after 24 h of continuous stimulation) in CHW cells, was sustained and reached 60% in Ltk-cells. The good correlation between the ß3AR mRNA reduction and the loss of binding sites observed in each cell type indicates that the ß3AR downregulation results primarily from regulatory control at the mRNA level. This regulatory mechanism was found to be cAMP-dependent, as the decline in both mRNA and ß3AR receptor number could be mimicked by membrane-permeant cAMP analogues and direct stimulation of adenylyl cyclase by forskolin. The difference in the extent of ß3AR down-regulation observed between CHW and Ltk-cells was thus attributed to the fact that the receptor stimulated cAMP production less efficiently in CHW than in Ltkcells. The reasons for this cell type-specific difference remain unknown, but could include different contingents of G protein subunits, adenylyl cyclase and phosphodiesterases isoforms. Taken together, these studies suggest that ß 3 AR down-regulation results primarily, if not exclusively, from the regulation of the mRNA steady-state levels, and that the agonist-promoted degradation that contributes to accelerate and amplify the down-regulation of the ß 2 AR is not a factor involved in ß 3 AR regulation. Differences in the extent of ß 3AR down-regulation observed in various cell types or tissues could then reflect different efficacy with which ß-adrenergic stimulation can activate the cAMP-dependent mRNA regulatory pathway. It should be noted, however, that even in cells in which significant ß 3AR down-regulation was observed, the extent of ß-adrenergic-stimulated adenylyl cyclase desensitization seen after 24 h of continued stimulation was considerably less than that for the ß 2AR (Nantel et al., 1998). This most likely reflects the absence, for the ß3AR, of the other mechanisms (uncoupling, sequestration) that contribute to the ß 2AR desensitization.

Regulation of the ß 3-adrenoreceptor signalling efficacy

33

The regulation of the ß3AR expression through modulation of its mRNA level is not unique to the process of homologous agonist-promoted down-regulation. Indeed, various stimuli including treatment with dexamethasone (Fève et al., 1992), insulin (El Hadri et al., 1998), phorbol ester (El Hadri et al., 1998) and butyrate (Krief et al., 1993) have been shown to promote heterologous down-regulation of both ß3AR transcript and binding sites in various cells.

2.6 Use of chimeric ß3/ß2-adrenoreceptors to delineate regulatory motifs The above sections have conveyed the notion that structural differences between the ß3AR and the ß2AR may underlie their subtype-specific down-regulation profiles. In order to test this hypothesis formally, chimeric ß 2/ß3 receptors in which specific domains of one subtype were transferred into the equivalent position within the other receptor were generated. The construction of such chimeric receptors offers a powerful tool to study the molecular determinants of subtype-specific regulation. In particular, it has the advantage of allowing a search to be made for a gain of regulatory phenotypes rather than for a loss, as is often the case in classical site-directed mutagenesis studies. Substituting the third intracellular loop (i3) and carboxyl tail (Ct) of the ß2AR for their counterparts within the ß3AR allowed confirmation to be made that the resistance of the ß3AR to rapid agonist-promoted desensitization resulted at least in part from the absence of PKA and GRK phosphorylation sites. Indeed, the substitution of the ß2ARderived i3 and Ct (that harbour the PKA and ßARK phosphorylation sites) within the ß3AR restored the ability of this receptor to become rapidly desensitized upon agonist stimulation (Liggett et al., 1993; Nantel et al., 1993a). Interestingly, however, the level of desensitization attained by the chimeric receptor was significantly less (by ~50%) than that observed for the ß2AR, suggesting that additional motifs would be required to fully restore a ß2-like desensitization profile. The construction of additional chimeric receptors including substitution of i1, i2, i3 and Ct individually or in various combinations allowed identification of the second cytoplasmic loop as being an important contributor to the desensitization phenotype (Jockers et al., 1996). The presence of the ß2AR i2 alone within the ß3AR restored approximately 20% of the desensitization normally observed with the ß 2 AR, whereas the simultaneous substitution of i2, i3 and Ct restored a rapid desensitization phenotype that was quantitatively indistinguishable from that of the ß2AR. Therefore, in addition to confirming the regulatory importance of the i3 and C t phosphorylation sites, the use of chimeric receptors allowed the proposal to be made that residues within the second cytoplasmic loop of the ß 2 AR also play a role in rapid agonist-promoted desensitization. However, the identity of the specific residues involved, and the mechanism by which they contribute to the desensitization process, remain to be determined. The chimeric receptor constructs also provided information concerning the determinants of agonist-promoted sequestration. Indeed, as reviewed above, the ß3 AR does not become sequestered upon agonist stimulation, but substitution of the ß2AR Ct within the ß3AR allowed some sequestration of the receptor (Liggett et al., 1993; Jockers et al., 1996). This result is consistent with the hypothesis that phosphorylation of the receptor by ßARK and the subsequent binding of ß-arrestin could play an important role

34

Bouvier

in agonist-promoted sequestration (see Section 2.2.2). This ß2AR domain was, however, not the only one that could confer a sequestration phenotype to the ß3AR. Indeed, substitution of i2 alone had similar effects to that of the substitution of the Ct on the sequestration profile. Furthermore, their simultaneous substitution had additive effects, and led to a sequestration phenotype very similar to that of the ß2AR. The fact that the i2 alone conferred agonist-promoted sequestration to the chimeric receptor is difficult to reconcile with the hypothesis that phosphorylation of the receptor by ßARK is a prerequisite for sequestration, but may suggest that this domain represent a binding site for ß-arrestin, even in the absence of phosphorylation. Although questions remain unanswered concerning the precise determinants involved in some aspects of desensitization and sequestration, there is no doubt that the use of chimeric receptors has confirmed the hypothesis that the resistance of the ß3AR to rapid desensitization is an intrinsic property of the receptor itself which results from the absence of specific regulatory motifs in its primary sequence.

2.7 Potential physiological consequences of the relative resistance of the ß3-adrenoreceptor to desensitization Teleologically, it has been proposed that the refractoriness of the ß3AR to desensitization could be the trademark of an emergency receptor that would become activated only in extreme situations upon a high level of stimulation, and thus should not undergo rapiddesensitization. This notion is supported by the fact that the affinity of the ß3AR for its known endogenous ligands, adrenaline and noradrenaline, is significantly lower that that of the other adrenoreceptors. One could then argue that the physiological contribution of the ß3AR to energetic metabolism would come into play only upon very high level of sympathoadrenal activation at a time when both ß1- and ß2AR have already been desensitized. The ß3AR-stimulated lipolysis would then proceed in adipose tissue for an extended period of time as an ultimate source of energy. In order for such a receptor to play a valuable role, resistance to the mechanism that normally leads to rapid desensitization would be an imperative feature. The relative resistance of the ß 3AR to desensitization may also have clinical implications. Indeed, the lipolytic action of the ß 3AR and its almost exclusive expression into adipose tissues, make it a prime target for the development of antiobesity agents. The observations that both the number and the signalling efficacy of the ß3AR are decreased in the ob/ob mice model of obesity (Collins et al., 1994; Begin-Heik, 1996) have reinforced this notion. It follows that selective ß3-adrenergic agonists have been developed with the idea of generating drugs that could promote weight loss with limited side effects. Treatment for 5–7 weeks with the selective ß3 agonist CL-316,243 has been shown to induce a significant weight loss associated with a reduction of abdominal fat in dogs. The fact that the ß3AR cannot undergo rapid desensitization may have important consequences in that context. It may suggest that agonists targeted at the ß3AR will not lead to the development of rapid tolerance that often limits the effectiveness of agonist drug therapy. However, as discussed above, long-term agonist-promoted down-regulation has been observed to some extent in cultured cell lines and animal tissues. Moreover, desensitization occurring down-stream of the receptor itself in the signal transduction pathway could lead to some tolerance. Because some of these processes have been shown to be cell type-specific, the tachyphilactic properties of ß 3-adrenergic agonists that

Regulation of the ß 3-adrenoreceptor signalling efficacy

35

could be clinically useful in humans, will need to be assessed directly in human adipocytes. If some form of desensitization is observed in these cells, a detailed understanding of the molecular processes that are involved will be required in order to develop therapeutic strategies that would minimize tolerance.

2.8 Conclusions Regulation of the G protein-coupled receptor’s signalling efficacy is a well-recognized phenomenon that has important pathophysiological implications. It may also have a direct impact on the therapeutic efficacy and undesirable effects of drugs which act through this class of receptor. Among the various regulatory mechanisms identified, agonist-promoted desensitization, that leads to a loss in tissue responsiveness to endogenous hormones in some pathological conditions and to the development of tolerance in the course of specific drug therapy, has been particularly well characterized. However, studies carried out during the past ten years have shown clearly that, despite the generality of this phenomenon, different receptors have distinct desensitization patterns. In particular, the ß3AR was found to be relatively resistant to agonist-promoted desensitization when compared with the other ßAR subtypes. Indeed, due to the lack of regulatory phosphorylation sites within its sequence, no rapid receptor uncoupling nor receptor internalization could be observed in any of the tissues or cell types studied, indicating that the desensitization refractoriness is an intrinsic property of the receptor that finds its source in its primary structure. In contrast, the extent of longer-term desensitization resulting in part from the regulation of the ß3AR gene expression via mRNA destabilization was found to be cell type-dependent. The role that this type of regulation might play in pathophysiological conditions or in limiting the efficacy of anti-obesity therapies based on the use of ß3AR agonists remains uncertain. Further studies carried out on physiologically relevant tissues and cells such as human adipose tissues and human adipocytes obtained from normal and obese subjects should help to clarify this important question.

3

Using Transgenic and Gene Knockout Techniques to Assess ß3-Adrenoreceptor Function BRADFORD B.LOWELL, VEDRANA S.SUSULIC1, DANICA GRUJIC2 AND MORIKO ITO 3 Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA 1 Present address: Wyeth-Ayerst Research, CN 8000, Princeton, NJ 08543, USA 2 Present address: Transkaryotic Therapeutics, 195 Albany St., Cambridge, MA 02139, USA 3 Present address: Novartis, Basel, Switzerland

3.1 Introduction Given that ß3-adrenoreceptors (ß3AR) are abundantly expressed on white and brown adipocytes of rodents, and that ß3AR-selective agonists have potent effects on the function of white and brown adipocytes, it has been presumed that ß3AR play a critical role in mediating effects of the sympathetic nervous system on white and brown adipocytes. However, it has been difficult to test this hypothesis critically, given the general unavailability of ß3ARselective blockers. As a means of assessing the functional importance of ß3AR, our laboratory (Susulic et al., 1995) and another group (Revelli et al., 1997) have used gene targeting to create mice which lack ß3AR. Like any method, the gene knockout approach has advantages and disadvantages. Since gene knockout mice have a complete and selective absence of ß3AR, there are no concerns regarding the incompleteness or non-specificity of ‘blockade’. However, because ß3AR are absent from the first day of embryonic life, questions regarding long-term compensation for loss of ß3AR arise. Despite this and other potential limitations, the gene knockout approach is one of many important tools for assessing adrenoreceptor function in vivo. By generating gene knockout mice lacking a given receptor, it is possible to establish unequivocally the relationship between a cloned receptor and pharmacologically defined activities—an issue which is sometimes a source of controversy.

3.2

3.2.1

Mice lacking ß3AR

Phenotype of mice lacking ß3AR

As shown in Figure 3.1, homologous recombination was used to create mice which lack ß3AR (Susulic et al., 1995). As expected, mice with targeted disruption of the ß3AR gene 36

Techniques to assess ß3-adrenoreceptor function

37

Figure 3.1 The ß3AR gene, targeting vectors and recombinant allele. Shown here is a partial restriction enzyme map of the ß3AR gene, the ß3-knockout (KO) targeting vectors and the predicted structure of the recombinant allele. The empirically determined map is consistent with a previously reported genomic map (Nahmias et al., 1991). The targeting vectors contain 12 kb of homologous ß3AR genomic DNA, with 5 kb located 5' and 7 kb located 3' of the PGKNEO-PolyA cassette. The PGK-NEOPolyA vector replaces 306 bp of ß3AR coding sequence between Nhel and Xhol, corresponding to ß3AR residue 120, in the middle of the third transmembrane domain, to residue 222, at the C-terminal end of the fifth transmembrane domain. Boxes refer to exons, the locations of which have been described previously (Granneman et al., 1992; van Spronsen et al., 1993). The translated segments are shown in black. Arrow refers to orientation of transcription. B, BamHl; H, Hind III; N, Nhel; P, Pstl; S, Sall; X, Xhol. (Figure reproduced from Susulic et al., 1995.)

lack intact ß3AR mRNA, and fail to respond to the ß3AR-selective agonist, CL-316,243 (to be discussed later in greater detail). From this it can be concluded that ß3AR gene knockout mice lack functional ß 3AR. Surprisingly, the phenotype of ß3AR gene knockout mice is relatively mild. Brown adipose tissue weight, protein content, DNA content and UCP1 protein content in mice maintained at 23°C or at 4°C is unaffected by the absence of ß3AR (Susulic et al., 1995). However, more recently (in unpublished observations), we have noted that the body temperature and UCP1 mRNA response to cold exposure is sometimes impaired in ß3AR gene knockout mice. This issue is presently being explored in greater detail. Total body fat content is slightly increased in ß3AR gene knockout mice (Susulic et al., 1995; Revelli et al., 1997). This increase in body fat supports the view that ß3AR play a role in maintaining energy homeostasis; however this effect—as observed in gene knockout mice—is not large. The lack of a major phenotype in ß3AR gene knockout mice suggests the following two possibilities: (i) that ß3AR play only a small role in regulating energy homeostasis; and (ii) that other gene products compensate for the loss of ß3AR. Given that ß1, ß2 and ß3AR all recognize the same extracellular ligands (noradrenaline and adrenaline) and signal via adenylyl cyclase to increase cAMP levels, and that ß1 and ß2ARs are expressed in white and brown adipocytes, it is reasonable to speculate that ß1 and/or ß2AR compensate for the absence of ß3AR. Indeed, we noted that ß1AR mRNA levels were up-regulated by 76% in brown fat and by 42% in white fat of ß3AR gene knockout mice (Susulic et al., 1995). However, these findings are in contrast to that of

38

Lowell, Susulic, Grujic and Ito

Revelli et al. (1997), where ß1AR mRNA levels were actually found to be 66% lower in brown fat of ß3AR gene knockout mice. The reason for this difference is unknown, but could be due to differences in background strain between the two studies. It is also possible that ß1 and ß2AR activity may have increased, independent of effects on gene expression. For example, it is known that agonist exposure results in the desensitization of ß1- and ß2AR. This raises the possibility that an absence of ß3AR, with its predicted decrease in adrenergic tone, might result in compensatory increases in the activity of ß1- and ß2AR.

3.2.2 Effects of ß3AR deficiency on catecholamine-mediated stimulation of adenylyl cyclase and lipolysis in adipocytes In normal mice, CL-316,243—a ß3AR-selective agonist—increases adenylyl cyclase activity in white and brown adipocyte membranes by 4- to 8-fold, and stimulates lipolysis in isolated white adipocytes by approximately 5-fold. These effects are completely absent in membranes and adipocytes isolated from ß3AR gene knockout mice (Susulic et al., 1995). Such results demonstrate that ß3AR gene knockout mice lack functional ß3AR. Isoproterenol is a ß1, ß2 and ß3AR agonist, and its effects on adenylyl cyclase activity and lipolysis in ß3AR gene knockout mice are of interest (Figure 3.2) (Susulic et al., 1995). The ability of isoproterenol to stimulate adenylyl cyclase activity maximally, was markedly impaired in membranes derived from ß3AR gene knockout mice. Specifically, maximally stimulated adenylyl cyclase activity was decreased by 70–80% in membranes derived from white and brown adipocytes, an observation which suggests that much of ßAR signalling in adipocytes is mediated, in large part, by ß3AR. With regards to a functional response such as lipolysis, however, the effect of ß3AR deficiency on adrenergic signalling is more complex, being heavily influenced by the presence or absence of negative effects on adenylyl cyclase activity (Susulic et al., 1995). Isolated adipocytes incubated in vitro normally produce adenosine, which activates adipocyte A1-adenosine receptors. These A1-adenosine receptors couple negatively with adenylyl cyclase, thus decreasing lipolysis. In order to avoid the confounding effects of adenosine during incubation of adipocytes, it is common to add adenosine deaminase, which degrades endogenously produced adenosine. When adipocytes were incubated in the presence of adenosine deaminase, isoproterenol-stimulated lipolysis was decreased by only 33% in adipocytes from ß3 AR gene knockout mice when compared with adipocytes from control mice. However, when adipocytes were incubated with adenosine deaminase and PIA (N 6[R-(–)-1-methyl-2-phenyl]adenosine), an A1-adenosine receptor agonist, isoproterenol-stimulated lipolysis was completely absent in ß 3 AR-deficient adipocytes. In other words, in white adipocytes isolated from control mice, PIA inhibited isoproterenol-induced lipolysis minimally, whereas in adipocytes isolated from ß3AR-deficient mice, PIA completely blocked isoproterenol-induced lipolysis. Thus, the ability of A1-adenosine receptors to inhibit catecholamine-induced lipolysis is critically dependent upon the abundance of ßAR (all three ßAR in control mice versus only ß1- and ß2AR in knockout mice). These findings demonstrate that the ratio of G i -coupled receptor activity to total ßAR activity has marked consequences for functional responses such as lipolysis.

Techniques to assess ß3-adrenoreceptor function

39

Figure 3.2 Adenylyl cyclase activity and lipolysis in response to CL-316,243 and isoproterenol. (A) Adenylyl cyclase activity. Membranes were obtained from isolated white adipocytes and brown adipose tissue of 8 to 12-week-old male wild-type (+/+) and ß3AR-deficient (-/-) littermates, and then assayed for adenylyl cyclase activity. Adenylyl cyclase response to maximally effective doses of CL-316,243 (CL) and isoproterenol (ISO) are shown. Results are expressed as the mean (±SEM) of 10 experiments. (B) Lipolysis in isolated white adipocytes. White adipocytes were isolated from epididymal fat pads of 8- to 12-week-old male wild-type (+/+) and ß3AR-deficient (-/-) littermates and then assayed for glycerol release as an indicator of lipolysis. Previous studies using wild-type adipocytes demonstrated that 10µM CL and 100µM isoproterenol produced maximal increases in lipolysis. Lipolysis. assays were performed in the presence of adenosine deaminase (ADA) and N6-phenyliso-propyladenosine (PIA) (left panel, +ADA, +PIA) or with adenosine deaminase only (right panel, +ADA, -PIA). Results are expressed as the mean (±SEM) of three experiments. (Figure reproduced from Susulic et al., 1995.)

These findings most likely relate to the observation that in mouse white adipocytes, ß1- and ß2AR mRNA transcripts are only 1/50 to 1/150 as abundant as ß3AR mRNA transcripts (Collins et al., 1994), implying that white adipocytes possess many more ß3AR than ß1- and ß2ARs (Figure 3.3). Consequently, maximal activation of ß1- and/or ß2AR produces a small stimulatory effect on adenylyl cyclase activity, and this smaller effect is more readily inhibited by negative influences on adenylyl cyclase activity, such as that induced by PIA treatment. This observation could have important implications for signalling in human white adipocytes, which possess few ß3AR and abundant a2AR (Lafontan and Berlan, 1995). a2AR, like A1-adenosine receptors, are negatively coupled to adenylate cyclase.

40

Lowell, Susulic, Grujic and Ito

Figure 3.3 Regulation of lipolysis in mouse adipocytes (a working model). ß3AR are significantly more abundant than ß1- and ß2AR in rodent adipocytes. In adipocytes from a wildtype mouse, combined stimulation of ß1, ß2 and ß2AR by isoproterenol easily overcomes the inhibitory influence of the A1-adenosine receptor on stimulation of lipolysis. In contrast, in gene knockout mice which lack ß3AR, inhibition via the A1-adenosine receptor easily overrides the stimulatory effect of the less abundant ß1- and ß2AR.

3.2.3

Effects of ß3AR deficiency on in vivo effects of CL-316,243

In normal mice, CL-316,243 increases whole body energy expenditure, increases insulin levels acutely, and decreases food intake acutely. CL-316,243 treatment also markedly reduces leptin gene expression in adipose tissue (Mantzoros et al., 1996). All of these actions are completely absent in ß3AR gene knockout mice (Susulic et al., 1995; Mantzoros et al., 1996). In fact, we have been unable to identify any response to CL316,243 which is preserved in ß3AR gene knockout mice. Thus, it can be concluded definitively that the actions of CL-316,243 are mediated exclusively by ß3AR. CL316,243 does not appear to interact with any additional receptors in causing the effects listed above; thus, there can be little doubt that CL-316,243 can be used as a reference standard for ß3AR in mice.

3.3 Role of ß3AR on white versus brown adipocytes in mediating effects of ß3-selective agonists on energy expenditure, insulin secretion and food intake As mentioned above, acute treatment of normal mice stimulates whole body energy expenditure 2-fold, increases insulin secretion 50- to 140-fold (Yoshida, 1992; Susulic et al., 1995) and decreases food intake by 45% (Tsujii and Bray, 1992; Himms-Hagen et al., 1994; Susulic et al., 1995). The relative role of ß3AR in white versus brown adipocytes, as well as ß3AR in other sites in mediating each of these effects has been unknown. In general, it has been difficult to determine the relative role of various target tissues in mediating complex physiological responses. Genetic engineering in mice, however, provides a means by which these issues can be addressed. In this respect, we

Techniques to assess ß3-adrenoreceptor function

41

Figure 3.4 Transgenic tissue-specific re-expression of murine ß3AR in knockout mice. ß3AR gene knockout mice were bred to produce embryos which are homozygous for the ß3AR gene knockout allele. These embryos were injected with either the aP2-ß3AR transgene or the UCPß3AR transgene to generate mice which express ß3AR in white and brown fat only (WAT+BATmice), or in brown fat only (BAT-mice), respectively.

have combined gene knockout and transgenic techniques to create mice in which functional ß3AR are completely absent (knockout mice; Susulic et al., 1995), or are expressed exclusively in selected tissues, namely white and brown adipose tissue (WAT+BAT-mice), or brown adipose tissue only (BAT-mice) (Grujic et al., 1997). To create WAT+BAT-mice and BAT-mice (Figure 3.4), transgenic constructs were generated in which murine ß3AR gene expression is driven by the tissue-specific promoter/ enhancers, aP2 for white and brown adipose tissue expression (Ross et al., 1990) and UCP1 for brown adipose tissue expression (Boyer and Kozak, 1991; Cassard-Doulcier et al., 1993). These transgenic constructs were then injected into fertilized mouse zygotes homozygous for the ß3AR gene knockout allele (Susulic et al., 1995), thus creating mice in which functional ß 3 AR are restricted to white and brown fat (WAT+BAT-mice), or brown fat only (BAT-mice). Control, knockout, WAT+BAT and BAT-mice were then used to investigate the relative role of ß3AR in white versus brown adipose tissue, as well as to rule out involvement of ß3AR in other sites, in mediating a number of responses to ß3-selective agonists. As will be summarized below, the effects of CL-316,243 on energy expenditure, insulin secretion and food intake were found to be mediated exclusively by ß3AR on white and brown adipocytes.

3.3.1

Role of white versus brown adipocyte ß3A R in mediating effects of CL-316,243 on energy expenditure

In general, it is assumed that the stimulatory effect of CL-316,243 on energy expenditure is mediated by ß3AR on brown adipocytes. In order to test this hypothesis, control, knockout, WAT+BAT and BAT-mice were treated with CL-316,243 and effects on energy expenditure (oxygen consumption) were assessed. As before, CL produced

42

Lowell, Susulic, Grujic and Ito

a large increase in oxygen consumption in control mice, and no response in knockout mice. In WAT+BAT mice, CL produced a full stimulation of oxygen consumption. In BAT-mice, however, CL produced only a partial stimulation of oxygen consumption (20% of control response). These findings indicate that the full thermogenic response to acute ß3-agonist treatment requires the presence of ß3AR on both white and brown adipocytes. The mechanism by which stimulation of white adipocytes greatly augments the thermogenic response is unknown, but could be related to mobilization of free fatty acids which are then used as substrates by brown fat. Alternatively, ß3agonist-stimulated white adipocytes, which express high levels of uncoupling protein2 (Fleury et al., 1997; Gimeno et al., 1997), might contribute directly to energy expenditure.

3.3.2

Role of white versus brown adipocyte ß3AR in mediating effects of CL-316,243 on Insulin secretion

The mechanism by which ß 3-selective agonists acutely cause large increases in insulin secretion has been unknown. This is especially true since pancreatic beta cells do not express ß3AR. In order to test the role of adipocytes in mediating this response, control, knockout, WAT+BAT and BAT-mice were treated with CL316,243 and effects on insulin secretion were assessed. CL produced a large increase in insulin secretion in control and WAT+BAT mice, but not in knockout or BAT-mice. This demonstrates that ß 3AR on white adipocytes are required for this response. Thus, a signal emanating from white adipocytes can, directly or indirectly, profoundly alter pancreatic beta-cell function. This signal may be free fatty acids, which are known to be secretagogues for insulin secretion (Prentki et al., 1992; Warnotte et al., 1994).

3.3.3

Role of white versus brown adipocyte ß3AR in mediating effects of CL-316,243 on food intake

Although acute treatment with ß3AR-selective agonists acutely decreases food intake (Tsujii and Bray, 1992; Himms-Hagen et al., 1994; Susulic et al., 1995; Mantzoros et al., 1996), the mechanism for this effect has been unknown. In order to test the role of adipocytes in mediating this response, control, knockout, WAT+BAT and BAT-mice were treated with CL-316,243 and effects on food intake were assessed. CL reduced food intake in control and WAT+BAT mice, but not in knockout or BAT-mice. Like the insulin secretion response, ß3AR on white adipocytes are required for inhibition of food intake. Since expression of ß3AR in adipose tissue (WAT+BAT-mice) completely restored the inhibitory food intake response, it can be concluded that ß3AR in other sites such as the brain are not involved. The nature of the signal emanating from adipose tissue which mediates the inhibitory effect of ß3-selective agonists on food intake is unknown. Leptin is a fat-derived protein that regulates appetite (Zhang et al., 1994); however, it is unlikely to meditate this response since leptin levels decrease substantially following ß3-agonist treatment (Giacobino, 1996; Mantzoros et al., 1996), and this would be predicted to have a stimulatory effect on food intake. Insulin is another factor that has been shown to suppress food intake (Woods and Gibbs, 1989; Woods et al., 1990). Since insulin levels rise acutely following ß3-agonist treatment, it

Techniques to assess ß3-adrenoreceptor function

43

could mediate decreased food intake. Finally, heat has long been known to have inhibitory effects on appetite (Strominger and Brobeck, 1953; Brobeck, 1960), and it has been postulated that heat generation by brown fat regulates food intake (HimmsHagen, 1995a,b). Increased heat production could be responsible for ß3-agonistinduced inhibition of food intake.

3.4

Creation of mice which express human, but not murine, ß3AR

Important similarities and differences exist between human and rodent ß3AR, and these differences have significant implications for the development of anti-obesity drugs. The receptors are similar in that they are both expressed predominantly in adipose tissue, their amino acid sequences are about 80% identical, and their pharmacological profiles are similar in that both receptors are relatively resistant to blockade by conventional ßAR antagonists and both are stimulated by ß3AR-selective agonists (Emorine et al., 1989; Granneman et al., 1991; Muzzin et al., 1991; Nahmias et al., 1991). Human and rodent ß 3AR differ, however, in two important ways. First, their relative expression in white versus brown adipocytes, and second, the degree to which they can be stimulated by specifie ß3AR-selective agonists. In rodents, ß3AR mRNA is abundant in WAT and BAT (Granneman et al., 1991; Muzzin et al., 1991; Nahmias et al., 1991), while in humans, ß 3AR mRNA is abundant in BAT only (Granneman et al., 1992; Granneman and Lahners, 1994; Granneman, 1995), with much less (Krief et al., 1993; Revelli et al., 1993; Berkowitz et al., 1995) or no (Thomas and Liggett, 1993) ß 3AR mRNA being found in WAT. In contrast with analyses of mRNA expression, some pharmacological studies support the existence of ß3AR in human white adipocytes (Lönnqvist et al., 1993; Enocksson et al., 1995; Hoffstedt et al., 1996a; Tavernier et al., 1996), though another study did not provide such support (Rosenbaum et al., 1993). For the most part, pharmacological evidence supporting the existence of ß3AR in human white adipocytes depends upon the demonstration that CGP-12,177, a ß1- and ß2AR blocker with partial ß3AR agonist activity, stimulates lipolysis in human white adipocytes. However, the significance of this finding in the absence of abundant ß3AR mRNA expression is unknown since CGP-12177 might possibly interact with another, as yet unidentified, receptor (Galitzky et al., 1997) (see below). Human ß3AR also differ from rodent receptors with respect to their ability to be activated by ß3AR-selective agonists. Many agonists which are extremely potent against the murine ß3AR, such as CL-316,243 and BRL-37,344, are only weakly effective against the human ß3AR (Blin et al., 1994; Dolan et al., 1994). As might be expected, these agents have not performed well as anti-obesity compounds in human clinical trials. Presently, cell lines expressing recombinant human ß3AR are being used to screen for compounds capable of potently activating the human ß3AR. In order to address differences in sites of expression and pharmacology between human and rodent ß 3AR, we have transgenically introduced a P1 genomic clone containing a large fragment of the human ß3AR genomic locus into ß3AR gene knockout mice (Ito et al., 1998) (Figure 3.5). Using a sensitive RNase protection assay, we observed that human ß3AR mRNA was expressed almost exclusively in BAT of mice transgenic for the human ßAR P1 genomic clone (Ito et al., 1998) (Figure 3.6), with little or no expression being

44

Lowell, Susulic, Grujic and Ito

Figure 3.5 Transgenic expression of human ß3AR in knockout mice. Embryos homozygous for the ß3AR gene knockout allele were injected with a P1-genomic clone bearing the human ß3AR genomic region.

Figure 3.6 RNase protection analysis of human (HU) ß3AR gene expression in multiple tissues. An RNase protection assay was used to detect human ß3AR mRNA in RNA samples isolated from brown adipose tissue (B), perigonadal white adipose tissue (W), liver (L), stomach (ST), small intestine (SI) and muscle (M). The RNA samples were isolated from tissues obtained from four different transgenic lines (H2, H3, H4 and H10). All RNase protection analyses were performed using 40 µg of total. K, ß3AR gene knockout mice; T, human ß3AR transgenic mice on the ß3AR gene knockout background. (Figure reproduced from Ito et al., 1998.)

detected in three WAT depots (perigonadal, inguinal and perirenal). Given the large size of the genomic clone employed in this study (~33 kb of 5' flanking sequence and ~44 kb of 3' flanking sequence), and that four out of four transgenic lines generated the same result, it is extremely likely that human versus rodent cis-regulatory elements within the ß3AR gene differ with respect to their ability to direct gene expression to white versus

Techniques to assess ß3-adrenoreceptor function

45

brown adipocytes. The human ß3AR cis-regulatory elements direct expression to brown, but not white, adipocytes while the rodent cis-regulatory elements direct expression to both brown and white adipocytes. Our finding that human ß3AR cis-regulatory elements are active in brown but not white adipocytes is in agreement with negative studies of mRNA expression in human WAT samples (Thomas and Liggett, 1993; Granneman and Lahners, 1994), suggesting that ß3AR do not exist or are rare in human white adipocytes. This conclusion then raises the possibility that CGP-12,177-stimulated lipolysis in human white adipocytes is mediated by another ‘atypical’ receptor. Functional evidence for an additional ‘atypical’ receptor, i.e. a ‘ß4AR’, has recently emerged, and will be discussed below in greater detail. Given that these transgenic animals express human but not murine ß3AR in a ‘humanlike’ pattern, i.e. in brown but not white fat, they should be useful in evaluating the potential efficacy of newly identified ß3AR-selective agonists which are potent for the human ß 3 AR. In order to assess this possibility, we have observed that acute administration of CGP-12,177 causes oxygen consumption in ‘humanized’ mice to increase by 91%, compared with an increase of only 49% in ß3AR gene knockout controls (Ito et al., 1998) (Figure 3.7). The stimulatory effect observed in the control animals (ß3AR gene knockout mice) is presumably mediated by an additional receptor (possibly the ‘ß4AR’ referred to earlier). Since the only difference between ‘humanized’ mice and gene knockout mice is the presence of the human ß3AR transgene, the larger effect of CGP-12,177 on oxygen consumption in ‘humanized’ mice strongly indicates that human ß3AR in these transgenic mice can effectively couple with increased energy

Figure 3.7 In vivo effects of CGP-12,177 on O2 consumption. ß3AR gene knockout mice (KO) and human transgenic mice (lines H3 and H4) on the ß3AR gene knockout background were treated with CGP-12,177 (s.c. injection of 1 mg/kg body weight) and effects on O2 consumption were assessed. Mice were awake and unrestrained for analysis. The results are expressed as % change in Vo2 over basal (i.e. pre-injection resting oxygen consumption), and are the mean (±SEM) for each group. Basal and post-injection Vo2 are the averages of seven consecutive measurements. Numbers of animals for each group were as follows: KO=11; H4=6. *=P900-fold versus ß1and ß2AR-mediated effects) inhibitor of rat distal colon that stimulates the human cloned ß3AR (Dow, 1997; Yamamoto et al., 1997a). FR-165914 is a less potent relaxant of rat intestinal preparations (Hattori et al., 1995). The Dainippon compound AD (or AJ)-9677 was a potent full agonist of the human cloned ß3AR in the system studied, and more than 100-fold less potent and a partial agonist at human cloned ß1- and ß2ARs (Harada et al., 1997). The Bristol-Myers Squibb compound BMS-187413 is approximately 10-fold more potent as a stimulant of the human cloned ß3AR than BRL-37344 (Sher et al., 1997), but in view of its similar structure it seems unlikely that it has significantly better efficacy at the human receptor. The structures of BMS-194449 (‘clinical candidate’), BMS196085 (‘selected for evaluation in clinical trials’) and BMS-210285 (‘pre-clinical backup to BMS-196085’) (Gavai et al., 1998; Sher et al., 1998; Washburn et al., 1998) seem more likely to give good efficacy at the human receptor.

Arch

66

The Merck compound L-757,793 has weak partial agonist activity at the human ß1AR, but only at concentrations 1000-fold higher than those that activate the ß3AR (EC50=0.43 nM). It displays 500-fold selectivity over binding to the ß2AR (Naylor et al., 1998; Weber et al., 1998b). L-770,644 is a partial agonist at both ß1- and ß2ARs and is less selective than L-757,793. L-771,047 is a potent (0.9 nM) full ß3AR agonist with more than 1000-fold selectivity over binding to ß1- and ß2ARs (Weber et al., 1998b; Mathvink et al., 1999). L-760,087 and L-766,892 (Parmee et al., 1999) and L-764,646 (Naylor et al., 1998) are less potent ß3AR agonists than L-771,047, and only L-764,646 approaches L-771,047 in selectivity over binding to ß1- and ß2ARs. The functional activity of these compounds at ß1- and ß2ARs has not been described. SM-11044 (Sumitomo) showed moderate selectivity as a relaxant of guinea-pig ileum via the ß3AR relative to its potencies in guinea-pig atrial and tracheal preparations. It was a full agonist of the human cloned ß3AR, but not selective relative to ß2AR stimulation (Sugasawa et al., 1992). Tecradine® induces rat oesophageal muscularis mucosae relaxation via the ß3AR, but the evidence for it being ß3AR-selective was based on lack of activity in atria and tracheal preparations from the guinea-pig (Barrionuevo et al., 1996). Guinea-pig atria are less sensitive than rat atria to ß1AR-agonists, and so it is possible that the ‘selectivity’ of Tecradine in these experiments was due to it having low efficacy. Salmeterol is a potent agonist of the human cloned ß3AR (Piétri-Rouxel and Strosberg, 1995), but it is an even more potent ß2AR agonist, characterized by a long duration of action (Coleman et al., 1996).

4.3.3

Aryloxypropanolamine ß3AR agonists

CGP-12177 and other ‘non-conventional partial agonists’ CGP-12177 (Figure 4.4) has become the standard aryloxypropanolamine ß3AR agonist. CGP-12177 is a highly potent ß1- and ß2AR antagonist, but the discoveries that it stimulates respiration in hamster brown adipocytes (Mohell and Dicker, 1989) and

Figure 4.4 Chemical structures of aryloxypropanolamines.

Figure 4.4 Continued

Figure 4.4 Continued

ß 3-Adrenoreceptor Ligands and the Pharmacology of ß3AR

69

Figure 4.4 Continued

lipolysis in white adipocytes of various species (Langin et al., 1991) led to the demonstration that it is an agonist of murine and human cloned ß3ARs (Granneman et al., 1991; Nahmias et al., 1991). CGP-12177 is, however, 1000-fold less potent as a ß3AR agonist than as a ß1- or ß2AR antagonist. In cell lines that express high numbers of the human cloned receptors it can also act as a potent full ß1AR agonist and as a potent weak partial ß2AR agonist (Pak and Fishman, 1996). It is therefore important to exclude the possibility that any effect of CGP-12177 is mediated by ß1- or ß2ARs by using an appropriate antagonist. CGP-12177 is one of a number of ß 1/2AR antagonists that Kaumann and his colleagues had found to stimulate cardiac function. The other ß1/2 antagonists were oxprenolol, alprenolol, carazolol, pindolol and derivatives of carazolol and pindolol (Figure 4.4). All these compounds have pD2 values for their cardiac effects that are lower than their pKB values for ß1- or ß2ARs, leading to their description as ‘non-conventional partial agonists’ (Kaumann, 1989; see also Arch and Kaumann, 1993). Their agonist activities reside exclusively in their (-)-enantiomers. (-)-RO-363 appears to have a similar pharmacological profile, except that it is a partial ß1AR agonist in cardiac tissues as well as acting at higher concentrations via another receptor (Molenaar et al., 1997b). As well as CGP-12177, all the other ‘non-conventional partial agonists’ and (-)-RO363 stimulate the cloned ß3AR (see Emorine et al., 1994). However, the cardiac effects of CGP-12177 in both rat and man, and its lipolytic activity in human adipocytes are antagonized with a pharmacology that differs from that expected for ß3AR-mediated responses (Galitzky et al., 1997; Kaumann, 1997; see Section 4.4.1). The pharmacology of the cardiac effects of the other non-conventional partial agonists has not been differentiated from ß3AR pharmacology, but it is probable that they too stimulate this ‘ß4AR’. Except for SB-226552, SB-236923 and SB-251023, which have no stimulant effect in human right atrial appendage (see below), it is possible that all the other compounds shown in Figure 4.4 also stimulate this putative receptor.

70

Arch

The disadvantages of CGP-12177 as a ß3AR agonist are therefore that it is a far more potent ß1- and ß2AR antagonist (and potentially ß1AR agonist), and that it stimulates the ‘ß 4 AR’. On the other hand, it has advantages over most phenylethanolamine ß3AR agonists of having much higher efficacy at the human ß3AR (Hoffstedt et al., 1996a; Wilson et al., 1996; see also comment in introduction to Fisher et al., 1998) and of stimulating human white adipocyte lipolysis via ß3- rather than ß1- or ß2ARs (Hoffstedt et al., 1996a; see Section 4.4.2). These features should also be borne in mind when judging the utility as pharmacological tools of other aryloxypropanolamines.

Other ß1/2AR antagonist/ß3AR agonists with small N-substituents The ß1/2AR antagonist carteolol has anti-obesity activity in mice (Takahashi et al., 1994). This effect is probably mediated by the ß3AR because propranolol is a weak antagonist of the stimulation of hamster brown adipocyte oxygen consumption by carteolol. However, carteolol appears to relax guinea-pig cecum via the ß2AR (Zhao et al., 1998). The a/ßAR antagonist arylthiopropanolamine arotinolol activates brown adipose tissue thermogenesis in vivo (Yoshida et al., 1994a). Although not demonstrated by detailed in vitro studies, it is probable that this effect is also mediated by the ß3AR. Propranolol, nadolol, carazolol and (—)-teratolol are ß1/2AR antagonists which have been shown directly to activate the human cloned ß3AR, but agonism has yet to be demonstrated in isolated tissues or in vivo (Méjean-Galzi et al., 1995; PiétriRouxel and Strosberg, 1995).

ZM-215001 and related molecules Prior to CGP-12177 being recognized as a ß3AR agonist, the aryloxypropanolamine ICI198157 was reported to have anti-obesity and anti-diabetic activity in obese (fa/fa) Zucker rats. Metabolic rate and brown adipose GDP binding in vivo (an index of brown adipose tissue thermogenesis) were stimulated at dose levels well below those that affected heart rate, and there were no ß2AR-mediated effects on muscle tremor or blood potassium (Holloway, 1989). However, ICI-198157, like some phenylethanolamines described in Section 4.3.2, is a methyl ester, and it is its acid metabolite ICI-201651, specifically the S enantiomer of this metabolite, ZM-215001, that is a ß3AR-selective agonist (Holloway, 1989; Holloway et al., 1991). Although ICI-201651 and ZM-215001 have slight ß1- and ß2AR agonist activity in some tissue preparations (Holloway, 1989), they are predominantly ß1- and ß2AR antagonists. ZM-215001 has pA2 values of 6.7 and 7.3 for antagonism of the effects of isoprenaline in guinea-pig atria and trachea (Tesfamarian and Allen, 1994). These values are similar to the pD2 value of ZM-215001 (7.4) for stimulation of rat white adipocyte lipolysis (Mayers et al., 1996). Thus, in contrast to CGP-12177, ZM-215001 can stimulate the ß3AR without totally blocking ß1- and ß2ARs, but some ß1/2AR antagonism will occur. Blin et al. (1993) found that ICI-201651 was a full agonist of the human cloned ß3AR, and Fisher et al. (1996) reported an intrinsic activity of 0.71 for ZM-215001 relative to isoprenaline. However, other workers (Fisher et al., 1998) commented that all the first-generation ß3AR agonists that they have tested in man are only weak partial agonists of the human ß 3 AR. It seems unlikely that a compound which lacks

ß 3-Adrenoreceptor Ligands and the Pharmacology of ß3AR

71

substituents in the phenyloxypropanol moiety can have good efficacy at the human receptor, and it is possible that the systems used by Blin et al. (1993) and Fisher et al. (1996) flatter these compounds. ZM-215001 must have poor efficacy at the rat ß3AR since it had an intrinsic activity of only 0.30 relative to isoprenaline or BRL-37344 as a stimulant of rat white adipocyte lipolysis (Mayers et al., 1996). Its pA2 value for antagonism of isoprenaline-stimulated lipolysis was 7.3. This value is similar to its pA2 values for antagonism of atrial and tracheal responses, showing that it has similar affinities for the three cloned ßARs. ICI/Zeneca chose to develop the amide ZD-7114 rather than the ester ICI-198157, presumably to achieve a better pharmacokinetic profile. The amide is also metabolized to the acid (Mayers et al., 1996). BMS-187257 (Bristol-Myers Squibb) has a similar profile at the human cloned ß3AR to BRL-37344 (Fisher et al., 1996). Like the ICI/Zeneca compounds, the substitution pattern in its aryloxypropanol moieties seems unlikely to provide good efficacy at the human ß3AR. The high intrinsic activity (0.72) reported for BMS-187257 may reflect the use of Strosberg’s cell line.

Recent SmithKline Beecham, Merck and Eli Lilly compounds When the first-generation ß3AR agonists and their prodrugs were evaluated in man, the results were largely disappointing (Table 4.8). These compounds either lacked adequate efficacy or their thermogenic, anti-obesity or anti-diabetic activity was associated with ß1- or ß2AR-mediated side effects (tachycardia or tremor). Poor oral bioavailability and rapid clearance was one reason for these failures (Section 4.3.5), but it was also recognized that the first-generation compounds lacked good selectivity and especially efficacy at the human ß3AR (Wilson et al., 1996; Fisher et al., 1998). Therefore, in recent years pharmaceutical companies have tried to identify ß 3AR agonists that are not only high selective but also have good efficacy at the human ß3AR. Table 4.8 Clinical experience with first-generation ß3AR agonists

[1] Arch and Wilson, 1996a; [2] Smith et al., 1990; [3] Toubro and Astrup, 1995; [4] Weyer et al., 1998

Arch

72

Sennitt et al. (1988) have described six aryloxypropanolamines that vary in their pharmacology at the human cloned ßAR. Four of these and a further compound, SB251023, are shown in Figure 4.4. SB-226552, SB-229432 and SB-251023 had intrinsic activities that were at least as high as that of CGP-12177 at the human ß3AR. Like CGP12177, they lacked agonist activity at ß1- and ß2ARs at the level of expression used. However, in contrast to CGP-12177, their binding affinities as antagonists (pKi values) at ß1- and ß2ARs were much lower than their pD2 values at the ß3AR. SB-236923 and SB246982 had higher intrinsic activities than isoprenaline at the ß3AR, but they were partial agonists at ß1ARs, though at higher concentrations than their ß3AR agonism. A key feature of these SmithKline Beecham aryloxypropanolamines is that Kaumann, the originator of the ‘ß4AR’ hypothesis, found that they either do not stimulate the force of contraction of human right atrial appendage (SB-226552, SB-229432, SB-251023), or any slight effect that they do have is blocked by 200 nM (-)-propranolol (SB-236923). Thus, they appear to have the advantage over CGP-12177 and other non-conventional partial agonists of not stimulating the ‘ß4AR’. The Merck compound L-755,507 is a potent agonist of human and rhesus monkey cloned ß3ARs (Fisher et al., 1998; Parmee et al., 1998). Its intrinsic activity at the human ß3AR is significantly higher than the intrinsic activities of ß3AR agonists that have been evaluated in man. It is a highly selective ß3AR agonist, but it does have partial agonist activity at both human and rhesus monkey ß1ARs. High intravenous doses of L-755,507 stimulate heart rate in rhesus monkeys, but it was suggested that this was a reflex response to ß3AR-mediated peripheral vasodilatation rather than a direct ß1AR- or ‘ß4AR’-mediated effect. Pyridines related to L-755,507, a phenol, have been described recently. L-749,372 and L-750,355 are somewhat less potent ß3AR agonists that have partial agonist activity at the ß1AR. Their ß3/ß1AR selectivity is more than 100-fold. They bind weakly to the ß2AR, presumably as antagonists (Weber et al., 1998a). LY-362884 is another potent agonist of the human cloned ß3AR that had a high intrinsic activity (0.98) relative to isoprenaline. Like SB-226552, SB-229432 and SB251023, it had only antagonist activity at the human ß1- and ß2ARs, but in contrast to the SB compounds, its ß3AR EC50 (30 nM) was similar to its IC50 values for antagonism of isoprenaline’s actions at ß1- and ß2ARs (Jesudason et al., 1998). A closely related compound, LY- 377604 is reported to be in Phase I clinical development (Cohen et al., 1999). There is no information on whether LY-362884 or LY-377604 have ‘ß4AR’ agonist-like activity.

4.3.4

Trimetoquinol and analogues

Trimetoquinol and its analogues constitute a third class of ß3AR agonists (Figure 4.5). These compounds lack the hydroxy group of arylethanolamines or aryloxypropanolamines, and the amino nitrogen is contained within a semi-rigid tetrahydroisoquinoline ring. Their large 3',4',5'-trimethoxybenzyl substituent resembles the large N-substituents of selective ß3AR agonists. (-)-Trimetoquinol and 3'-iodotrimetoquinol have similar potencies for stimulation of ß1AR- and ß3AR-mediated responses in rat tissues, with a slightly lower potency for ß2AR-mediated tracheal relaxation (Fraundorfer et al., 1994; Konkar et al., 1996) (see Table 4.4). 3',5'-Diiodotrimetoquinol shows, however, about 10-fold selectivity for stimulation of the ß3AR-mediated responses. All three compounds are also potent

ß 3-Adrenoreceptor Ligands and the Pharmacology of ß3AR

73

Figure 4.5 Chemical structures of trimetoquinols.

stimulants of cyclic AMP accumulation in Chinese hamster ovary cells expressing the rat ß3AR (see Table 4.5). The activities at the human ß3AR of trimetoquinol and some novel ß3AR-selective analogues have recently been reported (Zheng et al., 1999).

4.3.5

Metabolism and pharmacokinetics

Problems with the oral bioavailability or pharmacokinetics of ß3AR agonists have often limited their value, especially as therapeutic agents for humans. De-esterification of some of the first-generation compounds is beneficial to the extent that it produces the ß3AR-selective acid metabolites. In rats, the acids BRL-28410 and BRL-373444 are excreted in the bile and reabsorbed, resulting in sustained blood levels, but in man they are not excreted in the bile, and rapid renal excretion results in their having very short half-lives. The diacid CL-316,243, by contrast, has a very long plasma half-life in man (16 h), but only 10% of an oral dose is absorbed (Weyer et al., 1998). In rats, 12% of an oral dose is absorbed and in monkeys 3%. The 2,2-dimethylpropyl and 3-methylbutyl ester-type prodrugs of CL-316,243 had 2- and 3-fold enhanced oral bioavailabilities in monkeys. CP-114,271 had both poor oral bioavailability in the monkey (7%) and rat (11%), and a plasma half-life following intravenous administration of less than 30 min in these species (Wilson et al., 1995). Slightly better results were achieved in dogs (Wilson et al., 1995; Yee et al., 1995). The SmithKline Beecham aryloxypropanolamines shown in Figure 4.4 also encountered problems of oral bioavailability and short half-lives in the rat. L-755,507, a phenoxypropanolamine that is subject to rapid presystemic glucuronidation (Weber et al., 1998a), is one of a class of compounds that has poor oral bioavailability and relatively short half-lives in various animal species (Fisher et al., 1998). L-755,507 itself has an oral bioavailability of about 1 %, but the aminopyridinebased analogue L-750,355 has bioavailabilities of 4% in the rat and 47% in the dog, and a half-life of 13 h in the dog (Dow, 1997). The pyridine L-749,372, also has a better pharmacokinetic profile than L-755,507 (Weber et al., 1998a). Attempts to achieve oral bioavailability with 3-pyridylethanolamines have had limited success (Naylor et al., 1998, 1999; Parmee et al., 1999), except perhaps for L-770,644, which had 27% oral bioavailability in the dog. BMS-194449 has an oral systemic bioavailability in monkeys of less than 2% (Washburn et al., 1998). It is well absorbed, but is excreted in the bile as a monoglucuronide. BMS-194449 is one of three compounds (the others are BMS-196085

Arch

74

and BMS-201620) that have been described as transdermal agents that increase lipolysis, but whose development has been discontinued. BMS-196085 similarly has less than 5% oral systemic bioavailability (Gavai et al., 1998). Oral systemic bioavailability is not markedly improved in BMS-210285 (Sher et al., 1998). The Lilly compound reported to be in Phase I clinical trials, LY-377604, is described as having more than 20% oral bioavailability (Cohen et al., 1999).

4.4

4.4.1

Idiosyncrasies of ß3AR pharmacology

Relative agonist potencies vary with the nature of the assay

Studies conducted using the human cloned ß3AR have shown that agonists are more potent stimulants of whole-cell cyclic AMP accumulation than of adenylyl cyclase in membranes. Moreover, the relative potencies of the agonists differ in these two systems (Emorine et al., 1994; Arch, 1995; Wilson et al., 1996). In particular, the catecholamines and BRL-37344 are much more potent than aryloxypropanolamines, such as CGP-12177 or cyanopindolol, in the whole-cell assay, but not in the membrane assay. Wilson et al. (1996) showed that these findings extend beyond potencies to the functional affinities of the agonists (Table 4.9). They suggested that the aryloxypropanolamines interact with a signal tranduction pathway in the whole cell that impairs cyclic AMP production, but they were unable to identify what this other signalling pathway might be. For example, treatment of cells with pertussis toxin to prevent signalling via Gi did not affect the relative potencies of isoprenaline or CGP12177. One can speculate that interactions of aryloxypropanolamines with the ‘ß4’AR affects their ß3AR-mediated responses, or that the newly discovered RAMP proteins (McLatchie et al., 1998) differentially modulate responses to the agonists, but these influences would have to differ between whole cells and membranes to account for the findings. Whatever the reason, there must be at least two forms of the ß3AR with different absolute and relative affinities for agonists. Interestingly, Clarke and Bond (1998) have recently reviewed evidence that the relative efficacies of agonists can also vary according to the environment of the receptor. Table 4.9 Relative potencies of agonists at the human cloned ß3AR vary with assay*

*From Wilson et al., 1996

ß 3-Adrenoreceptor Ligands and the Pharmacology of ß3AR

75

Wilson et al. (1996) also showed that while isoprenaline was 229-fold more potent than cyanopindolol as a stimulant of whole-cell cyclic AMP accumulation, cyanopindolol was 365-fold more potent than isoprenaline in displacing [125I]-iodocyanopindolol from membranes that express the human cloned ß3AR (Table 4.9). The difficulties of interpreting such data were discussed in Section 4.3.1. It seems that cyanopindolol must bind with high affinity to the same population of ß3ARs as its close homologue [125I]-iodocyanopindolol, whereas isoprenaline binds with high affinity to only a fraction of the receptors that bind [125I]-iodocyanopindolol. The to membranes from untransfected cells was negligible. Discrepancies between binding possibility that the labelled ligand bound to the ‘ß4’AR can be excluded, since binding affinities and potencies for cyclic AMP accumulation have also been described for the rat ß3AR: the (–)-(S) isomer of trimetoquinol was 120-fold more potent than the (+)-(R) isomer in binding experiments, but 4700-fold more potent in functional experiments (Fraundorfer et al., 1994).

4.4.2

Prediction of human tissue pharmacology from cloned receptor pharmacology

Isoprenaline, noradrenaline and BRL-37344 each stimulate the human cloned ß3AR (Emorine et al., 1994). However, responses to BRL-37344 (if they occur at all), and to isoprenaline and noradrenaline in human gut and adipose tissue are mediated primarily by the ß1- or ß2- rather than the ß3AR (Langin et al., 1991; MacLaughlin and MacDonald, 1991; Lönnqvist et al., 1993; Rosenbaum et al., 1993; Sennitt et al., 1995), although responses of the gut preparations to isoprenaline and noradrenaline do involve a significant ß3AR-mediated component (MacLaughlin and MacDonald, 1991; De Ponti et al., 1996; Kelly et al., 1998). Indeed, of the arylethanolamines, only CL-316243, which has very low or zero efficacy at ß1- and ß2ARs, has been shown to elicit a lipolytic response in human white adipocytes that is totally resistant to antagonism by 10-7 M propranolol (Hoffstedt et al., 1996a). This compound has also been shown to stimulate insulin-mediated glucose storage in man by what must very probably be a ß3ARmediated mechanism, since no changes in heart rate, blood pressure or tremor could be detected (Weyer et al., 1998). This background led Sennitt et al. (1998) to investigate the lipolytic activities of two phenylethanolamines that had higher intrinsic activities than BRL-37344 in their human cloned ß 3AR assay. One of these compounds, SB-220646, appeared to elicit no stimulation of adenylyl cyclase in membranes that expressed human cloned ß1- or ß2ARs, and yet its lipolytic effect was antagonized by both nadolol and propranolol with KB values that indicated the involvement of one or both of these classical receptors. Moreover, the compound was almost a full agonist of human right atrial appendage contractility, an effect that was shown to be mediated by both ß1- and ß2ARs. Further studies using a cell line that expressed high numbers of ß2ARs revealed weak ß2AR agonist activity in SB-220646. This (and presumably weak ß1AR agonist activity) was apparently sufficient to subvert any ß3AR-mediated effect on lipolysis and elicit the inotropic activity. The second compound, SB-215691, had higher ß1- and ß2AR agonist activity than BRL-37344, but also higher intrinsic activity at ß3ARs. Low concentrations of SB215691 did have a nadolol-resistant component in its lipolytic effect in some experiments, but in every experiment high concentrations of SB-215691 stimulated lipolysis by a nadolol-sensitive mechanism.

Arch

76

The tendency of these phenylethanolamines to stimulate human white adipocyte lipolysis via ß1- or ß2ARs contrasts with data for CGP-12177 and the SmithKline Beecham aryloxypropanolamines described in Section 4.3.3 (Figure 4.4). The lipolytic effects of all these compounds were insensitive to nadolol, even in the case of the weak partial ß 1 - and ß 2 AR agonist SB-236923. The lipolytic effect of another aryloxypropanolamine (SB-248320; Sennitt et al., 1998) was sensitive to nadolol, but this compound was a strong partial agonist of the ß2AR. Since any atrial inotropic effects of the SmithKline Beecham compounds were sensitive to propranolol, and SB-251023 does not antagonize the inotropic effect of CGP-12177 (Arch et al., 1999), these compounds must stimulate lipolysis via the ß3- rather than the ‘ß4’AR. It seems that cross-talk between ß1/2ARs and ß3ARs tends to override signalling via the ß3AR conformation to which phenylethanolamines bind, but not signalling via the conformation to which aryloxypropanolamines bind. Consequently, for a phenylethanolamine to signal via ß3ARs in human white adipocytes, it must, like CL316243, be virtually devoid of efficacy at ß1- or ß2ARs.

4.5

Conclusions

The ß3AR is characterized by a low affinity for standard ß1- and ß2AR antagonists. In rat tissues, it distinguishes poorly between the more potent (-)- and less potent (+)enantiomers of these antagonists, but this feature has not been demonstrated using the rodent cloned ß3AR and is not found with the human cloned ß3AR. There are no potent and highly selective antagonists of the ß3AR, but SR-59230 displays moderate selectivity in the rat and isolated rat tissues, and L-748,328 and L-748,337 are moderately selective antagonists of the human cloned ß3AR. The ß3AR is selectively stimulated by certain phenylethanolamines, but many of these have low selectivity for and efficacy at the human ß3AR. Unless, like CL-316,243, they lack any efficacy at ß1- and ß2ARs, they tend to stimulate human white adipocyte lipolysis via ß 1- or ß 2 ARs. The ß 3AR is also selectively stimulated by certain aryloxypropanolamines, some but not all of which are potent ß1/2AR antagonists. Some of these compounds have good efficacy at human ß3ARs, and this class of compound generally seems more likely to elicit ß3AR than ß1AR or ß2AR-mediated responses in tissues that express all three receptors. Some aryloxypropanolamines, notably CGP-12177, appear to stimulate another receptor, now known as the ‘ß4’AR. This receptor shares with the ß3AR the property of insensitivity to ß1- and ß2ARs, but there are differences between ß3- and ‘ß4’AR pharmacology, notably that phenylethanolamines are not agonists for the ‘ß4’AR, at least in cardiac tissues (see Chapter 8). One must be cautious in assuming that a novel pharmacology implies a novel receptor, since the pharmacology of even cloned ß3ARs can vary, but ‘ß4’AR pharmacology has been demonstrated in atria and brown adipose tissue from ß3AR knockout mice. Recent work suggests that the ‘ß4’AR is a form of the ß1AR. ß 3AR agonists have potential for the treatment of human obesity and type 2 diabetes, but the first-generation compounds lacked selectivity and efficacy for the human ß3AR. Subsequent compounds have overcome this deficiency, but a compound that combines appropriate pharmacology with good oral bioavailability and sustained blood levels has not been described—at least not in any detail. This remains a major goal of this research area.

5

The Native Human ß3-Adrenoreceptor PETER ARNER AND FREDRIK LÖNNQVIST Department of Medicine at Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden

5.1

Introduction

Long before the ß3-adrenoreceptor (ß3AR) was characterized, it was implicated that certain catecholamine responses in man were mediated through an ‘atypical’ non-ß1AR, ß2AR. Using pharmacological experiments with selective and non-selective ß1AR and ß2AR agonists and antagonists, Zaagsma, Kaumann and their co-workers were the first to demonstrate the existence of such ‘atypical’ ßARs mediating lipolysis in human fat cells and metabolism and contractility in human heart (for reviews see Kaumann, 1989; Zaagsma and Nahorski, 1990). However, the role of this atypical receptor was largely unknown until the first cloning and molecular characterization of the human ß3AR (Emorine et al., 1989). This chapter focuses on the native human ß3AR and the possible physiological and pathophysiological role of the receptor. In the interest of space, review articles have been cited instead of original articles whenever p ossible.

5.2

The human ß3AR gene

While the structural aspects of the human ß3AR are discussed in detail in other chapters of this book, the clinically important factors are species differences in structure-function relationships. Most of our knowledge on ß3AR functions is derived from studies in nonhuman cells, and in particular cells or tissues from rodents. Some structural features predict that the human ß3AR gene may have unique functional properties in comparison with that of rodents. The mouse and rat ß3AR genes contain two introns and three exons, whereas the human gene contains only one intron and two exons (Granneman and Lahners, 1992; Van Spronsen et al., 1993). Furthermore, the ß3AR gene transcription differs markedly between human and rat with regard to the use of polyadenylation sites, promoter start sites and hormonal regulation (Granneman and Lahners, 1994). There are however, also species differences which cannot be predicted by a comparison of the structure of different ß3AR genes. The predicted primary structures of the rat and the human ß3AR are more than 90% similar, yet the receptor properties differ substantially in their pharmacological response to ßARagonists (Granneman et al., 1991). In contrast, the mouse ß3AR gene shows only 80% 77

Arner and lönnqvist

78

homology with the human ß3AR gene, although the pharmacological properties of the mouse and human receptors, when expressed in Chinese hamster ovary cells, are very similar (Granneman et al., 1991; Blin et al., 1994). As yet, our knowledge of the regulation of expression of the native human ß3AR gene is limited. However, it is likely that this knowledge will increase in the near future through results from experiments on transgenic animals, where the native gene product has been replaced by the human counterpart (Ito et al., 1998). Until then, caution should be exercised when data derived from experiments on laboratory animals or cell lines are extrapolated to the human ß3AR gene.

5.3

Tissue expression of mRNA for ß3AR

It is now evident that specific mRNA is present in a variety of human tissues (Table 5.1). The expression of mRNA has been documented with various techniques such as RTPCR, RNase protection and Northern blotting (Rodriguez et al., 1995; Deng et al., 1996). The measurement of ß3AR mRNA in man is mainly of a semi-quantitative nature, and therefore the true order of magnitude of gene expression in human tissues is unknown. It appears, however that brown adipose tissue and gallbladder have the highest levels of expression. In most tissues, mRNA for ß3AR co-exists with mRNA for ß1AR and ß2AR. The relative abundance of the three gene products in these various tissues is also unclear, but by using semi-quantitative techniques, it has been shown that quantities of ß3AR mRNA are less than those of ß1AR and ß2AR mRNA in both brain and brown adipose tissue (Krief et al., 1993; Berkowitz et al., 1995; Deng et al., 1996). For obvious reasons, it is likely that a direct ß3AR function is expected solely in tissues that express the corresponding gene, and the role of the receptor in these tissues will be discussed in the following text.

5.3.1

White adipose tissue

The major metabolic function of white adipose tissue (WAT) is to store and release energy-rich fatty acids that are mobilized through lipolysis in white fat cells. The role of ß3AR in human lipolysis has been examined in some detail (see Arner, 1996 for an overview of lipolysis regulation in man). The gene has been shown to be expressed in all human white adipose depots examined to date (Rodriguez et al., 1995; Deng et al., 1996). However, early studies of lipolysis in vitro in easy-to-obtain subcutaneous adipose tissue failed to show a significant lipolytic response following ß3AR stimulation, despite the same adipocytes responding to lipolytic stimulation of ß1AR and ß2AR (for details see Galitzky et al., Table 5.1 Human tissues expressing ß3AR

The native human ß 3-adrenoreceptor

79

1995). It is, however, likely that methodological factors contribute to the difficulties in demonstrating a functional ß3AR in these earlier in vitro studies on lipolysis. The lipolytic effect of ß3AR stimulation is dependent on a number of factors such as adipose tissue region (visceral fat is much more responsive than subcutaneous fat), the type of tissue preparation (isolated cells are more responsive than tissue pieces), and overall lipolytic activity in the in vitro preparation (Hoffstedt et al., 1995). Using a sensitive lipolysis assay it has been possible to demonstrate and characterize in detail the pharmacological properties of a ß3AR in omental (visceral) human fat cells with regard to lipolysis in vitro. ß3AR functionally co-exists with ß1AR and ß2AR (Lönnqvist et al., 1993), and its pharmacological properties clearly differ from those reported for the ß3AR in rat fat cells. A number of ‘rat-selective’ ß3AR agonists are either ineffective or non-selective on lipolysis in human fat cells (Hoffstedt et al., 1996a). However, the lipolytic properties of the receptor in omental fat cells is almost identical as in the mouse 3T3-L1 adipocyte cell line (Shimizu et al., 1996). So far, only two adrenergic agonists have been identified as selective ß3AR lipolytic agonists in human fat cells: CGP-12177 which is also a ß1AR/ß2AR non-selective antagonist (Lönnqvist et al., 1993), and CL-316,243 (Hoffstedt et al., 1996a), although some data suggest that the latter agent is non-selective (Umekawa et al., 1996). The strongest proof for a functional ß3AR in human WAT has been obtained from in vivo studies using microdialysis. This technique has proven very useful in pharmacological investigations of in situ lipolysis in humans (Lafontan and Arner, 1996), as it permits the monitoring and manipulation of local lipolysis and blood flow in vivo in human subcutaneous adipose tissue. In this tissue it was shown that all three ßAR subtypes regulate lipolysis in vivo, but ß2AR was shown to be the most important for lipid mobilization as only this receptor type stimulated lipolysis as well as local blood flow (Enocksson et al., 1995). The existence of a functional ß3AR in vivo in human subcutaneous adipose tissue was later confirmed by other microdialysis experiments (Barbe et al., 1996). The question remains as to whether the ß 3AR in white fat cells is subject to regulation. Extensive evidence exists to show that the adipocyte ß3AR is influenced by physiological and pathophysiological factors (Table 5.2), the most important factor probably being obesity. Subjects with upper-body obesity have a higher rate of catecholamine-induced lipolysis in visceral fat cells than non-obese subjects, this variation being ascribed mainly to enhanced lipolytic function of ß3AR in visceral adipocytes (Lönnqvist et al., 1995). Many metabolic and cardiovascular abnormalities found in upper-body obese subjects, which together form a so-called metabolic

Table 5.2 Modulation of ß3AR lipolytic function in white human fat cells

Arner and lönnqvist

80

(insulin resistance) syndrome, are related to the lipolytic function of ß3AR in visceral fat cells (Hoffstedt et al., 1996b). As discussed in detail (Arner, 1996), increased mobilization of fatty acids from the visceral fat depot may be a key factor behind many metabolic abnormalities observed in upper-body obesity. Only visceral fat is drained by the portal vein, so this fat depot has a unique direct link to the liver. Liver function is altered by increased ‘portal’ fatty acids, resulting in hyperinsulinaemia, dyslipidaemia, hyperglycaemia and liver insulin resistance, all of which form part of the metabolic syndrome in upper-body obese subjects (for details see Arner, 1996). Obese male subjects have higher lipolytic ß3AR function in visceral fat cells than obese women, which at least in part can explain the more marked metabolic aberrations in the obese males (Lönnqvist et al., 1997). The lipolytic ß3AR function is dependent on the adipose tissue region. In the two visceral depots, omental and mesenteric adipose tissue, the function is similar, but within these two regions ß 3 AR is more sensitive to stimulation than in the subcutaneous region (Van Harmelen et al., 1997). The regional differences in ß3AR function are much more attenuated in obese than in lean men (Hoffstedt et al., 1997). When all available data on lipolysis are considered together, it appears that the ß3AR is functional in all WAT regions, though ß3AR does not seem to play a leading role in lipolysis in the quantitatively dominating subcutaneous depot. In contrast, ß3AR appears to be of greater importance for lipolysis in visceral fat cells, and in particular in obese men. These subjects have very high rates of catecholamine-induced fatty acid mobilization from visceral fat, mainly owing to a high lipolytic function of ß3AR in their visceral fat cells. Thus, ß3AR may play a significant role in the pathogenesis of the metabolic syndrome, which often accompanies male obesity and is also seen in obese women who have an upper-body fat distribution (for detailed discussion see Arner, 1996). In these conditions, elevated ‘portal’ fatty acid levels could be the result of high ß3AR activity in visceral adipose tissue. In addition, ß3AR is responsible, at least in part, for the well-known regional variations in lipolytic activity between visceral and subcutaneous fat depots (Arner, 1995). The increased lipolytic activity in visceral as compared with subcutaneous fat cells is largely explained by the higher lipolytic function of ß3AR in visceral fat cells.

5.3.2

Brown adipose tissue

It is difficult to study brown adipose tissue (BAT) in humans because in general, only small amounts of tissue are present in adults. However, available data suggest that ß3AR is functional in human BAT. As in other species, the probable role of ß3AR is to stimulate thermogenesis through activation of uncoupling protein 1. ß3AR agonists increase uncoupling protein 1 mRNA levels in primary cultures of perirenal adipose tissue, which contains more brown fat cells than most other human fat depots (Champigny and Ricquier, 1996). Some of the thermogenic response in vivo to isoprenaline (a non-selective ßAR-agonist) infusion and to intravenous adrenaline administration is mediated by ß3AR (Wheeldon et al., 1993; Liu et al., 1995). Longterm oral treatment with CL-316,243, which is a selective ß3AR agonist in human fat cells (Hoffstedt et al., 1996a), increases thermogenesis in healthy volunteers (Weyer et al., 1998). Thus, it appears that the ß3AR is involved in the regulation of thermogenesis in BAT, although its role in relation to other adrenoreceptor sub-types in human brown fat cells is yet unknown.

The native human ß 3-adrenoreceptor

5.3.3

81

Heart

There is clear evidence that ß3AR co-exists with ß1AR and ß2AR and is functional in the human heart. For example, RO-363 (a ß1AR/ß3AR agonist) can increase atrial force in vitro through ß3AR (Molenaar et al., 1997b), while CGP-12177 mediates positive inotropic effects in human atrial myocardium in vitro (Kaumann, 1996). In addition, BRL-35135 and isoprenaline also mediate some of their cardiotropic effects in vivo through ß3AR (Wheeldon et al., 1993, 1994). Interestingly, cardiodepressive ß3AR effects which have been demonstrated in vitro in human heart and seem to be mediated by inhibition of cyclic AMP formation through a Gi-coupled receptor pathway (Gauthier et al., 1996). This must be a unique feature of human ß 3AR, since in man (and in other species) all other effects by this receptor subtype (as by ß 1AR and ß 2AR) are mediated by G s proteins. The negative cardiac inotropic effects of ß3AR vary markedly depending on the species, these being observed more readily in humans than in either rats or ferrets (Gauthier et al., 1999). Although a functional ß3AR is present in human heart, the physiological role of the receptor in relation to ß1AR and ß2AR remains unclear. Neither has the influence of ß3AR on pathophysiological conditions in the heart yet been determined.

5.3.4

Colon

The role of ß3AR in the regulation of colonie function has been demonstrated in humans in vitro (De Ponti et al., 1996; Roberts et al., 1997). The receptor is present in the colonie smooth muscle, but not in the colonie mucosa. ß 3AR mediates catecholamine-induced muscle relaxation, the role of ß3AR (and ß1AR) being to enhance mucosal blood flow in colon as a result of relaxation of vascular and nonvascular smooth muscle.

5.3.5

Brain

There is evidence for the presence of ß3AR mRNA in the human brain, although mRNA expression may be subject to regulation as its levels are much higher in the brain of children than in adults (Rodriguez et al., 1995). Although it remains to be established whether ß3AR is functional in human brain, a functional ß3AR has been reported in human neuroblastoma cells (Esbenshade et al., 1992).

5.3.6

Urinary bladder

mRNA for all three ßAR subtypes is present in the detrusor muscle of human urinary bladder (Takeda et al., 1999), and it is possible that ß3AR is of unique importance for the adrenergic regulation of urinary bladder motility. Only this ßAR subtype has been shown to have any marked functional effect on human detrusor muscle in pharmacological experiments (Igawa et al., 1999).

Arner and lönnqvist

82

5.3.7

Other tissues

mRNA for ß3AR is found in the gallbladder, small instestine and prostate gland of humans (Krief et al., 1993; Berkowitz et al., 1995), though as yet no evidence has been presented that ß3AR is functional in any of these tissues. However, a ß3AR protein has been shown to be present in gallbladder, as evidenced by the use of antibodies (Guillaume et al., 1994). Immunohistochemical studies have also suggested that a ß3AR protein is present in vascular smooth muscle in several regions of the gastrointestinal tract (Anthony et al., 1998).

5.4

Is there a fourth ßAR?

Some pharmacological data support the view that there is a fourth functional ßAR in human heart and white fat cells, and which is proposed to co-exist with the other three ßAR subtypes (Kaumann and Molenaar, 1997; Molenaar et al., 1997a; Galitzky et al., 1997, 1998; Kaumann et al., 1998). However, these data should be interpreted with some caution, since they are based on the use of the agonist CGP-12177 and the antagonist SR59,230A. The CGP compound is a partial ß3AR agonist, and its pharmacological behaviour is dependent on the amount and coupling of ß3AR. It has been shown in Chinese hamster ovary cells transfected with the cloned ß3AR that the sensitivity and potency of CGP-12177 is strongly dependent on receptor expression level and the nature of the pharmacological assay (Wilson et al., 1996). In the case of SR-59,230A, this compound is classified by the manufacturer as a ß3-selective antagonist. In our hands, however, the results of lipolysis experiments with human omental fat cells suggest that it is non-selective for all three known ßAR subtypes (P.Arner, unpublished results). Furthermore, unpredicted effects have been shown on human fat cells with regard to ßAR selectivity of novel agonists that are selective against ß3AR in cloned cells (Sennitt et al., 1998). Studies on the differentiation between native ß3AR and ß4AR in man have been hampered by lack of specific tools, and no full ß3AR agonist or truly selective ß3AR antagonist is available. However, data received suggest that it may be possible to develop selective antagonists for the human ß 3 AR (M.R.Candelore et al., unpublished results). There is no reliable radioligand available, although tritiumlabelled CGP-12177 and SB-206606 have each been used to monitor binding to ß3AR in human fat cells (Revelli et al., 1993; Deng et al., 1996). The CGP ligand binds to ß3AR with low affinity, but this is very difficult to differentiate from nonspecific binding. The SB ligand is a stereoisomer of BRL-37344, and the racemic form of the BRL compound is a non-selective ßAR ligand (Lönnqvist et al., 1993; Hoffstedt et al., 1996a). Difficulty also occurs in the correct pharmacological characterization of ß3AR in tissues that have a high number of ß1- and ß2ARs, but a low number of ß3AR (Sennitt et al., 1998). These unfavourable proportions of the three known ßAR subtypes exist in human adipose tissue. So far there are no genetic data in support of the existence of a ß4AR.

The native human ß 3-adrenoreceptor

5.5

83

Structural variations in the native human ß3AR

In 1995, three independent investigations demonstrated the existence of a coding polymorphism in the ß3AR gene that results in the replacement of tryptophan by arginine at position 64 (Trp64Arg) of the receptor (Walston et al., 1995; Clément et al., 1995; Widen et al., 1995). The less common Arg allele was associated with weight gain, early onset of non-insulin-dependent diabetes, and certain features of the metabolic (insulin resistance) syndrome. The polymorphism has attracted enormous scientific interest, and to date more than 100 studies have been published (MEDLINE) dealing with the genetic variance (see Walston et al., 1995; Widen et al., 1995 for detailed reviews). Some additional clinical features of the Arg allele have been presented (Table 5.3) such as decreased metabolic rate, increased fertility and increased risk of developing coronary heart disease. Furthermore, the association with obesity and increased weight gain seems gender-specific, as it is observed only in women. The phenotypic effect of the polymorphism is usually more marked in homozygotes than among heterozygotes. It should be stressed that the association between the Trp64Arg polymorphism and pathophysiology is controversial, as many studies have failed to demonstrate an influence of the polymorphism on obesity, weight gain, metabolic syndrome or noninsulin-dependent diabetes (Strosberg, 1997; Arner and Hoffstedt, 1999). One recent meta-analysis of 23 studies did not find any association between body mass index and the Trp64Arg polymorphism (Allison et al., 1998), though another meta-analysis did show such an association (Fujisawa et al., 1998). The reason for this divergence in results is not known, but it is not due to race, gender or population size. With regard to any association with obesity, evidence both for and against has been found in Finnish, Japanese, adult female and large populations. In contrast, most of the genetic studies have been performed on cross-sectional materials. In a paired sibling analysis, the Arg64 allele was associated with significant higher values in body mass index (Mitchell et al., 1998). An important aspect of a structural variance in a protein is whether it is functional or not. Again, conflicting results have been published regarding the Trp 64 Arg polymorphism. In Chinese hamster ovary cells transfected with the mutant ß3AR variant, receptor function has been either normal (Candelore et al., 1996) or reduced (Piétri-Rouxel et al., 1997) in comparison with cells carrying the ‘wild’ variant. In a small number of subjects the lipolytic function of ß3AR in omental fat cells was not significantly different between carriers and non-carriers of the Arg 64 allele (Li et al., 1996). However, in a larger study group the same investigators recently demonstrated that the lipolytic sensitivity of the ß3AR agonist CGP-12,177A was

Table 5.3 Phenotypes influenced by the Trp64Arg polymorphism in human ß3AR

Arner and lönnqvist

84

significantly and 10-fold reduced in omental fat cells of the carriers of the Arg 64 allele (Hoffstedt et al., 1999). Other investigators have also demonstrated that the Trp64Arg variant influences ß3AR function in visceral human fat cells (Umekawa et al., 1999). It is possible that ß3AR is a so-called ‘thrifty’ gene (Groop and Tuomi, 1997) which is important for survival among ancient tribes whose living conditions were dependent on the availability of food. As mentioned, the Trp64Arg variant of ß3AR is associated with rapid weight gain, low metabolic rate and improved fertility. All these features would be advantageous in a primitive society where food is sparse. Recent data suggest that the Trp64Arg polymorphism is an old genetic variant, as it is associated almost completely with several other (non-coding) structural variations in the ß3AR gene (Hoffstedt et al., 1999). When all available data are considered together, it is our opinion that the Trp64Arg polymorphism of ß3AR is functional and has an influence on body weight and on several metabolic parameters associated with obesity. However, the Arg64 variant cannot be considered as a major obesity gene abnormality.

5.6

ß3AR as a therapeutic target

Because of the central role of ß3AR in the regulation of thermogenesis in many animal models, selective ß3AR agonists have been considered suitable candidates for anti-obesity drugs (Arch and Wilson, 1996a; Lipworth, 1996; Weyer et al., 1999). A low metabolic rate is believed to be of pathophysiological significance for the development of obesity (Ravussin, 1995). Thermogenesis is mainly activated by ß3 AR agonists through stimulation of BAT (Lowell and Flier, 1997). As mentioned earlier, there is evidence that thermogenesis in humans can be stimulated by ß3AR and that this might occur in brown fat cells. However, a number of anti-obesity drug trials using selective ß3AR agonists have failed to induce weight loss and/or have been associated with ß 1AR or ß2AR-mediated side effects (Arch and Wilson, 1996a; Groop and Tuomi, 1997); these were most likely due to the agonists having been developed using ß 3 AR in rats as a pharmacological model. With reference to previous discussion in this chapter, the rat ß3AR differs greatly from its human counterpart, and thus most of the ‘rat-derived’ ß3AR agonists used in trials were either non-effective or non-selective on the native human ß3AR (Hoffstedt et al., 1996a). Several pharmaceutical companies have developed ß3AR agonists using the human ß3AR as a pharmacological tool in drug testing. For example, a highly potent human ß 3AR agonist, (L-755,507) developed by Merck (Parmee et al., 1998) increased metabolic rate in rhesus monkeys (Fisher et al., 1998). The compound appeared to be a selective ß3AR agonist on lipolysis in human fat cells (Zilberfarb et al., 1997). Several such second-generation ß3AR agonists are presently under test as anti-obesity drugs in clinical trials, but the outcome of these investigations is not yet known (Weyer et al., 1999). ß3AR agonists may also be beneficial in non-insulin-dependent diabetes (Arch and Wilson, 1996a; Lipworth, 1996; Weyer et al., 1998). The anti-diabetic effect can in part be secondary to weight reduction, but may also occur directly due to stimulation of the combustion of fatty acids, as the latter have several adverse effects on glucose metabolism. In addition, ß3AR agonists have been found to stimulate insulin secretion in some animal models (Arch and Wilson, 1996b; Lipworth, 1996), though

The native human ß 3-adrenoreceptor

85

Table 5.4 Possible clinical use of ß3AR agents

whether the same effect can be obtained in man remains to be established. The early ß3AR agonists have been used as anti-diabetic drugs, but results were disappointing (Arch and Wilson, 1996a; Lipworth, 1996; Weyer et al., 1999), probably for the same reasons that the agonists failed as anti-obesity drugs. Another possible indication for ß3AR agonists is that of anti-inflammatory agents for the gastrointestinal tract (Anthony, 1996). As mentioned above, the intestinal receptor is functional in man and may stimulate blood flow in the intestinal mucosa through the relaxation of smooth muscles. In addition to their spasmolytic properties, animal experiments have suggested that ß3AR agonists might heal gastric and small intestinal ulcers, though their clinical use in ulcero-inflammatory disorders of the gastrointestinal tract remains to be established. In theory, ß3AR antagonists might also be used as therapeutic agents, bearing in mind two points: first, the increased liploytic action of ß3AR in visceral adipose tissue of obese subjects; and second, the association between increased ß3AR function and the metabolic syndrome (see Section 5.3.1). In upper-body obese subjects with signs of the metabolic syndrome, ß3AR antagonists might preferentially inhibit fatty acid release from visceral adipose tissue and improve some of the metabolic abnormalities associated with the high ‘portal’ fatty acid flux (Lönnqvist et al., 1993; Arch and Wilson, 1996b; Arner, 1996). The putative clinical indications for ß3AR-directed agents are summarized in Table 5.4. Because the receptor is expressed mainly in adipose tissue it is an important target in anti-obesity and anti-diabetic drug programmes for the pharmaceutical industry. However, side effects from the intestine and heart—and perhaps also from the urinary bladder, gallbladder, brain and prostate gland, where the ß3AR is also functionally expressed—should be considered in both drug design and drug trials.

5.7

Conclusions

There is accumulating evidence that ß3AR is of functional importance in man, as the receptor has a clear role in promoting lipid mobilization from white adipose tissue— and in particular from visceral adipose tissue—because of its stimulatory effect on lipolysis in fat cells. The ß3AR appears to have a physiological role in the development of the metabolic (insulin resistance) syndrome because of its increased function in visceral adipose tissue of upper-body obese subjects, which is observed primarily in obese men. The receptor is also involved in the regulation of thermogenesis and contractility of the heart, urinary bladder and gallbladder, although its physiological

86

Arner and lönnqvist

importance and pathophysiological role in these processes is less well defined. A polymorphism, Trp64Arg, in the ß3AR has been described which has been shown to be associated with obesity and obesity complications in some, but not all, studies. This polymorphism may also alter the native receptor function; however, although it may influence body weight and metabolism, the polymorphism is probably not a major obesity gene abnormality. Although in the past ß3AR agonists and antagonists have been proposed as antiobesity and anti-diabetes drugs, the first-generation agonist drugs were either ineffective or had undesirable side effects. Consequently, second-generation agonists are currently being developed using the human ß3AR as a pharmaceutical model. Indeed, these agents might be useful in the future treatment of both obesity and non-insulin-dependent diabetes mellitus.

6

ß3-Adrenoreceptor-Mediated Responses in Heart and Vessels MAX LAFONTAN, DOMINIQUE LANGIN, JEAN GALITZKY, MICHEL BERLAN, CHANTAL GAUTHIER 1 AND GENEVIÈVE TAVERNIER Unité INSERM 317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangueil, 31403 Toulouse Cédex 4, France 1 Laboratoire de Physiopathologie et Pharmacologie Cellulaires et Moléculaires, INSERM CJF 96–01, Hôpital Hôtel-Dieu and Faculté des Sciences et Techniques, Université de Nantes, 44093 Nantes Cedex, France

6.1

Introduction

The cloning of the gene encoding the ß3-adrenoreceptor (ß3AR), rapidly followed by the delineation of its structural determinants in transfected cell lines, has opened a new avenue for our undertanding of the adrenergic regulation of a number of cells (Emorine et al., 1989, 1991; Piétri-Rouxel et al., 1995). The role and the regulation of the ß3AR have mainly been studied in white and brown fat cells, and in the gastrointestinal tract in various species, including humans. Other studies, based on ß3AR mRNA detection and/or functional studies with selective ß3AR agonists and antagonists have also revealed that its expression is probably not limited to these tissues (Arch and Kaumann, 1993). The majority of studies on the beta-adrenergic control of the cardiovascular system have focused on ß1- and ß3AR, and until now relatively little was known regarding ß3AR cardiovascular control. Here we present an overview of recent reports addressing the existence and role of the ß3AR in the cardiovascular system. Moreover, the existence of another putative ‘atypical’ ßAR—named the ß4AR—in heart will be discussed.

6.2

ßAR subtypes in the heart

A large number of studies have shown the co-existence of ß1- and ß2AR in the human heart. Their stimulation produces positive inotropic effects in in vitro human atrial and ventricular preparations (Brodde, 1991) as well as in single myocytes (Del Monte et al., 1993). However, a series of non-selective adrenergic antagonists with high affinity for myocardial ß1- and ß2AR have been shown to promote stimulant effects on atrium and ventricle. These actions were resistant to blockade by the highly selective ß1- and ß2antagonists. In 1989, Kaumann suggested that these partial agonists such as pindolol and related indoleamines may act through a third cardiac ßAR ressembling the ß3AR. These 87

88

Lafontan, Langin, Galitzky, Berlan, Gauthier and Tavernier

agents were causing stimulant effects at concentrations greatly in excess of those that usually caused blockade of the myocardial effects of catecholamines (Kaumann, 1989). This putative ßAR appeared to be coupled to adenylyl cyclase via a G protein of the Gs type. These preliminary studies have been followed by two major observations.

6.2.1

Functional ß3AR in the human heart

In isolated preparations of human ventricle, obtained from endomyocardial biopsies in patients having open-heart surgery or cardiac transplants, the ß3AR agonists promoted negative inotropic effects and reduction in the amplitude and duration of the action potentials (Gauthier et al., 1996). When considering the mechanical responses, the non-selective beta-adrenergic agonist, isoproterenol, at micromolar concentrations and under blockade of ß1- and ß2AR by nadolol, exerted negative inotropic effects. Nadolol is a ß1-/ß2-antagonist which has low affinity for the native and recombinant ß3AR (Bond and Clarke, 1987; Emorine et al., 1989; Galitzky et al., 1993a). The ß3adrenergic selective agonists (BRL-37,344, CL-316,243, SR-58,611A and CGP12,177), which are known to exert lipolytic and thermogenic effects in white and brown rodent fat cells respectively (Lafontan and Berlan, 1993), exerted concentration-dependent negative inotropic effects in human heart ventricle biopsies (Figure 6.1). They also induced a reduction in the amplitude and an acceleration in the repolarization phase of the action potential. The relative rank order of potency of the various ß 3 AR agonists (BRL-37,344>SR-58,611~CL-316,243>CGP-12,177) was similar to that observed in Chinese hamster ovary cells expressing human ß3AR (PiétriRouxel and Strosberg, 1995). CGP-12,177, the non-conventional ß 3-adrenergic agonist, having ß 1 - and ß 2 -adrenergic antagonist properties, also exerted a cardiodepressant action which was weaker than that elicited by the other ß3-agonists. The cardiodepressant effects of the various ß3AR agonists were unaffected by nadolol and metoprolol blockade, but were antagonized by bupranolol, which combines ß1-, ß2and ß 3AR antagonist properties. This pharmacological profile demonstrates the expression of a functional ß3AR in human heart. Treatment of endomyocardial biopsies with pertussis toxin blunted the cardiodepressant effect of BRL-37,344. This result is consistent with the coupling of the heart ß3-adrenoreceptor with a pertussis toxin-sensitive protein of the Go and Gi family. A more complete study is required to establish the Gi protein isoform activated by the ß3AR as well as the nature of the different steps of the receptor-effector pathway, downstream to Gi activation, involved in the control of electrical and mechanical responses. A recent report has shown that the decrease in human cardiac contractility promoted by activation of ß3AR operates through the activation of a nitric oxide synthase (NOS) pathway. Increases in nitric oxide (NO) production and intracellular cGMP levels were observed after treatment of endomyocardial biopsies with a ß3AR agonist. Pertussis toxin abolishes the effect of ß3AR stimulation both on cardiac contraction and on cGMP generation, suggesting the involvement of a Gi/o protein. Finally, immunohistochemical analysis have revealed abundant endothelial NOS (eNOS) but not intracellular NOS (iNOS) proteins in sections of human endomyocardial biopsies (Gauthier et al., 1998). Coupling of the ß3AR to Gi in heart is noticeable. At a basic research level, it is the first report implicating a ßAR whose primary biochemical function appears to be inhibitory and Gi-mediated. Most reports in native cell systems have shown that ßAR are coupled to Gs protein. However, it has

ß 3-Adrenoreceptor-mediated responses in heart and vessels

89

Figure 6.1 Effects of BRL-37,344 and SR-58,611 on the twitch tension of human endomyocardial biopsies. (A) Time-course of the effects of cumulative concentrations of BRL37,344 (left) and SR-58,611 (right) on twitch tension. After control value settlement (C), the ß3AR agonists were perfused for 10 min with each concentration to obtain a steady state of the effect. (B) Superimposed twitches obtained from the experiments illustrated in (A). (C) Concentration-response curves for the negative inotropic effect of BRL-37,344 and SR-58,611. Values are the means ±SEM of six to seven experiments. *Significant statistical difference (P