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VITAMINS AND HORMONES VOLUME 51

Editorial Board

FRANK CHYTIL

MARYF. DALLMAN JENNYP. GLUSKER

ANTHONYR. MEANS BERTW. O’MALLEY VERNL. SCHRAMM MICHAEL SPORN ARMENH. TASHJIAN,JR.

VITAMINS AND HORMONES ADVANCES IN RESEARCH AND APPLICATIONS

Editor-in-Chief

GERALDLITWACK Department of Pharmacology Jefferson Cancer Institute Thomas Jefferson University Medical College Philadelphia, Pennsylvania

Volume 51

ACADEMIC PRESS San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @ Copyright 0 1995 by ACADEMIC PRESS, INC. A11 Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495

United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NWI 7DX

International Standard Serial Number: 0083-6729 International Standard Book Number: 0- 12-709851-8 PRINTED IN THE UNITED STATES OF AMERICA 95 96 9 7 9 8 99 0 0 Q W 9 8 7 6

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4

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Former Editors KENNETH V. THIMANN ROBERTS. HARRIS Newton, Massachusetts

JOHNA. LORRAINE University of Edinburgh Edinburgh, Scotland

PAULL. MUNSON University of North Carolina Chapel Hill, North Carolina

JOHNGLOVER University of Liverpool Liverpool, England

GERALD D. AURBACH Metabolic Diseases Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland

University of California Santa Cruz, California

IRA G. WOOL University of Chicago Chicago, Illinois

EGONDICZFALUSY Karolinska Sjukhuset Stockholm, Sweden

ROBERTOLSON School of Medicine State University of New York at Stony Brook Stony Brook, New York

DONALDB. MCCORMICK Department of Biochemistry Emory University School of Medicine Atlanta, Georgia

This Page Intentionally Left Blank

Contents PREFACE .................................................................

xi

CAMP-Dependent Regulation of Gene Transcription by cAMP Response Element-Binding Protein and cAMP Response Element Modulator

JOEL F. HABENER, CHRISTOPHER P. MILLER,AND MARIOVALLEJO Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CAMP-Dependent Signal Transduction Pathway CAMP-ResponsiveTranscription Factors CREB, CAMP Response Elements ..................... . . . . . . . . . . . . . . . . . . . . . Mechanisms of Transciptional Transactivation . . . . . . . . . . . . . . . . . . . . . . . The CREB and CREM Genes Are Multiexonic in Structure: Alternative Exon Splicing Generates a Complex Array of Isoproteins That Are Either Transactivators or Transrepressors . . . . . . . . . . . . . . . . . . VII. CAMP-Dependent Autoregulation of the Expression of the CREB and CREM Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Roles of CREB and CREM in the Physiological Regulation of Gene I. 11. 111. IV. V. VI.

.......................................... on Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. X. Oncogenic Forms of CREB, CREM, and ATF-1 . . . . . . . . . . . . . . . . . . . . . . . XI. Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ..................................................

2 2 6 8 12

21 31 32 40 42 43 46

Multiple Facets of the Modulation of Growth by cAMP

PIERRE P. ROGER, SYLVIA REUSE,CARINEMAENHAUT, AND JACQUES E. DUMONT I. 11. 111. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Negative Control of Cell Cycle Progression by cAMP . . . . . . . . . . . . . . . . . Positive Control of Cell Cycle Progression by cAMP . . . . . . . . . . . . . . . . . . Relationship between Growth and Differentiation Controls by cAMP . .

vii

59 73 83 118

...

Vlll

CONTENTS

V. A Role for Cytoskeleton Changes in Control of Growth by CAMP? . . . . . VI . CAMP and the Growth of Cancer Cells .............................. VII . Conclusions and Perspectives ....................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

122 127 140 144

Regulation of G-Protein-Coupled Receptors by Receptor Kinases and Arrestins RACHELSTERNE-MARR AND JEFFREY L . BENOVIC I . Introduction .... ................... I1. GRK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... I11. Arrestins

........... .............

193 196 213 226 227

Vasopressin and Oxytocin: Molecular Biology and Evolution of the Peptide Hormones and Their Receptors EVITAMOHR.WOLFGANG MEYERHOF. AND DIETMAR RICHTER Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VP and OT Gene Expression and Regulation . . . . . . ............... VP and OT mRNA in Dendrites and Axons . . . . . . . ............... Somatic Recombination between the VP and OT Genes in Hypothalamic Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Evolution of the Vertebrate VP/OT Gene Family ..................... VI . Nonapeptide Receptors ............................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. I1. I11. IV.

235 237 242 247 249 251 258

Structure and Functions of Steroid Receptors M. G. PARKER I. I1. I11. IV. V. VI .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological Responses to Estrogens ................................ Intracellular Localization of Estrogen Receptors ..................... Hormone Binding and Receptor Dimerization ........................ Target Gene Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transcriptional Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

267 268 272 272 274 275

CONTENTS

VII. Specific Gene Transcription by the Estrogen Receptor . . . . . . . . . . . . . . . . VIII. Mechanism of Antiestrogen Action .................................. IX. Cross-coupling with Other Signaling Pathways ...................... References ........................................................

ix 276 277 280 282

Phosphorylation and Steroid Hormone Action WENLONG BAIAND NANCY L. WEIGEL Introduction ....................................................... Phosphorylation of Steroid Hormone Receptors ...................... Regulation of Steroid Hormone Receptors by Phosphorylation . . . . . . . . . Interaction between Steroid Hormone Action and Signal Transduction Pathways ......................................................... V. Summary: A Model of Steroid Hormone Action ...................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. 11. 111. IV.

289 290 296 301 305 307

Nucleocytoplasmic Shuttling of Steroid Receptors DONALD B. DEFRANCO, ANURADHA P. MADAN, YUTING TANG, UMAR. CHANDRAN, NIANXING XIAO,AND JUNYANC I. Introduction ....................................................... 11. Subcellular Localization of Steroid Receptors ........................ 111. Nucleocytoplasmic Shuttling of Steroid Receptors .................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

315 316 323 333

'Transcriptional Regulation of the Genes Encoding the Cytochrome P-450 Steroid Hydroxylases

KEITH L. PARKER AND BERNARD P. SCHIMMER I. 11. 111. IV. V. VI.

Introduction . . . . . . ..................................... Overview of Steroid Cell-Selective Expression . . . . . . . . . . . . . . . . . . . . . . . . . . Hormone-Regulated Expression . . . . ..................... Perspectives and Future Directio Summary ............................ .......... References .......................................

339 355 362 363

X

CONTENTS

Stress and the Brain: A Paradoxical Role for Adrenal Steroids CAMERON, BRUCES. MCEWEN,DAVIDALBECK,HEATHER HELENM. CHAO,ELIZABETH GOULD,NICOLASHASTINGS, YASUKAZU KURODA, VICTORIA LUINE,ANAMARIAMAGARINOS, CHRISTINA R. MCKITTRICK, MILESORCHINIK, CONSTATINE PAVLIDES, PAUL VAHER,YOSHIFUMI WATANABE, AND NANCY WEILAND I. Introduction ....................................................... 11. Adrenal Steroids and Hippocampal Neuronal Atrophy 111. IV. V. VI. VII. Stress Effects on Cognitive Perfo VIII. Deregulation of the HPA Axis in IX. Effects of Exogenous Glucocorticoid Treatment on Cognitive Performance in Humans ........................................... ..................................... X. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

371 374 377 380 387 388 391 393 394 395 396

Retinoids and Mouse Embryonic Development

T. MICHAELUNDERHILL, LORIE. KOTCH,AND ELWOOD LINNEY

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

459

Preface The contents of Volume 51 are divided into two sections. The first covers cyclic AMP, kinases, and polypeptide hormones and the second part covers steroid hormone receptors, related genes, and members of the gene family. The volume starts with a n in-depth summary of cyclic AMP regulation of gene transcription by CREB and CREM from members of the laboratory of J. F. Habener. This is followed by a lengthy discussion of the effects of cyclic AMP on growth by members of the J. E. Dumont laboratory. Next, a discussion of the G-protein-coupled receptors and their regulation by kinases and arrestins by R. Sterne-Marr and J. L. Benovic. The first section is completed by a contribution on the structural biology and evolution of vasopressin and oxytocin from D. Richter’s laboratory. The s e c d section begins with a summary of the structure and function of steroid receptors by M. G. Parker and is followed by a discussion on phosphorylation and steroid hormone action from W. Bai and N. L. Weigel. Next is a work on the nucleocytoplasmic shuttling of steroid receptors from the D. B. DeFranco laboratory. There is a report on the transcriptional regulation of genes encoding cytochrome P450 steroid hydroxylases by K. L. Parker and B. P. Schimmer and this is followed by coverage of adrenal steroid action on stress and the brain from the B. S. McEwen laboratory. This section ends with a summary of retinoids and their role in mouse development by the E. Linney laboratory. This is a rather large volume and should provide a great deal of upto-date information for the researcher and student on several topics of current interest. I thank members of the Editorial Board for suggesting some of these topics and authors. Academic Press continues to be supportive and prompt in the publication of assembled volumes. I trust that this fourth volume completed under my guidance will set the tone for future numbers in this serial. GERALD LITWACK

xi


VITAMINS AND HORMONES, VOL. 51

CAMP-Dependent Regulation of Gene Transcription by CAMPResponse Element-Binding Protein and CAMP Response Element Modulator JOEL F. HABENER," CHRISTOPHER P. MILLER,*?' AND MARIO VALLEJOt "Laboratory of Molecular Endocrinology Massachusetts General Hospital Howard Hughes Medical Institute Harvard Medical School Boston, Massachusetts 02114 'Reproductive Endocrinology Unit Massachusetts General Hospital Boston. Massachusetts 02114

I. 11. 111. IV. V.

Introduction CAMP-Dependent Signal Transduction Pathway CAMP-Responsive Transcription Factors CREB, CREM, and ATF-1 CAMPResponse Elements Mechanisms of Transcriptional Transactivation A. Kinases B. Phosphatases C. Other Transactivational Domains of CREB D. Adapter Proteins That Couple CREB Transactivation to the Basal Polymerase I1 Transcriptional Complex VI. The CREB and CREM Genes Are Multiexonic in Structure: Alternative Exon Splicing Generates a Complex Array of Isoproteins That Are Either Transactivators or Transrepressors A. Exons Encode Functionally Distinct Domains B. Repressor Isoforms of CREM Are Generated by Several Different Mechanisms C. Alternati.ve Exon Splicing Appears to Provide a Mechanism by Which to Modulate the Transactivational Activities of CREB and CREM D. Unphosphorylated CREB Can Repress Gene Expression Mediated by Phosphorylated CREB E. Exon-Deleted Repressor Isoform of CREM Down-regulates Expression of the c-ros and c-iun Genes VII. CAMP-Dependent Autoregulation of the Expression of the CREB and CREM Genes VIII. Roles of CREB and CREM in the Physiological Regulation of Gene Transcription A. Testes B. Anterior Pituitary Gland C. Brain: Hypothalamus and Pineal Gland D. Possible Role of CREB in Memory 'Present address: Genetics Institute, Inc., Cambridge, Massachusetts 02140 1

Copyright 1 ) 1995 by Academic Press. Inc All rights of' reproduction in any form reserved

2

JOEL F. HABENER et al.

IX. CREB/CREM Autoregulation Network X. Oncogenic Forms of CREB, CREM, and ATF-1 XI. Future Directions References

I. INTRODUCTION Forty years have elapsed since the initial discovery of CAMP, the important second messenger and mediator of cellular signal transduction (reviewed by Sutherland, 1972). Subsequently followed the discoveries of the cellular receptors that serve as sensors of hormones and other extracellular signaling molecules, the transducers consisting of stimulatory GTP-binding proteins (Gilman, 19891, and the effector protein kinases activated by CAMP(Krebs, 1989) involved in the signal transduction cascade ultimately leading to the regulation of gene expression. It has been only during the past 7 years that the final mediators in the CAMP-dependent signaling cascade have been identified. These are the CAMP-responsive transcription factors, DNA-binding proteins whose functions are to stimulate or repress the transcription of target genes. At least three CAMP-response DNA-binding proteins have been clearly identified so far: CREB (CAMPresponse elementbinding protein), CREM (CAMP-response element modulator), and ATF-1 (activating transcription factor 1). The intent of this chapter is to describe selected aspects of our current understanding about the workings of these CAMP-responsive transcription factors, with the main emphasis on CREB and CREM. Additional perspectives on CREB and CREM are given in other publications (Roesler et al., 1988; Habener, 1990; Montminy etal., 1990; Meyer and Habener, 1992,1993; Hoeffler, 1992; Foulkes and Sassone-Corsi, 1992; DeGroot and Sassone-Corsi, 1993; Vallejo and Habener, 1994; Sassone-Corsi, 1994; Lalli and Sassone-Corsi, 1994; Vallejo et al., 1995). 11. CAMP-DEPENDENT SIGNAL TRANSDUCTION PATHWAY In almost all living organisms cells communicate by sending and receiving chemical signals in the form of neurotransmitters and hormones. These signals induce specific cellular responses, for example, changes in plasma membrane properties (ion channels or receptors), cellular growth and metabolism, or gene expression, depending on the nature of the signal and the specific cell type involved (Herschman,

CREB AND CREM REGULATION

3

1989; Karin, 1992).To elicit their actions, the signal neurotransmitter and hormone molecules must first bind to specific high-affinity cellular receptors that reside in the cytoplasm (e.g., steroid hormone receptors), in the nucleus (e.g., thyroid hormone and retinoid receptors), or on the cell surface (e.g., plasma hormone receptors) of target cells. Small lipophilic molecules such as steroid hormones, thyroid hormones, and retinoids can readily diffuse across plasma membranes. After gaining access to the interior of the cells, these hormones bind to and activate nuclear or cytoplasmic receptors (Fuller, 1991; Gronemeyer, 1992; Simons et al., 1992; O’Malley, 1990; Beato, 1989). Often, the ligand-bound activated receptors are sequence-specific DNAbinding proteins that regulate the transcription of specific sets of target genes. Larger and more complex ligands such as peptide hormones cannot diffuse across plasma membranes. These molecules bind to and activate receptors located on the plasma membranes of target cells (Dohlman et al., 1991).The activated cell surface receptors then transduce signals to the interior of the cell by mechanisms that involve coupling to guanine nucleotide-binding proteins (G proteins) and/or phosphorylation cascades (Gilman, 1987; Dohlman et al., 1991; Simon et al., 1991; Yarden and Ullrich, 1988; Ullrich and Schlessinger, 1990). In many cases these signal transduction pathways lead to the nucleus in order to regulate gene transcription. There are several wellcharacterized pathways by which signals can be transmitted from the cell surface to the nucleus (Fig. 1). These pathways can be conceptualized as having four components, or “messenger systems.” The first messenger is the hormone ligand, a macromolecule that binds to the cell surface receptor coupled to the G proteins. This complex is responsible for sensing the signal (receptor) and transmitting the signal (G protein) across the plasma membrane. The first messenger systems regulate intracellular levels of second messengers such as CAMP,diacylglycerol, and calcium (Ca2+).These regulatory substances are responsible for the transfer of information from the interior of the plasma membrane throughout the cell. The second messengers regulate the activities of the third component, effector molecules such as CAMP-dependentprotein kinase A (PKA),protein kinase C (PKC), and calmodulin-dependent protein kinase (CaMK).These kinases regulate by phosphorylations the activities of the final component in the signal transduction pathways, DNA-binding proteins such as CREB. CREB is one of several closely related transcription factors capable of mediating transcriptional regulation by cAMP (see below). The cAMP signaling pathway is one of the most important intracellular signal transduction pathways in eukaryotic cells (Habener,

4

JOEL F. HABENER et al

CAMP-

PKA

RIPTION

FIG.1. The cellular signal transduction pathways that modulate gene transcription. The steps in the signaling pathways are designated as first through fourth component messengers (see text). The first components are hormones and ligands (H,, H,, and H,) that bind to and activate cell surface receptors (Rl, R,, and R3), leading to elevation of intracellular levels of second messenger mediator molecules and subsequent activation of effector proteins (third messengers). The effector proteins are typically kinases that phosphorylate the fourth messengers, DNA-binding proteins. The DNA-binding phosphoprotein CREB is activated by the cAMP signaling pathway. DAG, diacylglycerol; PKC, protein kinase C; PKA, protein kinase A; CaMK, calmodulin-dependent protein kinase; P, phosphate.

1993) (Fig. 2). Elevation of intracellular cAMP occurs when plasma membrane receptors coupled to stimulatory G proteins (G,) are bound and activated by their specific ligands, leading to the activation of adenylyl cyclase (AC). AC catalyzes the conversion of ATP to CAMP, which then binds and activates PKA. In the absence of CAMP, the inactive PKA holoenzyme is a heterotetrameric protein complex consisting of two regulatory subunits (R) and two catalytic subunits (C). Thus far, two classes of R subunits (RI and RII), each with two isoforms (RIa, RIP, RIIa, and RIIP), and three isoforms of the C subunit (Ca,CP, and Cr) have been described. cAMP binds cooperatively to two sites on the R subunits within the holoenzyme, thereby liberating free active C subunits. In addition to modulating the activity of the C subunits, the RII subunits bind proteins known as A kinase-anchoring proteins (AKAPs). AKAPs are a family of proteins responsible for tethering type I1 PKA holoenzyme to specific cellular structures, including the nuclear matrix (Scott and McCartney, 1994).Recent experimental evidence indicating the presence of PKA holoenzyme within the nucleus is described later (Section V1,D). Nuclear compartmentalization of type I1 PKA may play a n important role in regulating the phosphorylation of nuclear proteins. It appears that the transactivational ac-


5

mRNA

t

Protein

FIG.2. CREB-mediated CAMP-dependent gene transcription. Hormones and ligands (H) that bind plasma membrane receptors coupled to stimulatory GTP-binding proteins (G,) induce an increase in intracellular cAMP levels by activation of adenylate cyclase (Ac), which then catalyze the conversion of ATP to CAMP. Increases in cAMP levels cause dissociation of protein kinase A (PKA) holoenzyme to liberate inactive regulatory subunits (R)and active catalytic subunits (C); the latter are translocated to the nucleus to phosphorylate (P+)transcription factors such as CREB. Nuclear translocation of PKA C subunits is regulated, whereas translocation of CREB is constitutive. CREB binds to cAMP response elements (CREs; e.g., 5'-TGACGTCA-3'), leading to increased gene transcription and subsequent protein synthesis. R, receptor and regulatory subunit of PKA.

tivity of CREB may be regulated, in part, at the level of the reversible translocation of the free active catalytic subunit of PKA from the cytoplasm to the nucleus (Hagiwara et al., 1993). CREB itself is constitutively transported into the nucleus, where it awaits phosphorylation. This situation is different from that of several other transcription factors, such as the thyroid hormone T, and glucocorticoid receptors, Nuclear factor-&, and CAAT box/enhancer-binding protein p (C/EBP-p). The transport of these proteins from the cytoplasm to the nucleus is activated by phosphorylation, although the exact mechanisms for this regulation are not fully understood. CREB contains a strong consensus site (RRPSY) for phosphorylation by PKA. This site lies within a portion of CREB known as the phosphorylation region (P box), or kinase-inducible domain (KID). Phosphorylation of the serine residue within this sequence potently activates the transactivation functions of CREB (Gonzalez and Montminy, 1989). Within the nucleus CREB recognizes and binds to DNA sequences typified by the consensus palindromic cAMP response element (CRE), 5'-TGACGTCA-3' (Section IV).

6


111. CAMP-RESPONSIVE TRANSCRIPTIONFACTORS CREB, CREM, AND ATF-1 Regulation of gene transcription is accomplished by the interaction of DNA-binding proteins with DNA regulatory sequences and with proteins of the general transcription machinery (Maniatis et al., 1987; Mitchell and Tjian, 1989; Ptashne, 1988). Several DNA-binding proteins are recognized to mediate CAMP-regulated gene transcription. These include CREB (Hoeffler et al., 1988; Gonzalez et al., 19891,CREM (Foulkes et al., 1991b), and ATF-1 (Rehfuss et al., 1991; Liu et al., 1993) (Fig. 3).As discussed later (Section VI), CREB, CREM, and ATF-1 exist as multiple isoforms due to the alternative splicing of exons during processing of primary RNA transcripts to the mature mRNAs. For example, two isoforms of CREB are ubiquitously expressed in most tissues, CREB327 and CREB341, but differ by an alternatively spliced exon (exon D) encoding 14 amino acids (HoeMer et al., 1990) (Fig. 3). CREB and CREM are members of a larger class of transcription factors, known as bZIP proteins because they possess a basic region responsible for DNA binding and a n adjacent leucine zipper required for dimerization. Dimerization is an absolute requirement for the binding

7 rDNA binding 1

1-transactivation cAMP+PKA CREE

ATF-1

Q1

domain lbZlPl

+p P-BOX

76%

Q2

BR

91%

ZIP

86%

FIG. 3. CAMP-responsive bZIP proteins CREB, CREM, and ATF-1. These three transcription factors constitute a distinct subfamily of bZIP proteins characterized by highly conserved basic regions (BR), leucine zippers (ZIP),and phosphorylation regions (P Box, also known as the kinase-inducible domain). The percentages of amino acid (aa) similarities to those of corresponding regions of CREB are indicated. CREM I and CREM I1 are generated by alternative exon splicing, resulting in proteins with different DNA binding domains. Locations of the glutamine-rich regions (Q1 and Q 2 ) and the protein kinase A (PKA) phosphorylation site, Serl*g, are also shown. P, phosphate.


7

of bZIP proteins to DNA. The basic region is approximately 30 amino acids in length, is well conserved, and contains a relatively high proportion of the positively charged amino acids lysine and arginine. The leucine zipper lies immediately C-terminal to the basic region and consists of a region of amino acids with leucines occurring at every seventh position. The leucine zipper forms an amphipathic a-helix, with hydrophobic residues (including leucines) along one face of the helix and hydrophilic residues along the opposite face. Dimerization of bZIP proteins occurs by formation of a parallel coiled-coil structure with the hydrophobic surfaces of two leucine zipper a-helices facing each other (Landschultz et al., 1988; Vinson et al., 1989). Dimerization brings the positively charged basic regions into a configuration that facilitates recognition of target DNA sequences through contacts of the basic regions with nucleotides in the major groove of the DNA helix. Dimers of bZIP proteins are thought to assume a Y-shaped structure in which the stem of the Y is formed by the juxtaposed leucine zippers and the arms by the basic regions. This structure, first proposed as the “scissors grip” model by Vinson et al. (1989),appears to be basically correct according to results from X-ray crystallographic analysis of the yeast bZIP protein GCN4 (O’Shea et al., 1991). In addition to CREB-related DNA-binding proteins, the bZIP transcription factor family also includes C/EBP-related proteins, Fos/Junrelated proteins, and several more distantly related factors (Johnson and McKnight, 1989; Meyer and Habener, 1993). The C/EBP-related factors are expressed during terminal cell differentiation, whereas the Fos/Jun-related proteins mediate early transcriptional responses to the activation of PKC and growth factor-activated Ras-dependent signaling pathways. The C/EBP- and Fos/Jun-related bZIP proteins can also bind CREs and closely related motifs, but they do so generally with lower affinities than CREB, CREM, or ATF-1. The Fos/Jun-related proteins bind preferentially to the closely related tumor promoter agent response elements (typified by the sequence 5‘-TGACTCA-3’), whereas C/EBP and related proteins prefer sequence elements more closely related t o the CCAAT motif. The functional significance of Fos/Jun or C/EBP interactions with CREs is not understood. In some circumstances, however, C/EBP activates CRE-mediated transcription independent of cAMP signaling (Park et al., 1993; Vallejo et al., 1995). Among all proteins within the bZIP family, CREB, CREM, and ATF-1 are unique in that they mediate transcriptional responses via the modulation of cAMP signaling pathways. CREB, CREM, and ATF-1 are structurally similar (Fig. 3). The amino acid sequences are highly conserved in their basic regions and leucine

8


zippers, and all three have P boxes and glutamine-rich regions (Q1 and Q2) that function in transcriptional activation. The translational products of the CREM gene are unusual in that they consist of activators or inhibitors of CRE-mediated transcription, depending on whether exons C or G (encoding the glutamine-rich Q1 and Q2 regions; see Section VI,A) are spliced into the mature CREM mRNA (Foulkes et al., 1992). The absence of exons C and G results in CREM isoforms that bind CRE sequences but, since they lack the Q1 and Q2 regions, are unable to activate transcription, and therefore function as transcriptional repressors. The CREM gene products can also undergo alternative splicing of exons encoding two different bZIP domains (I and 11, Fig. 3) (Foulkes et al., 1992).The use of an alternative intronic promoter results in CAMP-inducible transcription of short mRNAs encoding potent repressors of CRE-mediated transcription known as inducible cAMP early repressors (ICERs) (Stehle et al., 1993; Molina et al., 1993). ICER proteins contain DNA binding and dimerization domains but lack glutamine-rich regions and P boxes. Consequently, they can bind CRE sequences and form homo- and heterodimers but cannot activate CRE-mediated transcription. To repress CRE-mediated transcription, ICER protein-s may bind CREs as nonfunctional homodimers or may dimerize with activators to form nonfunctional heterodimers. The CREM gene thus encodes most of the known repressors of CAMPinduced transcription. The role of alternative exon splicing in the interconversion of transactivator to transrepressor isoforms of CREB and CREM is discussed in more detail in Section V1,B. IV. cAMP RESPONSEELEMENTS Many genes whose transcription is regulated by cAMP contain the palindromic sequence 5’-TGACGTCA-3’, or close variations of it, in their promoter regions (Deutsch et al., 1988a,b; Roesler et al., 1988). This sequence is known as the CRE, and its integrity is required for transcriptional responses to CAMP. The perfect palindromic CRE sequence serves as a high-affinity binding site for CREB, CREM, and ATF-1. CREs also serve as binding sites for other bZIP proteins, including those related to C/EBP (Bakker and Parker, 1991; Park et al., 1990,1993; Vallejo et al., 19951, and Fos or J u n (Hai and Curran, 1991; Ryseck and Bravo, 1991). In these instances CREs may function as CAMP-independent transcriptional enhancers. Variant CREs include asymmetrical or atypical sequences that differ from the consensus motif by single or multiple nucleotide deletions


9

or substitutions. Examples of these include CRE-like sequences in the rat insulin (Philippe and Missoten, 19901,human enkephalin, and vasoactive intestinal polypeptide gene promoters (Deutsch et al., 1988a,b). Some of these sequences contain intact CRE half-sites (B’-CGTCA-3’) and are still capable of binding CREB and related proteins and mediating transcriptional regulation by CAMP, albeit to a lesser extent than for perfect palindromic CREs. Even among perfect palindromic CRE sequences there are variations in basal activities and relative responsiveness to CAMP-induced transcriptional activation. One reason for this variation is that sequences adjacent to the core palindromic octamer can influence the binding stability and/or transactivation functions of proteins bound to the core CRE (Deutsch et al., 1988a,b).The flanking sequences can serve as binding sites for additional DNA-binding proteins that interact with proteins bound to the core CRE octamer (Muro et al., 1992; Ikuyama et al., 1992; Miller et al., 1993). Additionally, flanking sequences can influence the stability of protein binding to the core octamer (Ryseck and Bravo, 1991). Results from DNase I footprinting and methylation interference experiments show that proteins in nuclear extracts and purified bacterially expressed CREB make base contacts that are well outside the core CRE octamer (Andrisani et al., 1988; Powers et al., 1989; Knepel et al., 1990; Vallejo et al., 1992). Consequently, it is not surprising that sequences flanking the core CRE can influence the binding or function of transcription factors bound to the CRE. All known bZIP transcription factors bind DNA as homodimers or heterodimers. Dimerization occurs in the absence of DNA, and results from experiments with synthetic peptides indicate that isolated leucine zippers of CREB form stable complexes similar to those of intact proteins (Yun et al., 1990). Additionally, from studies with Jun, Fos, GCN4, and C/EBP, it is known that the specificity and stability of the dimerization of bZIP proteins are determined by amino acids at particular positions within the leucine zipper a-helices (O’Shea et al., 1992; Scheurmann et al., 1991; Neuberg et al., 1991). Most bZIP proteins form homodimers capable of binding DNA. An important exception to this generalization is Fos, which does not form stable homodimers (Halazonetis et al., 1988; Smeal et al., 1989). In general, bZIP protein homodimers bind symmetrical CRE-like sequences, whereas heterodimers bind asymmetrical elements (Fig. 4). One criterion for establishing bZIP protein families (CREB, C/EBP, or Fos/Jun related) is the degree of amino acid similarity in the bZIP domains. To some extent, heterodimerization is permitted within these

10

qqp ($-()(-& JOEL F. HABENER et al.

TF-2

----c

--EX--

-TGACGNNN Asymmetric

Symmetric

CREE-P

CIEB

-

FIG. 4. Differential DNA binding of homo- and heterodimeric bZIP proteins to symmetrical and asymmetrical CAMP response elements (CREs). Homodimers of CREB, Jun, and ATF-2 bind to symmetrical CREs, whereas heterodimers of Jun:Fos, Jun:ATF-2, and CIATF:CIEBP preferentially bind asymmetrical or partial CREs. CREB:CREB homodimers also bind CRE half-sites. To bind asymmetric CREs CREB may have to be phosphorylated (P).

families because of the high similarity in the bZIP domains. Certain cross-family heterodimers can also form (Table I). For example, there is selective heterodimerization between members of the ATF and Fos/Jun families. Different heterodimers display DNA binding specificities distinguishable from each other and from those of their parental homodimers (Hai and Curran, 1991). CREB, ATF-2, and J u n bind symmetrical CREs as homodimers, and ATF-2 and ATF-3 form heterodimers with J u n . Ju n , but not Fos, heterodimerizes with ATF-2 (Macgregor et al., 1990). The binding specificity of ATF-2/Jun heterodimers is different from that of Jun/Fos heterodimers, indicating that the DNA binding specificity of J u n is modified by association with Fos or ATF-2. ATF-2iJun heterodimers have a preference for symmetrical CRE sites, but bind asymmetrical CREs and activator protein 1(AP-1) sites, whereas ATF-3/Jun heterodimers bind symmetrical and asymmetrical CREs and AP-1 sites with similar affinities (Hai and Curran, TABLE I TRANSCRIPTION FACTOR CROSS-TALK AMONG DIFFERENT bZIP FAMILIES OF PROTEINS Transcription factor Family 1

Family 2

Heterodimers

Jun CIEBP Jun Jun

Fos ATF C/EBP ATF

c-Jun:c-Fos CIEBP-PCIATF JunD:C/EBP-p c-Jun:ATF-2

11


1991). Recently, it has been shown that ATF-3 and JunD form heterodimers and act synergistically to stimulate CAMP-dependent transcription of the proenkephalin gene (Kobierski et al., 1991; Chu et al., 1994). In a similar manner different heterodimer combinations of Fos and J u n proteins bind with differing stabilities to various symmetrical and asymmetrical AP-1 elements and CREs (Ryseck and Bravo, 1991). Fos and J u n can heterodimerize with C/EBP-P to repress transcriptional activation by C/EBP-P (Hsu et al., 1994). In this circumstance, however, heterodimerization with Fos or J u n decreases the affinity of CIEBP-P for binding sites in the interleukin-6 promoter, leading to repression of transcription. One particularly interesting example of cross-family heterodimerization is the interaction between C/EBP-P and C/EBP-related ATF (C/ATF) (Vallejo et al., 1993) (Fig. 5). On the basis of the amino acid sequence of its bZIP domain, C/ATF is a member of the ATF/CREB family, yet it forms stable heterodimers with C/EBP-(3. Indeed, C/ATF was originally isolated in a search for novel CiEBP family members that could dimerize with C/EBP-P (Vallejo et al., 1993). C/ATF homodimers bind to symmetrical CREs and weakly activate transcription, but do not bind or transactivate asymmetrical CREs such as that of the gene encoding phosphoenolpyruvate carboxykinase, or CAAT-related DNA elements (Vallejo et al., 1993). C/ATF:C/EBP-P heterodimers bind to both symmetrical and asymmetrical CREs and activate transcription from both types of elements, but not from C/EBP sites. Therefore, C/ATF may function to redirect the binding of C/EBP-P from CAAT-related sequences to asymmetrical

Concentrations: Predominant: DNA-BPs: Elements

C/EBP>>C/ATF

CIATF=CIEBP

CIATFwwCIEBP

homodimers

heterodimers

homodimers

-1 CBS t

1

CRE-2r

CRE-1

f

TGCGCAAT TGACGCAG TGACGTCA Target genes containing different control elements

FIG. 5. Illustration of how DNA binding site preferences are determined by relative nuclear concentrations of CIEBP and C/ATF. When CiEBP levels exceed those of CIATF, formation of C/EBP hornodimers is favored, which preferentially bind CCAAT box-like DNA sequences (CBS). When CIATF levels are greater than those of CIEBP, C/ATF homodimers are favored, which bind symmetrical CRE sequences (e.g., CRE-1 of the rat proenkephalin gene promoter). At intermediate concentrations CiATF and CiEBP heterodirners are formed that preferentially bind asymmetrical CRE-like sequences (e.g., CRE-2 of the rat proenkephalin gene). BP, binding protein.

12


CREs. A hypothetical model has been proposed in which the relative levels of C/ATF and C/EBP proteins determine the relative amounts of homo- and heterodimers present, and consequently dictate the promoter binding sites that are occupied and the genes that are transcriptionally activated (Fig. 5). When C/EBP levels exceed those of C/ATF, the formation of C/EBP homodimers is favored, leading to the activation of genes containing canonical C/EBP recognition sites (CCAAT box sites). Conversely, when C/ATF levels are greater than those of C/EBP, the formation of C/ATF homodimers will be favored, resulting in the activation of genes containing symmetrical CREs. Finally, when levels of C/ATF and C/EBP are similar, the formation of heterodimers will be favored, resulting in the activation of genes containing asymmetrical CREs.

V. MECHANISMS OF TRANSCRIPTIONAL TRANSACTIVATION The transactivational activities of CREB, CREM, and ATF-1 are directly dependent on their state and extent of phosphorylation. Thus, the interplay between the phosphorylating protein kinases and the dephosphorylating protein phosphatases is critical in determining the relative transactivation potential at any given moment. A. KINASES The PKA phosphorylation motif shared by CREB, CREMT, and ATF-1, RRPSY, is located within a region of about 60 amino acids known as the KID, or P box (Fig. 6). It has been demonstrated that Ser133 in CREB341, and the corresponding Serllg in the CREB327 isoform, present in the RRPSY motif are phosphoacceptor sites for phosphorylation by PKA (Gonzalez and Montminy, 1989). Ser133 in CREB corresponds to Serll7 in CREMT and Ser68 in ATF-1, respectively. Phosphorylation of Ser133 (Serllg) by PKA is an absolute requirement for the generation of CREB transactivational activity in response to CAMP signaling, as assessed by mutational analyses of CREB expressed in transfected cells as well as by direct microinjection of recombinant CREB into the nuclei of fibroblasts (Gonzalez and Montminy, 1989; Alberts et al., 1994a). Although phosphorylation converts Ser133 from a n uncharged into a negatively charged amino acid, it appears that the gain of a negative charge per se is not sufficient for CREB activation, because Ser133 cannot be substituted for by other

13


I

I

/

I

\

I

I

1

\ \

I I I I

1 1 1 1 '

'"GTDGVQGLQTLTMTNAA~ 1-CBP

binding domln-1

kTARl,,pblndlnp domsln+

FIG.6. The structure of the CREB protein. Depicted are the glutamine (&)-rich regions located at either side of the kinase-inducible domain (KID),or phosphorylation box (P BOX); the basic region that contains the DNA binding domain; and the leucine zipper (L) domain (ZIP) that mediates protein dimerization. The KID (amino acids 108-132) contains the CAMP-dependent protein kinase A (PKA) phosphorylation site (A-kinase box), and is also the region that binds to CREB-binding protein (CBP). The amino acid sequence of the hydrophobic cluster (HC) that interacts with the TATA box-associated factor TAFII,,o is also shown.

negatively charged residues (Gonzalez and Montminy, 1989). Therefore, the mechanism by which phosphorylation by PKA activates CREB remains unclear. It has been proposed that phosphorylation induces a n allosteric transition that results in a conformational change of CREB from an inactive into a n active configuration (Brindle et al., 1993; Gonzalez et al., 19911, perhaps facilitating direct interactions with other transcription factors or coactivator proteins, such as the CREB-binding protein (CBP; see Section V,C). Increasing evidence indicates that Serl33 provides a common phosphorylation site at which different signal transduction pathways converge. Thus, CREB mediates some of the transcriptional changes observed after stimulation by membrane depolarization and calcium influx into cells as a result of phosphorylation of Serl33 by Ca2+/ calmodulin-dependent kinases (e.g., CaMK) (Enslen et al., 1994; Sheng et al., 1990, 1991; Dash et al., 1991; Matthews, et al., 1994; Schwaninger et al., 1993; Bading et al., 1993). ATF-1 has also been shown to mediate Caz+-induced transcriptional responses (Liu, 1993), and CREM can be phosphorylated by CaMK (DeGroot et al., 1993b). Therefore, CREB, CREM, and ATF-1 are common targets that integrate signals conveyed by the activation of two different transduction pathways, one CAMPdependent and the other Caz+/calmodulin dependent. In addition, CaZ+-induced gene transcription in neuronal cells is enhanced by nitric oxide through mechanisms that may involve CREB

14


phosphorylation by PKA (Peunova and Enikolopov, 19931, although the exact details of that interaction are unknown. Serl33 is phosphorylated in uitro by both CaMK-I1 and CaMK-IV (Matthews et al., 1994; Enslen et al., 1994). The cellular distribution of CaMK-IV is both cytoplasmic and nuclear, whereas CaMK-I1 is predominantly cytoplasmic. In addition, CaMK-I1 phosphorylates CREB on both Ser133 and Ser142, and phosphorylation at Ser142 inhibits the transcriptional capacity of CREB (Sun et al., 1994). These circumstances provide a n explanation for the observations that CREB-mediated transcriptional responses appear to be associated with CaMK-IV-dependent kinase activity (Matthews et al., 1994; Enslen et al., 1994). It is important to emphasize that both Ca2+- and CAMP-dependent pathways interact at different levels of the signal transduction cascade. For example, CAMP-induced phosphorylation regulates the activity of specific voltage-sensitive Ca2+ channels (Sculptoreanu et al., 19931, and in turn, Ca2+ affects the activity of certain phosphatases (Cohen, 1989) and AC isoforms (Katsushika et al., 1992; Yoshimura and Cooper, 1992). CREB is also phosphorylated by the activation of signal transduction pathways by growth factors such as transforming growth factor p l (Kramer et al., 1991) and nerve growth factor (NGF) (Ginty et al., 1994). Ginty et al. (1994) have recently reported that in PC12 cells, CREB mediates NGF-induced c-fos transcription via phosphorylation by a previously unidentified kinase. This CREB kinase (CREBK) is a single polypeptide with an apparent molecular mass of 105 kDa. The stimulation of CREBK by NGF (and related growth factors) is dependent on the stimulation of a Ras signaling pathway and does not involve the activation of Ca2+- or CAMP-dependent signaling pathways. The best-characterized Ras signaling pathway to date involves the activation of Ras after stimulation of cell surface receptor tyrosine kinases, which in turn results in the stepwise activation of the protein kinases Raf-1, mitogen-activated protein kinase (MAPK), and MAPK kinase (Blenis, 1993). NGF is only one of several growth factors that activate the Ras-MAPK pathway to phosphorylate several target proteins, including the transcription factors, c-Fos, c-Jun, and c-Myc (Blenis, 1993). However, NGF-induced Ras-dependent phosphorylation of CREB does not appear to involve activation of the MAPK pathway (Ginty et al., 1994). Therefore, the exact mechanisms of CREBK activation following NGF-induced stimulation of Ras remain to be determined. Interestingly, the CAMPand Ras-MAPK pathways interact at more proximal levels of the signal transduction cascade. Thus, it has been

15


observed in a number of cell types that activation of PKA by cAMP results in the inhibition of growth factor-induced MAPK activity (Burgering et al., 1993; Cook and McCormick, 1993; Graves et al., 1993; Sevetson et al., 1993; Wu et al., 1993). Studies carried out in uitro suggest that PKA does not directly inhibit MAPK and MAPK kinase activities (Graves et al., 1993). Rather, it appears that phosphorylation of Raf-1 by PKA is responsible for the observed blockade in the RasMAPK pathway (Cook and McCormick, 1993). Based on the observations described above, the following model is proposed (Fig. 7). Stimulation of cell surface receptor tyrosine kinases by NGF and other growth factors would result in the activation of Ras,

growth factor

depolarlzation

I cell membrane

I

I

I

I

SIGNALING

"

ZYS

FIG.7. Interactions among the Ras, Ca2+icalmodulin,and adenylyl cyclase-protein kinase A (PKA) signaling pathways. Signal transduction pathway cross-talk results in transcriptional responses from different sets of genes. Activation of growth factor receptors stimulates the Ras-mitogen-activated protein kinase (MAPK) pathway, resulting in the phosphorylation of transcription factors such as c-Jun, c-Fos, and c-Myc. In addition, a Ras-dependent CREB protein kinase (CREBK) that phosphorylates CREB is activated, although this pathway does not seem to involve MAP kinases. The Ras-MAPK pathway is also activated in response to Ca2+ entry triggered by membrane depolarization. Calcium entry activates Caz+icalmodulin-dependent protein kinases (CaMK), which phosphorylate CREB. In addition, Ca2+ alters the activity of some adenylyl cyclase (AC) isoforms, varying the amounts of cAMP that activate PKA to phosphorylate CREB. Finally, cAMP stimulation results in transcriptional responses mediated by transcription factors such as AP-2 and JunD, although direct phosphorylation of these transcription factors by PKA has not been demonstrated. MAPKK, MAPK kinase.

16


which would act as a branching point to distribute the signal through at least two different pathways: one leading to the phosphorylation of transcription factors such as c-Fos, c-Jun, and c-Myc via stimulation of MAPK activity, and the other leading to the phosphorylation of CREB (and perhaps its close homologs CREM and ATF-1) via stimulation of CREBK. In this manner growth factor activities would influence the relative transcription rates of a wide repertoire of genes important for cellular proliferation and/or differentiation. Subsequent activation of the CAMP-dependent pathway by different extracellular signals would attenuate the transcriptional activities of only a subset of growth factor-activated genes, without affecting (or perhaps potentiating) the transcriptional activities of genes regulated by CREB, which provides a point of convergence for both Ras-CREBK and CAMP-PKA signaling pathways. In addition, new genes would be recruited by the CAMPdependent activation of other transcription factors, such as AP-2, ATF-3, and JunD (Chu et al., 1994; Kobierski et al., 1991; Williams et al., 19881, although no direct evidence that PKA phosphorylates these transcription factors exists to date. Interestingly, the Ras-MAPK pathway is also activated by Ca2+ influx through voltage-sensitive channels (Rosen et al., 1994). All of these interactions at different levels among three different signaling pathways indicate the existence of a complex intracellular cross-talk that probably results in a fine regulation of gene expression. These precise mechanisms of response may allow cells to cope with the large constellation of environmental signals to which they are continuously exposed. Thus, by providing a common target for different intracellular signaling pathways, CREB may be directly involved in the regulation of a wide variety of cellular processes, including metabolic adaptations to the extracellular environment, proliferative responses, and differentiation events. In addition to phosphorylation of Ser133 by PKA, CaMK-IV, or CREBK, the KID also contains putative sites for phosphorylation by the processive protein kinases casein kinase I1 (CK-11) and glycogen synthase kinase-3 (GSK-3). Phosphorylation by these kinases is hierarchical, that is, it is facilitated by the prior phosphorylation of adjacent sites (Roach, 1991; Fiol et al., 1994). In the case of CREB, phosphorylation of Ser133 is the required first event. It has been suggested that CK-I1 and GSK-3 may play a functional role in regulating the transcriptional transactivation activity of CREB (Fiol et al., 1994). However, there is evidence suggesting that phosphorylation of CREM by CK-I and CK-I1 alters the binding capacity of CREMT (DeGroot et al., 199313).


17

B. PHOSPHATASES Under physiological conditions the degree of phosphorylation of a given protein in cells is the result of the regulated balance between the opposite effects of protein kinases and phosphatases. Accordingly, the transcriptional transactivation activity of CREB is down-regulated by phosphatase-mediated dephosphorylation. Protein phosphatase types 1 (PP-1)and 2A (PP-2A), two specific nuclear phosphatases, have been found to attenuate the CAMP-induced transcriptional activity of CREB (Wadzinski et al., 1993; Hagiwara et al., 1992). PP-1 dephosphorylates Ser133, and the attenuation by this phosphatase of the transcriptional responses induced by CREB is inhibited by a specific PP-1 inhibitor, 1-1(Hagiwara et al., 1992; Alberts et al., 1994b). However, different studies (Wheat et al., 1994; Wadzinski et al., 1993) have provided evidence that PP-2A is the primary enzyme involved in the inactivation of CREB by dephosphorylation. This notion is also supported by studies investigating the effects of phosphorylation on the binding of CREB to different high- and low-affinity CRE sites (Nichols et al., 1992). The notion that PP-2A is the enzyme that dephosphorylates CREB under physiological conditions derives from studies carried out by inhibiting PP-2A with simian virus 40 (SV40) small-tumor antigen, which inhibits the dephosphorylation of CREB and enhances CREB transactivation functions (Wheat et al., 1994). In contrast, 1-1,but not SV40 small-tumor antigen, was found to inhibit the dephosphorylation of and enhance transactivation by CREB (Alberts et al., 1994b). Thus, it remains unclear whether both PP-1 and PP-2A, or only one of them, is functionally important in the regulation of CREB activity induced by CAMP in cells. There is also evidence for the involvement of phosphatases in addition to PP-1 and PP-2A in regulating the phosphorylation of CREB. In uitro experiments show that Ca2+/calmodulin-dependentPP-2B (calcineurin) dephosphorylates CREB (Enslen et al., 1994). Calcineurin phosphatase activity may be required for CREB-mediated glucagon gene transcription, although this activity may occur via a n indirect mechanism (Schwaninger et al., 1993a,b). The overall functional significance of these apparently conflicting actions of protein phosphatases awaits further clarifications.

C. OTHERTRANSACTIVATIONAL DOMAINS OF CREB The transactivational domain of CREB contains two other regions with a relatively high content of glutamine residues. These regions,

18


termed Q1 and Q2, are located at either side of the KID (Fig. 6). Initial studies revealed that deletions of either of these two regions result in a marked reduction of CREB transcriptional activity (Brindle et al., 1993; Gonzalez et al., 1991; Quinn, 1993). In the absence of phosphorylation by PKA, the Q1 and Q2 domains are thought to be important for maintaining the basal activity of CREB. This activity may be due to interactions of the Q1 and/or Q2 domains with other transcription factors bound to neighboring sites located in proximity to a CRE. Consistent with this notion are the findings in the pancreatic islet cell lines Tu6 (Leonard et al., 1992) and RIN1027-B2 (Vallejo et al., 1995). CREB activity on the somatostatin gene promoter is dependent not on CAMP stimulation, but rather on interactions with another transcription factor(s) bound to the promoter located in the proximity of the CRE. The existence of glutamine-rich transactivation domains (Mitchell et al., 1989) has been documented in several transcription factors, such as S p l (Courey and Qian, 1988). These domains may provide interaction surfaces for the coupling of transcription factors with specific coactivator proteins associated with the RNA polymerase I1 complex and may result in the activation of transcription of target genes (Dynlacht et al., 1991; Hoey et al., 1993). Therefore, it is likely that the Q1 and Q2 regions in CREB are similarly involved. An interaction has been found between the Q2 domain of CREB and Drosophila TAF,,110 (Ferreri et al., 19941, one of the proteins associated with the basic transcriptional machinery (Hoey et al., 1993) (Fig. 8). It has been proposed that the Q1 and Q2 regions correspond to constitutive activator domains that become exposed for interactions with target coactivator proteins upon phosphorylation of the adjacent KID by PKA (Brindle et al., 1993; Ferreri et al., 1994; Quinn, 1993; Krajewski and Lee, 1994). Experiments carried out by Quinn (19931, in which the transcriptional transactivation activities of a number of deletion CREB mutants were tested, indicate that the transactivation domain of CREB is modular in structure, inasmuch as each of the activaticn domains (basal or inducible) can function independently of each other. Hybrid proteins consisting of the N-terminal transactivation domain of CREB (devoid of the DNA binding domain, amino acids 1248) fused to the DNA binding domain of B-cell activator protein (BSAP-1) that binds its regulatory element as a monomer, activate transcription constitutively independent of phosphorylation by PKA (Krajewski and Lee, 1994).By mutational deletion analyses, the transactivationai activity of the CREBIBSAP-1hybrid protein appears to be mediated by the glutamine-rich regions, not by the P boxlKID domain. Although the CREB/BSAP-1 fusion protein represents an artificial


inactive

19

e HC

P-BOX

Active

FIG. 8. The formation of protein complexes that mediate CREB basal transcriptional activity. CREB interacts via the hydrophobic cluster (HC) in the Q2 domain with the TATA box-associated factor TAF110. Note that this interaction can occur in the absence of CREB phosphorylation, hence generating basal transcriptional activity without CAMPstimulation. Contrast this situation (low-level transcriptional activity) with that illustrated in Fig. 9, in which CREB has been phosphorylated and interacts with the basal transcriptional machinery via a different set of basal proteins. TRX, transcription; TBP, TATA-box binding protein; TF, other transcription factors.

unphysiological model, the observations suggest that CREB monomers can couple to the basal transcriptional machinery, presumably by interactions of the glutamine-rich regions containing hydrophobic clusters with TAFlll10 (Fig. 9). Further, these findings suggest that CREB dimers may be required for productive transcriptional interactions with CBP (CREB-binding protein). D. ADAPTER PROTEINS THATCOUPLE CREB TRANSACTIVATION TO THE BASALPOLYMERASE I1 TRANSCRIPTIONAL COMPLEX

As discussed above, CREB may activate gene transcription not directly, but rather through interaction with another effector or coactivator proteins. Recently, a large (265-kDa) protein known as CBP that does not bind DNA has been identified (Chrivia et al., 1993) (Fig. 9A). Analysis of the amino acid sequence of CBP deduced from its cloned cDNA reveals the presence of at least three consensus phosphorylation sites for CaMK-I1 and one for PKA, as well as two putative zinc finger domains. In addition, the C-terminal region contains a glutamine-rich domain. The CREB binding domain, determined by deletional studies (Chrivia et al., 1993),is located within the N-terminal region (Fig. 9A).

20

JOEL F. HABENER et a1

xPKA

A ZF

ZF

y4

H2N-

COOH

1

2441

P-BOX

6 y2 ,~

TRX

Q1

P-BOX

Active (phosphorylate

RX

6 FIG. 9. Structure of the CREB-binding protein (CBP) and how CBP facilitates transcriptional activation by phosphorylated CREB. (A) The region of the molecule that contains the CREB binding domain (CREB BD) and the glutamine (Q)-rich region are shadowed. The location of the two putative zinc finger domains (ZF) and the protein kinase A (PKA) phosphorylation sites are also indicated. (B)The potential transcriptional adapter function of CBP. In the inactive state (top) unphosphorylated CREB is bound to the CRE in the promoter of the target gene, but cannot interact with the proteins that form the basic transcription (TRX) machinery assembled on the TATA box. Phosphorylation of CREB (and possibly also of CBP) by PKA triggers an interaction between CREB and CBP, which, in turn, interacts with TFIIB, forming a higher-hierarchy transcriptionally active complex (bottom). TBP, TATA-box binding protein; TF, other transcription factors; P, phosphate.

CBP interacts with CREB only when Ser133is phosphorylated. According to the proposed model that considers CBP as a coactivator, CBP is recruited to the CREB-DNA complex in the promoter of target genes upon phosphorylation of CREB. In this manner CBP mediates


21

CAMP-independent CREB-induced transcriptional responses. Microinjection of anti-CBP antibodies into the nuclei of cells inhibits transcriptional responses elicited by cAMP stimulation, suggesting that CBP is essential for the activation of transcription of CAMP-responsive genes. Interestingly, these studies also indicated that CBP interacts with c-Jun phosphorylated by Jun-kinase (Arias et al., 1994). Coactivator proteins are non-DNA-binding proteins that bridge DNA-bound transcription factors with protein components of the basal transcriptional machinery associated with RNA polymerase I1 (Dynlacht et al., 1991) As such a coactivator, CBP bound to phosphorylated CREB appears to interact directly with TFIIB (Kwok et al., 19941, one of the protein components of the basal RNA polymerase I1 transcription machinery (Buratowski, 1994) (Fig. 9B). The discovery of CBP has revealed interesting implications for the alterations in gene transcription elicited by certain viral proteins mediated via CRE-like elements. CBP contains regions with amino acid sequences homologous to those of equivalent regions in the protein p300, another recently identified nuclear protein that interacts with the adenovirus E1A oncoprotein and may mediate E l A-induced proliferation of cells (Arany et al., 1994). Both proteins share 85-95% similarity over several segments, one of which is also homologous to ADA2 (Arany et al., 1994; Chrivia et al., 19931, a coactivator protein found in yeast (Berger et al., 1992). Based on sequence similarities shared by p300 and CBP, it has been proposed (Arany et al., 1994) that interactions between CREB and p300, as well as between viral E1A (or its putative cellular counterpart) and CBP may occur in cells. In addition, CBP interacts with phosphorylated c-Jun (Arias et al., 19941, which activates gene transcription in response to mitogenic stimuli. Accordingly, these interactions among CREB, CBP, and p300 may provide a molecular substrate for the observed effects elicited by cAMP on cell proliferation and differentiation.

IN STRUCTURE: VI. THE CREB AND CREM GENESARE MULTIEXONIC ALTERNATIVE EXONSPLICING GENERATES A COMPLEX ARRAY OF ISOPROTEINS THATAREEITHER

TRANSACTIVATORS OR TRANSREPRESSORS The genes for the human and mouse CREBs have been isolated and partially sequenced. The genes are located on the long arm of human chromosome 2 mapped to 2q32.3-q34 (Hoeffler et al., 1990; Taylor et al., 1990) and to the proximal region of mouse chromosome 1 (Cole et

22


t

A exon: 5'-Flank A

B '? C

Alternatively Spllced Exons

D

Y

E

F

G

1 W H

I

GENE

PRO1rElN CREE341 4

ACTIVATION DOMAIN

I

1P b X , ] I '? I

CREE327 CREE-'?

,

*,STOP

CREB-Y CREE-W CREE4

S

FIG.10. The exonic organizaton of the CREB and CREM genes, mRNAs, and encoded proteins. (A) The CREB gene contains a t least 12 exons spanning more than 80 kb of DNA. The introns between exons A and B and H and I are very large (over 25 kb). The exons comprise functional modules in CREB. Exons E and F encode the phosphorylated domain and exons B, C, and G encode the glutamine-rich domains, important for tran-

23


al., 19921, which shares a large region of synteny with human chromosome 2q. The chromosomal location of the CREM gene has not yet been reported. The genes for ATF-1 and 2 ATF-2, related in structure to the CREB and CREM genes, are located on chromosomes 12q13 and 2q24.1-q32, respectively (Zucman et al., 1993; Diep et al., 1991).

FUNCTIONALLY DISTINCT DOMAINS A. EXONSENCODE The CREB genes consist of multiple exons, at least 12, spanning a n estimated 80-100 kb (Hoeffler et al., 1990; Ruppert et al., 1992). The structures of the CREM and ATF-1 genes have not yet been reported. However, several different cDNAs representing mRNAs encoded by these genes provide strong evidence that they are similar to CREB in their composite multiexonic structures (Foulkes et al., 1991b, 1992; Fkhfuss et al., 1991). The exons that make up the CREB and CREM genes are functionally modular in nature (Fig. 10). Exons E and F encode the P box, or KID, while exons C and G encode glutamine-rich regions important in mediating the transactivational activity imparted by phosphorylations in the P-box domain. Exons H and I encode the DNA binding domain consisting of the basic region and the leucine zipper dimerization sequences. The multiexonic structures of the CREB, CREM, and ATF-1 genes stand in sharp contrast to the intron-

~~

~~

~

~

~

~

scriptional transactivation. Transcription initiates from a non TATA box sequence (Inr). Exons H and I encode the bZIP domain involved in dimerization and DNA binding. LLLL, leucine repeat dimerization domain. Notably, exons 9,Y, D, and W are alternatively spliced in tissue- and development-specific patterns. The II sequence is not an alternatively spliced exon, but rather is an intron slippage alternative acceptor splice. The alternative splicing of exon D retains the open translational reading frame in CREB mRNAs, resulting in the formation of the two CREB isoforms, CREB327 and CREB341, differing by 14 amino acids. However, exons 9,Y,W, and the alternative II splice result in the premature termination of translation of the CREB mRNA. These isoforms of CREB are truncated a t the C-terminal end, and consist of CREBs devoid of the DNAbinding bZIP domain and the encoded nuclear translocation signal. It is believed that the formation of these isoforms serves to interrupt a positive autofeedback regulation of CREB on its own promoter. (B) The exons of CREM. Although the structure of the CREM gene is unknown, the multiplicity of alternatively spliced mRNAs (cDNAs) identified defines a multiexonic structure homologous to that of CREB. The CREM gene has two promoters: A 5' constitutively acting promoter, P1 (left), and a CAMP-regulated internal promoter, P2, located in the intron between exons G and X (y). The P2 promoter encodes a transrepressor isoform of CREM, ICER (inducible CAMP early repressor), consisting of the DNA-binding bZIP domain. Two bZIP domains are encoded by alternative splicing of exon I (I, and Ib). The nomenclature for the CREM isoforms is described in Greek letters on the left and according to their exonic composition on the right. S-CREM is formed by a n alternative translational initiation (ATG).

~

24


less genes encoding the related Jun and C/EBP subfamilies of bZIP proteins. The nomenclature describing the numerous CREB and CREM isoforms expressed as a consequence of alternative splicing of exons is confusing and requires clarification. Historically, the exons in the human CREB gene originally were reported and described by the letters A through I (Hoeffler et al., 1990). Subsequently, the peptide sequence corresponding to exon D of the CREB gene was designated a,because it was believed to be an a-helix (Yamamoto et al., 1990). In a later publication describing the structure of the mouse CREB gene, the authors designated the exons homologous to those of the human gene by the numbers 1 to 11 and Greek letters (Ruppert et al., 1992). The homology of the structure of the CREM gene to that of the CREB gene was apparently not recognized, so various forms of the cloned cDNAs representing alternatively spliced transcripts of the CREM gene were arbitrarily designated by Greek letters that bear no relationship t o the Greek letter designations given to the mouse CREB gene or to the corresponding letters A to I initially appended to the exons of the human CREB gene (see Fig. 10 for a partial explanation of the exon designations). Several of the exons of the CREB and CREM genes are alternatively spliced in the formation of mRNAs during the development and differentiation of specific tissue phenotypes. At least four alternatively spliced exons of the CREB genes have been identified so far (exons q , Y, D, and W) (Hoeffler et al., 1990; Waeber et al., 1991; Waeber and Habener, 1992; Ruppert et al., 1992). In addition, the mouse CREB gene undergoes an alternative splice site selection, known as intron slippage, between exons H and I, resulting in the translation of an R or H' sequence of amino acids (Ruppert et al., 1992). A remarkable property of the alternatively spliced exons in the generation of CREB mRNAs is that, with the exception of alternatively spliced exon D, which maintains the open translational reading frame of CREB, all of the other alternatively spliced exons (q,Y, and W) and the intron slippage isoform R introduce stop codons that result in the termination of translation. These alternative exon-splicing events cause termination of the translation of CREB proteins such that they do not have DNA-binding bZIP domains. Thus, these alternative splicing choices, resulting in the premature termination of translation, appear to be designed to inactivate the synthesis of transactivator forms of CREB. As discussed further below (Section VIII,A), it is believed that the premature termination of translation by the splicing in of exon W in Sertoli cells during spermatogenesis, and perhaps that of exons v' and


25

Y as well, may serve to interrupt a positive autofeedback loop of CREB acting on its own promoter. Transcripts of the CREM gene consist of combinations of alternatively spliced exons C, E, F, G, X, and I. Exon I consists of two alternatively spliced exons, I, and Ib, encoding alternative DNA binding and dimerization domains of CREM. The reasons for the two alternatively spliced bZIP domains are unknown. It is tempting to speculate, however, that they may provide diversity at the level of specificity of dimerization to other bZIP proteins and/or specificities in the recognition and binding to CREs with variations in their nucleotide sequences. Deletions of exons internal in the coding sequence markedly attenuate transactivational activities (Section V1.B). B. REPRESSOR ISOFORMS OF CREM ARE GENERATED BY SEVERAL DIFFERENT MECHANISMS An intriguing property ofthe expression ofthe CREB and CREM genes is the generation of multiple isoforms of the proteins due to mechanisms of alternative exon splicing and alternative utilization of translational start sites within the spliced mRNAs (Fig. 11).These mechanisms result in the interconversion of transcriptional activator to transcriptional repressor forms of the proteins and appear to be developmentally regulated in specific cell lineages, as discussed later (Section VIII). The internal exons in CREM, C through G, are all involved in encoding sequences important for the mediation of the transcriptional transactivation functions of CREM. Exons E and F encode P box, or KID, whose phosphorylation by CAMP-dependent kinases, Ca2f / calmodulin-regulated kinases, and Ras-activated kinases are critical for generating the transactivation functions. Exons C and G encode glutamine-rich regions with hydrophobic clusters that are also important in transactivation. Deletion of one or more of these exons results in a marked attenuation of the transactivational activities without alterations of the dimerization or DNA binding functions. Thus, these internally exon-deleted forms of CREM act as transrepressors by way of either competing for CRE sites as homodimers or forming less active heterodimers with transactivator isoforms of CREM and CREB (Foulkes and Sassone-Corsi, 1992; DeGroot and Sassone-Corsi, 1993). Alternative splicing of CREM takes place during the chronological maturation of spermatogenesis in the mouse (Foulkes et al., 1992). During the first 2 weeks of postnatal development, the repressor isoforms of CREM (CREMAC,G) are expressed in the maturing germ cells. Between 3 and 4 weeks of development, the C and G exons are

26


CREB

Alternative exon spllcing terminates translation and prevents translocation to nucleus.

SL- - - - - - - - - - - - ,-

P-BOX

CREM

.I-----?

:-Bl7-;-zlP--: NTS

Alternative exon splicing deletes Q-rich transactivation domains.

Q

Q (CREMa) BR I ZIP

Internal Translation (S-CREM) TX START

- - - - - - p.Box: ----- - - - r----I.

. I

1 BR I

ZIP

I

I

Internal Promoter (ICER)

FIG.11. Mechanisms for the generation of inactive and transrepressor isoforms of CREB and CREM. Splicing in of exons that terminate translation (exon W) inactivates CREB by preventing the synthesis of the DNA binding domain. In CREM, splicing out of exons important for transactivator functions (CREMa and CREMAC-G) creates repressor isoforms, as does alternative internal transcription (S-CREM) and/or translation (ICER). TRX, transcription; TX, translation; Q, glutamine; BR, basic region; ZIP, leucine zipper; NTS, nuclear translocation signal.

spliced into the CREM transcripts, resulting in the expression of transactivator isoforms of CREM. These important observations suggest that the target genes whose expression is regulated by CAMP signaling are switched from a repressed to an activated state during spermatogenesis by a mechanism of genetically and temporally programmed alternative splicing of CREM transcripts so as to switch the synthesis of CREM transrepressors to CREM transactivators of gene transcription. In addition to alternative splicing of exons in CREM, two other mechanisms are utilized to generate transrepressor isoforms of CREM: internal transcription/translationand internal translation. An alternatively used internal promoter exon that resides in the intron located between exons G and X is utilized to transcribe a 5’ truncated mRNA that encodes a small form of CREM called ICER (Foulkes et al., 1991b). ICERs consist of exons H and I, or 1, (with or without exon X) encoding the DNA binding domain devoid of any known transactivation do-


27

mains, and thereby are potent transrepressors of activator forms of CREM and CREB. The internal promoter of ICER, called P2, is upregulated by four closely located CREs activated by activator isoforms of CREB, CREM, and potentially other CRE-binding proteins, and is repressed by itself. As discussed in more detail in Section VIII,C, the temporally programmed interplay of the positive and negative regulation of CAMPresponsive target genes and the ICER promoter itself is important in controlling the 24-h circadian rhythm of melatonin synthesis in the pineal gland. The other mechanism, alternative internal translation, does not involve the alternative splicing of exons, but rather involves the alternative utilization of a n internal AUG codon to start translation, resulting in the synthesis of a n N-terminally truncated repressor similar to the exon-deleted and ICER CREM isoforms. Such alternative translation of CREM mRNA has been shown to take place during postnatal development of the rat brain (Delmas et al., 1992). It is curious that in the CREB gene alternative splicing in of exons q ,Y, and W, and the intron slippage splice, a,all result in the premature termination of the translation of the CREB mRNAs and the formation of C-terminal truncated forms of CREB lacking a DNA binding and dimerization domain with its encoded nuclear translocation signal. In contrast, all of the multiply alternatively spliced exons of CREM maintain the open translational reading frame. C. ALTERNATIVE EXONSPLICING APPEARS TO PROVIDE A MECHANISM BY WHICHTO MODULATE THE TRANSACTIVATIONAL ACTIVITIES OF CREB AND CREM As discussed earlier, most, if not all, isoforms of CREB and CREM, and also perhaps ATF-1, can freely dimerize with one another to form a large number of different combinations of CAMP-responsive DNAbinding bZIP proteins. Inasmuch as phosphorylation by CAMP-dependent PKA, and/or other protein kinases on the critical serine in exon E, and the integrity of the glutamine-rich exons C and G are required for the transactivation functions of CREB and CREM, impairment of phosphorylation or deletions of such exons that leave the DNA binding domain intact create repressor isoforms. These transactivationdeficient isoforms can dimerize with themselves and thereby serve as competitive inhibitors of the binding of transactivator forms of CREB and CREM to CREs. Alternatively, they can form heterodimers with transactivator isoforms, therefore reducing their relative transactivational activities when bound to DNA. Experimentally mutated forms

28


of CREB have been constructed in such a way as to impair transactivation (e.g., mutations in the PKA phosphorylation site) and to restrict heterodimerization combination pairs (e.g., mutations in the leucine zipper) (Loriaux et al., 1993). These studies show that a CREB dimer, in which one of the two monomeric members of the dimer is mutated in the phosphorylation site and is unable to be phosphorylated by PKA and the other monomer is not mutated and is phosphorylated, displays 50% of the activity of the wild-type fully phosphorylated homodimer. Similar experiments analyzing the relative transactivational activity of mutated dimers among CREB and CREM isoforms reveal similar additivity of activities: a dimer consisting of an exon-deleted CREM monomer and an intact CREB monomer gives 30-40% of the activity of the CREB homodimer (Loriaux et al., 1994). These observations underscore the complex nature of the combinatorial code at play in the regulation of gene transcription. Multiple different CRE-binding dimers consisting of various combinations of CREB and CREM isoforms can occur. The composition of the specific heterodimers, whether activator or repressor isoforms, determines their relative effectiveness to stimulate gene transcription. The prevalence of the specific heterodimers would be dependent on their relative concentrations in the nucleus, determined by the relative rates of their synthesis and degradation, which are likely to be influenced by both positive and negative autofeedback control on the promoters of the CREB and CREM genes.

D. UNPHOSPHORYLATED CREB CAN REPRESSGENEEXPRESSION MEDIATED BY PHOSPHORYLATED CREB Based on the considerations of the formation of heterodimers among different isoforms of CREB and CREM discussed above, at least three examples have been described in which unphosphorylated CREB (dephosphoCREB) antagonizes CREB and serves as a negative regulator of transcription (Fig. 12). Expression of dephosphoCREB in the pituitary glands of transgenic mice impairs their development (Struthers et al., 1991). A transgene consisting of a CREB isoform in which the Ser133 phosphorylated by PKA is mutated to an alanine targeted to the growth hormone-producing somatotrophs results in dwarfism due t o a marked developmental deficiency of somatotrophs, but not of other cell types. The mutated dephosphoCREB expressed by the transgene apparently antagonized the actions of wild-type CREB, resulting in a failure of the somatotrophs to develop. DephosphoCREB also represses transcriptional expression of the fos


P

29

P CREB I

CRE

TTRX I' CRE 1 '

PhosphoCREB strong activator

P

DephosphoCREB weak activator

P

Competition phosphoCREB vs. dephosphoCREB

Heterodimer of phosphoCREB I dephosphoCREB weak activator

FIG.12. Unphosphorylated CREB (dephosphoCREB) serves as a negative regulator of phosphoCREB. Because dephosphoCREB and phosphoCREB dimerize and bind CAMPresponsive element equivalently, and dephosphoCREB has markedly reduced transactivational activity compared to phosphoCREB, dephosphoCREB competitively inhibits transactivation of transcription. Inasmuch as protein kinase A activity in nuclei is believed to be rate limiting for the phospohrylation of CREB, the ratio of dephosphoCREB to phosphoCREB is an important determinant of the transactivational activity.

gene. An expression vector encoding CREB transfected into NIH 3T3 cells repressed the serum-stimulated expression of the fos gene (Ofir et al., 1991). This negative regulation by CREB was alleviated when CREB was phospohrylated on Ser133by cotransfection and expression of the catalytic subunit of PKA. Further, expression of the dephosphoCREB in which Serl33 was mutated to alanine also repressed serum stimulation of fos, but repression was not alleviated by PKA. These findings indicate that dephosphoCREB functions as a transrepressor of transcription of a gene (fos) encoding a transcription factor. Another example of negative regulation by dephosphoCREB was shown in its inhibition of the activation of transcription by C/EBP (Vallejo et al., 1995). The somatostatin gene is strongly up-regulated by cAMP via the interactions of CREB with a CRE located in the proximal region of its promoter. A somatostatin-producing cell line was discovered in which neither cAMP nor PKA could activate the somatostatin promoter. Rather, the unusually high constitutive basal transcription was driven by interactions of C/EBP with the CRE of the promoter. Further, it was shown that the PKA activity in these cells was severely inhibited by the overproduction of a n endogenous heat-stable inhibitor of PKA, resulting in a complete failure of the phosphorylation of CREB in response to the CAMP-PKA signaling pathway. Overexpression of

30


CREB transfected into the cells markedly attenuated transcription mediated by the CRE in the somatostatin promoter by competitively inhibiting the binding of C/EBP t o the CRE. These findings bring to light several potentially important insights. Namely, that transcription factors other than CREB, such as the C/EBP family of bZIP proteins, can bind to and activate CREs in the promoters of genes, that specific protein kinase inhibitors can markedly influence the activity of a signal transduction pathway, and that cells (the islet cell line) can survive and propagate in culture under conditions in which the nuclear CAMP-PKA pathway is essentially completely inactivated. Inasmuch as dephosphoCREB can serve as a negative regulator of phosphoCREB and other CRE-binding transactivators, the relative ratios of phosphoCREB and dephosphoCREB in the nucleus at any given moment will determine the relative magnitude of the CAMP-mediated transcriptional response. As discussed earlier, CREB is translocated constitutively t o the nucleus soon after completion of its synthesis ( t l i P of nuclear import, -15 min). In addition, nuclear CREB appears to be associated with nuclear chromatin at all times, presumably bound to CRE control elements of the DNA (Hagiwara et al., 1993). The regulated step in the phosphorylation of CREB is the availability of active catalytic subunit of PKA. The PKA holoenzyme type I1 is located in the perinuclear cytoplasm bound by specific AKAPs (Scott and McCartney, 1994). Upon activation by binding of cAMP to the regulator subunit, the catalytic subunit is released and translocates to the nucleus within 5-15 min (Hagiwara et al., 1993; Meinkoth et al., 1990; Nigg et al., 1985a).Holoenzyme probably also exists within the nucleus as well as in the perinuclear cytoplasm (Squint0 and Jungmann, 1989; Joachim and Schwoch, 1988; Schwoch and Freimann, 1986). AKAP-95 has been identified that is associated with the nuclear matrix and contains a zinc finger DNA binding domain (Coghlan et al., 1994). It seems likely that AKAP-95 tethers PKA holoenzyme to the nuclear matrix through interactions with the type I1 regulatory subunits (RIIa and RIIP). It has been observed that the addition of 8Br-CAMP to primary hepatocytes in uzuo results in the expression of nuclear PKA activity and the phospohrylation of CREB within 30-60 s (Gosse et al., 1995).These findings suggest that the stimulation of gene transcription in response to cAMP can occur very rapidly via activation of nuclear PKA, whereas longer, more sustained, responses occur by the recruitment to the nucleus of PKA catalytic subunits released from holoenzyme in the cytoplasm. The nuclear levels of PKA appear to be the rate-limiting step in


31

determining the ratio of phosphoCREB to dephosphoCREB. In studies of PC12 cells, it was estimated that a maximum of 40% of nuclear CREB is phosphorylated 30 min after the addition of forskolin (Hagiwara et al., 1993). Although the concentration of PKA in the nucleus (1.2 F M ) exceeds that of CREB (400 nM), the K , of CREB for PKA is relatively high (10 p M ) (Calbron et al., 1992). A tentative extrapolation of these findings suggests that in the absence or low levels of cAMP signaling, the vast majority of CREB is unphosphorylated and is serving a role as a repressor rather than as an activator of gene transcription. E. EXON-DELETED REPRESSOR ISOFORM OF CREM DOWN-REGULATES EXPRESSION OF THE c-fos AND c-jun GENES The alternatively spliced isoform of CREM that has the two glutamine-rich exons, C and G, deleted (CREMAC,G or CREMd is a repressor of cAMP stimluation of the c-fos gene in NIH 3T3 cells (Foulkes et al., 1991a). CREM does not antagonize serum-induced transcription of the c-fos gene, suggesting that dephosphoCREB and CREMAC,G act on the promoter of the c-fos gene by different mechanisms. Both CREB and CREMAG,C also down-regulate the expression of the c-jun gene in JEG-3 cells (Masquilier and Sassone-Corsi, 1992). The mechanism of the inhibition of the activation of c-jun is a direct competition by CREB and CREMAC,G of the binding of c-jun (and c-fos) to the TPA-response elements in the promoter of the human metallothionein IIA gene, not by dimerization of CREB and CREM with c-jun or c-fos. VII. CAMP-DEPENDENT AUTOREGULATION OF THE EXPRESSION OF THE CREB AND CREM GENES The transcription of both the CREB and CREM genes is autoregulated by their own encoded products, serving as important CAMPresponsive transcription factors. The CREB gene is positively autoregulated via three CREs that reside within the promoter consisting of 800 bp of sequence flanking the 5‘ end of the gene, that is, 5’ to exon A (Meyer et al., 1993; Walker et al., 1995) (Fig. 12). CREB produced by recombinant DNA techniques as well as CREB in extracts of cell nuclei bind to the CREs in the promoter of the CREB gene (Meyer et al., 1993; Walker et al., 1995). The CREM gene has an unusual dual-promoter structure. The 5’

32


A ATG

-1200

-600

+1

InR

B TRX

--I CRE HCRE

TRX

TX rMet

Ala Val

FIG.13. The human CREB and internal CREM (P2) gene promoters. (A) The 5’ flanking 1200 bp of the CREB promoter are enriched in G and C (70%). The transcription (TRX) is initiated from a n InR sequence and not a TATA box. Important control elements are three cAMP response elements (CREs) and three sites that bind the transcription factor Spl. The CREs lend autopositive regulation to the CREB gene. (B) The internal promoter (P2)of the CREM gene that encodes the transrepressor isoform (ICER) is upregulated by four closely spaced CREs. This mechanism provides an autoregulation as CREB and CREM transactivators activate the ICER promoter, resulting in the production of a dominant negative repressor (see also Fig. 10B). TX, translation.

flanking promoter (Pl) is constitutive and is not regulated by cAMP (Molina et al., 1993).Rather, the CREM gene contains a second promoter (P2) located within the intron between exons G and X that contains four closely spaced CREs [redesignated as CARES(Molina et al., 199311 (Fig. 13). The activation of the P2 promoter by transactivator forms of CREM and CREB results in the expression of a novel mRNA that encodes a transrepressor isoform of CREM called ICER, as mentioned earlier. Thus, in contrast to that of the CREB gene, cAMP signaling of the CREM gene results in the expression of a transrepressor of gene transcription. This fascinating interplay of positive and negative autoregulation of CREB and CREM transcription factors is discussed further in Section VIII in the context of their importance in the physiological regulation of gene expression. VIII. RQLESOF CREB AND CREM IN THE PHYSIOLOGICAL REGULATION OF GENETRANSCRIPTION Increasing evidence indicates that both CREB and CREM regulate certain cellular processes in several tissues of the intact organism, including the testes, the pituitary gland, and the brain. However, their exact physiological roles have not been completely established.


A.

33

TESTES

The CREB and CREM genes are highly expressed in the testes. The primary transcripts of these two genes undergo complex patterns of alternative splicing of exons during both the postnatal developmental maturation of spermatogenesis and the endogenous cycling of the seminiferous tubules in the adult animal. The alternative splicing results in the interconversions of transactivator, transrepressor, and inactive isoforms of CREB and CREM. The seminiferous tubules consist of the germ cells at various stages of development, the somatic Sertoli cells, and the intestinal Leydig cells. The pituitary gonadotropic hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH) stimulate CAMP-coupled receptors on the Sertoli and Leydig cells, respectively. The actions of FSH and LH, expressed from the pituitary gland during pubescence, activate the Sertoli cells to produce estrogenic hormones and other essential factors and activate the Leydig cells t o produce androgenic hormones, all of which are required to allow for the orderly maturation of the germ cells. Further, the dual actions of the FSH and LH gonadotropins are essential for driving the waves of germ cell differentiation in the testes of the adult animal. The program of germ cell differentiation takes 45-48 days to complete (Fig. 14A). Given the appropriate hormonal stimuli, the stem cell spermatogonia undergo three or four mitotic divisions to mature into spermatocytes, which then differerntiate through several additional stages (i.e., leptotene to zyotene to pachytene). The pachytene spermatocytes undergo two meiotic “divisions”: first segregation of diploid into haploid chromosomes, and then cell division into round spermatids, each of which contains a haploid complement of chromosomes. The round spermatids then undergo a process of morphogenesis to become elongated spermatids and finally mature spermatozoa that exit from the seminiferous tubules to the epididymis, where they are avaialable for fertilization. Spermatogenesis in the testes of rodents occurs in waves of approximately 12 days’ duration involving 14 cell association stages (stages I-XIV). Four such cycles (48 days) are required for the developmental maturation of stem cell spermatogonia to become mature spermatozoa (Fig. 14A). 1. Expression of the CREB Gene The CREB gene is expressed at high levels in both the Sertoli cells and the germ cells. Studies have been focused on the examination of the cyclical expression of CREB in Sertoli cells of the adult rat semi-

34


A I

i

'!

,

STAGES 12.9 days

I

+

B

4

z K

E 50 m

w K

0

I

11-111

+-12.9

VI VllC-d IX-XI XIII-: IV-V Vlla-b Vlll XI1 STAGE days-4

FIG.14. Cyclical expression of the CREB gene in Sertoli cells of rat seminiferous tubules during the 12-day spermatogenic cycle. (A) Depiction of the 14 germ cell association stages (I-XIV) in the seminiferous tubule of the adult rat. Shown is a diagrammatic cross-section of a tubule with the luminal and basal borders a t the top and bottom, respectively. The cells a t the bottom are the progenitor spermatogonia that give rise to spermatocytes above as they develop from left to right and progress up the staircase (bottom layer to top layer). The mature pachytene spermatocytes undergo two rapid meiotic divisions a t stage XIV (right, third cell layer from the bottom), resulting in the formation of the haploid round spermatids that then develop into mature spermatozoa. Approximately 12 days are required for each of the four layers of cells to traverse development from left to right (stages I-XIV). The entire time for development from

35


niferous tubule during the 12-day cell association cycles of maturation of the germ cells (Waeber et al., 1991) (Fig. 14). It has been proposed that cAMP generated by the action of FSH on Sertoli cells stimulates the expression of the CREB gene via CREs located in the promoter of the CREB gene. The positive autoregulation of the expression of CREB is interrupted by a switch in the processing of the CREB RNA transcripts, so as to splice an exon into the mRNA that terminates translation 5’ to the mRNA sequence encoding the bZIP DNA binding domain of CREB with its encoded nuclear translocation signal (Waeber and Habener, 1991).The C-terminal truncated isoforms of CREB, devoid of their nuclear translocation signal, become sequestered in the cytoplasm and are degraded. This mechanism of alternative splicing of exons serves as a means to inactivate the positive autoregulation of CREB. Such alternative splicing of at least two separate exons that result in the premature termination of translation of CREB mRNAs takes place in cell association stages V-VI of the seminiferous tubules of the rat (Waeber et al., 1991; Waeber and Habener, 1992). These exons appear to be spliced out during stages

“ f Bpp*r receptor

nucleus

y

\ -’u cytoplasm

A

STOP

---->

spermatogonia to fully mature spermatocyte takes approximately 48 days (four cycles of 12 days each). (B) CREB mRNA levels in Sertoli cells rise and fall during the 12-day cycle of spermatogenesis. The fluctuations in cAMP levels and follicle-stimulating hormone (FSH)binding sites precedes that of CREB mRNA, consistent with the autoregulation of CREB gene expression by FSH-mediated increases in CAMP.It has been proposed that the alternative splicing in of exon W, which stops translation before the DNA binding domain, interrupts the positive auto feedback loop of CREB gene transcription (see Fig. 10A). (C) Model depicting the FSH-regulated cyclical expression of CREB in Sertoli cells. PKA, protein kinase A; FSH-R, FSH receptor; NLS, nuclear localization signal; P, phosphoserine.

36


XIV-I, thereby allowing the synthesis of complete full-length CREB that can enter the nucleus and activate transcription (Fig. 14C).Notably, the mRNA encoding the FSH receptor in the testes appears also to be alternatively spliced by a similar mechanism, that is, the splicing in and out of exons that results in premature termination of translation N-terminal to the transmembrane-spanning domain that anchors the receptor in the plasma membrane. It has been proposed that the alternative splicing of the FSH receptor during the 12-day temporal cycles of the seminiferous tubule provides cyclical regulation of cAMP signaling in Sertoli cells (Walker et al., 1995). It is also possible that alternative splicing of the FSH receptor mRNAs may occur during the postnatal maturation of the testes and serve to activate FSH-mediated spermatogenesis at puberty. 2, Expression of the CREM Gene

As discussed above, the CREM gene encodes multiple regulators of the cAMP transcriptional response by alternative splicing (Foulkes et al., 1991b).In mice a developmental switch in CREM expression occurs during spermatogenesis, whereby alternative exon splicing converts CREM from a repressor to an activator (Foulkes et al., 1992).In premeiotic germ cells the CREMa (CREMAC,G)repressor isoform is expressed in low amounts. During the developmental transition of the spermatocytes through the pachytene stages VI-VIII, the glutamine-rich C and G exons are spliced into the CREM mRNAs, resulting in the translation of the CREMT(CREM I,) activator isoform (Fig. 10). The CREMTtransactivator accumulates to high amounts. During the early stages of spermiogenesis (stages X-XII), when the round spermatids elongate to spermatozoa, a new repressor isoform, CREMAC-G, is expressed (Walker et al., 1994). This repressor isoform of CREM (CREMAC-G) consists only of the N-terminal 38 amino acids of exon B and the 12 amino acids of exon X in frame with the exons encoding the DNAbinding bZIP domain (exons H and IJ. These circumstances suggest that during spermatogenesis, CREM gene expression undergoes a biphasic transition of isoforms: from repressor (prepachytene spermatocytes), to activator (postpachytene spermatocytes), to repressor (transition from round to elongated spermatids). These splicing-dependent changes in the transcriptional functions of CREM, along with the transitions in CREB splicing, are proposed to have an important role in programming the expression of CAMP-regulated target genes during spermatogenesis, although the detailed mechanisms involved are not yet understood (Walker et al., 1995). In the postnatal prepubertal rat FSH appears to be an important


37

functional switch necessary for the expression of CREM transactivator forms in the testes. Hypophysectomy of newly born rat pups, which eliminates production of FSH, results in the extinction of CREBT expression. Expression can be restored by the administration of exogenous FSH. The induction of CREMT by FSH appears to occur, to a large extent, by alternative utilization of a poly(A) addition site in the CREM mRNA, resulting in a marked stabilization of the mRNA (Foulkes et al., 1993). Analyses of CREM expression in the season-dependent modulation of spermatogenesis in hamsters have shown that FSH controls the switching on of CREM gene expression in the testes during the beginning of the experimental change from short to long photoperiods (summer), when FSH and LH levels are induced (Foulkes et al., 1993). B. ANTERIORPITUITARY GLAND In the anterior pituitary CREB may be involved in the regulation of the CAMP-dependentproliferation of somatotrophs. This notion is supported by loss-of-function experiments carried out by Struthers et al. (1991) in transgenic mice (described above) and by gain-of-function experiments carried out by Burton et al. (19911. These authors developed transgenic mice carrying a chimeric gene encoding an intracellular form of cholera toxin under the control of the growth hormone gene promoter. In these animals cholera toxin specifically expressed in somatotrophs irreversibly stimulates G, protein-mediated activation of AC, resulting in permanently elevated concentrations of CAMP.The phenotype of mice carrying this transgene is characterized by gigantism, hyperproliferation of somatotrophs, and pituitary hyperplasia (Burton et al., 1991).A clinical correlate of these observations is found in patients with pituitary adenomas due to constitutively active mutant forms of G, proteins (Landis et al., 1989).The possible existence of mutated CREB in other somatotroph adenomas has not been examined.

C. BRAIN:HYPOTHALAMUS AND PINEAL GLAND In the brain the distribution of transcripts encoding CREB and CREMT is diffuse, whereas the distribution of transcripts encoding repressor isoforms of CREM is restricted to specific areas (Mellstrom et al., 1993). This pattern of expression suggests that in the central nervous system the presence of CREM antagonists may determine region-specific differences in CREB- (or CREMT-)mediated responses

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to cAMP stimulation. Peripheral hyperosmotic stimulation results in CREB phosphorylation in the hypothalamic supraoptic and paraventricular nuclei (Borsook et al., 1994). In addition, osmotic stimulation results in the induction of CREMa and CREMp in neurons of the supraoptic nucleus (Mellstrom et al., 1993). These findings suggest that these transcription factors are involved in the control of hypothalamic homeostatic mechanisms that maintain plasma osmolality. CREB and CREM have been implicated in the physiological mechanisms of the regulation of circadian rhythms (Ginty et al., 1993; Stehle et al., 1993). A pacemaker that controls circadian rhythms resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. This nucleus receives inputs regarding light-dark cycles via direct retinohypothalamic projections that relay information received by the eye. In turn, the SCN controls the functional activity of noradrenergic inputs reaching the pineal gland, which produces the hormone melatonin. In this manner melatonin production and secretion follow the SCN-driven circadian rhythm. In the course of studies to elucidate the molecular mechanisms that synchronize the circadian pacemaker, it was found that in the SCN, CREB becomes rapidly phosphorylated in response to light (Ginty et al., 1993). This observation supports earlier studies indicating a n important role for cAMP in the regulation of circadian rhythms in the SCN (Murakami and Takahashi, 1983; Prosser and Gillette, 1989). At the level of the pineal gland, noradrenergic inputs controlled by the SCN activate AC-coupled p-adrenergic receptors, resulting in increased levels of cAMP in pinealocytes (Fig. 15). This elevation in cAMP levels induces transcription of the enzyme arylalkylamine N-acetyltransferase (NAT), which controls the synthesis of melatonin. Since CAMP-regulated NAT production is dependent on sympathetic stimulation controlled by the SCN, the concentration of NAT in pinealocytes exhibits a circadian rhymicity. What are the molecular bases of this circadian variation? One possible mechanism for this circadian variation is the CAMP-induced expression of ICER, a transcriptional repressor of CREB and CREM activator isoforms, under circadian control in the pineal gland. The expression of ICER in the pineal gland occurs in response to p-adrenergic stimulation, and consequently increases during the night and is lower during the day. Therefore, it is possible that elevations in cAMP levels in response to P-adrenergic stimulation at night results in the PKA-dependent phosphorylation of CREB (or CREMT),with subsequent transcriptional activation of the NAT gene. At the same time, CAMP-dependent activation of the CREM gene through the P2 promoter results in the production of ICER. After


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FIG.15. Generation of a circadian rhythm in the pineal gland. pl-Adrenergic receptor stimulation driven by the suprachiasmatic nucleus (through an indirect pathway involving the superior cervical ganglion) results in the activation of adenylyl cyclase (AC) and production of CAMP. Activation of protein kinase A (PKA)follows due to the dissociation of the catalytic subunits from the CAMP-bound regulatory subunits. The catalytic subunits enter the nucleus of the pinealocyte, where it phosphorylates CREB. As a result, transcription of the gene encoding the melatonin-synthesizing enzyme NAT is activated. At the same time, transcription of the P2 promoter of the CREM gene is also activated, resulting in the production of the transcriptional repressor ICER. The levels of ICER gradually increase until they are high enough to compete for CRE binding with CREB, resulting in transcriptional inhibition of the NAT and CREM genes. In this manner, the activation triggered by stimulation of pl-adrenergic receptor returns to basal levels before the following cycle. G, G-protein; CREBP, phosphorylated CREB.

a lag time the intracellular levels of ICER become sufficiently high to compete with CREB for binding to the CRE, resulting in transcriptional repression of the NAT and CREM P2 promoters. Although the target genes regulated by CREB and CREM in the brain are unknown, these observations indicate that both of these transcription factors participate in neuronal signaling mechanisms in the hypothalamus, and may play an important role in the circadian mechanisms that control neuronal and hormonal responses to lightdark cycles.

D. POSSIBLE ROLEOF CREB IN MEMORY Emerging evidence indicates that CREB is also required for the normal process of higher brain functions such as memory consolidation (reviewed by Frank and Greenberg, 1994; Stevens, 1994). In an

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effort to elucidate the physiological functions of CREB in uiuo, gene targeting via homologous recombination was used to inactivate the CREB gene (Hummler et al., 1994). Initially, the phenotype of homozygous mutant mice generated by this knockout of the CREB gene was found to be apparently normal, although these animals exhibit increased levels of CREM, indicating the existence of a considerable degree of functional redundancy to provide a back-up mechanism that compensates for the absence of CREB. A more detailed analysis of mice carrying the targeted mutation in the CREB gene, however, revealed a deficiency in long-term memory in these mice, although short-term memory is normal (Bourtchuladze et aZ., 1994). Interestingly, the fruitfly Drosophila homolog of CREB also appears to be involved in memory consolidation. This notion is indicated by experiments showing that the induced expression of a transgene that acts as a dominant negative regulator of Drosophila CREB results in a profound deficit in long-term memory associated with olfactory stimuli (Yin et al., 1994). This role of CREB in memory consolidation is presumably related to the CAMP-dependent transcriptional regulation of genes whose expression increases synaptic strengths in specific neuronal populations. It has been proposed that memories are encoded as patterns of synaptic strengths, and long-term potentiation (LTP) is a mechanism for the longlasting modification of synaptic strengths (Stevens, 1994). Consistent with the notion that CREB plays a role in memory consolidation by mediating LTP in the hippocampus, it has been found that LTP occurs via a CAMP-dependent mechanism (Huang et al., 19941, and that in mice carrying a targeted mutation of the CREB gene the stability of LTP is significantly reduced (Bourtchuladze et al., 1994). The hypothesis that CREB may be critically important for memory was initially derived from studies carried out in the sea slug Aplysia (Dash et al., 1990). Prolonged application or intracellular injection of cAMP into sensory neurons of Aplysia produced long-term increases in synaptic strength, suggesting that some of the gene products important for long-term facilitation are cAMP inducible. These findings in molusks, insects, and mammals identify CREB as a key component of an evolutionary conserved mechanism for memory consolidation.

IX. CREBXREM AUTOREGULATION NETWORK The promoters of the CREB and CREM genes contain several CRElike sequences. The binding of CREM and CREB proteins to these


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sequences supports the concept that these genes are part of an autoregulatory network that mediates both positive and negative feedback regulation (Fig. 16). Transcriptional transactivation of the CREB and CREM genes in response to cAMP stimulation (Meyer et al., 1993; Molina et al., 1993) results in the synthesis of mRNAs encoding proteins (CREB and CREM isoforms) that, in turn, may alter their own CAMP-dependent transcriptional responses by determining the relative levels of transactivator or transrepressor isoforms. This CAMPdependent autoregulatory network also provides a mechanism for the regulated control of transcription rates of different target genes after cAMP stimulation. The idea of a positive feedback mechanism that stimulates the CAMP-dependent transcription of the CREB gene is supported by the existence of CRE sequences in its promoter and by transient transfection studies using reporter plasmids bearing segments of the CREB gene promoter (Meyer et al., 1993; Walker et al., 1995). According to this model, activation of the cAMP signaling pathway results in the

FIG.16. CREB-CREM autoregulatory network. Membrane receptor stimulation results in the activation of adenylyl cyclase (AC) and production of CAMP,which, in turn, activates protein kinase A (PKA). After translocating to the nucleus, PKA phosphorylates CREB, triggering the transcriptional activation of target genes (TG), including the gene encoding CREB itself (positive autoregulatory loop) and the CREM gene through the P2 promoter. Subsequent production of ICER results in the transcriptional repression of CREB-activated genes by competition with CREB for binding to the CRE. G, G-protein; P, phosphorylation.

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stimulation of transcription of CREB and other target genes via phosphorylation of preexisting nuclear CREB or CREMT proteins. At the same time, transcription rates from the CREM gene P2 promoter can be also stimulated (Molina et al., 1993; Stehle et al., 19931, resulting in the production of the CREM repressor isoform, ICER. As the levels of ICER proteins increase, they occupy the CRE sites, repressing their own transcription, via an autoregulatory mechanism, as well as that of other CAMP-inducible genes, perhaps including the CREB gene (although this possibility has not been studied). Therefore, ICER acts as a delayed switch that resets the transcription rates of CAMP-inducible genes to basal levels. This hypothetical autoregulatory network may not be present in all cell types. Although the CREB gene seems t o be ubiquitously expressed, different CREM isoforms appear to be preferentially expressed in some tissues, including brain, pineal gland, and testes. Therefore, the relative concentrations of activator and repressor isoforms of these CREBs will determine the degree of expression of target genes in a spatial and temporal manner. In addition, these different isoforms interact to form homo- and hetoerdimers on the target CREs, adding another level of complexity to the regulatory pathway that controls CAMP-dependent gene expression.

X. ONCOGENEFORMS OF CREB, CREM, AND ATF-1 To date, the single example of an oncoprotein involving the CAMPresponsive factors is a fusion protein between ATF-1 and the EWS protein, an RNA-binding protein identified as a hybrid transcript in Ewing’s sarcoma that links its N-terminal domain to the ETs region of the DNA binding domain of the FLI-1 gene (Zucman et al., 1993) (Fig. 17).The expression of the EWS/ATF-1fusion gene results in a rare form of cancer called melanoma of soft parts. The expressed fusion gene consists of a balanced reciprocal translocation between chromosomes 12 and 22, which encode ATF-1 and EWS, respectively [t(12;22)(q13;q12)1. The N-terminal region of the EWS gene is fused to the C-proximal region of ATF-1 consisting of the DNA-binding bZIP domain. The fusion occurs in the intron between exons E and F that encode the P box of ATF-1. The N-terminal segment of the EWS RNA-binding protein is a potent transactivator of gene transcription, presumably targeted by the binding of the ATF-1 bZIP domain to CREs in the promoters of genes resulting in transformation. Several such fusion oncoproteins between

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CREB AND CREM REGULATION NTPEWS

656

EWS NHz

COOH

I bZlP I

EWS/ATF-1 NHz

COOH

t

65

ATF-1

ATF-llEWS

FIG.17. Reciprocal translocation between chromosomes 12 and 22 [t(12;22) 1(13;12)1 causes melanoma of soft parts. The translocation results in the expression of a fusion protein consisting of the transactivating region (NTD-EWS) of the EWS RNA-binding protein with the C-proximaliDNA-binding (bZIP) domain of ATF1-1. BD, Binding domain; PKA, protein kinase A.

RNA-binding proteins and DNA binding domains of transcription factors have been described (reviewed in Rabbits, 1994). It is notable that, as yet, no subjects with oncogenic isoforms of CREB or CREM have been uncovered, particularly since the multiply spliced exons and large introns in these genes would theoretically predispose the subject to miss-splice mutations or recombinatorial translocations. Interestingly, several other bZIP proteins related to CREB and CREM, such as c-Jun, c-Fos, and c-Myc,were originally discovered as their oncogenic variants, in the guise of “tumor viruses” in chickens and rabbits. XI. FUTURE DIRECTIONS Tremendous strides have been made during the past several years in the enhancement of our understanding of the mechanisms by which cAMP signaling regulates gene transcription. The CAMP-responsive DNA-binding proteins CREB, CREM, and ATF-1 appear to comprise the fourth and final components of the cAMP signaling cascade in the control of gene expression. However, many questions remain unanswered and much more investigative research remains to be done to resolve these issues. Certain of the important questions that still require further experimentation are considered in this section. One important area of investigation is to decipher the protein-DNA recognition code by which homodimer and heterodimer combinations

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of the CAMP-responsive DNA-binding proteins recognize and bind to composite CREs in the promoters of genes. Assuming that these proteins can freely dimerize with one another, the four known CAMPresponsive transcription factors, CREB, ATF-1, and the two alternatively spliced bZIP domains of CREM, can form 10 unique homoand heterodimer combinations (unique combinations = [ n x ( n + 1)]/2, where n represents the number of unique proteins). It is possible that there exist additional, as yet undiscovered, CAMP-responsive transcription families that can dimerize with CREB, CREM, or ATF-1. For example, the hypothetical discovery of two additional such proteins would increase the possible dimer combinations t o 21, thereby expanding even further the theoretical repertoire of different CRE motifs with which the dimers could interact. The number of dimer combinations is already theoretically even larger, given the evidence for the multiple alternatively spliced CREM isoforms (ICER, CREMa, CREMAC-G, and others; see Fig. 10B). The numbers could be even larger if circumstances are found in which CREB, CREM, and ATF-1 isoproteins are found to dimerize with other transcription factors, such as J u n and/or C/EBP members of the bZIP family of proteins. The formation of specific dimers of transcription factors within the nucleus predictably would depend on the concentrations of the proteins relative to one another, which, in turn, depend on their relative rates of formation and degradation. In many instances it has been established that the expression of genes encoding DNA-binding transcription factors is regulated by either autopositive or autonegative feedback control mechanisms, as has been found for CREB and for the ICER isoform of CREM. Important directions of investigation will be to decipher the mechanisms that control the complex autoregulatory networks of gene transcription, for example, elucidation of the feedback control mechanism by which targeted disruption of the CREB gene in mice leads to a 10-fold compensatory increase in the expression of the CREM gene. Current evidence indicates that, upon synthesis, CREB, and perhaps also CREM and ATF-1, is constitutively translocated to the nucleus. The nuclear translocation and activation of protein kinases and phosphatases appear to be the regulated steps in modulating the transactivation functions of CREB. An intriguing question regards the topographical compartmentalization of CREB and the kinases and phosphatases within the nucleus. Do all of these proteins comprise a ternary transcriptional complex? Is CREB bound more or less irreversibly to CREs of gene promoters and does it await interaction with protein kinases and phosphatases to regulate transcription? High-


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resolution confocal immunofluorescence microscopy may help to resolve these questions. Another question relates to the mechanisms of the interactions of CREB with CBP and with TAFII,,,, and how they couple to the basal RNA polymerase I1 factors, TFIIB and TFIID, respectively. How do phosphorylations of CREB and CBP by PKA lead to the formation of transcriptionally productive interactions? What are the changes in the structures of the proteins in response to phosphorylations? Eventually, the structures may be provided by solving X-ray diffraction patterns derived from cocrystals of CREB and CBP binding domains. The rationale for the existence of two apparently separate mechanisms for the coupling of CREB to the basal polymerase I1 complex requires further investigation. The coupling of the CREB P box to CBP is entirely cAMP dependent, whereas the coupling of the glutaminerich domains of CREB to TAFII,,, appears t o occur independently of cAMP signaling. Do both mechanisms occur simultaneously? Are they utilized independently during development and/or in cells of different phenotypes? The highly complex multiexonic structures of CREB and CREM are unusual among transcription factors and require further elucidation. Are there yet additional undiscovered alternatively spliced exons in the CREB and CREM genes? How extensive is the alternative exon splicing among different tissues? Is alternative exon splicing developmentally or metabolically regulated to interconvert transactivator to transrepressor isoforms? Further investigations are needed of the biological consequences of alternative exon splicing of the CREB and CREM transcripts. Transcription of the CREB gene is up-regulated by cAMP to provide potent transactivator isoforms. Up-regulation of CREB appears to be interrupted at the cellular level by the splicing in of one or more exons that terminate translation. No such exons that terminate translation appear in CREM. Rather, in CREM the expression of the transactivator isoforms appears to be constitutively regulated, and cAMP upregulates a second promoter located in an internal alternatively utilized and spliced exon to produce a potent transrepressor isoform (ICER). It will be important to determine how ubiquitous the CAMPregulated expression of ICER is. Also, the functional importance of the two alternatively spliced exons encoding two different DNA binding domains of CREM remains to be elucidated. Further, a major future direction of research will be to determine how the alternative splicing of CREB and CREM is regulated. What are the factors and determinants involved in the regulation of the

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specific RNA-splicing factors (splicosomes) that govern specific patterns of the alternative splicing of exons? Finally, it will be most exciting to determine the roles of CREB, CREM, and ATF-1 in development. Already, CREB functions have been implicated in spermatogenesis, pituitary development, and longterm memory consolidation. CREM is implicated as essential in spermatogenesis, the circadian regulation of melatonin synthesis in the pineal gland, and the cell division cycle. It is tempting to speculate that the ICER isoform of CREM may be a master down-regulator of the CAMP-dependent transcription of many essential genes in many different tissues in which gene expression in response to signaling is cyclical. ACKNOWLEDGMENTS We thank Doris A. Stoffers and Edward V. Maytin for critical reading of the manuscript and for helpful suggestions, and Townley G. Budde for preparation of the manuscript and the figures. J.F.H. is a n established investigator of the Howard Hughes Medical Institute. REFERENCES Alberts, A. S., Arias, J., Hagiwara, M., Montminy, M. R., and Feramisco, J . R. (1994a). Recombinant cyclic AMP response element binding protein (CREB) phosphorylated on Ser-133 is transcriptionally active upon its introduction into fibroblast nuclei. J . Biol. Chem. 269, 7623-7630. Alberts, A. S., Montminy, M. R., Shenolikar, S., and Feramisco, J . R. (199413).Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3T3 fibroblasts. Mol. Cell. Biol. 14, 4398-4407. Andrisani, 0. M., Pot, D. A,, Zhu, Z., and Dixon, J. E. (1988). Three sequence-specific DNA-protein complexes are formed with the same promoter element essential for expression of the rat somatostatin gene. Mol. Cell. Biol. 8, 1947-1956. Arany, Z., Sellers, W. R., Livingston, D. M., and Eckner, R. (1994). E1A-associated p300 and CREB-associated CBP belong to a conserved family of coactivators. Cell 77, 799-800. Arias, J., Alberts, A. S.,Brindle, P., Claret, F. X., Smeal, T., Karin, M., Feramisco, J., and Montminy, M. R. (1994).Activation of CAMP and mitogen responsive genes relies on a common nuclear factor. Nature 370, 226-229. Bading, H., Ginty, D. D., and Greenberg, M. E. (1993). Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways. Science 260,181-186. Bakker, O., and Parker, M. G. (1991). CAAT/enhancer binding protein is able to bind to ATFiCRE elements. Nucleic Acids Res. 19, 1213-1217. Beato, M. (1989). Gene regulation by steroid hormones. Cell 56, 535-544. Berger, S. L., Piba, B., Silverman, N., Marcus, G. A,, Agapite, J., Regier, J. L., Triezenberg, S. J., and Guarente, L. (1992). Genetic isolation of ADA2: A potential transcriptional adapter required for function of certain acidic activation domains. Cell 70,251-265. Berkowitz, L. A,, and Gilman, M. Z. (1990). ‘ h o distinct forms of active transcription factor CREB (CAMPresponse element binding protein). Proc. Nutl. Acad. Sci U.S.A. 87, 5258-5262.


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Multiple Facets of the Modulation of Growth by cAMP PIERRE P. ROGER, SYLVIA REUSE, CARINE MAENHAUT, AND JACQUES E. DUMONT Instituie of Interdisciplinary Research Campus Erasme Free University of Brussels B-1070 Brussels, Belgium

I. Introduction A. General Considerations on Cell Cycle Controls B. Probes of the cAMP System: Pharmacological and Genetic Tools 11. Negative Control of Cell Cycle Progression by cAMP A. Early Work on Continuous Cell Lines B. Normal Cells C. Conclusions 111. Positive Control of Cell Cycle Progression by cAMP A. Recent Examples of CAMP-Mediated Positive Growth Control B. Synergism between cAMP and Other Mitogenic Factors C. Positive Regulation of Cell Cycle Progression by cAMP D. Biochemistry of Positive Control of Cell Cycle Progression by cAMP E. CAMP-Dependent and -Independent Mitogenic Pathways IV. Relationship between Growth and Differentiation Controls by cAMP V. A Role for Cytoskeleton Changes in Control of Growth by CAMP? VI. cAMP and the Growth of Cancer Cells A. Negative Modulation B. Escape from Negative Modulation C. cAMP as a Tumor Promoter D. Oncogenes Related to the cAMP Signaling Cascade VII. Conclusions and Perspectives References

I. INTRODUCTION Recent successes in elucidating the functions of cell-transforming proteins encoded by various oncogenes and the demonstration of antioncogenes have shed new light on the assumption that cancers mostly develop from alterations of growth control mechanisms normally involved in homeostasis, tissue repair, and development (Hunter, 1991). Delineating processes that control proliferation and differentiation of normal cells and elucidating how these mechanisms are modified or subverted in neoplasia are critical to our understanding of carcinogenesis. 59

Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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A great part of our knowledge of growth control mechanisms is derived from the study of in vitro conventional models of cultured fibroblast-like cells, such as 3T3 cell lines (Baserga, 1985; Pardee, 1989; Rozengurt, 1986). These cells provide a well-defined and reproducible experimental material, although their immortality indicates that their regulatory circuits have been genetically altered. The proliferation of such cells is dependent on various external factors, including stimulatory or inhibitory growth factors, hormones, and neurotransmitters, and spatial restriction by cell-to-cellcontacts. Two groups of intracellular pathways have been shown t o relay the mitogenic signal brought by some of the primary stimuli. Membrane receptors for some growth factors (e.g., EGF,l PDGF, FGF, IGF-I, insulin and CSF-1) possess an intrinsic protein tyrosine kinase activity, as is the case for one class of transforming proteins encoded by oncogenes (Hunter, 1989; Cantley et al., 1991). Other membrane receptors are coupled via a GTP-binding protein to a phospholipase Cp that cleaves PIP, into diacylglycerol and IP, (Rozengurt, 1986; Berridge, 1987; Whitman and Cantley, 1988). Binding of some growth factors to their tyrosine kinase receptors also activates a phospholipase Cy by phosphorylation on a tyrosine residue. Diacylglycerol activates serinehhreonine PKCs and IP, mobilizes calcium from intracellular stores. The analogs of diacylglycerol, the tumor promoter phorbol esters, also activate C kinases and, in some cell types, proliferation. Intracellular Ca2+ has a well-accepted but poorly defined role in mitogenesis (Berridge, 1987; 'Abbreviations: ADF, actin depolymerizing factor; ACTH, adrenocorticotropic hormone; AKAP, A kinase-anchoring protein; AP, activator protein; ATF, activating transcription factors; bHLH-zip, basic region with a helix-loop-helix leucine zipper motif; C, catalytic subunit of PKA; CBP, CREB binding protein; CDK, cyclin-dependent kinase; CHO, Chinese hamster ovary; W P T , [8-(4-chlorophenyl)thio;CRE, CAMP-responsive element; CREB, CRE-binding protein; CREM, CRE modulator; CSF, colony-stimulating factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; FSH, folliclestimulating hormone; GH, growth hormone; GHF-1, growth hormone transcription factor 1; GIP, gastric inhibitory protein; GRF, GH-releasing factor; hCG, human chorionic gonadotropin; HIV, human immunodeficiency virus; IGF-I, insulin-like growth factor I; IL, interleukin; IP,, inositol 1,4,5-triphosphate; LH, luteinizing hormone; LRF-7, liver regeneration factor-7; MAP, mitogen-activated protein; MDCK, Madin-Darby canine kidney; MPF, maturation-promoting factor; MSH, melanocyte-stimulating hormone; NGF, nerve growth factor; ODC, ornithine decarboxylase; PCNA, proliferating-cell nuclear antigen; PDGF, platelet-derived growth factor; PGE, prostaglandin E; PI, phosphatidylinositol; PIP,, PI 4,5-biphosphate; PKA, CAMP-dependentprotein kinase, or protein kinase A; PKC, protein kinase C; PTH, parathyroid hormone; R, regulatory subunit of PKA; RB, retinoblastoma protein; RSK, ribosomal S6 kinase; SRE, serum-responsive element; TGF-P, transforming growth factor P; TPA, 12-0-tetradecanoylphorbol-13acetate; TRE, TPA-responsive element; TSH, thyroid-stimulating hormone; VIP, vasointestinal peptide.

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Whitfield et al., 19871, but in keratinocytes Ca2+ instead induces differentiation (Pillai et al., 1990).Activation of the PIP, cascade is by no means a universal way to induce proliferation (Choi et al., 1990; Dean and Boynton, 1990; Margolis et al., 1990; Rasp6 et al., 1992). Even though they are clearly distinct in their initial part (receptor, transducer, and first intracellular signal) (Berridge, 1987; Chambard et al., 19871, the protein tyrosine kinase pathways and the PIP, cascade rapidly converge on several events, such as activation of Na+/H+exchange and several transporters, and on a signaling cascade that involves in sequence, from cell membrane to nucleus, the activation of c-Ha-Ras, c-raf, MAP kinase kinase, and MAP kinase, then p62TCF and the transcription of protooncogenes c-jun, c-fos, and c-myc, the products of which are also modulated by phosphorylation (for reviews see McCormick, 1993; Crews and Erikson, 1993; Davis, 1993; Muller et al., 1993) (Fig. 1). These early events are assumed t o be necessary for the entry of a quiescent cell into the cell cycle, and are therefore often called “early mitogenic events.” However, causative relationships with late commitment to DNA replication and cell division remain unclear. After years of studying “early mitogenic events,” we discover that these events are not followed by DNA synthesis and trigger another program in many cells (Raspe et al., 1992; Seuwen et al., 1990a). cAMP is the first identified intracellular second messenger of hormone action. In the 1970s and early 1980s the hypothesis that it may govern (mostly negatively) cell proliferation was considered fascinating and prompted intense scientific activity. As generalized by Pastan et al. (19751, “It seems reasonably well established that treating cells with cyclic AMP analogs inhibits growth. . . . The observation that cyclic AMP inhibits the growth of many types of cells in uitro has obvious implications for the chemotherapy of cancer.” Parallel research led t o the opposite conclusion, which was also generalized; “After having examined most of the large amount of evidence that has accumulated during the past 20 years, we conclude that cAMP is programmed to stimulate an unknown event or events leading to DNA synthesis in a wide variety of cells and possibly a later event(s) leading to mitosis and division” (Boynton and Whitfield, 1983). These and other attempts to generalize the role of intracellular signals (e.g., Berridge, 1975) led to fruitless arguments and confusion. They were soon confronted by the failure to demonstrate such a universal role, raising disinterest in the whole field: “Because they constitute an excellent illustration of the problem of premature generalizations in assigning growth control functions to intracellular components. . . , we can now say that while cyclic nucleotides play a very important role in a cell’s life processes, they have little t o do with cell

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I+ Ca+ t

I+

MAPKK

:+

FIG. 1. Model of the transfer of CAMP-independent mitogenic signals from cell membrane to nucleus (McCormick, 1993; Crews and Erickson, 1993; Davis, 1993; Muller et al., 1993). Growth factors such as EGF and PDGF bind to their tyrosine kinase (TK) membrane receptors, which results in receptor dimerization and trans autophosphorylation on tyrosine residues. Receptor tyrosine autophosphorylation allows binding to other proteins through SH (src homology) 2 domains, including phospholipase Cy (PLCy) and the GRP-2 adapter, in turn bound through its SH, domains to the SOS GDP/GTP exchanging factor that activates RAS. RAS activation stimulates RAF kinase which initiates a cascade of protein kinase activations by phosphorylation, which involves MAP kinase kinase (MAPKK), MAP kinase (MAPK), and S6 kinase (RSK). Nuclear translocation of MAP kinase allows phosphorylation and activation of several transcription factors, including c-Jun, c-Myc, and p62TCF,and transcriptional control of other genes, such as c-fos. Other mitogenic hormones bind to receptors with seven membrane domains and activate a phospholipase Cp (PLCP) and then protein kinase C (PKC) which also activates the same protein kinase cascade initiated by RAF. This model is complicated by the existence of several isoforms at almost each level. The model does not provide explanations for recent observations that tyrosine kinase-negative EGF receptor can stimulate MAP kinases (Campos-Gonzalez and Glenny, 1992; Selva et al., 1993;Eldredge et al., 1994) and that EGF receptors lacking all of the known tyrosine autophosphorylation sites can signal a limited MAP kinase activation and mitogenesis (Decker, 1993). On the other hand, clues to other growth factor-elicited signaling pathways are now appearing almost every month. DAG, diacylglycerol.

proliferation” (Baserga, 1985). The sufficient role of any intracellular signal was even questioned: “Before oncogenes came on the scene there appeared to be a chance that activating a few second messengers like CAMPor inositol phosphates and diacylglycerol might be enough to get cells cycling. This was pure wishful thinking. . .” (Hall, 1991). Such


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negative conclusions explain why the role of cAMP in growth control was often ignored in recent general reviews on this subject [e.g., that by Muller et al. (1993) or published in a recent Cell volume on cancer and proliferation controls (64, 249-350, 1991)l. Obviously, the significance of an intracellular signal, like that of an electronic switch, varies depending on the network in which it is involved, that is, on the cell program (Dumont et al., 1981). The confusion in the field results not from demonstrating a role for intracellular signals in the proliferation of specific cells, but from the generalization of such conclusions. The stimulation of proliferation through mechanisms dependent on cAMP is now well demonstrated (Dumont et al., 1989). Our aim in this chapter is to critically review the last 12 years’ advances as to the positive and negative roles of cAMP on the multiplication of normal and cancer cells. The few available data on the possible mechanisms underlying these controls are examined in depth, showing that (and hopefully how) cAMP may have positive or negative effects, depending on the program, that is, the differentiation of the cell type involved. The relationships of these effects to differentiation and tumor development are considered. Previous data, mostly on negative cAMP control of growth, have been extensively reviewed (Pastan and Johnson, 1974; Pastan et al., 1975; Friedman, 1976; Rebhun, 1977). The reader is especially referred to the reviews by Boynton and Whitfield (1983) and Christoffersen and Bronstad (1980) for a critical assessment of the evidence of negative and positive roles of cAMP in various stages of the cell cycle progression. The definitive evaluation of the overall roles played by cAMP in the proliferation of various cell types, as considered here, depends on the precise knowledge of their cycle (Section 1,A) as well as on carefully controlled manipulations of the cAMP signaling cascade (Section 1,B). A. GENERAL CONSIDERATIONS ON CELLCYCLE CONTROLS The basic mechanisms of cell division are probably well conserved in eukaryotic cells, as shown by the finding of homologous and similarly acting genes in yeasts and humans [Nurse, 1990; Nasmyth, 1990; Pelech et al., 1990; Koff et al., 1991; Muller et al., 1993; and several reviews on cell cycle regulation in Cell (79,547-582,1994)l. However, the controls exerted on these basic mechanisms may vary greatly among species, cell types, etc. (Baserga, 1990). The cell cycle of most eukaryotic cells has been operationally divided into four phases, generally described as successive, but which, in fact,

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are overlapping to an extent depending on the system and the experimental conditions. In the classical description a cell goes through a G, (gap-1)phase, during which it grows and prepares to replicate its chromosomes, then through an S (DNA-synthetic) phase, during which it replicates its chromosomes;then it pauses in a second gap, or G, phase, during which it prepares for the next phase: mitosis, cytokinesis, and division into two new cells. Some cells lack a detectable G, or even G, phase, indicating that the events normally performed during these phases could be achieved during the previous ones. The V79-8 tumor cell lacks a G, phase (Prescott, 19871, implying that the preparation for DNA replication is executed during the previous cycle (Cooper, 1979). In general, cells stop growing with a G, content of DNA, although, more rarely, G, arrest could also occur [it is the rule in some lower metazoans (David and Campbell, 197211. The duration of the G, phase is the most variable, and cell cycle progression is mostly dependent on external controls during this phase. Normal cells can rest in a quiescent, or resting, state (often called Go) outside the cell cycle, with a G, DNA content, from which they can be stimulated to reenter the cell cycle. Such quiescent cells undergo DNA replication after a long “prereplicative phase,” including the time necessary for entry into the cell cycle and the G, phase of the cell cycle (Baserga, 1985). G1 phases and prereplicative phases can be classified into at least four main categories (Boynton and Whitfield, 1983).Type 1is the basic minimum prereplicative phase of continuously cycling cells of established lines. Some of these cell lines, mostly of tumoral origin (HeLa, etc.), are unable to enter into the quiescent Go-like state. Like some prokaryotes, they cycle or they die. Type 2 is the prereplicative phase that follows stimulation of quiescent (Go) cells in starved or confluent culture of “normal” cell lines, which continuously cycle with a type l-like G, phase under unrestricted growth conditions (with serum or growth factors) (3T3 cell lines). The duration of the prereplicative phase of Go cells exceeds that of the type 1 G, phase of the same cells when continuously cycling. Clearly, the longer the cells stay in Go, the more labile and other components needed for the initiation of DNA synthesis are degraded and the longer it takes to initiate DNA synthesis upon stimulation (Baserga, 1985).In the model BALB/c-3T3 mouse embryo cell cultures the progression into the prereplicative phase is dependent on the synergistic cooperation of at least three hormones or growth factors present in serum: a short exposure to PDGF makes the Go cells “competent” to progress through the prereplicative phase in response t o the sequential addition of EGF and IGF-I, the latter controlling commit-

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ment for DNA replication (O’Keefe and Pledger, 1983). Only IGF-I is necessary for supporting a further type 1G, phase in cycling BALB/c3T3 cells, but the presence of PDGF and EGF during the previous cycle is required in order to prevent cells from entering Go after mitosis (Campisi and Pardee, 1984). Although the assignment of different roles for different growth factors might be of restricted application [in human fibroblasts PDGF and EGF are interchangeable, each being able to support all of the prereplicative development (Westermark and Heldin, 198511, the model supports a general concept of the prereplicative phase as a sequence of major regulatory events and points out that Go-G, transition, that is, entry into the cell cycle, requires specific events not involved in type 1 G, phase progression. A good example of events induced during GoG, transition is the rapid stimulation of the expression of protooncogenes such as c-fos, c-jun, and c-myc (Kaczmarek and Kaminska, 1989). Although c-myc expression is sharply and transiently induced by growth factors in quiescent cells, c-myc mRNA levels do not vary during cell cycle progression in continuously cycling cells (Thompson et al., 1985). Moreover, it is clear that continuously cycling cells just after mitosis still contain various relatively stable proteins required for cell cycle progression, DNA replication, and mitosis. Such proteins need resynthesizing during the type 2 prereplicative phase but are subject to only modest fluctuations during type 1 cycling [e.g., DNA polymerase a-associated primase (Tseng et al., 1989); PCNA/cyclin, the auxiliary protein required for DNA polymerase 6 activity (Morris and Mathews, 1989); and p34cdc2 kinase (Draetta et al., 1988)l. Whether type 2 prereplicative development is a good model for the stimulation of quiescent highly differentiated cells in uiuo, such as hepatocytes and kidney and thyroid cells, is still not clear. It is quite possible that the long prereplicative phase of such cells includes specific controls that reflect their differentiation. These cells, when stimulated, are already transcribing differentiation-related genes. Thus, their prereplicative phase does not require a triggering of protein, RNA, and lipid synthesis but includes a reorientation of the genetic expression program from differentiation toward more general activities required for cell cycle progression. This is simply a reformulation of the old concept of growth and differentiation as exclusive activities of the cell. On the other hand, in such differentiated cells repetitious stimulation by functional activators also leads to growth. These adaptive processes enhancing the functional capacity of the tissue might well follow distinct pathways in order to obey different constraints; that is, they may imply a third class (type 3).

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A fourth type is obviously the very long prereplicative phase following the activation of the small lymphocyte. This cell consists mainly of a small nucleus with highly condensed chromatin and a greatly reduced cytoplasm with few mitochondria, a poorly developed Golgi apparatus, and few if any, polyribosomes. Unlike the other types of prereplicative phase, the first stage of type 4 prereplicative phase includes decondensation of the chromatin, activation of the genome, and de nouo build-up of the mitochondria and the protein-synthetic apparatus. It is obvious that such different models of cell cycle progression and activation of quiescent cells might be regulated differently and at different levels.

B. PROBESOF THE cAMP SYSTEM: PHARMACOLOGICAL AND GENETIC TOOLS So far there is no evidence of cAMP growth regulation in mammalian cells by other means than its intracellular receptors, the PKAs. The demonstration that a given effect of an extracellular signal is mediated by cAMP and PKAs uses pharmacological arguments and genetic manipulation of the CAMP-PKA cascade. As reviewed earlier (Doskeland et al., 19911, if an effect B of agent A is mediated by cAMP and PKAs, 1. A should increase the activity of PKAs in a relevant compartment before or concomitantly with the appearance of B. A simply measurable index is the increase of cAMP concentration at the whole-cell level. It should be noted that while a lack of measurable increase of cAMP excludes a massive general PKA activation, it does not exclude a slight or local activation. Direct measurement of PKA activation within single cells is still at the pioneering study level (Adams et al., 1991, 1993). 2. If A acts by stimulating adenylate cyclase, B should be enhanced by inhibitors of cyclic nucleotide phosphodiesterases, and the action of A should be mimicked by agents that activate endogenous PKAs and by overexpression of the C subunit of PKA. 3. The action of A should be blocked by agents interfering with its stimulation of cAMP production or agents that lower cellular CAMP,by inhibitors of PKAs, and by agents acting downstream of the PKA. However, it should be considered that A may signal via another pathway than the cAMP system, but B may be observed only if the PKA retains its basal activity (i.e., cAMP has a


67

permissive effect). In this case the effect of A will be counteracted by agents that inhibit the PKA below basal levels. Molecular mechanisms may be substrate-directed control (the residue phosphorylated by PKA becomes more or less accessible due either to phosphorylation of another residue or to the binding of a ligand) o r the control of phosphatases. Pharmacological and genetic tools used to manipulate the CAMPPKA cascade are summarized in Table I. Forskolin, a plant diterpene, activates vertebrate adenylate cyclase directly and enhances its response to activated G, (the GTP-binding protein transducer that stimulates adenylate cyclase), thus to the receptors that regulate G, (Seamon and Daly, 1986). Extracellular forskolin acts rapidly and is easy to wash off. It is thus particularly useful in inducing pulses of high-level cAMP of defined duration (Seamon and Daly, 1986; Roger et al., 1987a). However, this compound has other effects (Laurenza et al., 1989). For instance, independently of adenylate cyclase, it inhibits insulin-stimulated glucose transport (Joost and Steinfelder, 1987) and CAMP-stimulated L-type Ca2+ current (Boutjdir et a1., 1991);it influences Golgi apparatus function (Lippincott-Schwartz et al., 1991) and the gating of voltage-dependent K+ channels (Hoshi et al., 1988);and it desensitizes the acetylcholine nicotinic receptor (Wagoner and Pallotta, 1988). Analogs of forskolin may help to distinguish the effects on cyclase: 1,9-dideoxy-forskolin does not activate cyclase but reproduces other effects (Laurenza et al., 1989). Inhibition of glucose transport may be counteracted by raising glucose concentrations. Cholera toxin binds to membrane gangliosides through its B subunits. Its A unit is injected into the cytosol, from which it “ADPribosylates” the a, subunit of G,. ADP ribosylation inhibits the GTPase activity of a,,thus leading to permanent activation of G, and of adenylate cyclase (Moss and Vaughan, 1988). Cholera toxin is a useful agent to induce, after a delay, long-term steady elevations of CAMP.However, the B subunit of the toxin, binding to GM, ganglioside, may induce gene expression (Qureshi et al., 1991) or enhance or decrease proliferation per se, independently of cAMP (Spiegel et al., 1985; Spiegel and Fishman, 1987; Tetsumoto et al., 1988). Even the toxinmediated ADP ribosylation may not be entirely specific for G, (McCloskey, 1988). Pertussis toxin, after its internalization, ADP-ribosylates the subunit ai of Gi (the GTP-binding transducing protein that negatively controls adenylate cyclase).ADP-ribosylated Gi becomes inactive, thus relieving cyclase of any negative control (Foster and Kinney, 1985). In

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PIERRE P. ROGER et al

TABLE I TOOLS USEDTO ASSESSTHE RQLEOF THE cAMP CASCADE Pharmacological Modulation of adenylate cyclase Activation Hormones, neurotransmitters, etc. Cholera toxin Forskolin Pertussis toxin Inhibition Hormones, neurotransmitters

Genetic

Activation Expression of constitutive activating receptor (A,) Expression of constitutive a , Expression of A subunit of cholera toxin

Inhibition Expression of mutated inactive a, (dominant negative phenotype) Antisense oligonucleotides of a, Modulation of cAMP phosphodiesterases Inhibition Activation Expression of yeast cAMP phosphodiesterase RO-201724, methylxanthines, etc. Modulation of PKAs Activation Activation Antisense oligonucleotides of R subunit of 8-chlorophenylthio-CAMP Sp CAMPS PKA Overexpression of C subunit of PKA Pairs of site-specific kinasespecific analogs C subunit of PKA Inhibition Inhibition Antisense oligonucleotides of C subunit Rp CAMPS Expression of mutated cAMP binding domain Inhibitors of protein kinase (inactivating C subunit) R subunit Overexpression of R subunit of PKA Overexpression of Walsh PKA inhibitor Activation Expression of constitutively activated transcription factor cDNA (CREB) Antisense oligonucleotides of CREM Inhibition Antisense oligonucleotides of CREB Expression of dominant inactivating mutations of CREB Expression of CREM

cells with such a tonic control, pertussis toxin therefore induces a prolonged elevation of cAMP levels. However, pertussis toxin also inhibits other Gi effects, for example, the activation in the PIP, phospholipase C cascade, and may inactivate other proteins (Sontag et al., 1991).


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The invasive adenylate cyclase toxins of Bordetella pertussis and Bacillus anthracis are powerful but rarely used tools to specifically elevate cellular cAMP levels in eukaryotic cells. The B . pertussis toxin seems to require activation by calmodulin and is rapidly proteolyzed by a process dependent on ATP binding. Removal of the toxin from the target cell medium results in a rapid loss of its intracellular activity, leading to a decay in the level of cellular cAMP (Hanski, 1989). Inhibitors of cyclic nucleotide phosphodiesterases by inhibiting the catabolism of cAMP also raise cAMP levels. However, due to the negative cooperativity of the phosphodiesterase system (Erneux et al., 19851, these inhibitors are much more efficient in increasing the cAMP response to activators of adenylate cyclase than in increasing basal levels of CAMP. Moreover, they also have other important CAMPindependent pharmacological effects: methylxanthines inhibit adenosine receptors and glucose transport (Steinfelder and Petho-Schramm, 1990), enhance Ca2+ release from sequestration sites in some cells (Huddart and Syson, 19751, and inhibit the induction by TSH and cAMP of ODC in thyroid cells (Mockel et al., 19801, a crucial step in proliferation induction. Preferably, more than one phosphodiesterase inhibitor should be tested, and it should be shown that the potencies of the drugs correlate with their known potencies against the major phosphodiesterases rather than, for example, their activity as adenosine antagonists. Analogs of CAMP are currently used mostly t o mimic-in some cases to inhibit-the effects of CAMP.Ideally, they should penetrate cells, be potent activators of PKA, and be resistant to hydrolysis without blocking the degradation of endogenous CAMP.For initial studies 8-CPTcAMP is preferred by many investigators because it has a generally high potency and is easily available commercially. However, it is also a potent inhibitor of cGMP-specific phosphodiesterase and therefore cGMP may increase in cells treated with this analog (Connolly et al., 1992). The effects of similar concentrations of the noncyclic nucleotides should be checked. Analogs with an S in the P ring come closest to meeting the above criteria. They activate (Sp CAMPS)or inhibit (Rp CAMPS)the action of cAMP (Rothermel et al., 1984; Erneux et al., 1986). However, sometimes, due to low penetration, high concentrations must be used. Commercial preparations of Rp CAMPSshould be used cautiously, since they have been reported to contain biologically active amounts of adenosine (Musgrave et al., 1993). A new Sp cAMP analog has been designed (Sp-5,6-dichloro-l-~-~-ribofuranosylbenzimidazole-3',5 '-monophosphorothioate), which associates the interesting properties of being a very potent and specific activator of PKAs

70


with a high lipophilicity and a strong resistance to phosphodiesterases (Sandberg et al., 1991). Butyrylated derivatives (mono- and dibutyryl-CAMP) have often been used in cell cycle control studies. The monobutyryl-CAMP derivative activates PKAs and inhibits some phosphodiesterases (Hsie et al., 1975). However, preparations are often contaminated by butyrate and anyway these analogs are hydrolyzed in the cells releasing butyrate (O’Neill et al., 1975; Coulson and Harrington, 19791, which has effects per se on cell proliferation and differentiation (Herlyn et al., 1988). Experiments with such analogs should therefore test butyrate and the nucleotide itself as controls. Martin and Kowalchyk (1981) have shown that in several cell lines the growth inhibition by cAMP derivatives does not require the 3’3’phosphodiester linkage. Phosphodiesterase and 5’-nucleotidase, which are present in serum-supplemented culture media and at the external surfaces of cells, hydrolyze cAMP and cAMP analogs generating the respective AMP and then adenosine derivatives, which have their own multiple receptors (Niles et al., 1979; Hargrove and Granner, 1982; Weisman et al., 1988; Van Lookeren Campagne et al., 1991). These derivatives are frequently toxic or cytostatic, even at low concentrations. When this occurs, the effects of cAMP and cAMP analogs are prevented by the addition of adenosine deaminase (Hargrove and Granner, 1982; Van Lookeren Campagne et al., 1991), inhibitors of adenosine transport such as dipyridamole (Weisman et al., 19881, or uridine (Niles et al., 1979; Hargrove and Granner, 1982; Weisman et al., 1988) and deoxycytidine (Albert et al., 1991). Adenosine toxicity has been ascribed to an inhibition of pyrimidine synthesis, probably by inhibiting ribonucleotide reductase activity and synthesis (Albert et al., 19911, which is bypassed by uridine addition (Hargrove and Granner, 1982). N6-benzyl-CAMP (Zorn et al., 19931, 8-amino-cAMP, and 7-deaza-cAMP, like 8-Cl-cAMP, are readily broken down into extremely toxic derivatives. The very strong growth-inhibiting properties of 8-C1-CAMP on several cancer cell lines, which is claimed to act through binding to type I1 PKA (Cho-Chung, 1989), were, in fact, ascribed to the toxicity of 8-C1-adenosine (Van Lookeren Campagne et al., 1991). 8-Br-cAMP, often cited as being hydrolysis resistant, is much less resistant than other analogs, such as N6-benzoyl-CAMP and N6-monobutyryl-c AMP. Recently, analogs of cAMP specific for the two sites of the two types of PKAs have been synthesized. If general activation of PKAs is desired, pairs of such analogs with preferential affinity for each of the


71

cAMP sites (A and B) on the R subunit of PKA should be used for synergy, one member of the pair binding preferentially to site A and the other binding to site B. Among commercially available analogs, N6-benzoyl-CAMP and N6-monobutyryl-CAMP are site A selective and 8-methylamino-CAMP and 8-aminohexylamino-CAMP are site B selective. Demonstration of synergism suggests that the analogs really act via PKA activation. Other analogs allow specific stimulation of either PKA I or 11(Cho-Chung, 1989; Beebe et al., 1988; Van Sande et al., 1989). Although interpretation of the results in intact cells is complicated by the unknown penetration and degradation rates of these analogs, the great specificity of these compounds makes it very likely that results obtained with a specific pair do involve the corresponding PKA. No synergism would be expected if the analogs acted by phosphodiesterase inhibition, cGMP-dependentkinase activation, or adenosine action on its receptors. Inhibitors of specific protein kinases are commercially available (Hidaka et al., 1991). In general, the specificity of these compounds is only weak, a moderate increase in concentration (factor 5 ) being sufficient t o inhibit other kinases. An effect of such inhibitors might at least suggest that protein kinases, not other proteins such as channels, are involved (Kaupp, 1991). Investigation of the role of the cAMP cascade can also be carried out by the direct microinjection of the purified subunits of the PKAs themselves. Injection of the C subunit should reproduce the activation of the cascade (McClung and Kletzien, 1984; Roger et al., 1988a; Lamb et al., 1988), while injection of the R subunit should inhibit it. Such effects are indeed observed, but their duration is reduced by the fact that uncoupled subunits are degraded very quickly in the cell, which tends to reestablish the R and C subunit concentrations at their normal levels (Richardson et al., 1990). Exogenous free R and C subunits are thus short-lived, and for this reason will not induce the effects of longterm stimulation or inhibition of the cascade. The heat-stable inhibitor of PKA (Walsh and Glass, 1991; Fernandez et al., 1991; Kupperman et al., 1993) and antibodies against the C subunit of PKAs can also be used (Browne et al., 1987). When available, genetic tools might be the best to modulate, in the long term, the activity of the cAMP cascade. Mutations conferring constitutive activation of the cascade, whether at the level of receptors coupled to adenylate cyclase, such as the adenosine A2 receptor (Maenhaut et al., 19901, or at the level of G, (Landis et al., 1989; Zachary et al., 1990), have been shown to confer the proliferation phenotypes to

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transfected cells in which cAMP is a positive regulator of growth. To block CAMP-dependent responses, antisense oligonucleotides of the subunits of PKAs or CAMP-regulated transcription factors (CREB and CREM) and dominant negative mutated forms of these proteins have been used. For instance, antisense oligodeoxynucleotides targeted against the RII subunit of PKA I1 decrease the level of the protein and result in a great decrease in the differentiating antiproliferative action of cAMP analogs on HL60 leukemia cells (Tortora et al., 1990). Specific expression of mutated “killer” CREB in the somatotrophs of transgenic mice does not allow these cells to proliferate and induces dwarfism (Struthers et al., 1991). Expression of active recombinant fragments of protein kinase inhibitor or dominant mutations of RI has also been used to block “CAMP-dependent”gene transcription (Groveet al., 1987) and mitogenesis (N. Huang et al., 1994). Expression of mutated inactive a, also exerts a dominant negative phenotype (Osawa and Johnson, 1991). cAMP responses, including CAMP-dependent cell proliferation, are suppressed in mammalian cells expressing high levels of the yeast low-K, CAMP-phosphodiesterase gene (Kessin et al., 1992; N. Huang et al., 1994). Finally, cells with a mutation in one of the proteins of the cAMP cascade demonstrate, on the contrary, the role of the cascade in their proliferation. Tumor cells with mutations in the PKAs have been shown to be resistant to the inhibitory effects of the cAMP cascade (Bourne et al., 1975). Mutations that confer constitutive activity to the a, and ai2 subunits of GTP-binding proteins, which activates or inhibits adenylate cyclase, have been found. The fact that such mutations have been demonstrated in tumors suggests that in different cells the relief of either activation or inhibition of the cAMP cascade has an oncogenic potential (Lyons et al., 1990). However, constitutive ai2 and a, have other effects than the modulation of adenylate cyclase. Moreover, the effects of genetic tools might also be indirect. A mutation in a protein kinase might influence many features in the cell that are not directly involved in growth control but could indirectly influence it. In conclusion, there are many pharmacological and genetic tools to investigate the role of the cAMP cascade in the control of cell proliferation. None of these allows unambiguous conclusions. To be reached, such conclusions require the convergence of as many different experimental results as possible. Such results should preferably be obtained in one species, at one phase of development. Indeed, control pathways may vary from one species and from one stage of development to another (Dumont et al., 1981).


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11. NEGATIVE CONTROL OF CELLCYCLE PROGRESSION BY cAMP

A. EARLY WORKON CONTINUOUS CELLLINES The larger body of evidence in the 1970s suggesting a major negative regulatory role for cAMP on cell growth was derived from the study of neoplastic or nonneoplastic fibroblastic cell lines, mostly of rodent origin (types 1 and 2 prereplicative phases). The evidence has been extensively and critically reviewed (Pastan et al., 1975; Friedman, 1976; Rebhun, 1977; Boynton and Whitfield, 1983; Christoffersen and Bronstad, 1980). Thus, we restrict ourselves here to a brief outline of some major features of these studies. cAMP had been proposed as the intracellular mediator signaling the cell to stop growing under conditions that indeed limit its proliferation (e.g., culture confluence, cell starvation, or growth-inhibitory hormones) (Otten et al., 1972). The following criteria were generally accepted in order to establish cAMP as a specific negative regulator of cell cycle progression (Friedman, 1976). 1. The intracellular cAMP level should rise coincidentally with the entrance of cells into the quiescent state and, conversely, should fall as cells exit quiescence and enter the growing state. 2. Experimental elevations of cellular CAMP, at the appropriate time of the cell cycle, should induce cells to enter the quiescent state and prevent quiescent cells from entering the growing state in response to a growth stimulus. The first criterion was based on the now untenable assumption that cAMP could be the sole mediator of quiescence/cell cycle transitions. It depends on the accurate measurement of low basal cell cAMP concentration, which still remains a difficulty: cAMP basal contents vary little under conditions in which stimulated cAMP levels are shown to be inhibited; moreover, to evaluate concentrations, cell mass should be precisely estimated. In agreement with this criterion, the addition of serum, growth factors, or mitogenic proteases to quiescent cells with type 2 prereplicative phase often transiently decreases the levels of cAMP (Pastan et al., 1975). However, as thoroughly examined by Friedman (1976), Rebhun (19771, and Boynton and Whitfield (19831, these studies were challenged by other reports in which such changes were not observed and even opposite changes were demonstrated. Until recently, satisfying the second criterion also presented major difficulties. As discussed in Section I,B, the problem was to obtain

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elevations in intracellular cAMP in a specific manner and for the desired duration. Prolonged or too high an increase in cAMP concentration could inhibit cell cycle progression, even in systems in which cAMP is unequivocally mitogenic (Boynton and Whitfield, 1983). It should be emphasized that while most effects of cAMP are obtained by an increase in concentration by a factor of 2-4, cAMP enhancements by a factor of 20 are easily reached in the presence of the commonly used phosphodiesterase inhibitors (Miot et al., 1984). Elevating cellular cAMP levels does slow down or block cell cycle progression in a variety of cell lines. Moreover, many studies used brominated or butyrylated cAMP analogs with too often inadequate controls. Some results might be explained by non-CAMP-specific effects. For example, 8-Br-CAMP-resistant variants of GH pituitary cells are deficient in adenosine kinase (Martin and Fbnning, 19811,suggesting that these normal cells are, in fact, inhibited by the nonspecific generation of adenosine derivatives. Nevertheless, the fact that in some tumor cell lines mutants with altered PKAs are resistant to cAMP growth inhibition suggests that in these cells cAMP indeed acted through its normal effector IS49 lymphoma cells (Bourne et al., 1975), CHO cells (Singh et al., 19811, and Y1 adrenocortical tumor cells (Schimmer et al., 198611. Analyses of the kinetics of cell cycle progression in the presence of CAMP-elevating treatments showed that, depending on the experimental system, cAMP inhibits cell cycle progression at different phases. 1. Inhibition during S phase [as found in Reuber H35 hepatoma cells (Van Meeteren et al., 1982)l or at the initiation of S phase has little physiological meaning and might instead reflect a nonspecific perturbation of the balance between nucleotide precursors affecting DNA synthesis. It is reproduced by noncyclic analogs and reversed by uridine (Hargrove and Granner, 1982; Van Lookeren Campagne et al., 1991). 2. Inhibition of G, progression by high cellular cAMP seems to be widespread (Friedman, 1976). In some cell lines with a type 1 cell cycle, such as HeLa cells, it is the only effect of CAMP,whereas in other cell types (with a type 1 or 2 prereplicative phase) it coexists with inhibition (Weidman and Gill, 1977) or even stimulation of GI progression (Baptist et al., 1993). It gives sense to the decrease in endogenous cAMP often observed in late G,/mitosis (Friedman, 1976). These observations were extended recently, and some clues to the mechanism were provided (see Section II,B,6). The physiological meaning of a G, control by cAMP in response to external factors


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should be questioned in somatic cells, as, in general, these cells possess a G, DNA content. 3. In various cell lines cAMP has been reported to inhibit G, progression (Friedman, 1976). When analyzed, this inhibition was found at the different stages of the prereplicative phase that are the targets of positive regulation by serum or growth factors: at the GoG, transition (Heldin et al., 1989), in mid-GI (Leof et al., 1982)’or at the restriction or commitment point for DNA replication in late GI (Gill et al., 1980). In BALB/c-3T3 mouse embryo fibroblast-like cells, cAMP blocks the progression into the prereplicative phase at point V, 6 h before the onset of DNA synthesis. This negative control coexists with a positive effect of cAMP on the earlier acquisition of competence (O’Keefe and Pledger, 1983; Leof et al., 1982; Smets and Van b o y , 1987). Again, this illustrates very well that cAMP may exert both positive and negative influences at different stages of cell cycle progression. The established cell lines used for such analysis are often neoplastic and therefore defective in their proliferation controls. Therefore, in the next section we concentrate our attention on some well-described models of normal cells in which cAMP inhibition of growth might have a physiological meaning (Table 11). B. NORMAL CELLS

1. Fibroblasts PGE, inhibits the restimulation of Go-G, transition in quiescent lung fibroblasts by serum, PDGF, or macrophage-derived growth factor. This effect is reproduced by dibutyryl-CAMP (Fine and Goldstein, 1987). In agreement with such in uitro findings, a suppressive factor released by macrophages, which stimulates endogenous fibroblast PGE, production and cAMP formation, has been suggested to limit bleomycin-induced pulmonary fibrosis in hamsters (Clark et al., 1982, 1983). The inhibition of proliferation by cAMP has also been observed in CCL39 Chinese hamster fibroblasts (Magnaldo et al., 1989b), 10 T 1/2 cells (Matsukawa and Bertram, 1988)’ Syrian hamster embryo cells (Cowlen and Eling, 19921, and normal diploid human fibroblasts (Espinoza and Wharton, 1986). In these systems the inhibition is less potent in exponentially growing cells and mainly affects the restimulation of quiescent cells or the growth of the cells at high density (Magnaldo et al., 1989b; Matsukawa

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TABLE I1 "NORMAL" VERTEBRATE CELLSIN WHICHcAMP HASBEENFOUNDTO MEDIATE GROWTH INHIBITION Growth inhibitors PGE

PGE, adenosine, VIP, P-adrenergic PGE PGE, p-adrenergic, adenosine PGE PGE, interferon a1p ? ACTH Vasopressin, p-adrenergic ? ? ?

Glucagon

Cell systems Human fibroblasts,".* 10 T 112 cells,b rat-1 cells,b CCL39 Chinese hamster fibroblasts,b and BALBc-3T3 mouse embryo cells* Bovine aortic and rat cerebral microvascular endothelial cellsb Rat arterial smooth muscle cells* Human and rabbit aortic smooth muscle cellsh Murine and human T 1ymphocytesa.b Murine and human B lymphocytesh Mouse macrophage& BC3Hl muscle cell line and L6 myoblasts" Bovine and rat adrenocortical cells in culture" Rabbit kidney cellsb Human and rat astrocytes" Rat glial cells* Rat placental cells* Rat hepatocytes*

"Primary references can be found in the review by Boynton and Whitfield (1983). bSee text for details and references. c ? , Physiological growth inhibitor, not yet known.

and Bertram, 1988; Espinoza and Wharton, 1986). cAMP could affect mainly the Go-G, transition, but not the progression through G I . Different mechanisms have been proposed. In NIH 3T3 and rat-1 fibroblasts, cAMP inhibits the mitogenic signaling cascades converging on MAP kinase activation (Wu et al., 1993; Cook and McCormick, 1993; Hordijk et al., 1994). Analysis of various signaling intermediates indicates that cAMP interferes at a site downstream of p2lra5, but upstream of raf-1 kinase (Cook and McCormick, 19931,possibly involving a direct phosphorylation by PKA of Ser43 in the regulatory domain of raf-1 (Wu et al., 1993; Hafner et al., 1994). The inhibition by cAMP of the Go-G, transition also involves an inhibition of protooncogene expression: c-myc in human fibroblasts stimulated by PDGF (Heldin et al., 1989), c-myc and c-jun induced by TPA in 3T3 cells (Mechta et al., 1989), and c-jun in Syrian hamster embryo cells (Cowlen and Eling, 1992). However, in the latter two systems cAMP also inducesjunB.


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Induction of interferon pz (IL-6) gene expression by cAMP in human fibroblasts (Heldin et al., 1989; Zhang et al., 1988) and in other cells (Ray et al., 1989; Spangelo et al., 1990) could also contribute to explaining an inhibition of growth, sometimes by c-myc down-modulation (Zullo et al., 1985; Forsberg et al., 1988). However, antibodies neutralizing interferon p, fail to prevent the growth-inhibitory effects of cAMP agonists in human fibroblasts (Heldin et al., 1989). In AKR2B fibroblast cells cholera toxin inhibits TGF-P,-stimulated c-sis (PDGF) expression, and consequently its induction of c-myc expression (Howe et al., 1989).Another likely mechanism is the suppression of the expression of cyclin D1 by cAMP in human fibroblasts (Sewing et al., 1993). Whether this is a consequence of the inhibition by cAMP of raf-1 activity or c-myc expression remains unknown. On the other hand, the role of the several CAMP-dependent phosphorylations of cyclin D1 in human fibroblasts (Sewing and Muller, 1994) has not been defined. Pouyssegur and collaborators have even suggested that an inhibition of adenylate cyclase could contribute to the mitogenic activity of serotonin on CCL39 cells (Seuwen et al., 1988) or of a,-adrenergic agonists on CCL39 cells transfected with an a,-adrenergic receptor gene (Seuwen et al., 1990b). Similarly, lysophosphatidate induces proliferation of human fibroblasts and rat-1 cells seemingly by activating Gi2 (Van Corven et al., 1989). Oncogenic mutations activating G,2 inhibit cAMP accumulation (Wong et al., 1991) and induce high-density proliferation and neoplastic transformation of rat-1 fibroblasts (Pace et al., 1991) and NIH 3T3 cells (Hermouet et al., 1991). However, the activation of Gi2 may have other effects than inhibiting adenylate cyclase, such as the activation of phospholipase C, phospholipase A,, channels, or p2lras (Gupta et al., 1990; Van Corven et al., 1993). It is now clear that adenylate cyclase inhibition is not sufficient to explain the effect of ai2 on fibroblast growth (Hermouet et al., 1993; Stephens et al., 1993; Pouyssegur and Seuwen, 1992). 2. Vascular Endothelial and Smooth Muscle Cells Forskolin, cAMP analogs, and various phosphodiesterase inhibitors, including dipyridamole, at their therapeutic plasma concentrations, markedly inhibit the proliferation of bovine aortic (Leitman et al., 1986) and rat cerebral microvascular endothelial cells in culture (Kempski et al., 1987). However, in newborn human dermal microvascular endothelial cells (Davison and Karasek, 1981) and in fetal bovine aortic endothelial cells (Presta et al., 1989) cAMP is reported to be stimulatory.

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The serum- and PDGF-induced multiplication of vascular smooth muscle cells is inhibited by cAMP (Kempski et al., 1987; Nilsson and Olsson, 1984; Jonzon et al., 1985; Orekhov et al., 1986; HultgardhNilsson et al., 1988; Fukumoto et al., 1988) during the early (Nilsson and Olsson, 1984) and/or late prereplicative phase (Fukumoto et al., 1988). In rat arterial smooth muscle cells cAMP may mediate the growth-inhibitory effects of PGE (Nilsson and Olsson, 19841, adenosine (Jonzon et al., 19851, VIP (Hultgardh-Nilsson et al., 1988), and norepinephrine through P-receptors (Nakaki et al., 19861, as well as the growth-inhibitory effects of PGE in human aortic intima cells in primary culture (Orekhov et al., 1986). In these cells, as in fibroblasts (Cook and McCormick, 19931, cAMP inhibits the PDGF-induced MAP kinase and MAP kinase kinase cascade (Graves et al., 1993). However, inhibition of DNA synthesis cannot be attributed solely to this mechanism, since DNA synthesis is also blocked when forskolin is added several hours after PDGF (Graves et al., 1993). Moreover, unlike the situation in human fibroblasts, the inhibition of DNA synthesis by cAMP in adult rat smooth muscle cells is hardly explained by a n inhibition of early-response nuclear protooncogenes, including c-fos, c j u n , and c-myc (Hultgardh-Nilsson et al., 1994). 3. Immune System As reviewed by Kammer (1988), cellular cAMP in cells of the immune system has long been studied and considered as a potent and possibly physiologically activated immunosuppressive factor, a t least in part because of its negative influence on lymphocyte proliferation. Impairment of T-lymphocyte proliferation by HIV proteins was recently proposed to be mediated by the activation of the CAMP-PKA pathway in these cells (Hofmann et al., 1993). The view that CAMP-increasing factors or cAMP analogs inhibit the mitogenic activation of T lymphocytes is now well accepted. Prostaglandins of the E type and p-adrenergic factors, through CAMP,potently suppress the mitogenic activation by lectins or phorbol esters in murine thymic lymphocytes (Novogrodsky et al., 19831,various murine T-cell clones (Kim et al., 1988), and enriched preparations of human T lymphocytes (Chouaib et al., 1985;Iwaz et al., 1986;Maca, 1984; Ravid et al., 1990; Bartik et al., 1993). Adenosine, via CAMP, also inhibits mitogenesis in mice splenic T lymphocytes (Dos Reis et al., 1986).DNA synthesis stimulation by IL-2 is inhibited by cAMP in many systems (Maca, 1984; Dos Reis et al., 1986; Beckner and Farrar, 1986; Farrar et al., 19871, but it is reported to be relatively insensitive to cAMP in comparison to the activation by TPA in other T-cell systems (No-


79

vogrodsky et al., 1983; Kim et al., 1988; Friedrich et al., 1989). PKA I, but not PKA 11,was reported to mediate the inhibitory effects of CAMP on human T-cell proliferation induced through the antigen-specific T-cell receptor-CD3 complex (Skalhegg et al., 1992). cAMP might inhibit T-lymphocyte activation and cell cycle progression by acting at different levels: on the mobilization of intracellular calcium elicited by lectins in human T cells (Chouaib et al., 1987; but see Lingk et al., 19901, on the CAMP-dependent phosphorylation of phospholipase Cy 1, which leads to inhibition of the activating tyrosine phosphorylation of this enzyme induced by the T-cell antigen-receptor complex (Park et al., 1992),on the activation of the Na+/H+antiport in murine thymocytes (Grinstein et al., 19871, etc. Interestingly, PKA I, but not PKA 11, interacts with the CD3 antigen-T-cell receptor complex (Skalhegg et al., 1994).These authors thus proposed a mechanism whereby CAMP, through PKA I-dependent phosphorylation of the T-cell receptor-CD3 complex or associated proteins (e.g., phospholipase Cyl), could inhibit antigen-activated T-cell proliferation. In all T-cell systems IL-2 production, which amplifies the activation process, is potently inhibited by cAMP (Jonzon et al., 1985; Chouaib et al., 1985; Iwaz et al., 1986; Averill et al., 1988; Mary et al., 1987;Novak and Rottenberg, 1990). This is not sufficient to explain the mitogenic inhibition by CAMP,since it is not completely overcome by the addition of IL-2 (Jonzon et al., 1985; Chouaib et al., 1985; Iwaz et al., 1986). Moreover cAMP suppresses the induction by IL-2 and colony-stimulating factors of c-myc protoncogene (Farrar et al., 1987) and ODC (Farrar et al., 1988; Farrar and Harel-Bellan, 1989) in the murine CT6 T-cell clone, and the late expression of the required transferrin receptor in human T cells (Chouaib et al., 1985). However, CAMP,like IL-2, induces the accumulation of IL-2 receptor mRNA (Farrar et al., 1987; Narumiya et al., 1987; Shirakawa et al., 1988) and the expression of fos and myb nuclear protooncogenes (Fsrrrar et al., 1987). In marked contrast with these studies, Takeshita et al., (1990) established a CAMP-dependent growing human T-cell line from an IL-2dependent cell line, and Shirakawa et al. (1988) found that IL-1 induces an increase in cAMP levels in murine thymocytes and in several cell lines and that forskolin and cAMP analogs mimic the comitogenic effect of IL-1 in the presence of lectin in thymocytes. As an apparent paradox, the mitogenic activation of human peripheral T lymphocytes involves the formation of new nuclear CREB complexes. This could imply a positive role for cAMP in the normal physiological regulation of T-lymphocyte activation (Wollberg et al., 1994). Similarly, the transcription of PCNA (the DNA polymerase S auxiliary protein) is in-

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duced by IL-2 in murine T lymphocytes, at least in part, through CREBs (D. Huang et al., 1994). Mitogenic and antimitogenic influences of CAMP or CAMP-controlled intermediates might well coexist in T lymphocytes and be diversely expressed, depending on the lymphocyte clone and the regulatory context. cAMP also suppresses the mitogenic activation of B lymphocytes (Kammer, 1988).Forskolin, like PGE and dibutyryl-CAMP,inhibits the stimulation by B-cell growth factor of DNA synthesis in human B lymphocytes (Muraguchi et al., 1984).This inhibition is a general finding, although some controversy remains as to the period of the cycle and the metabolic step involved (Hoffman, 1988; Simkin et al., 1987; Blomhoff et al., 1987; Holte et al., 1988).Inhibition of c-myc expression by a CAMP-induced block in transcription initiation was involved in several B-cell systems (Holte et al., 1988; Blomhoff et al., 1988; Slungaard et al., 1987; Andersson et al., 1994). However, some mitogenic pathways (e.g., IL-4) may escape the CAMP-induced inhibition (Vasquez et al., 1991; Kolb et al., 1993). Interferon a/pincreases cAMP levels and inhibits proliferation in a mouse macrophage-like cell line. Many interferon-resistant variants are also resistant to cholera toxin and have a defect in adenylate cyclase [Nagata et al., 1984); however, cAMP is not the mediator of the growth-inhibitory effects of interferon in mouse fibroblasts (Ebsworth et al., 1984) or smooth muscle cells (Fukumoto et al., 1988)I. The growth of murine macrophages is also reversibly inhibited by PGEz and cAMP (Vairo et al., 1990; Jackowski et al., 1990; Rock et al., 1992). The inhibition occurs in mid- or late G, and involves the inhibition of CDK4 activation and RB protein phosphorylation (Kato et al., 19941, which was caused either by a suppression of CYLUcyclin D gene expression (Cocks et al., 1992) or by an increased expression of the CDK inhibitor p27kW (Kato et al., 1994). In contrast with observations in B and T lymphocytes, early responses to mitogens, including Na+/H+ exchange and c-myc induction, are not affected by cAMP (Vairo et al., 1990; Rock et al., 1992). Thus, analysis of the recent literature confirms that cAMP inhibits lymphocyte and macrophage proliferation, but reveals that this activity is quite complex, may bear at different levels, affects differentially some activation pathways, and presents considerable variability in different systems. Positive effects of cAMP are even observed under certain conditions. 4. Other Systems Cholera toxin and forskolin inhibit the serum-stimulated proliferation of a nonfusing muscle cell line (BC3H1) (Kelvin et al., 1989).


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Forskolin inhibits the DNA synthesis in rat glial cells (Kempski et al., 1987) and rat placental cells (Soares et al., 1989) and mitosis of pig epidermal cells in a n in vitro outgrowth system (Takeda et al., 1983). In rabbit kidney vasopressin, isoproterenol, and dibutyryl-CAMP inhibit the synthesis of DNA in both cortical collecting tubules and cortical thick ascending limbs (Wilson and Horster, 1983). In rat hepatocytes in primary culture, although cAMP positively influences early GI progression, glucagon, forskolin, and cAMP analogs, in synergy with dexamethasone, transiently block DNA synthesis at a late GI stage (Vintermyr et al., 1989, 1993a; Thoresen et al., 1990). In contrast to what was observed in T lymphocytes (Skalhegg et al., 19921, this inhibition is mediated by both PKA isozymes (Vintermyr et al., 1993b). The late G, block by cAMP in hepatocytes is associated with a n inhibition of RB protein phosphorylation (Okamoto et al., 1993). 5. CAMP-Dependent Prophase Block of Meiosis in Oocytes There is at least one example of a well-demonstrated and welldocumented physiological G, inhibition by CAMP:the block of meiosis in amphibian oocytes (reviewed by Maller, 1987). The administration of progesterone, which induces meiotic maturation of Xenopus oocytes, induces a rapid decline in cAMP levels. Cholera toxin and other cAMP enhancers completely block the action of progesterone. The microinjection of the active C subunit of PKA inhibits the progression of meiosis, while a protein inhibitor of PKA induces meiosis per se (Darr et al., 1993), which demonstrates the role of the activated PKA. In fact, CAMP,through PKA activation, prevents the activation of p34cdc2 kinase by cyclin B (Rime et al., 1992) and the depolymerization of nuclear lamins (Molloy and Little, 1992). Thus, the decline of cAMP appears both necessary and sufficient t o overcome the prophase block. cAMP also maintains meiotic arrest before nuclear membrane breakdown in mammalian oocytes (Racowsky, 1984; Freter and Schultz, 1984; Bornslaeger et al., 1986; Homa et al., 1991; reviewed by Sato and Koide, 1987; Smith, 1989). 6. CAMP-Dependent Inhibition of G,-Mitosis Transition in Other Systems

The negative influence of cAMP on meiosis events in oocytes might well reflect a more widespread CAMP-dependent G2-mitosis block that also concerns somatic cells (see also Section 11,A).The microinjection of inhibitors of PKA in rat embryo fibroblasts induces early mitosis events such as chromatin condensation and, in cooperation with p34cdc2, nuclear envelope disassembly (Lamb et al., 1991). PKA inhibits the mitotic p34cdc2 kinase activity in fibroblast cell-free extracts

82

PIERRE P. ROGER et ~ l .

(Hohmann et al., 1993). In activated Xenopus egg extracts oscillations of PKA activity control the oscillations of p34cdc2 activity and successive mitoses (Grieco et al., 1994). The inhibition of p34cdc2 activity by PKA is due not to lack of cyclin expression but to a stabilization of Qr15 inhibitory phosphorylation of p34cdc2, which depends on an inhibition of cdc25 phosphatase activity through its dephosphorylation by a serine phosphatase (Grieco et al., 1994). In canine thyroid epithelial cells the potent effect of TSH and cAMP on cell cycle entry and S-phase initiation coexists with a relative inhibition of the cell cycle in G2 phase (Baptist et al., 1993). This block is at least partly dependent on continuous elevation of cAMP levels, since cessation of adenylate cyclase activation, once cells have reached S phase, hastens their entry into mitosis (Baptist et al., 1993). It is associated not only with a stabilization of the Tyr15 phosphorylated form of p34cdc2, but also with an especially high nuclear accumulation of both cyclin A and CDK2 (Baptist et al., 1995). Similar observations have been made in the case of G, delay induced by DNA-damaging treatments (O’Connor et al., 1993) and might explain inhibition of mitosis entry, since cyclin A (likely as a complex with CDK2) is required not only for S-phase progression, but also to ensure the dependence of mitosis on completion of DNA replication (Walker and Maller, 1991). Inhibition of PKA in response to unknown intracellular mechanisms could therefore be a necessary step at late stages of the cell cycle. The physiological meaning of a G, control by cAMP and PKA is speculative. G2 delays, by allowing DNA repair, enhance cell resistance to mutagenic treatments and ionizing radiations (Jung et al., 1994). Whether cAMP could contribute to signal G2 delays induced by DNA damage, or unreplicated DNA (as recently speculated by Grieco et al., 1994) is not known. C. CONCLUSIONS The existence of a negative control of the cell cycle by cAMP is well supported in a limited number of cell systems (Table 11). Both the Go/G, inhibition in some fibroblasts, vascular endothelial and smooth muscle cells, some lymphocytes, and macrophages and the prophase block prior to meiosis in the very special case of oocytes might have a physiological meaning. However, even in normal somatic cell systems, no generalization can be drawn about the mechanisms of this inhibition. In different systems cAMP can inhibit the cell cycle at each of its main control points: not only at the early Go-GI transition (i.e., on the initiation of the cell division program) involving the inhibition of early


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events such as the raf-1-MAP kinase signaling cascade and c-myc expression, but also in mid-G, or at the restriction point in late G, involving inhibition of cyclin D expression, CDK4 activity, and RB phosphorylation. The overall physiological significance of such latter effects, which do not involve inhibition of early mitogenic events, is not well understood. They may represent “safety exits” from the program of cell proliferation for cells exposed to unfavorable conditions. In this case the cells would revert to a Go stage. The late decision point in GI might also be a stage at which cAMP can change the definitive interpretation by the cell of early “mitogenic” events as truly mitogenic rather than functional or even apoptotic signals. Finally, in other cases the effects of cAMP bear on only some mitogenic cascades and should be regarded as belonging to the multiple cross-signaling between cascades existing in the cell.

111. POSITIVE CONTROL OF CELLCYCLE PROGRESSION BY cAMP Aside from the models discussed in Section II,B, most of the examples of negative growth control by cAMP are derived from established cell lines, mostly of tumoral origin. A strikingly converse situation is found for positive control of growth. Table I11 lists many examples of systems in which hormones, neurotransmitters, or growth factors produce an elevation of cAMP before enhancing proliferation and in which pharmacological activators of the cAMP production and cAMP analogs reproduce the mitogenic stimulation. Many of these cells are differentiated epithelial cells in primary culture or normal epithelial cell lines maintaining differentiation characteristics. A CAMP-positive control is also demonstrated for some differentiated nonepithelial systems. It should be pointed out that CAMP-mediated stimulations of proliferation are generally obtained with concentrations of cAMP that are within the physiological range (see, e.g., Ethier et al., 19891, while inhibitions are often found with high “pharmacological” doses of cAMP analogs. Although, in general, evidence is derived from in uitro culture models, in some cases evidence is also available that a mitogenic effect of cAMP could be effective in uiuo. Recently, a strong overall proliferative effect of VIP has been reported in whole incubated mouse embryos (Gressens et al., 1993).It is still not known whether this is mediated by CAMP,like most VIP actions, nor whether it involves the direct activation of regional VIP receptors or a growth factor secretion by the central nervous system. Previously, a stimulation of DNA synthesis by

TABLE I11 VERTEBRATE CELLSIN WHICHCAMPMEDIATES THE ACTION OF GROWTH-PROMOTING FACTORS Stimulants

p-Adrenergic Glucagon, P-adrenergic

Glucagon, PGE, P-adrenergic

Glucose

?

FSH TSH, hCG, thyroid-stimulating immunoglobulin P-Adrenergic, PGE

FSH, LH GRF ? ?

VIP MSH, ? P-Adrenergic, VIP P-Adrenergic ?

ACTH PTH, PGE ? 7

P-Adrenergic, PGE, IL-I ?

PGE PGE, adenosine, VIP PGE

Cell systems Rat parotid acinar cells in primary culture and in vivoa Rat hepatocytes in primary culture" (Miyazaki et al., 1992), T51B rat liver cells," and chick embryo liver cells. MDCK canine kidney cells,a mouse epithelial kidney cells in primary culture," and chick metanephric kidney cells in primary culture" Rat pancreatic islet B cells in primary culture (Rabinovitch et al., 1980) Rat embryo pancreatic epithelial cells (Filosa et al., 1975) Mouse primordial germ cells (De Felici et al., 1993) Immature rat Sertoli cells" Canine, rat, and human thyroid epithelial cells in primary culturec and FRTL5 rat thyroid cell line.' Murine, bovine, and human mammary epithelial c e l l s ~ ~ . ~ and rat prostate epithelial cells" (McKeehan et al., 1984; Nishi et al., 1988) Human, bovine, porcine, and rat ovarian granulosa cells.Murine and human somatotrophsc Rat and guinea pig Schwann Guinea pig enteric glial cellsc Rat embryo sympathetic neuroblasts' Mouse and human melanocytes in primary culturee and S91 mouse melanoma mutant cell line0 Mouse and human keratinocytes in primary culturea (Haegerstrand et al., 1989) Human epithelial cells from foreskin, cornea nasopharynx," and trachea (Willey et al., 1985) Mouse palatal epithelial cells in primary culture (Grove and Pratt, 1984) Rabbit adrenocortical cells in primary culture" Chick osteoblasts in culturec Rat chondrocytes in culture" Human dermal microvessel" and fetal bovine aortic (Presta et al., 1989) endothelial cells Rat thymic lymphoblastsa,c Canine marrow erythroid cells (Brown and Adamson, 1977) BALBc-3T3 mouse embryo cellsc Swiss 3T3 mouse embryo cells",c A10 rat embryo smooth muscle cell line.

"Primary references can be found in the review by Boynton and Whitfield (1983). b?, Physiological growth factor, not yet known. CSee text for details and references.


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cholera toxin or cAMP analogs was obtained in vivo for rodent epidermis (Kuroki, 19811, mammary cells (Silberstein et al., 1984), rat thyroid gland (Pisarev et al., 1970; Lewinski, 1980), adrenal cortex (Hornsby, 1985), rat parotid gland (Tsang et al., 1980), and small oviduct endometrium (Laugier et al., 1988). Such experiments do not prove that cAMP directly stimulates proliferation. cAMP may merely induce the secretion by one type of cell of a mitogenic factor for another type of cell. Indeed, the proliferative effect of cAMP on the adrenal cortex as a second messenger for ACTH seems to be indirect (reviewed by Hornsby, 1985; see also Mesiano et al., 1991). Nevertheless, constitutive activation of the cAMP cascade by genetic means in transgenic mice suggests that cAMP may be a more direct mitogenic stimulus in somatotrophs (Burton et ul., 1991) and thyroid cells (Ledent et ul., 1992). The consequences of an inappropriate cAMP increase in several human tissues can be deduced from the clinical pattern of McCuneAlbright syndrome (Levine, 19911, which is caused by a somatic mutation of the G,a gene in the early embryo and the expression of an activated G p protein that constitutively stimulates adenylate cyclase in multiple tissues (Weinstein et al., 1991; Schwindinger et al., 1992; Shenker et al., 1994). Abnormalities in McCune-Albright syndrome include GH-producing pituitary adenoma, hyperthyroidism and thyroid nodules, autonomous maturation associated with hyperplasia of ovarian follicles resulting in sexual precocity, some hyperplasia of testis Leydig cells, adrenocortical adenoma, thymic hyperplasia, gastrointestinal polyps, and polyostotic fibrous dysplasia. A. RECENTEXAMPLES OF CAMP-MEDIATED POSITIVE GROWTH CONTROL Much of the evidence of CAMP-mediated growth stimulation in the systems listed in Table I11 was already acquired before 1983 and was extensively reviewed and discussed by Boynton and Whitfield (1983). Therefore, we restrict our task here to updating their effort in a discussion of recent significant advances using present methodologies reported for only some systems. Some of the best-confirmed and most popular examples of CAMP-mediated stimulation of growth are no longer recalled in this section (including keratinocytes, hepatocytes, and parotid cells), in the absence of such new data. 1. Thyrocytes TSH, mainly through CAMP,stimulates the synthesis and secretion of thyroid hormones by thyroid follicular epithelial cells (thyrocytes),

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but also controls tissue growth. By relieving the feedback exerted by thyroid hormones on the pituitary, any treatment that decreases thyroid secretion induces a secretion of TSH. TSH, in turn, causes the hyperfunction, hypertrophy, and hyperplasia of the thyroid gland (Dumont, 1971). In hypophysectomized rats the injection of TSH increases the mitotic activity of the thyroid gland. This effect is reproduced by the injection of dibutyryl-CAMP (Pisarev et al., 1970; Lewinski, 1980). TSH also stimulates DNA replication and the proliferation of canine thyrocytes in primary culture as cell monolayers with serum and triggers DNA synthesis in the absence of serum (Roger et al., 1982, 1983; Roger and Dumont, 1984). Binding of TSH to receptors coupled to adenylate cyclase results in a rapid and sustained elevation of cellular cAMP levels. In this system the adenylate cyclase activators-cholera toxin and forskolin-and various cAMP analogs perfectly mimic the mitogenic effects of TSH (Roger et al., 1983; Roger and Dumont, 1984; Van Sande et al., 1989) as well as its functional and differentiation effects (Dumont, 1971; Passareiro et al., 1985; Roger et al., 1985; Gerard et al., 1989). This has been confirmed in a model of rat thyroid follicles in suspension culture (Wynford-Thomas et al., 1987) and, despite earlier negative reports (Valente et al., 1983a,b), in the FRTL5 rat thyroid cell line (Dere and Rapoport, 1986; J i n et al., 1986; Yun et al., 1986; Ealey et al., 1987; Tramontano et al., 1988). In the WRT rat thyroid cell line the TSH-induced DNA synthesis is inhibited by microinjection of a n antibody to G,a (Meinkoth et al., 1992). The direct mitogenic effect of TSH via cAMP was confirmed in normal human thyrocytes in primary culture (Roger et al., 1988b). In the absence of serum, in these different systems the mitogenic effects of TSH and cAMP require the comitogenic influence of insulin or IGF-I (Roger et al., 1983, 198713, 198813; Tramontano et al., 1988; Smith et al., 1986). The role of CAMP in thyroid growth in uitro and in uiuo has now been confirmed by manipulating the expression of the adenosine A2 receptor positively coupled to adenylate cyclase. This receptor, recently cloned in our laboratory (Libert et al., 1989), appears to be physiologically constitutive (Maenhaut et al., 19901, the concentration of adenosine normally present in many tissues being sufficient to activate it. Microinjection of the mRNA of this receptor in dog thyrocytes is sufficient to elicit DNA synthesis as well as differentiation expression (Maenhaut et al., 1990). Expression of this receptor in transgenic mice under the control of the thyroid-specific thyroglobulin promoter elicits goiter and hyperthyroidism and greatly enhances cell proliferation, that is, hyperfunction, differentiation expression, and growth (Ledent et al., 1992). In some human hyperfunctioning thyroid ade-


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nomas a mutational constitutive activation of G,a has been found (Lyons et al., 1990; Suarez et al., 1991; O’Sullivan et al., 1991). In patients affected by McCune-Albright syndrome, this mutation is associated with a nodular adenomatous goiter and hyperthyroidism (Weinstein et al., 1991). Moreover, in seven of 12 hyperfunctioning thyroid adenomas, new mutations of the TSH receptor, conferring constitutive activation of adenylate cyclase but not of phospholipase C, have been found (Parma et al., 1993). Thus, in thyroid cells the elevation of cAMP provides a sufficient relay for the induction of cell proliferation by TSH. 2. Somatotrophs cAMP has been proposed as a mediator for the stimulation by the hypothalamic GRF of GH secretion and gene transcription in pituitary somatotrophs (Barinaga et al., 1985). Forskolin mimics the mitogenic effect of GRF in rat somatotrophs in primary culture, while somatostatin, which reduces the GRF-stimulated rise in cAMP (Bilezikjian and Vale, 19831, attenuates this growth effect (Billestrup et al., 1986). In 40% of hyperfunctioning adenomas of the pituitary gland, an activating somatic mutation of the G,a has been found (Vallar et al., 1987; Landis et al., 1989, 1990; Lyons et al., 1990). This mutation also produces GH-secreting pituitary adenoma in McCune-Albright syndrome (Weinstein et al., 1991). Moreover, expression of the activating subunit of cholera toxin in somatotrophs of transgenic mice causes hyperfunctioning adenomas (i.e., hyperfunction, differentiation expression, and mitogenesis) (Burton et al., 1991). As in the thyroid gland, chronic constitutive activation of the cAMP cascade leads to stimulation of function, differentiation, and growth. Moreover, transgenic mice overexpressing in the somatotrophs a transcriptionally inactive mutant of CREB, which cannot be phosphorylated, exhibit atrophy of the pituitary gland, depletion of somatotrophs, and dwarfism (Strutters et al., 1991). 3. Mammary Epithelial Cells In mouse mammary epithelium ovariectomy reduces cAMP levels and DNA synthesis (Silberstein et al., 1984). Implants releasing cholera toxin, cAMP analogs, or forskolin or systemic injections of cholera toxin stimulate growth and morphogenesis of mouse mammary ducts (Silberstein et al., 1984; Sheffield et al., 1985). Cholera toxin also reinitiates growth in senescent mammary epithelium (Daniel et al., 1984). The earlier findings by Yang et al. (1980) and Taylor-Papadimitriou et al. (1980) involving cAMP in synergy with other factors in the stimula-

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tion of proliferation of cultivated mammary cells have been largely confirmed. PGE,, mimicked by cholera toxin, cAMP analogs, or phosphodiesterase inhibitors, potently synergizes with EGF, insulin, or IGF-I and induces in serum-free cultures the rapid proliferation of mammary epithelial cells from mice (Imagawa et al., 1988), rats (Ethier et al., 1987,1989),humans (Stampfer, 1982; Hammond et al., 1984), and calves (Shamay et al., 1990).

4. Bone Cells PTH stimulates DNA synthesis and proliferation in various osteoblastlike cells and chondrocytes in in vitro models (van der Plas et al., 1985; McDonald et al., 1986; Koike et al., 1990; Somjen et al., 1990).Whether the PTH mitogenic effect is direct and mediated by cAMP or Ca2+ remains controversial. cAMP analogs stimulate DNA synthesis in rat cartilage segments in uitro (Bomboy and Salmon, 1980). cAMP analogs and forskolin reproduce the PTH effect on the DNA synthesis of chick osteoblast-like cells in culture (van der Plas et al., 1985). These effects could be indirect, since in fetal rat osteoblasts in culture (McCarthy et al., 1990), PTH and cAMP stimulate the synthesis of IGF-I, a known mitogen for these cells. DNA synthesis and cAMP responses to PTH have recently been dissociated. In embryonic rabbit and chick chondrocytes PTH increases cAMP at 100-fold higher concentrations than those required for stimulation of cell division (Koike et al., 1990). Moreover, different fragments of the PTH molecule elicit the growth of cAMP responses in various models of chondroblasts and osteoblasts (Somjen et al., 1990). This evidence seems to exclude a direct mitogenic effect of cAMP in these cells. 5. Ovarian Follicular Granulosa Cells There is no doubt that gonadotropins acting through CAMP, such as FSH, enhance granulosa cell proliferation and differentiation in uiuo. In McCune-Albright syndrome the activating mutation of the G,a is found in hyperplastic ovarian follicles that undergo maturation independently of gonadotropins, resulting in the development of cystic ovaries and sexual precocity (Weinstein et al., 1991). However, in vitro studies are contradictory. No mitogenic effect of FSH or LH was demonstrated on dissociated human, bovine, porcine, or rat granulosa cells in culture (Savion et al., 1981; Hammond and English, 1987; Dorrington et al., 1988). Nevertheless, FSH very strongly potentiated the small stimulation of DNA synthesis by TGF-f3 in rat granulosa cell


89

culture (Dorrington et al., 1988).By contrast, in intact hamster ovarian follicles incubated or cultivated in uztro, FSH or LH induced within 2 h an increase in DNA synthesis (Roy and Greenwald, 1986,1988, 1989). This peculiarly rapid effect on DNA synthesis, also observed by others (Pedersen, 1972; Monniaux, 19871, indicates that some granulosa cells are naturally arrested in late GI. It is reproduced by 8-Br-CAMP (Roy and Greenwald, 1988). Dibutyryl-CAMP also increases the granulosa cell number in isolated mouse follicles cultured in collagen gels (Carroll et al., 1991) and promotes the proliferation of chicken granulosa cells in culture (Yoshimura and Tamura, 1991). Whether the proliferation effects of FSH in uiuo or on intact follicles in uitro represent direct mitogenic effects remains unclear. It has been suggested that the stimulation of IGF-I production by FSH and cAMP in porcine granulosa cells could mediate, in part, the growth effects of these factors (Hsu and Hammond, 1987). However, FSH and cAMP synergize with IGF-I in the induction of DNA synthesis in serum-free cultures of rat granulosa cells (Bleg et al., 1992). On the other hand, a neutralizing antibody against EGF inhibits the stimulation of DNA synthesis by FSH and cAMP in hamster ovarian follicles, which indicates that it is mediated by an enhanced synthesis of EGF (Roy and Greenwald, 1991). 6. Adrenocortical Cells ACTH stimulates cAMP synthesis, hypertrophy, and hyperplasia of adrenocortical cells in animals (Hornsby, 1985). Macronodular hyperplasia of adrenal cells and adrenocortical adenoma that contain the activating mutation of G,a have been described in McCune- Albright syndrome (Weinstein et al., 1991). However, most authors have reported that ACTH and cAMP inhibit the proliferation of adrenocortical cells both in short-term cultures and in established lines from adrenocortical tumors (Hornsby, 1985). In contrast, Menapace et al., (1987) have reported that ACTH can directly stimulate DNA replication and multiplication of the parenchymal cells in primary cultures of rabbit adrenal cortex in a serum-free medium. The mitogenic effect of ACTH does not seem to be completely mimicked by CAMP,since cholera toxin and dibutyryl-CAMP stimulate DNA replication but not entry into mitosis in these cells (Menapace et al., 1987). Here, as in bone cells and ovarian granulosa cells, whether the mitogenic effect of cAMP is direct, or indirect through the induction of a growth factor such as FGF, remains an open question. FGF is induced by ACTH and cAMP analogs in the human fetal adrenal gland (Mesiano et al., 1991).Species differences in the mitogenic pathway of a hormone remain a possibility.

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7. Melanocytes Cultures of melanoma cells have long been established, providing interesting information in some lines on how MSH and its second messenger cAMP can stimulate cell multiplication (Pawelek et al., 1975). Since 1982 pure cultures of normal melanocytes have been established and used for analysis of growth and differentiation controls. Eisinger and Marco (1982) found that TPA and cholera toxin synergize to support the proliferation of normal human melanocytes. This was confirmed using a variety of substances increasing cAMP (Halaban, 1988; Abdel-Malek et al., 1992). cAMP is also permissive for the mitogenic activity of FGF (Halaban et al., 1988; Abdel-Malek et al., 1992). In fact, optimal growth of normal human melanocytes in serum-free conditions requires PKC activation , FGF, insulin, and MSH or other cAMP increasing factors (Herlyn et al., 1988; De Luca et al., 1993). Dibutyryl-CAMP also induces DNA synthesis and proliferation of isolated mouse embryo melanocytes (Mayer, 1982). The sustained proliferation of mouse melanoblasts in a serum-free medium depends on the presence of keratinocytes and is stimulated synergistically by dibutyryl-CAMP and FGF (Hirobe, 1992). 8 . Peripheral Nervous System Like melanocytes, Schwann cells and glial cells are derivatives of neural crest cells that appear in the early stages of the embryogenesis of vertebrates. In rodent embryos the proliferation of Schwann cells that provide support for axons is a n important part of peripheral nerve development. In adult rodents Schwann cells are stimulated to divide when a nerve is injured. The first observations by Raff et al. (1978), as expanded by Sobue et al. (1986), demonstrated that cAMP analogs, cholera toxin, and forskolin are potent inducers of DNA replication and proliferation of neonatal rat Schwann cells of the sciatic nerve cultured in the presence of serum. The increase in cellular cAMP is necessary to observe the mitogenic effects of PDGF and FGF and potentiates the growth effects of a glial growth factor and TGF-p (Ridley et al., 1989; Davis and Stroobant, 1990). In the neonatal guinea pig cholera toxin and dibutyryl-CAMP are mitogenic for Schwann cells, but also for enteric glial cells cultured with or without serum (Eccleston et al., 1987). The question of what controls Schwann cell cAMP levels in vivo is still unanswered, but a n axon-bound molecule could be involved, thus providing a mechanism by which axons can stimulate the proliferation of their support cells (Eccleston, 1992). VIP, through CAMP, stimulates the proliferation and survival of


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embryonic rat sympathetic neuroblasts cultured in a serum-free medium (Pincus et al., 1990a,b). 9. Thymocytes In sharp contrast with the well-demonstrated inhibitory effect of cAMP on lymphocyte activation and IL-2-induced mitogenesis, Shirakawa et al. (1988) reported that the comitogenic effect of IL-1 on phytohemagglutinin-induced murine thymocytes can be mediated by CAMP.IL-1 rapidly induces a sharp cAMP rise and a 4-h treatment with forskolin completely mimics the mitogenic effect of IL-1. 10. Nonepithelial Continuous Cell Lines Contrasting with data on normal diploid human fibroblasts and CCL39 Chinese hamster fibroblasts, cAMP positively influences the proliferation of fibroblast-like cell lines from mouse embryo. In different BALBc-3T3 cell clones the amplitude of the mitogenic response to PDGF or other growth factors is positively correlated with the basal levels of cAMP (Olashaw et al., 1984). In addition, although cAMP inhibits the progression of BALBc-3T3 cells in mid-G, (O’Keefe and Pledger, 19831, it potentiates the induction of competence by PDGF and TPA (Wharton et al., 1982; Smyth et al., 1992). As demonstrated by Fbzengurt (1986) and collaborators, the mitogenic effect of cAMP is still more conspicuous in some Swiss 3T3 mouse embryo cell lines. A variety of agents that promote cAMP accumulation in these cells, including PGE, (Rozengurt et al., 1983a), an adenosine agonist (Rozengurt, 1982a),ATP (N. Huang et al., 19941, and VIP (Zurier et al., 19881, induce DNA synthesis, acting synergistically with insulin, phorbol esters, and other growth factors. Interestingly, VIP also functions as a growth factor on whole mouse embryos in uitro (Gressens et al., 1993). Their mitogenic effects in Swiss 3T3 cells are mimicked by cholera toxin, forskolin, and cAMP analogs and potentiated by phosphodiesterase inhibitors (Fbzengurt et al., 1983a; Rozengurt, 1982a; Zurier et al., 1988). Moreover, these effects are inhibited in cells transfected with expression vectors encoding a mutated R subunit of PKA and a yeast low-K, phosphodiesterase (N. Huang et al., 1994). CAMP-elevating agents, among other factors, are required to support the sustained proliferation of Swiss 3T3 cells in a serum-free medium (Brooks et al., 1990). In these cells PGE, synthesis and secretion may mediate, in part, the growth effects of PDGF (Rozengurt et al., 1983b1, bombesin (Mehmet et al., 1990a), mastoparan (a peptide from wasp venom) (Gil et al., 19911, and IL-1 (Burch et al., 1989).These factors increase the synthesis and secretion of E-type prostaglandins,

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which, in an autocrine fashion, would stimulate cAMP synthesis (RQzengurt et al., 1983b; Mehmet et al., 1990a; Burch et al., 1989; Gil et al., 1991); however, the CAMP-mediated mitogenic activity of IL-1 in thymocytes appears to be direct (Shirakawa et al., 1988), and in FRTL5 thyroid cells IL-1 stimulates proliferation independently of cAMP (Mine et al., 1987). In contrast with observations of the primary culture of vascular smooth muscle cells, PGE, via CAMP,induces DNA synthesis in the quiescent A-10 rat embryo vascular smooth muscle cell line (Owen, 1986). However, it is inhibitory when these cells are asynchronously cycling in the presence of serum (Owen, 1986).

B. SYNERGISM BETWEEN cAMP AND OTHERMITOGENIC FACTORS Some form of cooperation is generally needed between different growth factors in order to achieve the stimulation of division of normal cells (de Asua et al., 1977; Pledger et al., 1978).This is not surprising, as the decision to divide should be submitted to several restrictions, that is, to several independent controls. A marked synergism among different growth factors often indicates that they control different mitogenic events, which must be executed in the right sequence to allow progression into the prereplicative phases and commitment to DNA replication and cell division. By analogy, the stepwise progression of tumors could reflect successive bypasses at different levels of growth regulation, that is, escape from different restrictions and controls. A different classification of multiple signals required for mitogenesis was presented by Rozengurt (1986). In the Swiss 3T3 cell line two or three synergistic signals may also be required for entry into S phase, but no temporal distinction is observed between them, and in some cases a single factor, such as PDGF or bombesin, is sufficient to elicit growth as it activates different signaling cascades (Rozengurt, 1986). The positive role of cAMP in cell cycle progression provides interesting examples of such synergistic cooperation. The full mitogenic effect of glucagon through cAMP requires both insulin and EGF in rat hepatocytes (McGowan et al., 1981; Friedman et al., 1981; Miyazaki et al., 1992). In melanocytes the growth effect of MSH and cAMP requires insulin and phorbol esters or FGF (Halaban, 1988). In human bronchial epithelial cells isoproterenol, through CAMP,potentiates the mitogenic stimulation by EGF and a pituitary extract (Willey et al., 1985). In human mammary epithelial cells cAMP stimulates growth only in the presence of EGF or serum (Taylor-Papadimitriou et al.,


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1980). The CAMP-dependent mitogenic activity of glucagon or PGE, in MDCK cells in a serum-free medium is observed in the presence of insulin or IGF-I (Taub et al., 19791, as are the effects of TSH in canine, rat, and human thyroid epithelial cells (Tramontano et al., 1988; Roger et al., 1987b, 1988b; Smith et al., 1986). In canine thyrocytes (Roger et al., 1987b) and mouse mammary cells (Imagawa et al., 1988; Ethier et al., 1987) a supplementary synergism is provided by EGF. The combination of EGF, insulin, and cAMP (cholera toxin) is also active in rat prostate epithelial cells (McKeehan et aZ.,1984; Nishi et al., 1988). Thus, the optimal proliferation of many different epithelial cells is obtained with similar combinations of a CAMP-stimulating hormone with general growth factors such as EGF or insulin (IGF-I).Such similitude could denote similar growth control mechanisms in these different epithelial cell types, as in the nonepithelial Swiss 3T3 cell line, in which the synergistic interactions between cAMP agonists and either EGF or insulin have been most thoroughly examined (Rozengurt, 1986). In these cells (Rozengurt, 198213) and in most experiments with canine thyrocytes, both insulin and cAMP are required t o initiate the prereplicative phase. However, in Swiss 3T3 cells, unlike canine thyrocytes, other growth factors, such as EGF, vasopressin, or phorbol esters, can also synergize with cAMP in the absence of insulin (Rozengurt, 1986). The increase in cellular cAMP also potentiates the mitogenic activity of several growth factors in rat Schwann cells (reviewed by Eccleston, 1992). In the analysis of such synergisms, we must distinguish the real signals triggering proliferation in uiuo from ubiquitous necessary comitogenic factors, that is, prove that a factor is involved in physiological control. For example, in the thyroid gland TSH is regulating; insulin, although necessary, is not. Glucose may be necessary for cell division in most systems, but it is only a signal for islet cells. Conversely, mice in which either the IGF-I or IGF-I1 receptor gene has been “knocked off” develop harmoniously, but at 60% of the normal rate.

C. POSITIVE REGULATION OF CELLCYCLE PROGRESSION BY cAMP As briefly discussed earlier, progression throughout the prereplicative phase and triggering of the deterministic part of the cell cycle (late GI, S, G,, and mitosis) depend on the sequential execution of several potentially rate-limiting events. Only some of these events may be regulated by CAMP, and their apparent importance for cell cycle progression may depend on both the cell type and its environment (experimental conditions imposed on the cell, that is, the condi-

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tions used for maintaining the cell in a quiescent state). In general, however, we are interested in the final influence on the commitment of cell cycle progression resulting from the integration of the controls a t the various restriction points, that is, on the overall proliferation effect. In density-inhibited BALB/c-3T3 cells cAMP potentiates the acquisition of competence induced by PDGF or FGF (Wharton et al., 1982), but sustained cAMP elevation impedes progression into the rest of the prereplicative phase [the cells are arrested at the V point in mid-G, (Leof et al., 1982)l. A 4-h elevation of CAMP also potentiates the induction of DNA synthesis by phytohemagglutinin observed after 48 h in rat thymocytes (Shirakawa et al., 1988). In cultured rat hepatocytes glucagon and CAMP-elevating agents enhance early in G,, but delay late in G,, the progression of the cycle (Vintermyr et al., 1989, 1993a; Thoresen et al., 1990; Miyazaki et al., 1992; Friedman et al., 1981). The latter effect requires higher cellular cAMP concentrations (Thoresen et al., 1990; Vintermyr et al., 1993a). By contrast, Boynton and Whitfield (1983) have reviewed the evidence for a late surge in cAMP preceding the onset of DNA replication. In T51B liver cells and in thymic lymphoblasts arrested near the end of G, by calcium deprivation, cAMP enhancers and cAMP itself can overcome this block and trigger DNA synthesis (Boynton and Whitfield, 1983). In some systems such as Swiss 3T3 cells (Rozengurt, 1982b1,Schwann cells (Sobue et al., 1986), and canine thyrocytes (Roger et al., 1987a), not only does cAMP initiate the prereplicative development, but a sustained elevation of cAMP is required before inducing DNA replication. In the latter system a late commitment point to DNA synthesis is still dependent on cAMP elevation by forskolin. Before this commitment point interruptions in forskolin presence, as short as 2h, delay the onset of DNA synthesis. Thus, in the presence of forskolin, canine thyrocytes progress toward S phase, but if this factor is withdrawn before the cells reach the commitment point, they rapidly regress to an earlier part of G,, from which they can be rescued by forskolin readdition (Roger et al., 1987a). Thus, in these cells the CAMP-dependent events, which are crucial for the progression through G, phase and for the commitment to DNA synthesis, are peculiarly labile. The fact that pulses of cycloheximide also bring canine thyrocytes back to the resting state (Roger et al., 1987a) shows that the proteins associated with passage through the restriction point in late GI are also labile, as was suggested by Pardee (1989). Canine thyrocytes should be considered as a system in which all of the major control points of prereplicative phase can be regulated by CAMP.


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Thus, the major control points in the prereplicative phase (initiation, progression, and S-phase commitment in late GI) can be dependent on CAMP augmentation, but many cells might be defective at some of these control points. It is interesting that in some non-CAMP-responsive cells clones of cells responding by proliferation to CAMPelevation may arise. If the positive control by CAMPat some steps may be lost in some cases, it can be induced de nouo in others (Pawelek et al., 1975; Takeshita et al., 1990). In contrast with the large body of evidence of the positive effects of CAMPon GI-phase progression and with the frequent inhibition of G, transit in established cell lines, there are sparse reports of a CAMPdependent G2 control of mitosis in uiuo. The p-adrenergic blocker propranolol prevents regenerating rat liver cells from entering mitosis without affecting their ability to initiate or complete DNA replication. It also inhibits the early prereplicative surge of cAMP that occurs shortly after partial hepatectomy, and CAMPinjection 2 h after operation reverses the mitosis-inhibiting action of propranolol. Rixon and Whitfield (1985) therefore proposed that an early CAMP-dependent prereplicative event determines mitosis rather than DNA replication. By contrast, Bybee and Tuffery observed a 3-fold increase in the appearance of metaphases in follicular cells of the thyroid gland and in acinar cells of the parotid and submaxillary glands, as early as 5 min after injection of TSH or isoproterenol in the rat (bdmond and Tuffery, 1981; Bybee and Tuffery, 1988, 1989). These results implicate the immediate recruitment by hormones acting through CAMPof a cell population resting in late G, or early mitosis in these tissues. However, we were unable to observe such an effect in primary cultures of thyrocytes stimulated by TSH (P. P. Roger, unpublished observations). On the other hand, Browne et al. reported that inhibition of PKAs by microinjection of a protein inhibitor delays the formation of the mitotic spindle in PHKl cells (Browne et al., 1987) and in fertilized sea urchin eggs (Browne et al., 1990). An increase in CAMPalso initiates meiosis in brittle star oocytes (Yamashita, 1988). Such effects would only have physiological relevance as controls in cells blocked in G, in their quiescent state. OF POSITIVE CONTROL OF CELLCYCLE D. BIOCHEMISTRY

PROGRESSION BY CAMP 1. Protein Kinase Activation PKAs are the major cellular receptors for CAMPin eukaryotes, and most of the effects of CAMPare assumed to be mediated through PKA

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activation and phosphorylation of specific protein substrates (Lohmann and Walter, 1984; Taylor et al., 1988). In the inactive form isozymes of PKA are tetramers composed of two C and R subunits. Two major types of isozymes, differing from each other only with respect to their R subunits (RI and RII) are found in most mammalian cells, but their relative amounts vary considerably from tissue to tissue or in different species for the same tissue and are very sensitive to physiological or pathological processes. Regulation is different in the two kinases, R being phosphorylated when PKA I1 is activated, while ATP mostly stabilizes PKA I and thus antagonizes activation. Isoforms of the RI and RII subunits, and of the C subunit, have been documented. The RIa, RIIa, and Ca subunits are expressed in most tissues, whereas RIP and Cp are found mainly in the brain and the testes. RIIP is most abundant in the brain, ovaries, and testes, whereas Cy has so far been found only in the testes. The biological significance of the coexistence of these distinct isozyme forms with a common C subunit is still largely unknown (Doskeland et al., 1993). However, differential anchoring of the R subunits leads to nonhomogeneous partitioning of C subunits and could cause a different protein substrate targeting of the two kinases. RI isoforms are primarily cytoplasmic, while RII isoforms are mainly localized on membranes, subcellular organelles, or the cytoskeleton through binding t o different AKAPs (for recent references see Scott et al., 1990; Ndubuka et al., 1993; Keryer et al., 1993). Distinct compartmentation of PKA isoenzymes should also be considered in view of recent evidence of compartmentalized accumulation of CAMP, which could explain the differential activation of PKA subtypes in response to different hormones (Scott and McCartney, 1994). Upon dissociation some C subunits migrate to the nucleus (Meinkoth et al., 1990). Heat-stable protein kinase inhibitors (aand P), which bind to a dissociated active C subunit and inhibit it, can suppress the effects of basal levels of cAMP on the activity of the protein kinases (Walsh et al., 1990). They also inhibit the nuclear translocation of the C subunits (Fantozzi et al., 1992). cGMP-dependent protein kinase can also be activated by cAMP in some special situations, explaining the cAMP effect on smooth muscle relaxation (Jiang et al., 1992). Although in the yeast Saccharomyces cereuisiae, the dependence of cell cycle progression upon activation of PKA is well demonstrated (Toda et al., 1987), there is little direct evidence that the activation of PKA mediates the CAMP-positive control of growth in mammalian cells. In several established cell lines marked variations of PKA isozyme relative amounts and activations were observed during cell cycle progression (reviewed by Boynton and Whitfield, 1983; Lohmann and


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Walter, 1984). This led some authors to postulate a specific role for either PKA I or I1 in the control of the cell cycle (Byus et al., 1977). However, these purely associative relationships can by no means indicate a causal link between the activation of PKA and replication controls. Furthermore, in these systems there is often no demonstration that cAMP plays a positive role in growth regulation. In some tumor cell lines PKAs were firmly implicated in the inhibition of proliferation by cAMP by the biochemical analysis of CAMPresistant mutants (see Section 11,A). Such evidence is lacking in the case of the positive control. Nevertheless, the elevated requirement for cAMP of a mutant melanoma cell line, as compared to its parental counterpart, is related to an elevated activation constant of PKA I for cAMP (Pawelek, 1979). Antisense oligonucleotides to the C subunit of PKA inhibit DNA synthesis of mouse mammary epithelial cells (Sheffield, 1991).The microinjection of the heat-stable PKA inhibitor inhibits, in part, the induction of DNA synthesis by TSH and 8-Br-CAMP in WRT rat thyroid cells (Kupperman et al., 1993). Martin and collaborators, by means of direct measurements of the activation of each PKA isozyme, showed that some hormones may selectively activate type I or I1 kinase. They correlated the stimulation of DNA synthesis by PGE, in the UMR 106 osteosarcoma cell line to the preferential activation of PKA I and the inhibition of proliferation in normal calvarial cells and in the human breast cancer cell line T47D to the specific activation of type I1 kinase by PGE, (Livesey et al., 1985; Livesey and Martin, 1988; Ng et al., 1983). In canine thyrocytes in primary culture, we more directly addressed the question of the involvement of PKA isozymes in the induction of DNA replication, using pairs of specific cAMP analogs that differentially modulate the activities of PKA I and 11. Before stimulation these cells contain comparable amounts of both kinase isozymes (Breton et al., 1989). DNA synthesis, as well as function and differentiation expression, present the same synergistic dependence on cAMP analogs as does the activation of PKA; this suggests that PKAs indeed mediate the cAMP action on these processes (Van Sande et al., 1989). Furthermore, in contrast to stimulation of function and differentiation expression, DNA replication seems more sensitive to PKA I activation (Van Sande et al., 1989). Consistently, the specific desensitization of this growth response (but not of the CAMP-dependent differentiation expression) is accompanied by the disappearance of PKA I, but not PKA 11, after several days of culture with TSH or forskolin (Breton et al., 1989; Roger et al., 1991). In the FRTL5 rat thyroid cell line it was also suggested that the TSH-regulated cell cycle progression involves mostly PKA I (Tortora et al., 1993).

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Thus, a predominant role of PKA I activation in the CAMPdependent stimulation of growth in several cell types is suggested. Recently, it was even reported that retroviral vector-mediated overexpression of RIa, but not RIIP, or C subunits of PKA enables a mammar y epithelial cell line to grow in a serum-free medium (Tortora et al., 1994). However, in some cells activation of PKA I instead inhibits proliferation [T lymphocytes (Skalhegg et al., 199211 or even leads to programmed cell death [myeloid leukemia cell line (Lanotte et al., 1991; Vintermyr et al., 1993b)l. Suggestions for specific roles of the subunits RI and RII of PKAs included different compartmentations, translocation or turnover of kinase isozymes (Nigg et al., 1985; Meinkoth et al., 1990; Weber and Hilz, 1986; Doskeland et al., 19931,or other functions of R subunits, unrelated to PKA activation, such as inhibition of protein phosphatases for RII (Khatra et al., 1985; Jurgensen et al., 1985; Vereb et al., 1986) or indirect activation of cGMP-dependent protein kinase for RI (Geahlen and Krebs, 1980). The work of Whitfield and collaborators on the peculiar model of T51B rat liver cells blocked in late G, by Ca2+ deprivation has been eventually explained by a clearly distinct feature of the involvement of PKA in cell cycle control. The early intriguing observation was the rapid initiation of DNA synthesis in such arrested cells by the addition of low concentrations of CAMP,which is membrane impermeant, as well as the finding of CAMPbinding sites at the outer face of the cell membrane (Boynton et al., 1985).These CAMPbinding sites are probably the R subunits of PKAs, since the ability of external CAMP to initiate DNA synthesis is mimicked by the addition of the C subunit of PKA and inhibited by the protein inhibitor of PKA. Kleine and Whitfield (1987) further showed that endogenous PKAs are accumulated at the outer surfaces of these cells during their progression into prereplicative phase initiated by serum factors. They proposed that Ca2+deprived cells fail to accumulate CAMP, thus preventing membrane PKA activation. CAMPwas presented as an autocrine G, progression factor with the external PKAs as its receptors (Kleine and Whitfield, 1987). This model clearly differs from the generally observed requirement for an elevation of intracellular CAMPin the CAMP-dependent cell proliferation. 2. Protein Phosphorylation Little is known about the nature and function of the proteins phosphorylated in response to the CAMPmitogenic stimulus. The fact that any interruption in CAMP signal introduces great delays in the onset of DNA synthesis in canine thyrocytes (Roger et al., 1987a) suggests


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that the phosphorylation of a specific protein(s) is necessary at the different stages of the prereplication phase. An 85-kDa CAMPdependent phosphoprotein was associated with late G, phase in B lymphocytes (Feuerstein et al., 1991). Various cytoskeleton proteins are phosphorylated in response to cAMP enhancers (see Section V), and the CAMP-dependent phosphorylation of vimentin was suggested to have a role in the stimulation of proliferation by cAMP in Swiss 3T3 cells (Escribano and Rozengurt, 1988). Upon dissociation of the cytoplasmic PKA I holoenzyme with CAMP, free C subunit progressively appears in the nucleus (Meinkoth et al., 1990) as a rate-limiting step for CAMP-dependent transcriptional induction (Hagiwara et al., 1993). Some transcription factors, such as the c-erbA-encoded thyroid hormone receptor (Goldberg et al., 19881, c-Fos (Abate et al., 1991; Tratner et al., 19921, and CREB (Yamamoto et al., 1988;Gonzalez and Montminy, 19891, are phosphorylated by PKA. The phosphorylation of c-Fos in its C terminus (either by PKC or by PKA) is required for transrepression activity of Fos on its own promoter and a mutation affecting this phosphorylation site enhances the c-fos transforming potential to a level comparable to that of v-fos (Tratner et al., 1992; Ofir et al., 1990). This phosphorylation could therefore be involved in the negative control of proliferation. By contrast, the CAMP-dependent phosphorylation of CREB and ATF-1 (Rehfuss et al., 1991) induces the transcription of genes containing CRE elements in their promoters. The induction of cell cycle-related proteins may thus take place a t the level of transcription and involve phosphorylation of CREB. Expression in the somatotroph of mutated nonphosphorylatable CREB indeed leads to pituitary atrophy and somatotroph depletion (Struthers et al., 1991). The same dominant negative mutant of CREB reduces, to some extent, PHIthymidine incorporation and proliferation in FRTL5 cells (Woloshin et al., 1992). PKA also phosphorylates the nuclear cyclin D1 at several sites, but the role of these phosphorylations remains to be determined (Sewing and Muller, 1994). 3. Gene Expression Balanced cell division requires duplication of all of the elements composing the cell. Therefore, in the preparation of cell division, all of the proteins should accumulate and be synthesized up to twice their initial level. It is not this aspect of protein metabolism that interests us in the mechanisms of cell proliferation, but rather the synthesized proteins that could represent signals or limiting factors in the whole process. a. Expression of Nuclear Protooncogenes. The protooncogenes of the

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myc, fos, and j u n families that encode nuclear phosphoproteins functioning as transcription factors play important roles in the regulation of cell growth, differentiation, and development (reviewed by Spencer and Groudine, 1991; De Pinho et al., 1991; Angel and Karin, 1991; Studzinski et al., 1991). Some of them, including c-myc, c-jun, and c-fos, pertain to the class of so-called immediate-early mitogenic genes, which are rapidly induced by growth factors and tumor promoters independently of protein synthesis (Herschman, 1991) during the mitogenic stimulation of a variety of cell types. They are also induced in nonmitogenic cascades (e.g., in the nervous system) and are therefore better called early-response genes. In fibroblast-like or leukemic cell lines antisense strategies or microinjections of neutralizing antibodies have suggested that c-myc (Spencer and Groudine, 1991; De Pinho et al., 1991),c-fos (Herschman, 19911, and thejun gene family (Kovary and Bravo, 1991) are necessary for the Go-S transition. While c-Fos and FosB activities are required mostly during the Go-G, transitions of Swiss 3T3 cells, Fra-1 and Fra-2 (Fos-related antigens) seem to be involved both in the Go-G, transition and in asynchronous growth (Kovary and Bravo, 1992). Fos and J u n proteins contain a leucine repeat that can mediate protein dimerization by the formation of the leucine zipper structure necessary for binding to DNA (Landschulz et al., 1988). The AP-1 transcription factor (Angel and Karin, 1991) is formed by a dimer of FosJ u n or Jun-Jun proteins. However, both Fos and Jun proteins may dimerize with other leucine zipper proteins. The c-Myc protein, whose C terminus contains a bHLH-zip, is also a DNA-binding transcription factor. The c-Myc protein dimerizes with another bHLH-zip protein, termed MAX, always present at significant levels in the cells, and the Myc-MAX complex, which is the active species of Myc, binds to DNA in a sequence-specific manner (Blackwood and Eisenman, 1991). Besides the function of these protooncogene proteins as transcription factors, it was suggested that c-Myc (Studzinski et al., 19911, and more recently c-Fos and c-Jun (AP-1 factor) (Murakami et al., 1991; Carter et al., 1991), may have more direct roles in DNA replication. CAMP-elevating factors induce c-fos mRNA transcription and accumulation in a wide variety of cell systems. In the FRTL5 thyroid cell lines (Colletta et al., 1986; Tramontano et al., 1986; Isozaki and Kohn, 1987) and in canine and human thyrocytes (Reuse et al., 1990; S. Reuse, unpublished observations) TSH, via CAMP,rapidly induces a transient elevation of c-fos mRNA. The effect of TSH in FRTL5 cells is transcriptional (Damante and Rapoport, 1988). Antisense c-Fos inhibits the TSH-dependent FRTL5 cell proliferation, suggesting that ex-


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pression of c-Fos is indeed necessary (Foti et al., 1990). Isoproterenol induces accumulation of c-fos mRNA and immunoreactive protein and acinar cell division in mouse submaxillary glands in uiuo (Barka et al., 1986). GRF induces c-fos expression and proliferation in rat somatotrophs (Billestrup et ul., 1987). cAMP also triggers c-fos expression and proliferation of H4IIE hepatoma cells (Squint0 et al., 1989). The expression of c-fos is rapidly induced in response to gonadotropins in rat ovarian granulosa cells (Delidow et al., 1990; Ness and Kasson, 1992) and porcine Leydig cells (Hall et al., 1991). 8-Br-CAMP is an inducer of c-fos in Swiss 3T3-Ll preadipocytes (Cornelius et al., 1991). In BALBc and Swiss 3T3 cell lines cAMP is a relatively poor inducer of c-fos mRNA and/or protein, but it potentiates the action of other CAMP-independent growth factors (Ran et al., 1986; Tsuda et al., 1986; Mehmet et al., 1988, 1990b; Mechta et al., 1989). In these different systems the CAMP-dependent induction of c-fos can therefore be part of a CAMP-dependent process of mitogenic activation. However, c-fos is similarly induced by cAMP in systems in which cAMP does not affect or even inhibit cell proliferation, including macrophages (Bravo et al., 1987b; Vairo et al., 19901,T lymphocytes (Farrar et al., 1987),PC12 pheochromocytoma cells (Greenberg et al., 19851,the R-5HT A5 epithelial cell line (Yeh et al., 19881, astrocytes (Gabellini et al., 19911, and NIH 3T3 cells (Fisch et al., 1989).The expression of c-fos has also been associated with CAMP-dependent induction of differentiation processes [e.g., in HL60 promyelocytic leukemia cells (Tsuda et al., 1987; Nakamura et al., 19901, a mastocytoma cell line (Goulding and Ralph, 1989), and the BC3H1 muscle cell line (Hu and Olson, 198811. Therefore, c-fos is more a gene of general early response to any stimulus than a specific mitogenic signal. The mechanism of the control of c-fos transcription in response to cAMP and other factors has been especially well studied (for reviews see Herschman, 1991; Angel and Karin, 1991). The major element responsive t o cAMP in the c-fos promoter is centered at position -60 and contains the sequence TGACGTTT, which, although different from the canonical CRE consensus (TGACGTCA), binds CREB (Foulkes et al., 1991b). CREB binds as a dimer to a CRE(s) and exerts its transcriptional regulatory function when phosphorylated by PKA. However, in corticotropic cells PKA regulates c-fos transcription by CREB-dependent and -independent mechanisms (Boutillier et al., 1992). Other sequences more upstream in the fos promoter (Fisch et al., 1989; Berkowitz et al., 1989) or in the transcribed region of the fos gene (Hartig et al., 1991) could also confer cAMP inducibility. A mechanism of down-regulation of the CAMP-dependent transcription of c-fos was

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recently proposed: the transcriptional antagonist CREM can bind to c-fos CRE and heterodimerize with activator CREB, thereby blocking cAMP induction (Foulkes et al., 1991b). The structure of the fosB gene is very similar to that of c-fos, including the CRE, SRE, and AP-1 elements in the 5’ upstream region, suggesting that c-fos and fosB are subjected to quite similar controls (Lazo et al., 1992). Data on the cAMP control of c-jun transcription are more disparate. In fact, in THP-1 monocytic leukemia cells, cAMP uncouples the transcription of c-fos and c-jun components of the AP-1 factor, c-jun being unresponsive to cAMP (Auwerx et al., 1990). It was proposed that the inhibition by cAMP of the induction by growth factors and TPA of c-jun transcription in HeLa cells, 3T3 cell lines, and Syrian hamster embryo cells could be related to growth-inhibitory effects of cAMP (Mechta et al., 1989; Angel et al., 1988; Janet et al., 1992; Cowlen and Eling, 1992). However, a similar inhibition of c-jun by cAMP was also observed during the mitogenic activation of canine thyrocytes (Reuse et al., 1991) and WRT rat thyroid cells (Tominaga et al., 1994) by TSH or forskolin. A similar inhibition of c-jun transcription was shown in rat Sertoli cells stimulated by FSH (Hamil et al., 1994). In porcine Leydig cells c-jun mRNA accumulation, unlike that of other protooncogenes, is not responsive to gonadotropins and cAMP (Hall et al., 1991). In contrast, there have also been some reports of a positive modulation of c-jun mRNA accumulation by CAMP.A delayed response was observed in 3T3-Ll preadipocytes (Cornelius et al., 19911, and a rapid induction was reported in PC12 cells (Wu et al., 1989), HL60 leukemia cells (Nakamura et al., 1990), and rat granulosa cells (Ness and Kasson, 1992). At variance with observations in canine thyrocytes, c-jun was reported to be precociously induced during the TSH and cAMP stimulation of proliferation in the thyroid FRTL6 cell line (Colletta and Cirafi, 1992). Unlike the case in c-fos, the c-jun promoter does not contain a CRE-like element (Angel et al., 1988).Nevertheless, in transfection experiments in 3T3 cells (Lamph et al., 19901, CREB can bind to the AP-1 site of c-jun and block the TPA-induced expression of a n exogenous c-jun promoter. This repression by CREB can be alleviated by its phosphorylation by PKA (Lamph et al., 1990). It remains unclear why PKA activation has opposite effects on the transcription from endogenous (Mechta et al., 1989) and transfected c-jun promoters in 3T3 cells. JunB and JunD proteins display strong homology in their C-terminal region to the DNA binding domain of c-Jun. They can form dimers with Fos proteins and recognize similar DNA sequences. However, JunB might inhibit the trans-activating and transforming properties


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of c-Jun (Chiu et al., 19891, and it could be involved in negative regulation of c-jun expression. Like c-fos, the transcription ofjunB appears to be induced by cAMP in all of the systems, irrespective of the effect of cAMP on growth. junB mRNA is rapidly and transiently accumulated in response to cAMP in BALB/c-3T3 cells (Mechta et al., 19891, 3T3L1 preadipocytes (Cornelius et al., 19911, porcine Leydig cells (Hall et al., 19911, adrenocortical cells (Viard et al., 19921, rat Sertoli cells (Hamil et al., 19941, and canine thyroid cells (Pirson and Dumont, 1994). The mouse j u d promoter contains a previously undescribed inverted repeat, which is necessary for cAMP induction, confers cAMP inducibility to heterologous promoter, and binds a 110-kDa protein (de Groot et al., 1991).junD is also observed to be positively regulated by cAMP in BALB/c-3T3 cells (Mechta et al., 1989) and canine thyrocytes (Reuse et al., 19911, but in some cells it seems to act as an inhibitor of proliferation and transformation (Pfarr et al., 1994). At present, the definitive influence on late gene expression of the different combinations of Fos and J u n proteins, the activities of which are also modulated by phosphorylation, is especially difficult to evaluate. For instance, as recently discussed (Tratner et al., 19921, PKA can be involved, directly or indirectly, in both activating and repressing Fos activity, either at the transcriptional level or in phosphorylation of the Fos protein involved in the down-regulation, or even by allowing its nuclear translocation (Roux et al., 1990). As detailed in Section II,B, an inhibition of c-myc expression is associated with the inhibition of growth by cAMP in several systems. It is thus interesting that, conversely, c-myc mRNA accumulation is rapidly induced in systems in which cAMP stimulates cell proliferation. In FRTL5 thyroid cells TSH, forskolin, and cAMP analogs trigger a marked increase in c-myc mRNA levels (Dere et al., 1985; Tramontano et al., 1986; Isozaki and Kohn, 1987). In canine (Reuse et al., 1986, 1990) and human thyrocytes (M. Taton, unpublished observations) the kinetics of CAMP-dependent c-myc expression, with a n early increase followed by a n abrupt decrease, are explained by a stimulation followed by a n active mechanism of termination of the myc response that is triggered by cAMP and depends on protein synthesis (Reuse et al., 1990). By contrast, in porcine thyrocytes TSH and cAMP are not mitogenic and do not enhance c-myc gene expression (Heldin and Westermark, 1988). cAMP triggers c-myc expression and DNA synthesis in H4IIE hepatoma cells (Squint0 et al., 1989). Although their mitogenic activity in uitro is controversial, gonadotropins, through CAMP,induce c-myc expression in ovarian granulosa cells (Delidow et al., 1990) and porcine Leydig cells (Hall et al., 1991). c-myc is potently induced by

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cAMP in Swiss 3T3 cells (Yamashita et al., 1986; Mehmet et al., 1988). In BALBc-3T3 cells, in which cAMP levels positively modulate the response to growth factor (Olashaw et al., 1984) and potentiate competence formation (Wharton et al., 19821, cholera toxin also potentiates the EGF effect on c-myc mRNA accumulation (Ran et al., 1986). Aside from the exception of the c-myc induction by cAMP in PC12 cells (Greenberg et al., 1985) and BC3H1 muscle cells (Hu and Olson, 19881, the c-myc protooncogene thus appears as the only “immediateearly gene” whose expression in response to cAMP is predictive of the effect of cAMP on growth in different normal cells. The CAMPdependent induction of c-myc could be necessary in the stimulation of proliferation by CAMP,while the CAMP-dependent inhibition of c-myc might suffice to explain a growth-inhibitory effect of CAMP. The mechanism involved in the dual effects of cAMP on c-myc mRNA transcription and/or stability is unknown. In canine thyrocytes inhibitors of protein synthesis potentiate the early induction by cAMP of c-myc mRNA accumulation, but prevent the late CAMP-dependent down-regulation (Reuse et al., 1990). However, the induction of c-myc expression is not sufficient to provoke CAMP-mediated induction of DNA synthesis, since it is too transient to explain the continuous requirement for high cAMP levels during progression into GI phase in canine thyrocytes (Roger et al., 1987a), and overexpression of transfected c-myc in thyroid cell lines does not diminish TSH dependence for growth (Fusco et al., 1987). b. Other Events. Although generally considered to be constitutively expressed, the CREB gene promoter contains three CRE elements, which might explain its up-regulation by FSH and cAMP in Sertoli cells (Meyer et al., 1993). LRF-1, a transcription factor induced early during the possibly CAMP-dependent liver regeneration, complexes with J u n proteins and activates CRE-containing promoters (Hsu et al., 1992). Other events associated with mitogenic stimulation include the induction of ODC activity and polyamine biosynthesis in canine thyrocytes (Mockel et al., 1980) in response to TSH or various cAMP enhancers, or in bronchial epithelial cells in response to a P-adrenergic comitogenic stimulation (Willey et al., 1985). The ODC mRNA accumulates 4 h after stimulation of FRTL5 thyroid cells by TSH or forskolin (Colletta and Cirafi, 1992). The promoter of the ODC gene contains a CRE (Fitzgerald and Flanagan, 1989). TSH, through CAMP, also transcriptionally induces the expression of hydroxymethylglutarylCoA reductase, a key enzyme of the synthesis of many isoprenoids required for cell proliferation (Grieco et al., 1990). Statin, a nuclear protein reported to specifically identify quiescent Go cells, rapidly dis-


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appears in rat thyroid gland stimulated by TSH (Bayer et al., 1992). The expression of a statin-related gene is also down-regulated in the rat parotid gland in response to the mitogenic stimulation by isoproterenol (Ann et al., 1991). An increase in ras protooncogene mRNA content was reported during TSH-induced G, phase progression in FRTL rat thyroid cells (Dere et al., 1986). In canine and human thyrocytes TSH, through CAMP,stimulates the synthesis of several proteins prior to stimulation of DNA synthesis, including PCNA/cyclin (Baptist et al., 1993; Lamy et al., 1989, 19901, the auxiliary protein required for DNA polymerase 6 activity. CDK2 is subjected to nuclear translocation and phosphorylation just before DNA synthesis initiation, and cyclin A and p34cdc2 progressively accumulate during S and G, phases in canine thyrocytes stimulated by TSH (Baptist et al., 1995). Therefore, distal to protein kinase activation, cAMP induces several of the well-known pleiotypic biochemical markers of cell progression to DNA replication. Thus far, almost all of the data concerning the biochemistry of cell cycle control by cAMP were obtained in highly differentiated thyroid epithelial cells and in the Swiss 3T3 fibroblast-like cell line. They confirm the kinetic data revealing an action at the major control points of G, phase progression. However, the biochemical description of the mitogenic cAMP pathways remains very sketchy. cAMP might also positively control the cell cycle or modulates the activity of other growth-promoting substances by quite diverse and perhaps unknown mechanisms.

E. CAMP-DEPENDENT AND -INDEPENDENT MITOGENICPATHWAYS In the late 1980s there was great interest in the realization that growth factors, acting through their tyrosine kinase receptors or through the activation of phospholipase C, separately trigger early events assumed to be important in mitogenesis (Chambard et al., 1987; Rozengurt, 1989). Thus, cell proliferation can be regulated by distinct mitogenic pathways. However, it is also manifest that these pathways interact and converge very early in the long path preceding the commitment for DNA replication (Fig. 1). Hence, it is often expected that the progression into the prereplicative phase induced by different growth factors involves a necessary sequence of events, which are common, since they are obligatory for the execution of the cell multiplication program. As in the previous sections, cAMP could act only as an accelerator of each of the tyrosine kinase or phospholipase C mitogenic pathways, or be able to activate its own mitogenic cascade.

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1. Cross-signaling between CAMP-Dependent and -Independent Mitogenic Stimulations There are many possibilities of cross-signaling between the cAMP cascade and other mitogenic pathways. Among the proteins synthesized in response to CAMP,a certain number could function as relays in other mitogenic cascades. In bone cells PTH and cAMP induce the synthesis and secretion of IGF-I, a growth factor for these cells (McCarthy et al., 1990). FSH and hCG have the same effect in the testes (Naville et al., 1990) and in ovarian granulosa cells (Hsu and Hammond, 1987). Growth factor receptors are also induced by CAMP. For example, in porcine thyrocytes (in sharp contrast to what is found in canine, rat, and human cells) CAMP,on its own, is unable to trigger DNA synthesis or c-myc oncogene expression (Gartner et al., 1985; Heldin and Westermark, 1988). However, it provokes a n increase in the availability of EGF receptors and potentiates the mitogenic effect of EGF (Westermark et al., 1986). The promoter of the EGF receptor gene contains a CAMP-responsive enhancer (Hudson et al., 1990). IGF-I receptors (Adashi et al., 1986) and FGF receptors (Shikone et al., 1992) are induced by FSH and cAMP in rat granulosa cells. cAMP induces PDGF receptor mRNA and protein, as well as the PDGF response in rat Schwann cells (Weinmaster and Lemke, 1990). Low concentrations of gonadotropin (hCG) and 8-Br-CAMP enhance estrogen receptor mRNA levels in a Leydig cell line (Ree et al., 1990). The potentiation by cAMP of competence acquisition in BALB/c-3T3 cells is perhaps due to the fact that EGF can activate the phospholipase C cascade in the presence of high cellular CAMP,but not in its absence (Olashaw and Pledger, 1988). cAMP also potentiates the stimulation of phospholipase C by vasopressin in rat hepatocytes (Pittner and Fain, 1989). These effects are not due to the phosphorylation of phospholipase Cy by PKA (Olashaw et al., 1990). A recent report claimed that TSH up-regulates the expression of the “mitogenic” G i a l in human thyroid cells (Selzer et al., 1993). Moreover, agents enhancing cAMP levels stimulate the translocation of PKC to the nuclei of B lymphocytes (Cambier et al., 19871, where they could modulate gene expression. TSH and cAMP were reported to potentiate the IGF-I-dependent phosphorylation of proteins on tyrosine in FRTL5 thyroid cells (Takahashi et al., 1991). CAMP-dependent phosphorylations of p60src (Roth et al., 1983) or EGF receptor tyrosine kinase (Ghosh-Dastidar and Fox, 1984), or activation of tyrosine phosphatase (Brautigan and Pinault, 19911, provides other possibilities of modulation. Conversely, cAMP may also contribute to the growth response in-


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duced by PKC activators in Swiss 3T3 cells, since these potentiate the adenylate cyclase activation (Rozengurt et al., 19871, likely through direct phosphorylation of type I1 adenylate cyclase by PKC (Yoshimasa et al., 1987; Yoshimura and Cooper, 1993). In the same cell line the potent mitogenic activity of PDGF and bombesin is also partly mediated by the synthesis of PGE,, which, in turn, increases the cellular cAMP levels (Rozengurt et al., 1983b; Mehmet et al., 1990a). In a few systems EGF activates adenylate cyclase or potentiates its activation by other factors (Ball et al., 1990; Nair et al., 1990; Magnaldo et al., 1989a). In rat H4IIE hepatoma cells (Squint0 et al., 19891, primary rat hepatocytes (Skouteris and Kaser, 19911, and the rat mammary gland (Lavandero et al., 1990) this mechanism may, in part, explain the mitogenic activity of EGF. EGF stimulation of adenylate cyclase could be through G, activation (Nair et al., 1990) or synthesis of prostaglandins (Skouteris and Kaser, 1991). FGF also potentiates the stimulation of adenylate cyclase in CCL39 fibroblasts (Magnaldo et al., 1989a1, but displays the opposite effect in BALB/c-3T3 cells (Logan and Logan, 1991). Many examples of the interdependence of Ca2+ and cAMP also exist, with calcium activating adenylate cyclase (Whitfield et al., 1987) or cAMP inducing Ca2+ influx (Rasmussen and Barrett, 1984; Penner et al., 19881, or calcium decreasing cAMP levels by activation of the Ca2+/calmodulin-dependent phosphodiesterase (Dumont et al., 1984). Moreover, CAMP,via PKA activation, enhances the responsiveness to IP, of intracellular Ca2+ pools in guinea pig hepatocytes (Burgess et al., 19911, which could be mediated by the phosphorylation of the IP, receptor by PKA (Ferris et al., 1991). The transcription factor CREB is activated by phosphorylation not only by PKA but also by Ca"/ calmodulin-dependent protein kinase (Dash et al., 1991; Sheng et al., 19911, and the induction of c-fos transcription by Ca2+ is mediated by the CRE in its promoter (Sheng et al., 1990). It is now increasingly evident that CREB and CREM are activated by phosphorylation not only by PKA but also by several other kinases in response to growth factors (Ginty et al., 1994; de Groot et al., 1994). The signaling cascades of PKC and PKA also interact on the control of gene promoter activity in a n extremely complex manner. AP-2 transcription activators are responsive to both cAMP and PKC inducers (Imagawa et al., 1987). Moreover, PKC phosphorylates in uitro and stimulates the dimerization of CREB (Yamamoto et al., 1988). On the other hand, the Fos/Jun AP-1 factor induced by phorbol esters is negatively controlled by a n IP, factor, which itself is inactivated by CAMPmediated phosphorylation (Auwerx and Sassone-Corsi, 1991, 1992).

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Although Fos/Jun and ATF/CREB were initially thought to interact preferentially with different regulatory elements (the AP-1/TRE and ATF/CRE sites, respectively), several members of both transcription factor families form cross-family heterodimers with distinguishable DNA binding specificities (Hai and Curran, 1991; Lamb and McKnight, 1991). For example, while the dimer Fos/Jun preferentially binds to AP-1 sites, the CREBP2/Jun dimer binds to the CRE (Ivashkiv et al., 1990). Changes in the expression of CREBP2, c-fos, and c-jun by altering the ratio of CREBP2lc-Jun to c-Fos-c-Jun complexes, would thus affect the relative expression of PKA- and PKC-responsive genes. Moreover, the similarity between AP-1 and CRE sequences may itself involve an interplay in transcriptional regulation and “cross-signaling” between PKA and PKC pathways. Thus, c-jun efficiently transactivates CRE sequences and c-Fos and c-Jun efficiently bind and cooperate in activating CRE promoter elements (Sassone-Corsi et al., 1990; Ryseck and Bravo, 1991).Conversely, CREB binds to an AP-1 site in the c-jun promoter (Lamph et al., 1990). An even more complex situation has been reported for the CAMP-dependent regulation of the proenkephalin promoter at the CRE-2 site, which is mediated by binding of JunD but inhibited by JunB (Kobierski et al., 1991). These examples provide support for combinatorial models of gene regulation, whereby protein-protein interactions, which alter the DNA binding specificity of protein complexes, can expand the flexibility of cellular transcriptional responses (Ivashkiv et al., 1990).However, these different studies performed in vitro or using cotransfection models should be interpreted cautiously. It remains to be established that such interplays of transcription factors and regulatory elements are also functioning on the regulation of endogenous genes at the concentrations of transcription factors normally found in intact cells. The extremely various possibilities of cross-signaling between the different levels of the few major signal transduction pathways and their combinations are essentially cell type specific. Their diversity could greatly contribute to providing the cell type specificity of proliferation controls. 2. cAMP Can Induce Its Own Mitogenic Pathway Which is Quite Distinct from the CAMP-Independent Ones At variance with these various possibilities of positive interaction and intervention of cAMP in tyrosine kinase and phospholipase C pathways, a defect in cAMP phosphodiesterase, causing elevated cellular cAMP levels in a variant of MDCK cells, abolishes the growth requirement for PGE, but does not affect the mitogenic dependence on


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EGF (Taub et al., 1983, 1984). Other indications for the coexistence of self-sufficient CAMP-dependent and -independent stimulations of proliferation have been obtained in keratinocytes (Tongand Marcelo, 1983), mammary epithelial cells (Imagawa et al., 19881, and differentiated epithelial thyroid cells and Swiss 3T3 fibroblast-like cells. Comparative studies of the mitogenic activation through both types of mechanisms were performed only in these latter systems. In primarily cultured canine thyrocytes, which are quiescent in an insulin-supplemented serum-free medium, DNA replication is induced not only by TSH, via cAMP and PKA activation, but also by EGF and phorbol esters (Roger and Dumont, 1984;Roger et al., 1986,198713).The stimulation of DNA synthesis by EGF requires the comitogenic action of high insulin concentrations that activate IGF-I receptors, while very low insulin concentrations are sufficient to permit the growth response to TSH (Roger et al., 1987b).In the presence of insulin, EGF synergizes with cAMP enhancers to induce DNA synthesis, but its effects are not additive to those of phorbol esters (Roger et al., 1986,198713).This was a first indication that EGF and phorbol ester act through a somewhat common mechanism that is different from the mode of action of cAMP in thyrocyte mitogenesis. TSH and cAMP enhancers at mitogenic concentrations do not activate the PI cascade (Graff et al., 1987; Berman et al., 1987; Raspe et al., 1992)or protein tyrosine phosphorylation (Contor et al., 1988; F. Lamy, unpublished observations). The activation of the PI-calcium cascade by acetylcholine decreases cAMP levels (Dumont et al., 1984). Phorbol esters that are potent activators of PKC do not increase cAMP levels (Mockel et al., 1987; Roger et al., 1991). EGF, which activates protein tyrosine phosphorylation, does not modify cAMP levels and does not activate the PI cascade (Raspe et al., 1992). Thus, despite the great number of possible interactions between different second messenger systems, the primary effects of mitogenic agents acting on the three pathways are distinct at the level of intracellular signals in canine thyrocytes. In the FRTL5 rat thyroid cell line phorbol esters and IL-1 are also mitogenic without increasing cAMP levels, in contrast to TSH (Lombardi et al., 1988; Mine et al., 1987). Similarly, in the Swiss 3T3 cell line increasing cellular cAMP levels neither activates PKC nor mobilizes calcium, releases arachidonic acid (as a result of phospholipase A, activation), or increases Na+ influx (Paris and Rozengurt, 1982) and cellular pH (Hesketh et al., 1988), whereas mitogens that activate tyrosine kinase receptors (EGF)or PKC do not increase basal CAMP levels (Rozengurt and Mendoza, 1985).None of these events is thus obligatory for mitogenic activation. In these cells inhibition of the CAMP-PKA pathway by genetic expression of a mu-

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tated subunit of PKA or yeast CAMP phosphodiesterase affects the CAMP-dependent mitogenic signaling, but not the activity of CAMPindependent growth factors (N. Huang et al., 1994). Rozengurt and Mendoza (1985) also suggested that in Swiss 3T3 cells the CAMPdependent pathway is clearly separate from other mitogenic pathways with which it acts synergistically. This conclusion was extended in canine thyrocytes by the analysis of the phosphorylation of intact cell proteins separated by two-dimensional gel electrophoresis, which gives a high-resolution picture of the activation state of protein kinase-dependent regulatory networks (Contor et al., 1988). As in fibroblasts (Cooper et al., 1984; Kohno, 19851, EGF rapidly induces the phosphorylation of five proteins, including the phosphorylation on serine of the 28-kDa heat-shock protein (L. Contor, unpublished observations) and the phosphorylation of the two related 42- and 44-kDa MAP kinases on tyrosine, threonine, and/or serine residues (Lamy et al., 1993). Identical activating phosphorylations of MAP kinases are a common response to all mitogenic factors examined so far by several groups in fibroblasts (Cooper et al., 1984; Nakamura et al., 1983; Rossomando et al., 1989). They are also phosphorylated during MPF activation of meiosis in Xenopus oocytes (Cooper, 1989; Lohka et al., 1987; Posoda and Cooper, 1992). MAP kinase substrates include microtubule-associated protein 11, the S6 kinase I1 (RSK), and other proteins on serine and threonine. S6 kinase 11, in turn, via the phosphorylation of the ribosomal protein S6, could play a major role in quantitative and qualitative changes in protein synthesis associated with cell cycle progression (Rossomando et al., 1989; Thomas, 1992). It also phosphorylates lamin C, which may play a role in the nuclear breakdown at mitosis (Ward and Kirschner, 1990). Moreover, MAP kinases are translocated to the nucleus (Lamy et al., 1993; Chen et al., 1992; Lenormand et al., 1993) and phosphorylate two serine residues in the N-terminal A 1 transactivation domain of c-Jun and a serine in the N-terminal domain of c-Myc, which positively regulates their transacting activities (Pulver et al., 1991; Alvarez et al., 1991).Activation of MAP kinases by phosphorylation is therefore assumed to play major roles in cell cycle control (Lenormand et al., 1993). Phorbol esters via PKC activation also cause MAP kinase phosphorylation and nuclear translocation in canine thyrocytes (Lamy et al., 19831, although they do not activate the EGF receptor kinase (Davis and Czech, 1985; F. Lamy, unpublished observations). In addition, they induce the phosphorylation of another set of proteins that could be related to the acute functional and morphological effects specific to PKC activation (Contor et al., 1988). This strongly suggested that tyrosine kinase- and


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PKC-dependent mitogenic pathways converge early on in a cascade of protein kinases and common MAP kinase phosphorylations. In sharp contrast, TSH, the most potent mitogen in canine thyrocytes, induces, via CAMP,the phosphorylation of a completely different set of proteins that includes neither the 42- and 44-kDa MAP kinases, the 28-kDa heat-shock protein, nor any phosphorylation on tyrosine residues (Contor et al., 1988; Lamy et al., 1993; F. Lamy, unpublished observations). Moreover, MAP kinases are not translocated to the nucleus in response to cAMP (Lamy et al., 1993). None of these events is thus necessary in the CAMP-dependent mitogenic pathway. Our data, based on 32P incorporation in NaOH-washed twodimensional gels and Western blots with antiphosphotyrosine and anti-MAP kinase antibodies from primary canine thyrocytes, differ from a recent report of a stimulation by TSH of tyrosine phosphorylations in the FRTL5 rat thyroid cell line (Takahashi et al., 1991). It was recently claimed that the activation of ras could be involved in the CAMP-dependent mitogenic pathway, since microinjection of a rasdominant negative mutant inhibits, in part, the induction of DNA synthesis by TSH and cAMP in the WRT rat thyroid cell line (Kupperman et al., 1993). We believe that this inhibition instead bears on the comitogenic (permissive) insulin-IGF-I signaling pathway of such cells (Brandi et al., 19871, which could even be activated through autocrine mechanisms. In canine thyrocytes the complete absence of MAP kinase phosphorylation in the cAMP pathway rules out any involvement of ras, since it is activated upstream of MAP kinases in signaling cascades. In Swiss 3T3 cells Rozengurt and collaborators also did not find any overlap between markers of PKC and PKA activation during mitogenic stimulation via these different pathways (Escribano and Rozengurt, 1988). The expression of c-fos and c-jun, encoding components of the AP-1 transcription factor, as well as c-myc is also generally considered a common necessary response in all of the mitogenic activation processes. Indeed, c-fos and c-myc mRNAs rapidly and transiently accumulate in response to both CAMP-dependent mitogens, as well as CAMP-independent factors such as EGF, phorbol esters, and serum. This was observed in Swiss 3T3 cells (Tsuda et al., 1986; Mehmet et al., 1988; Yamashita et al., 19861, FRTL5 rat thyroid cells (Tramontano et al., 1986; Isozaki and Kohn, 19871,primary canine thyrocytes (Reuse et al., 1986, 19901, rat hepatoma cells (Squint0 et al., 19891, and porcine Leydig cells (Hall et al., 1991).However, in Swiss 3T3 cells, while c-myc is similarly induced by cAMP and PKC activators, the c-fos response to cAMP is only 5-10% of c-fos mRNA levels reached after stimulation

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by PKC (Mehmet et al., 1988). In this system a large c-fos induction is not necessary in the CAMP-mediated mitogenesis. In comparison to PKC activators, the c-fos response to cAMP is also weaker in canine thyrocytes (Reuse et al., 1990; M. Baptist, unpublished observations), porcine Leydig cells (Hall et al., 19911, and adrenocortical cells (Viard et al., 1992). The c-fos gene transcription in response to CAMP-dependent or -independent mitogens involves independent mechanisms. (1)cAMP and EGF are synergistic on c-fos mRNA accumulation in several systems (Reuse et al., 1990; Hall et al., 1991; Mehmet et al., 1990b). (2) c-fos inductions in response to a number of CAMP-independent ligands (serum, TPA, EGF, FGF, NGF, and PDGF) converge at the SRE site of the c-fos promoter and involve the phosphorylation of the of the transcription factor P62TCF by MAP kinases (Gille et al., 1992), which are not activated in the cAMP cascade (Lamy et al., 1993).Mutations in the SRE abolish c-fos induction by these factors (for a review see Herschman, 1991), but not c-fos induction by agents that elevate cAMP or Ca2+, which act on the CRE at position -60. (3) The stimulation of c-fos transcription by PKC and tyrosine kinase activators is rapidly repressed by a retrocontrol mechanism involving the SRE and the c-Fos protein. On the contrary, more stable kinetics of c-fos expression were observed in response to cAMP (Bravo et al., 198713; Reuse et al., 1990), and c-Fos is unable to down-regulate the CAMP-dependent transcription of c-fos (Foulkes et al., 1991b). (4) The CREM protein specifically down-regulates the induction of c-fos mediated by the CRE but not the SRE (Foulkes et al., 1991b).Thus, the mechanisms involved in both the induction and the down-regulation of c-fos transcription in response to CAMP-dependent and -independent ligands are completely distinct. This suggests that CAMP-dependent or -independent mitogenic pathways separately control events that could be necessary for growth stimulation. In canine thyrocytes TPA and EGF increase the mRNA levels of c-jun andjunD. The effects are especially well observed in the presence of cycloheximide. Increasing cellular concentrations of cAMP by TSH or forskolin also stimulate junD expression, but inhibit c-jun expression (Reuse et al., 1991). Similarly, the TPA or EGF stimulation of c-jun expression is inhibited by cAMP (Reuse et al., 19911, as in fibroblasts in which cAMP inhibits proliferation (Mechta et al., 1989). The inhibition by cAMP of c-jun expression was confirmed in WRT rat thyroid cells (Tominaga et al., 1994). CAMP,unlike PKC activators, also fails to induce c-jun in porcine Leydig cells (Hall et al., 1991) and


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in ovine and bovine adrenocortical cells (Viard et al., 1992).In contrast to what has been postulated in fibroblasts, the expression of c-jun is not universally correlated with the stimulation of cell proliferation. It is not necessary in the CAMP-dependent mitogenic pathway, and thus represents another of the very different responses elicited by the cAMP cascade as compared to the other mitogenic pathways in canine thyrocytes. c-Jun is a substrate of MAP kinases that are activated in the EGF and PKC cascades, but it is not phosphorylated by PKA (Baker et al., 1992). In the CAMP-dependent mitogenic pathway in canine thyrocytes, it is tempting to connect the absence of effect on c-jun mRNA and presumably on c-Jun phosphorylation, both involved in AP-1 factor activity. This suggests a poor AP-1 activity in the cAMP pathway, and indeed there is some evidence that AP-1 activity is even repressed by cAMP in WRT thyroid cells (Tominaga et al., 1994).CREB and AP-1 activities depend on a common coactivator, CBP, the intracellular level of which might well be limiting (Arias et al., 1994). This may help to explain the specificity of, or even the competition between, the transcriptional responses to cAMP or CAMP-independentmitogens, despite the multiple possibilities of positive cross-signaling at this level (see Section III,E,l). Considering the opposite effects of cAMP and EGF or TPA on differentiation in thyroid cells (Roger et al., 1985, 19861, it is interesting that high cellular AP-1 activity can inhibit epithelial cell differentiation (Jehn et al., 1992) and that c-Jun alone could also function as a repressor of differentiation, as exemplified by its direct interaction with MyoD, inhibiting myogenic differentiation (Bengal et al., 1992). In primary canine thyrocytes the kinetics of c-myc mRNA accumulation are very different in response to either TSH or forskolin, or in response to EGF or TPA, again suggesting different mechanisms of control. c-myc mRNA levels are still enhanced over basal levels 9 h after EGF or TPA stimulation. By contrast, after the cAMP stimulation c-myc expression is biphasic, with an enhancement at 1h followed by a rapid down-regulation (Reuse et al., 1990). The effects of EGF and cAMP on c-myc are additive at 1h, in correlation with additive effects on DNA synthesis, but at 3 h cAMP even markedly inhibits the stimulation of c-myc expression by EGF (Reuse et al., 1990).The early stimulatory effects of cAMP and EGF are independent of protein synthesis, but the secondary inhibitory effect of cAMP is blocked by the protein synthesis inhibitor cycloheximide (Reuse et al., 1990). We hypothesize that the biphasic character of the cAMP action on c-myc is related to

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the opposite requirements of the stimulation of both proliferation and differentiation expression by CAMP, whereas EGF and TPA induce mitogenesis but inhibit differentiation. The expression of MAX (the c-Myc heterodimeric partner) is also subjected to some modulation in canine thyrocytes. EGF and TPA, but not TSH, produce a delayed increase in max mRNA content, which could modify the cell sensitivity to c-Myc as a transcription factor (Pirson et al., 1994). Uncoupling of c-fos and c-jun expression and transient c-myc expression in the cAMP cascade implies that the pattern of late gene expression could be different in the CAMP-dependent and -independent mitogenic pathways. Indeed, the patterns of protein synthesis (as analyzed by two-dimensional gel electrophoresis) induced by activation of canine thyrocytes by TSH and CAMP,on the one hand, and EGF, phorbol esters, and serum, on the other, are entirely different during G, phase progression (Lamy et al., 1986, 1989). The transient induction of the synthesis of a n 80-kDa protein in mid-G, constitutes a marker of G, phase progression stimulated by CAMP-independent mechanisms (EGF, serum, and phorbol esters), but not of the activation by cAMP (Lamy et al., 1986, 1989). The synthesis and nuclear accumulation of PCNA/cyclin, the auxiliary protein required for DNA polymerase 6 activity (Prelich et al., 1987; Bravo et al., 1987a), are already stimulated by TSH via cAMP in the second part of the prereplicative phase, but they are detected only just before the onset of DNA synthesis phase in response to CAMP-independent mitogenic treatments (Lamy et al., 1989; Baptist et al., 1993). Kinetic experiments suggested that the accumulation of PCNA to threshold levels could be a rate-limiting event for the initiation of DNA replication in the case of CAMPindependent mitogenic stimulation, but not in the case of TSH (Baptist et al., 1993). Nevertheless, as expected, CAMP-dependent and -independent mitogenic pathways converge before G1-S transition a t the late stage of proteins that control the cell cycle machinery. Convergence includes common changes of subcellular localization and phosphorylation of CDK2 and cdc2, and common induction of cyclin A and cdc2 expression (Baptist et al., 1995). Altogether, these studies of discrete biochemical events suggest that the CAMP-dependent and -independent mitogenic pathways may remain partly separated throughout the prereplicative phase. Kinetic experiments on the onset of DNA synthesis in canine thyrocytes also support this conclusion (Roger et al., 1987a,b). Although a late elevation in cAMP is still required for the commitment of DNA synthesis of cells already in late G, in response to cAMP (Fig. 2A), it is without any

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FIG. 2. (A) Time dependence of commitment to DNA synthesis in quiescent canine thyrocytes exposed to forskolin. Canine thyrocytes cultured in a serum-free medium supplemented with insulin were given forskolin (10-6M ) and [3Hlthymidine. At the time intervals indicated on the abscissa, cultures were washed free of forskolin. The cumulative labeling index was determined after continuous labeling for 48 h. (Inset)The kinetics of the accumulation of [3Hlthymidine-labeled nuclei after a continuous incubation with forskolin (10-6 M ) are shown for comparison. The experiment shows that in the CAMP-dependent mitogenic pathway, late rate-limiting events for the onset of DNA synthesis are dependent on continuous activation of adenylate cyclase. cAMP acts on late prereplicative events. (From Roger et al., 1987a.) (B) Kinetics of the cooperation of EGF and forskolin (FORSK) a t suboptimal concentrations on the stimulation of DNA synthesis in quiescent canine thyrocytes. Canine thyrocytes cultured in a serum-free medium supplemented with insulin received the following additions: (3, 1.6 F M forskolin a t time 0; 0 , 1 . 6 ng/ml EGF at time 0; and 0 , 1 . 6 W Mforskolin and 1.6 ngiml EGF added together a t time 0, or 1.6 (IM forskolin added a t 24 h to cells incubated since time 0 with EGF, or 1.6 ngiml EGF added a t 24 h to cells incubated since time 0 with forskolin. Kinetics of the accumulation of PHIthymidine into acid-insoluble material were determined as done by Roger et al. (1987b). When either forskolin or EGF is added 24 h after the other factor, their synergy on DNA synthesis is delayed by a time (17 h) that corresponds exactly to the duration of the prereplicative phase. The same 17-h lag time is thus necessary for the effect of each factor alone and for the establishment of its synergy with the other factor. Even though forskolin acts on late prereplicative events in the CAMP-dependent mitogenic pathway (A), it cannot interact with the late prereplica(continued)

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10 h Forsk

12

24

36

40

TIME (HOURS) tive development controlled by EGF without having executed its own mitogenic sequence. (From Roger et al., 198713) (C) Influence of a forskolin pretreatment on the kinetics of DNA synthesis induced by EGF in canine thyrocytes. Quiescent canine thyrocytes in a serum-free medium supplemented with insulin were pretreated (*, x ) or not (0,O) with 1 p M forskolin (Forsk)for 10 h, before stimulation at time 0 with EGF in the absence of forskolin (*, 0).Kinetics of the accumulation of [SHIthymidine into acidinsoluble material were determined as done by Roger et al. (198713).The preincubation of cells for 10 h with forskolin [a treatment too short to induce DNA synthesis by itself (A)] does not reduce the lag time for initiation of DNA synthesis stimulated by EGF. Thus, a 10-h progression into prereplicative phase controlled by cAMP is not helpful in the EGFdependent mitogenic cascade. (D) Phenomenological model of cooperation of distinct CAMP-dependent and -independent mitogenic cascades in canine thyrocytes. The triggering of S phase (S) in dog thyrocytes could require the induction of necessary early events such as c-fos and c-myc expression and the execution of a sequence of regulatory events. The sequence induced by TSH vis cAMP is partially distinct during the major part of the prereplicative phase from the sequence common to EGF and TPA actions. Examples of X early events involved in the EGFiTPA sequence but not in CAMPdependent mitogenesis are the activation of MAP kinases and the induction of c-jun.

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D

TS H

1

GAMP

0

20h

Examples of common steps are the early induction of c-myc and the induction of c-fos and jurzD. cAMP controls its sequence a t both early and late stages [Roger et al. (1987a); (A)]. Some cooperation between events of the separate CAMP-dependent and -independent sequences is demonstrated by their synergy, but it is possible only when both sequences have progressed to similar stages, as suggested by the kinetics of the synergism of EGF and forskolin on DNA synthesis [Roger et al. (1987b); (B)]. For example, the stimulation of the synthesis of a 80-kDa protein by EGF, which is potentiated by TSH (Lamy et al., 1989), has the characteristics of a Y event. The stimulation ofthe synthesis of PCNA in GI by TSH, which is potentiated by EGF (Lamy et al., 19891, has the characteristics of a C event. The coexistence of these partially separate mitogenic sequences, together with the fact that TSH only very transiently induces c-myc expression, could explain how TSH could stimulate growth and differentiation as compatible programs, a t the difference of EGF and TPA which stimulate growth while inhibiting differentiation. Such a working model integrates the presently known data on the phenomenology and kinetics of canine thyrocytes proliferation. It should help in the design of experiments to define the intermediate steps of both proliferation cascades.

rapid potentiating effect on the rate of entry into the DNA synthesis phase of cells already in late GI in response t o EGF (Fig. 2B). Late GI cells are therefore different, depending on whether they receive mitogenic stimulation by EGF or by CAMP.In fact, the experiment in Fig. 2B is not compatible with models in which early events triggered by either cAMP or EGF synergistically interact with later rate-limiting events dependent on the other factor. For example, a rapid CAMPdependent phosphorylation could not increase the activity of a tardily induced protein, on which would depend the rate of DNA synthesis in

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the mitogenic sequence triggered by EGF. Furthermore, the CAMPdependent progression into prereplicative phase induced by a 10-h pretreatment with forskolin is without any influence on the kinetics of DNA synthesis induction by EGF (Fig. 2C). CAMP-dependent mitogenic events (even those that are also induced by EGF, such as c-fos and c-myc expressions) are not helpful in the sequence stimulated by EGF; they do not correspond to the late rate-limiting events of the EGF-induced prereplicative development. Therefore, distinct ordered sequences of mitogenic events stimulated by either EGF or cAMP should be self-sufficient and, in parallel, lead to commitment for DNA replication (Fig. 2D). In the rat thyroid gland in uiuo, an irreversible desensitization mechanism limits the TSH-dependent growth, but not the growth induced by tissue wounding (Wynford-Thomaset al., 1983; Smith et al., 1987). In canine thyrocytes, during the CAMP-independent cell division, a dominant repressor factor is generated that specifically inhibits the TSH (CAMP)-dependent mitogenic pathway but not the growth response to EGF (Roger et al., 1992). Similarly, TGF-p strongly inhibits the CAMP-dependent proliferation of these cells, but weakly affects the CAMP-independent mitogenesis elicited by various factors. (F. De Poortere and P. P. Roger, unpublished observations). This raises the concept that growth suppressor mechanisms could specifically affect the CAMP-dependentproliferation. These results also differentiate the cAMP and the growth factor mitogenic cascades.

IV. RELATIONSHIP BETWEEN GROWTH AND DIFFERENTIATION CONTROLS BY cAMP cAMP plays a major mediator role in the action of many hormones that stimulate or induce specialized functions related to differentiation in their target cells (Friedman, 1976). Such a situation is very frequent, as exemplified by the stimulation of melanogenesis in melanocytes, of steroidogenesis in adrenocortical cells or ovarian granulosa cells, and of thyroid hormone synthesis and secretion in thyroid follicular cells. In all of these cases, cAMP is a positive modulator of the function and “expression” of differentiation by acting via various mechanisms on the tissue-specific transcription of genes encoding proteins responsible for specialized functions. It is by far less clear that cAMP could also affect the process of “determination,” that is, the irreversible commitment of undifferentiated cells to become specialized cells. cAMP directs the differentiation of F9 embryonal carci-


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noma cells toward a parietal endoderm phenotype (Strickland, 1981)) and it participates in the induction of the adipose conversion of 3T3L1 fibroblasts. However, in most cases cAMP seems to be unable to change the tissue specificity of the expression of genes, but it has a very widespread role in maintaining the full expression of differentiated properties conferred during determination. According to a n old but still current view, growth and differentiation are often considered mutually exclusive processes in cell life. Terminal differentiation processes involve a n irreversible cessation of growth. In addition, several examples of initially highly differentiated cell types present a temporal loss of differentiation expression during the proliferative phase. This was reported very early for iris pigmented cells (Doljanski, 1930) and more recently for retinal pigment cells (Rodesch, 19731, hepatocytes (Leffert et al., 1978; T. Y. Nakamura et al., 1984; Nakamura and Ichihara, 19851,hepatoma cells (T. Nakamura et al., 19841, thyroid medullary carcinoma cells (Berger et al., 1984; de Bustros et al., 1986))ovarian granulosa cells (Epstein-Almog and Orly, 19851, and prostatic epithelial cells (Chevalier et al., 1981). In many tumor cell lines in which cAMP inhibits cell proliferation, it stimulates the expression of differentiation, thus restoring a n apparent normal phenotype (Pastan and Johnson, 1974; discussed more thoroughly in Sections V and VI). Conversely, growth factors (e.g., EGF) acting through tyrosine kinase receptors or tumor promoters acting through PKC are potent inhibitors of differentiation expression in a variety of systems, including mammary epithelial cells (Taketani and Oka, 19831, granulosa cells (Ascoli, 19811, Leydig cells (Welsh et al., 19841, adrenocortical cells (Simonian et al., 19821, keratinocytes, and thyroid epithelial cells (Roger et al., 1985, 1986; Bachrach et al., 1985). However, in the hematopoietic system hormones such as erythropoietin and CSF can induce both proliferation and differentiation. Hormones that stimulate growth through cAMP in normal specialized cells also often stimulate the expression of their differentiation. A nonexhaustive list of examples includes hepatocytes (Miyazaki et al., 1992; van Roon et al., 19891, Schwann cells (Sobue et al., 19861, keratinocytes (Tong and Marcelo, 19831, melanocytes (Halaban, 1988; Abdel-Malek et al., 19921, pituitary somatotrophs (Billestrup et al., 19861, MDCK epithelial cells (Lever, 1979; Taub et al., 19831, and thyroid epithelial cells (Roger and Dumont, 1984; Roger et al., 198813).The apparent paradox between the antagonism of growth and differentiation and the stimulation of both processes by cAMP has been addressed in canine thyroid epithelial cells in primary culture. While EGF and TPA stimulate proliferation and inhibit differentiation (in-

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cluding thyroglobulin gene expression), TSH, through CAMP, induces both processes (Roger and Dumont, 1984; Roger et al., 1985, 1986, 198813). Study of the induction of thyroglobulin and thyroperoxidase mRNA accumulations by in situ hybridization demonstrates that they are fully compatible with cell cycle progression when they are promoted by CAMP.Thus, cAMP can induce mitosis and differentiation expression at the same time, in the same cells (Pohl et al., 1990, 1993). Both the differentiation induced by TSH and cAMP and the dedifferentiation induced by EGF or phorbol esters are independent of the effects of these agents and cascades on proliferation, as they are obtained in the absence of insulin (which is required for proliferation) or in cells at confluence that do not proliferate anymore (Roger and Dumont, 1984). A molecular mechanism of the dissociation of these two types of effects, even when mediated by the same transcription factor, is suggested by the case of MyoD, in which separate domains of the protein are involved in the induction of differentiation and in the inhibition of proliferation (Crescenzi et al., 1990). Suppression of differentiation expression in thyrocytes therefore is not a consequence of cell cycling itself, but could be related to the activation of some mitogenic signaling cascades. Considering the evidence of the coexistence of largely separated controls of cell cycle progression in thyrocytes, it appears quite plausible that the CAMPdependent mitogenic pathway, by opposition to the CAMP-independent ones activated by EGF and TPA, might have characteristics that make it compatible with differentiation expression (Pohl et al., 1990; Dumont et aZ., 19891, such as a poor AP-1 activity, as discussed in Section III,E,2. Another clue to such a possibility is the observation that the c-myc response to EGF and TPA in canine thyrocytes is a relatively stable process, while its response to TSH and cAMP is very transient, due to a specific CAMP-dependent inhibitory mechanism (Reuse et al., 1990). A more stable induction of c-myc by cAMP is associated with the inhibition of the differentiation of the mouse muscle cell line BC3Hl (Hu and Olson, 1988). c-myc protooncogene expression prevents differentiation in most systems (reviewed by Dang, 19911, in addition to appearing to be a prerequisite for growth stimulation. Therefore, the rapid termination of c-myc induction in canine thyrocytes stimulated by cAMP might be necessary in order not to interfere with the maintenance of differentiation expression that occurs together with growth stimulation (Reuse et al., 1990; Pohl et aZ., 1990). At variance with the case of thyrocytes, the synthesis of myelin proteins and late differentiation responses in postnatal rat Schwann cells are induced by cAMP only in the fraction of nonproliferating cells


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or in conditions that do not allow cell division (Morgan et al., 1991). Stimulation of proliferation by cAMP and CAMP-dependent induction of late differentiation markers seem to be incompatible in this system. However, in Schwann cells, contrary to what we have shown in thyrocytes, it is not clear that cAMP can promote proliferation by acting on a separate cascade. The mitogenic effect of cAMP is dependent on other factors, such as PDGF, and cAMP induces the appearance of PDGF receptors and PDGF responsiveness in Schwann cells (Weinmaster and Lemke, 1990). Differentiation processes generally include modifications of growth characteristics. Various models of cell differentiation consider the concept of two kinds of cell cycle, either preserving or changing differentiation phenotypes (Lajtha, 1979; Yamada, 1989). The adipose conversion of 3T3-Ll fibroblasts is often considered a differentiation process that needs postconfluence cell cycle progression (Kuri-Harcush and Marsh-Moreno, 1983; Schmidt et al., 1990).According to Schmidt et al. (19901, the promoting effects of CAMP on postconfluent mitoses and on differentiation cannot be dissociated and are both potentiated by EGF together with IGF-I. However, the mitogenic effects of FGF and PDGF, which are not potentiated by CAMP,are not associated with adipose conversion (Schmidt et al., 1990). The CAMP-dependent and -independent cell cycles could thus differently modify the differentiated phenotype of 3T3-Ll cells, only the former inducing the adipose conversion. Also, in the canine thyrocyte experimental model CAMP-dependent and -independent cell cycles are not equivalent for the maintenance of the cell phenotype. While the TSH (CAMP)-dependentdivision of thyrocytes preserves their responsiveness to both TSH (CAMP)and EGF mitogenic pathways, cells that have divided in response to CAMPindependent stimuli (EGF and serum) lose mitogenic sensitivity to TSH and cAMP but retain sensitivity to EGF (Roger et al., 1992). Cell fusion experiments suggest that the extinction of the CAMP-dependent mitogenic pathway is due to the induction during the CAMP-independent cell cycle of a specific diffusible intracellular inhibitor (Roger et al., 1992). Differentiated functions are generally suppressed in heterokaryons or somatic hybrids formed by the fusion of a cell expressing this function with a nonexpressing cell (Harris, 1990). The TSH (CAMP)dependent mitogenic pathway, which is repressed in cell fusion experiments (unlike the EGF mitogenic pathway) (Roger et al., 19921, could thus be analyzed as a differentiated trait of thyrocytes. Together with differentiation markers, the TSH (CAMP)growth pathway is also specifically lost in somatic hybrids of FRTL5 thyroid cells and BRL rat

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liver cells (Veneziani et al., 1990) or in hybrids of FRTL5 and undifferentiated FRT thyroid cells (Zurzolo et al., 1991). The CAMP-dependent mitogenic pathwayk) is a tissue-specific characteristic of a set of cell types, including many types of differentiated epithelial secretory cells. “It is possible that secretory cells whose secretory functions are positively regulated by cAMP may also develop under the positive control of CAMP.. . . The [CAMP-dependent mitogenic] response . . . may also represent a characteristic of differentiating systems, that is that high levels of cAMP are required for differentiative transition. Thus proliferation of cells undergoing differentiation may have a different regulatory mechanism than the various cell cycles normally studied in cell cultures” (Filosa et al., 1975). This prophetic speculation about embryonic development of pancreatic cells is very well supported by recent investigations suggesting that the CAMPdependent growth stimulation and differentiation are intimately related processes in certain cell lineages, such as thyrocytes (see above) and somatotrophs. Overexpression of a dominant negative CREB in the pituitary of transgenic mice leads to ablation of the somatotroph lineage and dwarfism (Struthers et al., 1991). The Snell dwarf mutations, which cause both deficiency in GH synthesis and pituitary hypoplasia (Li et al., 1990), reside in GHF-1. GHF-1 is a tissue-specific CAMP-dependentgene under the control of CREB. Inhibition of GHF-1 synthesis with antisense oligonucleotides leads not only to a marked decrease in GH expression, but also to a marked inhibition of proliferation of somatotrophic cell lines (Castrillo et al., 1991).

V. A ROLE FOR

CYTOSKELETON CHANGES IN OF GROWTH BY CAMP?

CONTROL

There is still a continuously increasing body of evidence indicating that cell growth and differentiation are especially dependent on cell shape, direct cell-cell interactions, and mechanical as well as chemical properties of cell substratum (reviewed by Bissel and Barcellos-Hoff, 1987; Ben-Ze’ev, 1989; Ingber and Folkman, 19891, as is well illustrated by the anchorage dependence of proliferation of normal fibroblasts, which is relieved in cancerous or transformed cells. Extracellular matrix proteins influence morphogenesis, differentiation, and growth control characteristics (Juliano and Haskill, 1993). Their receptors (integrins) are directly coupled to the cytoskeleton through talin and vinculin (Carraway and Carothers Carraway, 1989). The cytoskeleton, providing a protein continuum between the cell


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membrane and the nucleus (Georgatos et al., 1987), could also convey various signals affecting gene expression. Different, but nonexclusive, hypotheses are envisaged to explain how the complete cell structure (or at least the cytoskeleton as a whole) may modulate the expression of genes involved in morphogenesis, differentiation, and growth control: (1) as most mRNAs are associated with the cytoskeleton, any change in the cytoskeleton organization could affect the translability of mRNA and macromolecular synthesis (Farmer et al., 1983); (2) since there is evidence for a connection between transcribed genes and the nuclear matrix, changes in the interaction of the cytoskeleton with the nuclear lamina could cause exposure of particular genes and sequestration of others (Bissell and Barcellos-Hoff, 1987; Ashall et al., 1988; Cook, 1989); (3) several protein kinases [including some A and C kinases (Miller et al., 1982; Papadopoulos and Hall, 1989) and growth factor receptors (Payrastre et al., 199211 and calmodulin are associated with the cytoskeleton, and reorganization of some cytoskeleton elements could be necessary for their translocation (e.g., toward the nucleus in order to phosphorylate transcription factors); and (4)cell adhesion or integrin clustering, as well as mitogenic neuropeptides and PKC activators, induce the tyrosine phosphorylation and activation of the p125fak kinase (Juliano and Haskill, 1993; Zachary and Rozengurt, 1992; Guan and Shalloway, 1992). The neuropeptide effect is independent of Ca2+ or PKC, but requires the actin cytoskeleton integrity (Sinnett-Smith et al., 1993). Cell adhesion could also affect other signaling cascades [Ca2+, inositol lipids (McNamee et al., 19931, and cAMP (Juliano and Haskill, 199311. Since the demonstration of a direct correlation between the degree of cell spreading or change in cell shape and the growth rate by Folkman and Moscona (1978), circumstantial evidence is accumulating that suggests a major role of the reciprocal relationship between cell shape and the cytoskeleton in the control of growth and its alterations associated with cell transformation. Thus, (1)the main identified substrates of oncogenic tyrosine kinases are components of adhesion plaques and cytoskeleton, such as integrins, vinculin, talin, and calpactin (Sefton et al., 1981; Pasquale et al., 1986; Glenney, 1986; Volberg et al., 19921, and growth factors and tumor promoters often produce rapid changes in cell morphology and cytoskeleton organization that resemble those associated with cell transformation (Bockus and Stiles, 1984; Herman and Pledger, 1985; Werth and Pastan, 1984; Nishida and Gotoh, 1992). (2) Various genes encoding extracellular matrix or cytoskeleton proteins are rapidly induced together with protooncogenes upon mitogenic stimulation in fibroblasts (Rollins and Stiles,

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1989). (3) On the other hand, one of the most prominent markers of cell transformation by a number of oncogenic viruses is the repression of the synthesis of some tropomyosin isoforms with high avidity for actin (Hendricks and Weintraub, 1984; Matsumura and YamashiroMatsumura, 1985; Cooper et al., 1985; Galloway et al., 1990). The repression of these tropomyosins is associated with the in uiuo tumorigenicity developed by some fibroblasts after transfection with a p-actin gene bearing a mutation frequently found in tumors induced by chemical carcinogens (Leavitt et al., 1987). p-actin could thus have the characteristics of a protooncogene activated by mutation. (4) While EGF and insulin disorganize microtubules and vimentin filaments during progression of BALBc-3T3 fibroblasts toward S phase (Bockus and Stiles, 19841, the disruption of the microtubule system by various microtubule poisons is sufficient to activate DNA synthesis in the absence of serum (Crossin and Carney, 1981) or to potentiate the DNA synthesis stimulation by serum of growth factors (reviewed by Otto, 1982). Microtubule inhibitors such as colchicine induce fos and myc protooncogene expression (Miura et al., 1987) and MAP kinase phosphorylation (Shinohara-Gotoh et al., 1991). How important is this conspicuous but still somewhat puzzling framework for the understanding of the role of cAMP in growth controls? cAMP profoundly affects cell morphology and the organization of the three main cytoskeleton systems in various ways in different cells. Hence, the early findings of cAMP effects on growth were already considered t o be intimately related to the CAMP-dependent morphological alterations (Pastan et al., 1975; Puck, 1987; Lockwood et al., 1982). Various drugs increasing cAMP and cAMP analogs restrict growth and restore “normal” fibroblastic morphology in spontaneously transformed CHO cells, src gene-transformed vole fibroblasts, and H-rastransformed NIH 3T3 cells (Puck, 1987; Meek, 1982; Lockwood et al., 1987). Although some of these drugs seem to produce this so-called “reverse transformation” partly as a result of their CAMP-independent side effects (Rajaraman and Faulkner, 1984), the involvement of CAMP-dependent protein kinases is well established by analysis of CHO mutants defective in PKA (Singh et al., 1981).The morphological changes follow an expansion of the cytoplasmic microtubule network, the assembly of microfilament bundles, and a redistribution of myosin into these bundles (Lockwood et al., 1982; Meek, 1982; Puck, 1984; Osborn and Weber, 19841, as well as rapid restoration of a wellorganized vimentin network (Chan et al., 1989).The CAMP-dependent reverse transformation also involves an increase in fibronectin depos-


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its (Puck, 1987). Puck proposed that the major but specific changes in the exposure or sequestration of genes associated with the reverse transformation and growth arrest are mediated by the CAMP-dependent changes in cytoskeleton organization (Ashall et al., 1988; Puck, 19871, since this gene exposure reaction is prevented by microtubule or microfilaments inhibitors such as colcemid and cytochalasin B. The CAMP-dependent reverse transformation is not general and is not specific to the cell type or the transforming factor. In src-transformed CHO cells CAMP,on the contrary, potentiates the tumorigenic action of the oncogene (Roth et al., 1983). On the other hand, the growth arrest provoked by cAMP in adrenal tumor cells is associated with a completely different morphological change (Hall et al., 1979). These cells rapidly retract and round up as a consequence of the CAMP-induced disruption of the microfilament bundles (stress fibers). This very rapid and dramatic morphological change also occurs in a variety of normal cells, including epithelial cells such as thyrocytes (Westermark and Porter, 1982; Tramontano et al., 1982; Nielsen et al., 1985; Roger et al., 19891, melanocytes (Preston et al., 1987), granulosa cells (Ben-Ze’evet al., 19891, Sertoli cells (Spruill et al., 1981), or some fibroblasts (Aubin et al., 1983; Lamb et al., 1988) and arterial muscle cells (Chaldakov et al., 1989). It presents obvious similarities with cytoskeleton changes associated with cell transformation (reviewed by Ben-Ze’ev, 1985) or provoked by PKC-activating tumor promoters, including the fact that this acute microfilament disorganization precedes decreases in the synthesis of actin (Cheitlin and Ramachandran, 1981; Passareiro et al., 1985) and of the high-molecular-weight “transformation-sensitive” tropomyosin isoforms (Roger et al., 1989; Ben-Ze’ev et al., 1989). The mechanisms involved in the early reorganization of cytoskeleton networks induced by cAMP are not well understood. The cAMP effect on actin cable disruption is mediated by the subunit of PKA (Roger et al., 1988a;Lamb et al., 1988),which, in uitro or in intact cells, phosphorylates the myosin light-chain kinase (Lamb et al., 19881, hence reducing its activity (Nishikawa et al., 1984). As the interaction of actin and myosin involved in microfilament assembly seems to be dependent mainly on the phosphorylation of the myosin light chain, it is plausible that the decrease in myosin light-chain phosphorylation observed in chick embryo fibroblasts microinjected with PKA C subunit (Lamb et al., 1988) or in thyrocytes treated with cAMP agonists (Ikeda et al., 1986; Contor et al., 1988) destabilizes microfilaments and provokes the disruption of stress fiber (Lamb et al., 1988; Roger et al., 1989; Baorto et al., 1992). However, a decrease in myosin light-chain phosphorylation has also been involved in the induction of stress fiber

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formation during the CAMP-dependent reverse transformation of CHO cells (Lockwood et al., 1982). A CAMP-dependent dephosphorylation of ADF/destrin might also be involved in the actin microfilament breakdown (Baorto et al., 1992; Saito et al., 1994). Similar confusion arises from comparison of the analysis of the CAMP-dependent phosphorylation of vimentin in different systems. In uitro, the phosphorylation of vimentin by the CAMP-dependent protein kinase disassembles the vimentin filaments (Inagaki et al., 1987).This phosphorylation, which resembles that occurring at mitosis, is associated with the collapse of vimentin filaments (Lamb et al., 1989). In Swiss 3T3 cells it was associated with CAMP-dependent mitogenesis (Escribano and Rozengurt, 1988). It is puzzling, however, that during the reverse transformation of CHO cells by CAMP,a phosphorylation of vimentin also precedes its reassembly in a well-expanded filamentous network (Chan et al., 1989). The CAMP-dependent disruption of actin-containing filaments was observed not only in cells that proliferate in response to CAMP, such as thyrocytes (Roger et al., 1989) or melanocytes (Preston et al., 19871, but also in unresponsive cells or in cells whose growth is inhibited by cAMP (Hall et al., 1979; Hornsby, 1985; Chaldakov et al., 1989). However, as cAMP may affect cell proliferation by acting on different intracellular targets in different systems, it cannot be excluded that some CAMP-induced cytoskeleton changes would be involved in the proliferative response in only some systems. Cytoskeleton changes could also be instrumental in some, but not all, of the mitogenic pathways. In Swiss 3T3 cells the mitogenic effect of cAMP does not involve changes in the organization of microtubules, although antimicrotubule drugs are mitogenic in these cells (Wang and Rozengurt, 1983). The mitogenesis of these cells in response to growth factors acting via CAMP-independentmechanisms is well correlated with increased motility (dependent on cytoskeletal changes), whereas CAMP-dependent growth-promoting treatments, on the contrary, inhibit cell migration (O’Neill et al., 1985). Similarly, canine thyrocytes that round up in response to TSH and cAMP have poor motility compared to cells treated with serum, EGF, or TPA (P. P. Roger, unpublished observations). In fact, the mitogenic effect of TSH via cAMP is similarly observed both in thyrocytes cultured in monolayer and as follicles in suspension (Roger et al., 1983; WynfordThomas et al., 1987). In human thyrocytes the stimulation of DNA replication and synthesis of PCNA/cyclin by TSH and cAMP occurs independently of culture configuration (monolayers or dense cell aggregates), although many other changes in gene expression induced


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by TSH are mimicked by culturing unstimulated cells as dense aggregates instead of monolayers (Roger et al., 1988b; Lamy et al., 1990). These observations do not favor a determinant role of cell shape and the cytoskeleton in the positive control of proliferation by CAMP, at variance with the possible major involvement of cytoskeleton changes in the CAMP-dependent restoration of restricted growth characteristics in some transformed cells. The positive effects of cAMP in some normal cells and its negative effect in some transformed cells might be exerted at quite different levels and may represent unrelated phenomena. cAMP stimulates growth by an action on key regulatory events of cell cycle progression, while in some transformed cells cAMP may affect proliferation characteristics by altering cytostructural changes involved in their mitogenic cascades. Clearly, despite 20 years of suspicion of an involvement of the cytoskeleton in growth control by CAMP, we are still looking forward to more direct evidence of this. Experimentation is difficult, because the significance of the message delivered by the cAMP system, including the activation of protein kinases and the effects of protein phosphorylation, is partly dependent on the complexity of the whole cytostructure (itself being affected by CAMP), which is lost in broken subcellular preparations. VI. cAMP AND

THE

GROWTH OF CANCERCELLS

A. NEGATIVE MODULATION Early reviews emphasizing a negative influence of cAMP on cell proliferation bear mostly on tumor-derived cell lines (Pastan et al., 1975). As pointed out in the more recent review by Boynton and Whitfield (1983) and in this chapter, cAMP is, on the contrary, a positive modulator for the multiplication of many normal differentiated cells. This might suggest that a change in the cAMP effect on growth is associated with cancerous transformation in many instances. The best examples are the inhibition of proliferation by cAMP in various melanoma cell lines (Wong and Pawelek, 1973; Slominski et al., 1989; Niles and Loewy, 19911, breast cancer cell lines (Cho-Chung et al., 1981, 1983; Fentiman et al., 1984; Iwasaki et al., 1983; Fontana et al., 1987; but see Kung et al., 1983; Welsch and De Hoog, 1983; Sheffield and Welsh, 19851, and Schwann-like cells from neurofibromas (Sobue et al., 19851, whereas cAMP is unequivocally demonstrated to be a mitogenic factor for their normal counterparts. Pregnancy-dependent

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mouse mammary tumors maintain the cAMP dependence of growth, but at the later ovarian-independent stage of progression, the growth is inhibited by cAMP (Imagawa et al., 19921. In the case of human melanoma cells, the sensitivity to growth inhibition by CAMP was closely correlated with the metastatic activity in different variants of the same parental cell line (Ormerod and Hart, 1989). Furthermore, transformation by Ha-ras oncogenes markedly increases the sensitivity to growth inhibition by cAMP in NIH 3T3 cell lines (Lockwood, et al., 1987; Davies et al., 19891, and cAMP specifically blocks the proliferation of rat 3T3 cells after their transformation by polyoma virus (Kamech et al., 1987). TSH via cAMP inhibits the growth of metastatic transformed cells that spontaneously arise from the TSH (CAMP)-dependentFRTL cloned rat thyroid cell line (Endo et al., 1990). Other recent examples of human cancer cell lines inhibited by CAMP include medullary thyroid carcinoma (de Bustros et al., 1986), salivary gland adenocarcinoma (Azuma et al., 19881, renal adenocarcinoma (Kinoshita et al., 1985), the gastric carcinoma cell line Kato I11 (Nakamura et al., 19891, HT29 colon adenocarcinoma cells (Garnet et al., 19921, small-cell lung carcinoma (Francis et al., 19831,and colon cancer cells (Tagliaferri et al., 1988a). cAMP mediates, in part, the antiproliferative effect of interferon a in PC3 prostate carcinoma cells (Okutani et al., 19911,but not in an astrocytoma cell line (Hubbel et al., 1991). Nevertheless, in the latter system cAMP can mediate the direct growth-inhibitory effect of double-stranded RNA (Hubbel et al., 1991). The apparent inversion of proliferative response in cancer cells is not unique to the CAMPcontrol of growth. Tumor-promoting phorbol esters, through PKC activation, in fact inhibit the growth of many tumor cells (Gescher, 19851, and the proliferation of several carcinoma cells is inhibited by EGF. In this case the EGF inhibition of growth has been correlated with the overexpression of EGF receptors (Gill and Lazar, 1981; Chen and Lin, 19931. EGF and TPA were reported to exert their inhibitory effects on the G2-mitosis transition (Kaszkin et al., 1992), as is the case for the CAMP-dependent inhibition of proliferation in many tumor cell lines (Friedman, 1976). Depending on the cell system and the putative factor responsible for cancerous transformation, different mechanisms have been proposed to explain how cAMP might specifically inhibit the growth of cancer cells. The hypothesis implicating the cytoskeleton in the “CAMPdependent reverse transformation” has been discussed in Section V. Another hypothesis relies on alterations in the permeability of the gap junctions that permit direct communication between adjacent cells by conducting low-molecular-weight cytoplasmic molecules, including


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small signal molecules. This junctional communication is profoundly altered in many transformed cells and cells from various cancers (reviewed by Sheridan and Atkinson, 19851, which may contribute to their autonomy. cAMP is a positive modulator of the junctional communication (Sheridan and Atkinson, 1985; Munari-Silem et al., 19901, even in neoplastic cells, in which this was correlated with growth inhibition by cAMP (Mehta et al., 1986; Murray and Taylor, 1988). Phosphorylation of the gap junction protein connexin 32 by PKA, as observed in hepatocytes (Saez et al., 19901, or CAMP-dependent transcription of connexin 43 (Saez et al., 1993) provides clues to the mechanism, as connexins are found to have tumor-suppressing activity. Quite interestingly, Mehta et al. (1986)observed that CAMP-increasingdrugs inhibit the proliferation of chemically and virally transformed 10 T 1/2 cells, only when promoting their communication through gap junctions with normal 10 T 1/2 cells. They suggested that cAMP promotes the transfer of small growth-inhibitory molecules from normal cells to cancerous ones. Thus, cAMP can inhibit the growth of cancer cells by restoring normal junction communication properties with normal cells. Also, in normal 3T3-Ll cells Shiba et al. (1989) found a correlation between CAMP-inhibitedgrowth stimulation by serum and CAMPinduced enhancement of gap-junctional communication. While MSH, through CAMP, stimulates the growth of normal melanocytes, it inhibits the proliferation of Cloudman S91 mouse melanoma cells. However, Pawelek et al. (1975) isolated variants of these cells that present a cAMP dependence of growth. Examination of these variants revealed that their type I CAMP-dependent protein kinases required a higher concentration of cAMP for their activation (Pawelek, 1979). It seemed that these cells required an optimum level of CAMP for proliferation. If the intracellular levels of CAMP are above or below this optimum, the cell cycle is lengthened, but this optimum may be modified by mutations of PKAs that alter their sensitivity to cAMP (Pawelek et al., 1983). This leads to the quite different hypothesis that the direction of the cAMP effect on growth is dependent on the characteristics of PKAs. A variation of this “kinase hypothesis’’was first proposed by Russell and collaborators (Byus et al., 1977) and is still receiving considerable support from a few groups, among which Cho-Chung’s (1989) is the most active. While type I PKA could be responsible for positive effects of cAMP on growth in some systems (see Section III,D,l; Livesey and Martin, 1988; Van Sande et al., 19891, type I1 kinase might mediate the inhibitory effects. Thus, the growth of several cell lines derived from hormone-dependent metastatic breast cancers, including MCF7 and

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T47D cells, is inhibited by cholera toxin, forskolin, and cAMP analogs (Cho-Chung et al., 1981, 1983; Fontana et al., 1987; Katsaros et al., 1988) and hormones increasing cAMP levels, such as calcitonin (Ng et al., 1983) and PGE (Iwasaki et al., 1983). Calcitonin was reported to activate only type I1 kinase (Ng et al., 19831, and site-specific cAMP analogs that preferentially activate type I1 kinase are the most potent growth inhibitors (Tagliaferri et al., 1988a). Furthermore, the growth arrest parallels an increase in type I1 PKA activity (Cho-Chung et al., 1981, 1983) and concentration (Ogreid et al., 19871, together with a decrease in type I PKA (Katsaros et al., 1988). A parallelism between RI/RII mRNA ratios and growth is also found in transformed versus control cells and in response to the inhibitor 8-C1-CAMP (Ciardiello et al., 1990). Cho-Chung (1974; Cho-Chung et al., 1983) also reported evidence that dibutyryl-CAMP and cholera toxin are potent inhibitors of the growth of hormone-dependent DMBA-induced mammary tumors in the rat in uiuo. Interestingly, autonomously growing rat mammary tumors that fail to regress after dibutyryl-CAMP treatment differ from CAMP-sensitive tumors by displaying another subtype of PKA I1 (Ogreid et al., 1987). Cho-Chung’s group generalized their findings to other tumor systems. The type I1 kinase-specific cAMP analog 8-C1-CAMP also inhibits the growth of human colony cancer cell lines (Katsaros et al., 1987; Tagliaferri et al., 1988a) and the clonogenic growth of leukemic blast progenitors (Pinto et al., 1992). In the LS174T colon cancer cell this inhibition involves the nuclear translocation of RII cAMP receptor protein and both an increase in RII and a decrease in RI gene transcription (Ally et al., 1988). The transformation of NIH 3T3 fibroblasts by Harvey murine sarcoma virus is reversed by cAMP analogs that predominantly activate PKA I1 (Tagliaferri et al., 1985). As in mammary tumor cells, the growth inhibition is accompanied by an increase in RII cAMP receptor protein and a decrease in RI protein (Tagliaferri et al., 1988b). Inhibition of RI and induction of RII gene expression precede the inhibition of growth and induction of megakaryocytic differentiation by 8-C1-CAMP in the K562 human leukemic cell (Tortora et al., 1989). A similar shift between the expression of RI and RII PKA subunits was also reported during the inhibition of growth of human lung carcinoma cells by 8-C1-CAMP in athymic mice (Ally et al., 1989). Similar effects of 8-Cl-cAMP, including growth inhibition (with no accumulation of cells at a specific cell cycle phase), a decrease in RI binding activity, and nuclear translocation of RII, have also been reported for some gastric carcinoma cell lines (Takanashi et al., 1991l.In HL60 leukemia cells antisense oligonucleotides against RIIp of PKA


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relieve the antiproliferative differentiating effects of cAMP analogs (Tortora et al., 19901, while antisense oligonucleotides against RIa induce an increase in RIIp mRNA, growth inhibition, and monocytic differentiation (Tortora et al., 19911, thus bypassing the effects of exogenous cAMP analogs. Similar findings were reported for different human cancer cell lines (Yokozaki et al., 1993). 8-Cl-cAMP, as a specific activator of PKA I1 and inducer of the RII nuclear translocation and transcription, was thus proposed as a potentially therapeutic agent in a wide spectrum of cancers (Katsaros et al., 1987; Cho-Chung, 1990; Pinto et al., 1992). The data of Cho-Chung and colleagues are obviously of important therapeutic significance. However, they are not undisputed and their interpretation recently raised major controversies. (1) Other groups even reported that cholera toxin and cAMP analogs stimulate the growth of MCF7 and T47D mammary carcinoma cells in uitro and in uiuo (Kung et al., 1983; Sheffield and Welsh, 1985) and promote rather than inhibit, the growth of MNU-induced rat mammary carcinomas (Welsch and De Hoog, 1983; Shefield and Welsch, 1988). (2) On the other hand, it is puzzling that the growth-inhibitory effects of 8-C1cAMP are not associated with an accumulation of cells at a specific phase of the cell cycle (Tagliaferri et al., 1988a; Takanashi et al., 1991; Pepe et al., 1991). In HL60 cells this is in sharp contrast with the accumulation of cells in G,/G, induced by N6-benzyl-CAMP (Pepe et al., 1991). Astonishingly, in HL60 cells and colon carcinoma cells, Sp and Rp phosphorothioate derivatives of 8-C1-CAMP (Sp. 8-C1-CAMPS and Rp 8-Cl-cAMPs)-agonist and antagonist, respectively, of PKAs-are both inhibiting growth (Yokozaki et al., 1992).The growth inhibition by Rp 8-C1-CAMP should be independent of PKA activation (Yokozaki et al., 1992). Van Lookeren Campagne et al. (19911, Langeveld et al. (19921, and Lange-Carter et al. (1993) ascribed the very strong inhibiting properties of 8-C1-CAMP on several cancer cell lines to the especially cytostatic properties of its metabolite &Cl-adenosine, which is formed in the culture medium [see also the published correspondence between Y. S. Cho-Chung and M. M. Van Lookeren Campagne and colleagues on this subject (Cancer Res. 51, 6206-6208, 199111. The growth-inhibitory effects of 8-Cl-CAMP are completely prevented by adenosine deaminase and even by the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (by inhibiting the conversion of 8-C1-CAMP into 8-C1-adenosine) (Van Lookeren Campagne et al., 1991; Langeveld et al., 1992; Lange-Carter et al., 1993).Even the downregulation of the RIa and C subunits is mediated by 8-C1-adenosine through a CAMP-independent mechanism in normal and neoplastic

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epithelial cells (Lange-Carter et al., 1993). (3) Finally, Otten and McKnight (1989) demonstrated, using ras-transformed NIH 3T3 cells, that overexpression of the type I1 R subunit of PKA, which leads to elimination of type I holoenzyme, does not reverse the transformed phenotype. This indicates that the 8-C1-CAMP-induced reverse transformation reported by Tagliaferri et al. (1988b) in this system could not be caused by the shift in PKA isozyme expression. Such a shift could be simply a general consequence of the activation of PKAs (McKnight et al., 19881, which could also occur in cells whose growth is stimulated by CAMP.In Section III,D,l we discussed the possible significance of a similar shift between PKAs I and I1 during the CAMPdependent proliferation of thyroid cells (Breton et al., 1989; Roger et al., 1991). A clear means by which cAMP could specifically inhibit the growth of transformed or cancer cells is by interacting with the expression of the transforming genes (oncogenes).Indeed, Cho-Chung and collaborators also reported that cAMP analogs that activate type I1 PKA inhibit the synthesis of the p2lras protein (either cellular or viral) in NIH 3T3 cells transfected with Harvey murine sarcoma virus DNA (Tagliaferri et al., 1985) and in mammary carcinomas and MCF7 cells, restoring to a normal level their high cellular p21H-1-a~ expression (Huang and ChoChung, 1984; Tagliaferri et al., 1988a). Similarly, Azuma et al. (1988) observed that dibutyryl-CAMP suppresses p21ras expression and cell proliferation in a human salivary gland adenocarcinoma cell line. However, these observations were not confirmed in other systems. The CAMP-induced reverse transformation in ras-transformed BALB/c3T3 cells (Ridgway et al., 1988) and the induction of differentiation by cAMP in ras-transformed MDCK cells (Wu and Lin, 1990) occur without changed p2lv-ras levels. Whether ras overexpression is directly responsible for the formation of these various CAMP-sensitive tumors and how cAMP could negatively control both cellular and viral ras oncogene expressions remain important questions. Also, it is not known whether the p2lras function is altered by its CAMP-dependent phosphorylation in intact cells (Saikumar et al., 1988). The fact that dibutyryl-CAMP partially restores PDGF-dependent signaling events in ras-transformed NIH 3T3 cells (Olinger et al., 1989) is, however, supportive of the suggestion that cAMP can antagonize the ras function. A mechanism was recently provided by the demonstration that, in several cell types, including the H-ras-transformed NIH 3T3 cells (Chen and Iyengar, 1994), cAMP inhibits the raf-l-MAP kinase cascade activated by ras (see Section 11,B). Other human tumor cell lines, including glioma and osteosarcoma


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cell lines, express the c-sis oncogene encoding the PDGF, which may contribute to carcinogenesis by autocrine or paracrine mechanisms. Indeed, various cAMP enhancers and analogs block the transcription of this protooncogene in these cells (Harsh et al., 1989). Conversely, cAMP can also arrest growth by inducing the secretion of autocrine growth inhibitors, such as TGF-P, in the PC3 prostate carcinoma cell line (Bang et al., 1992). Besides the evidence that cAMP may specifically inhibit the growth of various cancer cells, it is obvious that cells whose growth is normally inhibited by cAMP (fibroblasts and lymphocytes) may maintain this negative control after neoplastic transformation. This is especially true for various leukemic cells in which cAMP inhibits growth and induces differentiation. This reverse transformation process seems to involve the inhibition of c-myc expression also observed in normal lymphocytes (Slungaard et al., 1987; Tortora et al., 1989). Whatever cellular mechanisms are implicated in the inhibition of growth of cancer cells by CAMP,the potentiality of using it as a therapeutic means to control the progression of tumors in uiuo has been considered very attractive by several authors. The B. pertussis invasive adenylate cyclase (Slungaard et al., 1983), cholera toxin (Cho-Chung et al., 1983; Lanotte et al., 19861, and 8-C1-CAMP (Cho-Chung, 1990; Pinto et al., 1992) have thus been proposed as tools to manipulate the cAMP cascade in tumor cells in uiuo. This enthusiasm should be moderated when considering the following caveats: (1) cAMP is a ubiquitous second messenger involved in almost all of the vital functions of the organism, which could be perturbed as cAMP is systematically increased: (2) the mechanisms involved in the cytostatic and cytotoxic properties of 8-C1-CAMP and its metabolite, 8-Cl-adenosine, are still poorly understood and are less specific than was initially claimed; (3) by limiting the proliferation of lymphocytes, cAMP enhancers and 8-C1-CAMP are potential immunosuppressive factors and thus could favor neoplastic development; and (4)as discussed in Section VI,C, CAMP,by stimulating the growth of some tumor cells, could act as a tumor promoter. B. ESCAPE FROM NEGATIVE MODULATION The hypothesis that cAMP is a ubiquitous inhibitory factor involved in normal growth control prompted a series of studies based on the notion that cancer could result from defects in the CAMP-PKA cascade (Pastan and Johnson, 1974; Chlapowski et al., 1975). Some recent studies still attempt to support this view, which contrasts sharply with

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that discussed in the previous section. Tumor cells lose hormone receptors coupled to adenylate cyclase, such as PGE receptors during the progression of rat mammary tumors (Abou-Issa and Minton, 1986) and in a culture of tumorigenic rat urothelium (Chlapowski and Nemecek, 19851, or P-adrenergic receptors in another rat urothelium cancer cell line (Chlapowski and Nemecek, 1985)and in NIH 3T3 cells upon transformation by N-ras (Davies et al., 1989).Moreover, a decrease in adenylate cyclase activity was observed in ras-transformed NIH 3T3 cells (Tarpley et al., 1986; Davies et al., 19891, in ras-transformed FRTL5 rat thyroid cells (Colletta et al., 19881, and in some rat urothelium tumor cells (Chlapowski and Nemecek, 1985).It was proposed as a marker to differentiate colon cancer from benign cells in culture (Nelson and Holian, 1988). The GTP binding t o the adenylate cyclase-activating subunit Gsa is defective in mouse lung tumors (Droms et al., 19871, leading to decreased hormone responsiveness (Droms et al., 1989). Whether tumor progression depends on such defects in cAMP metabolism remains doubtful, as they could be secondary or could reflect the general loss of differentiation linked t o transformation. In fact, Chlapowski and Nemecek (1985) did not observe any correlation between in vitro growth rate or tumorigenicity and the various abnormalities in cAMP metabolism (ranging from a strong reduction to an excess of adenylate cyclase activities) they found in urothelium tumor cells. The ras-transformed NIH 3T3 cells with reduced adenylate cyclase activity are paradoxically more sensitive to growth inhibition by cAMP (Davies et al., 1989). Similar reservations should be made for the interpretation of studies reporting alterations in PKA responses to cAMP in tumor cells. In mouse lung urethane-induced tumors the high-affinity binding of cAMP to the RII PKA subunit is strongly reduced, not because of a structural alteration of RII but possibly due to a modified conformational state or interaction with other cytosolic molecules (Butley et al., 1984, 1985). In other lung tumor cells lower PKA I expression as well as deficiencies in PKC activity were reported (Nicks et al., 1989). The decrease in PKA I expression is due to a decrease in RI mRNA content, likely at the transcriptional level (Lange-Carter et al., 1990; LangeCarter and Malkinson, 1991). Whether such modifications are causal in the tumorigenic process is not known. More significant could be the study by Hiwasa et al. (19871, who reported that some carcinogeninitiated clones (i.e., growing in soft agar with TPA) derived from BALB/c-3T3 fibroblasts are resistant to CAMP,while clones selected for cAMP resistance after carcinogen treatment behave like initiated


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clones in a two-stage carcinogenesis model. In both categories of clones, the cAMP resistance of growth is correlated with defects in PKA activities (Hiwasa et al., 1987). However, the alterations in PKA activities are only quantitative and might depend on several factors. C. cAMP AS

A

TUMOR PROMOTER

Since the activation of the cAMP cascade stimulates the growth of many differentiated cells, it is likely that cAMP could also stimulate the proliferation and thus the clonal expansion of some cells initiated by oncogene activation. Again, the thyroid gland provides the best illustration of this assumption. The classical experimental protocols to induce thyroid tumorigenesis all involve chronic stimulation by TSH (Christov and Raichev, 1972; Dumont et al., 1980). Thyroid follicular cell malignancies (mostly follicular carcinomas) frequently develop during TSH-dependent goitrogenesis (Konig et al., 1981; Williams, 1990), and TSH suppression by treatment with thyroid hormones delays the growth of many thyroid tumors (Clark, 1981).Follicular carcinomas likely progress from follicular adenomas, many of which could be caused by the mutational activation of ras oncogenes (Lemoine et al., 1989). Human thyroid follicular adenoma cells often retain the TSH dependence of growth in uitro (Williams et al., 1988) and in explants in nude mice (Dralle, 1989). Adenylate cyclase sensitivity to TSH is even greater in some follicular carcinomas (Clark and Gerend, 1985) and the proliferation effects of TSH on normal human thyrocytes are mediated by cAMP (Roger et al., 1988b). Therefore, the promotion and progression of many follicular tumors of the thyroid gland seem to be dependent on the mitogenic effects that TSH exerts via intracellular cAMP elevation. By contrast, papillary carcinomas of the thyroid gland are frequently associated with a high-iodine diet and thus could have progressed despite low circulating TSH levels (Konig et d., 1981; Williams, 1990). The activation of ret (Grieco et d., 19901, TRK (Bongarzone et al., 19891, and MET (DiRenzo et al., 1991) tyrosine kinase oncogenes, but not ras oncogenes (Lemoine et al., 19881, is often found in this second thyroid tumor type. Overexpression of the erb-B2 protooncogene was also reported in papillary carcinomas (Aasland et aZ., 1988). Thus, the two main thyroid malignancies not only seem to have been determined by different hormonal promoting environments, but also are associated with different oncogenes. We speculate that the activation of the distinct CAMP-dependent or -independent mitogenic

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pathways (as discussed in Section 111,E,2) could have promoted-and selected-the clonal growth of cells initiated by the activation of different oncogenes, thus leading to different tumor phenotypes. Whether a similar tumor promoter role could be ascribed to cAMP and CAMP-enhancing factors in other differentiated tissues, the growth of which is also positively controlled via CAMP,remains poorly documented and awaits further studies. The proliferation of some mammary tumor cell lines (Taylor-Papadimitriou et al., 1980; Imagawa et al., 1992) and of a rat bladder carcinoma cell line (Boyer and Thiery, 1993) is stimulated by cAMP in synergy with other factors. There has been recent interest in finding VIP receptors coupled to adenylate cyclase in several tumor types (Ruellan et al., 1986; Siperstein et al., 1988; Gespach et al., 1988; Yu et al., 1992; Moody et al., 1993), as this neurotransmitter was proved to be mitogenic via cAMP in some normal cells in culture (Haegerstrand et al., 1989; Pincus et al., 1990a; Zurier et al., 19881, as well as in Lewis lung carcinoma cells, a mouse mammary carcinoma cell line (Scholar and Sudhor, 19911, and a human colon carcinoma cell line (Yu et al., 1992). It was hypothesized that VIP could serve as an autocrine growth factor in neuroblastoma (O’Dorisio et al., 1992). A VIP antagonist inhibits the accumulation of cAMP and growth of non-small-cell lung cancer in uitro and in uiuo (Moody et al., 1993). Gastrin, possibly via CAMP, is a potent promoter of the growth of a xenotransplantable human gastric carcinoma (Ochiai et al., 1985; Sumiyoshi et al., 1984; Yasui et al., 1986; but see Takanashi et al., 19911, but cholera toxin has no tumor-promoting activity in mouse skin, although it is a potent inducer of epidermal hyperplasia (Kuroki et al., 1986).

D. ONCOGENES RELATEDTO THE cAMP SIGNALING CASCADE Until 1987 there was no report of an oncogene product related to the cAMP signaling cascade (Gottesman and Fleischmann, 1986). This probably justified the relative lack of interest in the CAMP-dependent mitogenic cascade, as compared to tyrosine kinase-dependent mitogenic pathways that involve the cellular homologs of many oncogenes. Hyperactivation of the cAMP signaling cascade should lead to the generation of hyperfunctioning tumors in many systems of differentiated endocrine cells (Dumont et al., 1989).Our first biochemical analysis of the cAMP pathway in hypersecreting thyroid adenomas (“hot nodules”) did not demonstrate a major constitutive activation of the cAMP cascade (Van Sande et al., 1980, 1988). However, recently we


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have found new somatic mutations of the TSH receptor conferring constitutive activation of adenylate cyclase but not of phospholipase C in such adenomas (Parma et al., 1993). In late 1987 Vallar et al. reported that many pituitary adenomas that hypersecrete GH do carry autonomously active G, protein and adenylate cyclase. Extracts of metastatic B16 melanoma cells also confer increased adenylate cyclase sensitivity in reconstituted ,949 cyccell membranes (Lester et al., 1987). Inasmuch as the secretion and growth of somatotrophic cells were known to be cAMP dependent (Billestrup et al., 19861, the alteration of G, was assumed to be the direct cause of both high secretory activity and autonomous growth of the tumors in which it occurs. As a, also activates some Ca2+ channels, an additional role of Ca2+ in the pathologies caused by its constitutive activation cannot, however, be completely excluded (Hamilton et al., 1991). Further analysis of the pituitary tumors led to the demonstration of three somatic mutations of the G, protein a-chain at codons 201 and 227 (ArgzOlCys,ArgZOlHis, and Gln227Arg), which cause constitutive activation of a, by inhibiting its GTPase activity (Landis et al., 1989).Interestingly, the importance of a, GlnZz7mutations for a, activity had already been suggested by deliberate site-directed mutational analysis (Masters et al., 1989; Graziano and Gilman, 1989) and by its analogy with the Glu61 mutation, which inhibits GTPase activity of p2lras. On the other hand, Argzol is precisely the site of a,that is ADPribosylated by cholera toxin, also resulting in GTPase inhibition and block of Gs inactivation (Freissmuth and Gilman, 1989).These activating mutations of G,a were predicted t o convert G, into a dominant oncogene [anticipatively called gsp (Landis et al., 198911 in cells programmed to proliferate in response to CAMP.This prediction was confirmed in some cases of autonomously functioning thyroid adenoma (hot nodule) (Lyons et al., 1990; Suarez et al., 1991; O'Sullivan et al., 19911, in 50% of somatotroph adenomas, and in other hypophyseal tumors (Lyons et al., 1990; Landis et al., 1990; Spada et al., 19901, but not in a large panel of tumors including melanomas, ovarian, and adrenal cortical tumors (Lyons et al., 1990). However, ArgZolCys and ArgzolHis mutations of a, were recently demonstrated by Weinstein et al. (1991) and Scheidinger et al. (1992) as the probable cause of McCune-Albright syndrome, a disease characterized by polyostotic fibrous dysplasia, caf6 au lait pigmentation of the skin, sexual precocity, and hyperfunction of multiple endocrine glands. The somatic mutation should appear at an early postzygotic stage in order to explain its occurrence in multiple tissues and the

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mosaic distribution of cells bearing or not bearing the dominant mutation. In McCune- Albright syndrome hyperplasia and formation of functional nodules and adenomas associated with a, mutations are not restricted to thyroid cells and somatotrophs, but are also found in the ovaries, resulting in “autonomous” follicle maturation, and in the adrenal cortex (Weinstein et al., 19911, two tissues for which the growthstimulatory role of cAMP has been questioned and whose tumors were associated with mutational activation of ai2 rather than a, (Lyons et al., 1990). Thymic hyperplasia, gastrointestinal adenomatous polyps, and Leydig cells hyperplasia in the testes were also observed (Weinstein et al., 1991). The transfection of constitutively active G,a with the Glu227Leu mutation increases the mitogenic responsiveness of Swiss 3T3 cells (Zachary et al., 1990). Heterotypic expression of adenylate cyclase-controlling receptors could, depending on the role of cAMP in the proliferation of the cells involved, lead to growth or atrophy. A remarkable model for this has been provided by the adenosine A2 receptor. This newly cloned receptor, which positively controls adenylate cyclase, is in many cells physiologically constitutive (Maenhaut et al., 1990). This is, at least in part, due to the fact that the adenosine normally produced by these cells is sufficient to activate the receptor. When expressed in canine thyroid cells by microinjection of mRNA, this receptor induces constitutive proliferation (Maenhaut et al., 1990). When expressed in transgenic mice with a thyroid-specific thyroglobulin promoter its cDNA induces an hyperfunctioning goiter and thyrotoxicosis (Ledent et al., 1992). There are also some clues that overexpression of P-adrenergic receptors could be associated with thyroid neoplasia. In some cases of autonomous thyroid adenomas, the cAMP response to norepinephrine was found to be enhanced compared to that of normal tissue (Van Sande et al., 1980). In the FRTL5 thyroid cell line, which normally does not possess P-adrenergic receptors, introduction of a P,-adrenergic receptor by transfection (Hen et al., 1989) or infection with a retroviral construct (Tsuzaki et al., 1991) confers an isoproterenol-sensitive growth. Interestingly, in the latter case the overexpression of P,-receptors (105receptors per cell) also markedly increases the basal cAMP levels and causes an autonomous (i.e., in the absence of TSH or isoproterenol) proliferation (Tsuzaki et al., 1991).A spontaneously transformed FRTL5-derived cell line that is malignant in nude mice also has elevated basal cAMP levels and has acquired P,-adrenergic receptors (Endo et al., 1990). P,-Adrenergic receptor mRNA was preliminarily reported to be overexpressed in some neoplastic human thyroid tissues (Ling et al., 1992). Heterotypic expression of GIP receptors in the adrenal gland and the


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consequent activation of the cAMP cascade in these cells after each meal has now been shown to be responsible for some Cushing’s syndromes with adrenal hyperplasia and adenoma (Lacroix et al.,1993). Many other adenylate cyclase-activating receptors were previously found to be ectopically expressed in adrenocortical tumors, including receptors to TSH, LH, FSH, glucagon, vasopressin, and adrenaline (reviewed by Lacroix et al., 1993). As was first shown by Lefkowitz’s group, G protein-coupled receptors with seven transmembrane-spanning domains can also be activated by mutations and thus constitutively turned on (Lefkowitz et al.,1993).In tissues growing in response to CAMP,constitutive activation of adenylate cyclase by such mutations should cause tumors. Indeed, somatic Ala623 and Asp619 mutations of TSH receptors cause TSH-independent activation of adenylate cyclase, but not phospholipase C, and hyperfunctioning thyroid adenomas (Parma et al., 1993). Other adenylate cyclase-activating mutations of LH receptors of Leydig cells were found to be responsible for male precocious puberty, a disorder caused by Leydig cell hyperfunction and hyperplasia (Shenker et al., 1993). These observations suggest that G protein-coupled receptors that activate adenylate cyclase may behave as protooncogenes. In the yeast S. cerevisiae, the growth of which is dependent on CAMP, any mutation activating the CAMP-PKA signaling cascade leads to deregulated proliferation and escape from nutrient control (Dumont et al.,1989). By analogy, it could be inferred that other mutations overactivating this pathway will prove to be responsible for tumor formation in tissues in which growth is CAMP-dependent. For example, a mutation of the C subunit of PKA disrupts inhibition by the R subunit without altering substrate recognition in yeast and leads to autonomous growth (Levin et al.,1988; Levin and Zoller, 1990).This mutation affects the interaction between R and C subunits in a region that is well conserved, structurally and functionally, in mammals (Levin and Zoller, 1990). The homologous mouse mutant Thrl97Ala also remains partially active in the presence of excess R subunits (Orellana and McKnight, 1992). Even greater unregulated activities of the mouse C subunit are observed in the Hiss7Glu and Trpl96Arg mutants (Orellana and McKnight, 1992). A mutation of the mouse RII subunit was also described (ArgArg92,93AlaAla),which does not alter the holoenzyme formation but confers full enzymatic activity even in the absence of cAMP (Wang et al., 1991). Such mutations likely lead to functional tumors of thyroid cells or somatotrophs. Inactivation of elements negatively controlling the cAMP cascade should have the same result. Alteration of cAMP phosphodiesterase

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renders MDCK cells independent of the CAMP-dependent growth stimulation by glucagon or PGE, (Taub et al., 1983). cAMP phosphodiesterase could thus be a potential target for recessive oncogenic mutations in differentiated epithelial cells. In the thyroid gland cold adenomas (i.e., adenomas that do not take up iodide) have been found to have defects in iodide trapping and iodide oxidation (DemeesterMirkine et al., 1984). Considering that iodide through an oxidized intermediate inhibits the TSH-CAMP cascade, such adenomas could result from the relief of this negative control (Maenhaut et al., 1991). In cells in which cAMP is a signal for growth, any negative element in the cAMP cascade can thus be considered as a potential antioncogene, whose inactivating mutation could cause tumorigenesis; any positive element or gene in the cascade could be a potential oncogene when a mutation causes constitutive activation. While many undifferentiated tumors are associated with the activation or overexpression of oncogenes related to the tyrosine kinase and Ca2+ phospholipid pathways, further studies in differentiated hyperfunctioning adenomas should define the extent to which these and other alterations of the cAMP system account for some of these benign tumors. A second G-protein oncogene, g i 2 , results from somatic point mutations in the gene for the a-subunit of Gi2 (Lyons et al., 1990). gip2 is found in endocrine tumors of the adrenal cortex and the ovary (Lyons et al., 1990), and it induces neoplastic transformation of rat-1 fibroblasts (Pace et al., 1991) and, to a lesser extent, NIH 3T3 cells (Hermouet et al., 1991). Unlike gsp, gip2 inhibits cAMP accumulation (Wong et al., 19911, which was proposed to exert a mitogenic influence in some fibroblasts (Seuwen et al., 1988; Van Corven et al., 1989). However, it remains doubtful that this mechanism could explain the oncogenic effects of Gi2 mutations, since Gi2 can be coupled t o other cascades (Letterio et al., 1986; Zachary et al., 1991; Gupta et al., 1992). In particular, dissociation of Gi2 can activate phospholipase C by its py-subunits and ai2 might lead to p2lras activation independently of adenylate cyclase inhibition or phospholipase C stimulation (Van Corven et al., 1993). The fact that pertussis toxin inhibits the growth effects of gip2 in rat-1 cells (Pace et al., 19911, but not its inhibitory effects on adenylate cyclase, also suggests that its mitogenic effects are not due to this inhibition (Wong et al., 1991). VII. CONCLUSIONS AND PERSPECTIVES

A main drawback of previous analyses of the role of cAMP in proliferation has been the tendency of authors to define a “universal role”


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for the intracellular signal in cell proliferation. No such generalization is possible. It is quite evident that cAMP is a negative regulator of proliferation in some cells, is a positive regulator in others, and has a biphasic effect in a third category. Cells of the latter type can evolve clones in which only the stimulatory effects are seen (S91 melanoma cells) (Pawelek et al., 1975). On the other hand, cells positively regulated by the cAMP cascade (FRTL5 cells) can produce clones in which cAMP has only a negative effect (Endo et al., 1990). It is therefore apparent that animal cells possess in their genes both positive and negative CAMP-dependent proliferation cascades and that, depending on the program (differentiation) of the particular cell type, either one or both-or parts of both intervening at different steps of the mitogenic pathway-may coexist. Depending on this program, activation of the cAMP cascade will lead to the triggering of proliferation, proliferation arrest, or synergism with other factors eliciting either of these opposite outcomes. It is obvious that the CAMP-dependent machinery to induce growth must be very sophisticated: cAMP as a sufficient activator of mitogenesis must support all stages of the prereplicative phase. On the other hand, the machinery for negative control needs to operate on only one step (preferably Go-G, transition) of the mitogenic activation process. To add a supplementary level of diversity, the growthinhibitory effects of cAMP in normal cells such as fibroblasts or lymphocytes may use different strategies. Interaction with the signaling cascade of mitogens and/or inhibition of cell cycle progression at different restriction points, ranging from an inhibition of the raf-l-MAP kinase cascade and c-myc expression at the Go-G, transition to a block at the S-phase commitment point in late G,, have been convincingly reported. These mechanisms should be distinguished from much less specific inhibitory effects of CAMP,as they are observed in many cancer cells: they require cAMP clamping at levels never reached in physiology, involve abnormal blocks of the cell cycle in S or G, phase, involve neutralization of the effects of activated oncogenes, or are secondary to effects on cytoskeleton organization, cell-cell communication, or differentiation. The growth stimulation by cAMP is also produced by quite different mechanisms. As we have shown in thyroid cells, cAMP may trigger its own mitogenic cascade, but positive interactions with the signaling cascades of other mitogens are common. The effects of cAMP on proliferation may also be indirect, as in the case of the adrenal gland and ACTH, in which the cAMP cascade in viuo stimulates function, proliferation, and differentiation expression, but the effect on proliferation is not reproduced in uitro, as it is secondary t o the activation of an

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autocrine loop. The various strategies of cAMP growth stimulation are not exclusive and may cooperate in one system. In Schwann cells cAMP seems to exert a direct mitogenic influence together with induction of PDGF receptors (Weinmaster and Lemke, 1990) and inhibition of the production of an autocrine negative factor (Muir et al., 1990). The role cAMP plays in one type of cell depends on its position in the regulatory network. Vertebrate cells contain genes that can complement yeast cells defective in cell cycle control. The general machinery of the cell cycle is therefore probably remarkably conserved through evolution. That CAMP-dependent protein kinases are not involved in this basic machinery is suggested by the normal proliferation behavior of S49 kinase-deficient mutants (Coffin0 et al., 19751, although type I1 CAMP-dependent protein kinase was found to be associated with p34cdc2 kinase (Tournier et al., 1991). However, the physiological controls that operate on this machinery are specific for each cell type and may even evolve during cell cycle progression, as exemplified by the cell cycle-dependent coupling of the calcitonin receptor to different G proteins (Chakraborty et al., 1991). They must therefore themselves control the core machinery. Between these levels operate the different intracellular regulatory cascades. However, although many extracellular influences operate on any cell, the number of transducing cascades is remarkably small and their role varies in the different cells. It was therefore to be expected that each cascade, depending on its role on the general physiology of the cell, would either stimulate or inhibit cell proliferation. As we have shown, this is obviously the case for the cAMP cascade. The very interesting problem this raises is how the same cascade could act positively in some cells and negatively in others and what determines the wide diversity of the mechanisms of these positive and negative modulations in various systems. The solution to this problem could lie, in part, in the black box that links protein phosphorylation and protooncogene expression, and in the multiple cell type-dependent interactions between the major signaling cascades. An interesting example of how this could be achieved is the expression of isoenzymes with the same function but different regulation characteristics. For example, isozyme p of PKC inhibits cAMP accumulation, while isozyme y enhances it (Gusovsky and Gutkind, 1991). Another clue derives from the finding that a splicing event generates a switch of CREM function from an antagonist to an activator during spermatogenesis (Foulkes et al., 1992). Similar developmental switches by alternative splicing (Foulkes et al., 1991a) or alternative usage of initiation codons in mRNA (Delmas et al., 1992) could explain the differ-


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ential regulation (positive or negative) of the c-myc, ODC, and perhaps cyclin D1 genes by cAMP in some cells stimulated o r inhibited by CAMP, whereas the expression of fos and j u n protooncogenes is induced or inhibited by cAMP irrespective of the final effect on growth. To answer such questions, it will be necessary to study a few welldefined systems in depth to obtain coherent answers. Most often, in the cells in which it is a positive control on proliferation, cAMP is also a positive signal for function, and conversely, a similar conclusion can be drawn for the role of the PIP,-Ca2+ cascade: in the cells in which this cascade activates function, it also stimulates growth, for example, in lymphocytes stimulated by IL-4, in hepatocytes, and in smooth muscle. This makes physiological sense, as a tissue stimulated repeatedly would develop its capacity to respond to stimulation, that is, would grow. In adult tissues growth is the longterm adaptation to functional stimulation. In the thyroid and somatotroph paradigms and probably in many other cell types in which function is controlled by CAMP,the CAMP-dependent stimulation of cell proliferation appears as a differentiation characteristic. It has been suggested recently that in somatotrophs the lineage-specific transcription factor GHF-1 induced by cAMP is necessary not only for CAMP-dependent transcription of the GH gene, but also for lineagespecific cell proliferation (Castrillo et al., 1991). As a specialized function the CAMP-dependent mitogenic pathway in thyroid cells possesses various odd characteristics that make it unexpectedly dissimilar from the more convergent CAMP-independent growth-stimulatory mechanisms. Unlike the tyrosine kinase and PKC growth-signaling pathways, the cAMP mitogenic cascade of thyrocytes does not utilize the phosphorylation of proteins on tyrosine, the activation of MAP kinases, or the induction of c-jun. Kinetic experiments indicate that the events associated with the prereplicative development supported by cAMP are not helpful in the CAMP-independent mitogenic cascades or vice versa. It is recognized, sometimes with difficulty, that different cell types may utilize different genes (Baserga, 1990) in their regulation of cell cycle progression. Comparison of CAMP-dependent and -independent modes of cell cycle control in thyroid cells suggests that, in a given cell, different strategies utilizing different genes and sequences of regulatory events may coexist, separately leading to cell division. Far-reaching consequences ensue, opening new research perspectives. For instance, unique characteristics of the CAMP-dependent pathway in thyrocytes might explain how CAMPdependent growth and differentiation can be compatible. CAMPdependent and -independent cell division might also have different

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consequences on the cell phenotype, which might be important in differentiation processes. Intracellular inhibitors of cell cycle progression might well be instrumental on only some particular mitogenic pathways. They might be involved in pathway-specific growth desensitization mechanisms and senescence-like processes, and may constitute “conditional antioncogenes.” While perturbations of the tyrosine kinase and phospholipase C-PKC mitogenic pathways are often found in dedifferentiated tumors, hyperactivation of the cAMP signaling cascade (the gsp oncogene and the TSH receptor) is now well documented in several examples of hyperfunctioning tumors. It might now be fruitful to search for the genes and proteins specifically involved in the cAMP mitogenic cascade and for new oncogenic mutations that might occur at various levels of this cascade. NOTEADDED IN PROOF The promoter of the gene encoding cyclin A, a pivotal regulatory protein involved in the S phase, is inducible by activation of the cAMP signaling pathway in human fibroblasts (Desdouets et al., 1995). This is mediated via a CRE and cell-cycle-regulated phosphorylation of CREB and CREMT and transient disappearance of the inducible CAMPearly repressor (ICER) (Desdouets et al., 1995). This is reminiscent of evidence of CREB involvement in the mitogenic activation (Wollberg et al., 1994) and in the induction of the PCNA gene (D. Huang et al., 1994) in T lymphocytes. Thus, even in systems in which an overall negative effect of cAMP on cell cycle was shown, intermediates of the CAMP-signaling cascade might be positively involved in the sequential expression of cell cycle regulators in response to PKA activation or the activation of other kinases by growth factors (Ginty et. al., 1994; de Groot et al., 1994). ACKNOWLEDGMENTS We thank Stein 0. Doskeland for helpful suggestions and D. Leemans for secretarial assistance. This work was supported by the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Federal Service for Science, Technology and Culture (scientific responsibility is assumed by the authors), the Fonds National de la Recherche Scientifique, Fonds de la Recherche Scientifique Medicale, Caisse GBneral d’Epargne et de Retraite, Euratom, Televie and Association contre le Cancer. P.P.R. is a Research Associate of the Fonds National de la Recherche Scientifique (Belgium). REFERENCES Aasland, R., Lillehaug, J. R., Male, R., Josendal, O., Varhaug, J. E., and Kleppe, K. (1988). Expression of oncogenes in thyroid tumours: Coexpression of c-erbB2ineu and c-erbB. Br. J. Cancer 576, 358-363. Abate, C., Marshak, D. R., and Curran, T. (1991). Fos is phosphorylated by ~34~11~2, CAMP-dependent protein kinase and protein kinase C at multiple sites clustered within regulatory regions. Oncogene 6,2179-2185. Abdel-Malek, Z., Swope, V. B., Paleas, J.,Krug, K., and Nordland, J. J. (1992).Mitogenic, melanogenic, and cAMP responses of cultured neonatal human melanocytes to commonly used mitogens. J . Cell. Physiol. 150, 416-425.


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Regulation of G Protein-Coupled Receptors by Receptor Kinases and Arrestins RACHEL STERNE-MARR AND JEFFREY L. BENOVIC Department of Pharmacology Jefferson Cancer Cancer Thomas Jefferson University Philadelphia, Pennsylvania 19107

I. Introduction 11. GRKs A. Cloning B. Tissue Localization C. Membrane Localization D. Regulation E. Receptor Specificity F. Distinguishing Features of Members of the GRK Family 111. Arrestins A. Cloning B. Polypeptide Variants C. Tissue, Cellular, and Subcellular Localization D. Mechanism of Retinal Arrestin Binding to Rhodopsin E. Mechanism of Nonvisual Arrestin Binding to Receptors F. Receptor Specificity IV. Conclusions References

I. INTRODUCTION Signal transduction via seven-transmembrane domain or serpentine receptors, which is mediated by guanine nucleotide-binding (G) proteins, accounts for a significant fraction of all signaling in the body. Heterotrimeric G proteins modulate the activities of multiple effectors, including cGMP phosphodiesterase, adenylyl cyclase, phospholipases C and A,, and potassium and calcium ion channels, which alters the levels of second messenger molecules and ultimately leads to a variety of cell-specific events (Birnbaumer et al., 1990). One ubiquitous feature of signaling through G protein-coupled receptors (GPRs) and other cell surface receptors is the rapid loss of cellular sensitivity following presentation of a stimulus, a phenomenon alternatively referred to as desensitization, adaptation, deactivation, tolerance, tachyphylaxis, or quenching (Hausdorff et al., 1990; Dohlman et al., 193

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1991).Two receptor systems have served as useful models to dissect the mechanism of desensitization: visual transduction by the light-sensitive receptor rhodopsin in retinal rod cells and chemical transduction via the catecholamine-sensitive P2-adrenergic receptor (P,AR). Absorption of a photon of light by a molecule of rhodopsin residing in the disk membrane of rod outer segments results in the isomerization of the chromophore 1l-cis-retinal to the all-trans conformation, yielding the active form of rhodopsin, metarhodopsin I1 (MII) (Chabre and Deterre, 1989; Hargrave and McDowell, 1992). Activated rhodopsin catalyzes the exchange of GTP for GDP on the retinal-specific G protein, transducin (GT),and GTP-bound GT activates cGMP phosphodiesterase. The decrease in cGMP concentration closes cGMP-gated cation channels in the plasma membrane of rod cells, generating an electrical signal which is propagated along the visual pathway. In order to perceive continuous changes of light in the environment, the rod cell must recover from light activation; therefore, a rapid (highmillisecond-second time scale) “turnoff ” mechanism has evolved. Upon light activation MI1 becomes a substrate for a retinal-specific kinase, rhodopsin kinase (RK), which phosphorylates rhodopsin in a light-dependent fashion at multiple serine and threonine residues on the C-terminal tail of the receptor (Bownds et al., 1972; Kuhn and Dreyer, 1972; Frank et al., 1973; Wilden and Kuhn, 1982).Phosphorylation of MI1 lowers the affinity of the receptor for transducin while dramatically increasing its affinity for a 48-kDa protein called arrestin (also called S antigen for its immunogenic properties). The binding of arrestin to the light-activated and phosphorylated rhodopsin physically occludes G protein interaction with the receptor, thereby uncoupling the receptor from the G protein and “arresting” transmission of the visual signal (Wilden et al., 1986). An analogous system of desensitization by the tandem action of a kinase and an arrestin is operative in the hormonal regulation of adenylyl cyclase. Binding of catecholamine to the P2AR causes the “stimulatory” G protein, Gs, to activate adenylyl cyclase (Hausdorff et al., 1990; Dohlman et al., 1991). The newly synthesized CAMP activates CAMP-dependent protein kinase to phosphorylate various target proteins, which, in turn, leads to numerous physiological responses, such as increased heart rate and smooth muscle relaxation. This system is also rapidly desensitized within a period of seconds to minutes. A kinase that phosphorylates residues in the C-terminal tail of the P2AR in an agonist-dependent fashion was initially identified in S49 lymphoma cells and named P-adrenergic receptor kinase (PARK) (Benovic et al., 1986). A molecular search for an arrestin homologue resulted in

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the identification of a bovine brain protein, called p-arrestin (parr), which could effectively uncouple PARK-phosphorylated p2AR from G, in uitro (Lohse et al., 1990). A t least three mechanisms for uncoupling G proteins from receptors are recognized: (1)homologous or agonist-specific desensitization requiring the kinase-arrestin duo as described above, (2) agonist-dependent sequestration of the receptor away from the plasma membrane into endocytic vesicles, and (3) heterologous or nonagonist-specific desensitization occurring in an arrestin-independent manner and requiring receptor phosphorylation by second messenger-dependent kinases such as the CAMP-dependent protein kinase and protein kinase C. The extent to which each of these three mechanisms is responsible for desensitization is cell type and receptor specific. For example, in rod cells rhodopsin is present not in the plasma membrane but in the disk membrane, and therefore endocytosis is unlikely to play a role in rhodopsin deactivation. Conversely, 95% of the thrombin receptors in HEL (and CHRF) cells are internalized within minutes of thrombin treatment, perhaps obviating the necessity for other desensitizing mechanisms (Brass, 1992; Hoxie et al., 1993). Since the model of homologous desensitization mediated by kinases and arrestins was elucidated in uitro with purified components (Benovic et al., 1987a; Lohse et al., 1992; Pitcher et al., 1992a1,it is important to ask whether this pathway occurs in uiuo. Clearly, in DrosophiZa a mutational analysis has verified that arrestins are required for the regulation of rhodopsin function (Dolph et al., 1992).Unfortunately, no vertebrate cell lines or animals that lack functional kinases or arrestins have been generated to enable an assessment of their role in uiuo. Nevertheless, several lines of experimentation support an in uiuo role for receptor kinases and arrestins. First, transfection of CHW cells with forms of the P2AR that lack serine and threonine residues normally present in the tail of the receptor abrogates both agonistdependent phosphorylation and rapid agonist-induced desensitization of the receptor (Bouvier et al., 1988). Second, expression of PARK or parr increases the agonist-stimulated desensitization of coexpressed p2AR in CHO cells (Pippig et al., 1993).Third, when a kinase-deficient form of PARK that retains its regulatory domains, a so-called dominantnegative mutant, is overexpressed in bronchial epithelial BEAS-2B cells, homologous desensitization of the P2AR is attenuated (Kong et al., 1994). In the last few years it has become clear that RK and PARK are members of a multigene family in which six mammalian members have been identified to date. Members of the mammalian family are

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now referred to as G protein-coupled receptor kinases (GRKs), where RK is GRKl and PARK is GRK2. Likewise, visual arrestin and Parr are members of a family that now includes four mammalian homologs, three of the four being expressed as more than one polypeptide form. These observations raise questions of the receptor specificity of these proteins: Does receptor specificity occur in uiuo? Is it defined by cell type? Is it defined by unique interactions of GRKs and arrestins with receptors? Since G protein-coupled receptors are the targets of many drugs in clinical use today, defining the receptor specificity of GRKs and arrestins could have an enormous impact on pharmaceutical efficacy. In this chapter we focus on the cloning, tissue (and cell) localization, characterization, structure-function, and specificity studies of mammalian GRKs and arrestins. Since several similar reviews have recently been published (Hausdorff et al., 1990; Dohlman et al., 1991; Palczewski and Benovic, 1991; Inglese et al., 1993; Lefkowitz, 1993; Wilson and Applebury, 1993; T. Haga et al., 19941, our goal here is to provide complementary and updated information.

11. GRKs A. CLONING The first member of the GRK family to be cloned was bovine PARK. Oligonucleotides designed from the sequences of two bovine PARKderived peptides were used to probe a bovine brain cDNA library (Benovic et al., 1989). Cloning of the other five members took advantage of techniques that are reflective of the status of molecular biology in the early 1990s: low-stringency hybridization, polymerase chain reaction (PCR), rapid amplification of cDNA ends (RACE),and positional cloning in combination with exon amplification. PARK2 (GRK3) was cloned by hybridizing a bovine brain cDNA library with a fragment from the catalytic domain of PARK as a probe under low-stringency conditions (Benovic et al., 1991a). Like the cloning of PARK, initial isolation of the cDNA for RK utilized oligonucleotides based on the sequence of the purified bovine retinal protein (Lorenz et al., 1991). After probing two bovine retinal cDNA libraries, the 5’ RACE procedure (using retinal RNA) was required to identify cDNA sequences encoding the N terminus of RK. Serendipity played a role in identification of the fourth mammalian member of the family. In a search for genes that may be responsible for the dominant defect in Huntington’s


197

disease, IT11 (GRK4) was cloned using cDNA derived from the exon amplification procedure to probe a human frontal cortex cDNA library (Ambrose et al., 1992). Unlike other members of the GRK family, GRK4 may exist as polypeptide variants that differ by the absence or presence of 32 amino acids near the N terminus (Sallese et al., 1994). Variation near the C terminus of GRK4 may exist as well (Inglese et al., 1993). In 1988 Hanks (1988) compared 65 different members of the protein kinase superfamily and defined 11 catalytic “subdomains” that contain identical or conserved amino acids in these positions in each of the kinases. In a n attempt to isolate Drosophila GRKs, oligonucleotides designed from residues in subdomains VI and VIII and biased toward the PARK and PARK2 sequences were used in PCR amplification of fly retinal cDNA. PCR products that showed highest homology to bovine PARK were then used to probe Drosophila cDNA and genomic libraries to determine the structure of the full-length proteins. Two PARK homologs, designated GPRK-1 and GPRK-2, were identified in this manner (Cassill et al., 1991). In some subdomains the sequences of PARK, PARK2, RK, GPRK-1, and GPRK-2 are conserved among the five proteins but differ significantly from other kinases in the database. Taking advantage of this observation, degenerate oligonucleotides designed from subdomains I1 and VIII of the GRKs were used in PCR amplification of human heart cDNA, while subdomains I and VIII were used as primers to amplify sequences from bovine circumvillate papillae. Both primer pairs led to the identification of GRK5 (Kunapuli and Benovic, 1993; Premont et al., 1994). Finally, the catalytic domains of PARK and PARK2 were used as probes to screen a human heart cDNA library at low stringency. A partial clone for GRKG was obtained and subsequently used to isolate a full-length clone from a human fetal brain cDNA library (Benovic and Gomez, 1993). GRK5 and GRKG were also isolated from human neutrophil RNA using the sequences from subdomain VI common to GRKs and several other protein kinases for one forward primer, sequences unique to GRK subdomain I for a second forward primer, and sequences derived from GRK subdomain VIII for the reverse primer in PCR amplification (Haribabu and Snyderman, 1993). While the GRKs are most closely related to the protein kinase C and CAMP-dependent kinase families, three features distinguish the cloned GRKs from other mammalian kinases (Figs. 1 and 2). GRKs are serinehhreonine kinases with a central 263- to 266-amino-acid catalytic domain flanked by large amino (186- to 190-amino-acid) and carboxy (82- to 236-amino-acid) regulatory domains. [The catalytic domain is

198 HUMAN IIARK HUMAN O W 2 HUMAN GRKS HUMAN GRX6 HUMAN GRK4 BOVINE RK

RACHEL STERNE-MARR AND JEFFREY L. BENOVIC ..MADLEAVL ..MRDLEAVL MELENIV MELENIV MELENIV MDFGSLEW

... ...

...

00

--

ADVSYLHRME ADVSYLHRME ANNLLKIRE ANNLLKIRE ANSLLLKARQ ANSAFIAARG

-

0

KSFATPRIRh KSKATPW ..GGGGKRKG ..GGGGNRKG ..GGYCKKSG SFDASSGPAS

SKK.....IL SKR.....IV KSKKWKEILK KSKKWRQMLQ RSKKWKEILT RDRKYLARLK

-

0

-

LPEPSIRSVM LPEPSIRSVM FPHISQCEDL FPHISQCEEL LPPVSQCSEL LPPLSKCEAL 0

-

0

QKYLEDRGEV QKYLAERNEI RRTIDRDYCS RLSLERDYHS RHSIEKDYSS RESLDLGFEG

-

GYLLFRDFCL GFLLFKDFCL GRLLFRQFCE GRLLFREPCA GRRLFRQFCD GKRLFQQFLR

CQN1,RGDVFQ CESLRGDZFC HEYLRGFPFN HEYLSVAPFA HNYLRGEPFE LAHLGQAPFQ 0 0

UF:ESDKFTR kCOHKWELN 141T!54DtSY 1 9 3 KFMFSDKFTR FCOYKSVELN I'11TT4LfSV 1 9 3

...

- 0

NHLEERReLV NEINEAVPQV TR.PGLECYI TR.PELSRCV TK.PTLKRH1 TH.EQHGPAL

-

0

00

EFYEEIKKYE KFYEEIKEYE QFLDSVAEYE AFLDGVAEYE EFLDAVAEYE QLWKDIEDYD

93

TFEKIFSQKL TFDKIFNQKI LCDK...QPI LCER HA1 LCDK...QPI MCLE ...QPI

-

93

91 91 91 96

0-

""* ... HUMAN OARK HUMAX OARK2

HUMAN GRKS HUMAN GRK6 HUMAN G R K 4 BOVINE RK

KLETEEERVA RSRElFDSYl MKELUCSHP KLDIEEDRLC RSRQlYDAYl MKELLSCSHP .YTPnFY.GF Y G Y F . I M T K I I TPKSPWIAO ...~..~ .VTPDDKRKA CGRHVTQNFL SHTGPDLIPE .VMDEDRSD CGLSILDRFF N D K W L P E .TMDALPPQ XAQALRRAYL EPQAQLFCSF

- -

ISLSAIEHVC PSKQAVCHVQ VGO2LVSOTE VPRQLVTNCT IPPDWTECR LDAETVW

SHLGKKQVPP SHLSKKOVTS EKL.LOKPCK QRL.EQGPCK LGLKEENPSK AGAGD

~-~~ ~-~~ ~

~

~-

~~

.....

DLF3PYlEEl lLFQPYlEEl ELFSACADSV DLFQELTRLT KAFEECTRVA GLFQPLLRAV

-~

~~~

-0

~~~

~

EKLDSWkDil FLOWXILEHO . . .I'VIKN:FH3

DYLDSIYFNR FLQHKWLERQ .PVTKNTFRQ EYQESSYFSQ FLQWKWLERQ .PVTKNTFRH EFLDSLYFLR FLQWKWLEAQ .PMGEDWFLD -

4

0

t t i o # t i # t # n # i n i # w n i # i # i t t # #~ Y # # R # # Y Y # # # # # # I # # # # # # u # t w + i it # t # t # # # wi in n a n n t # n # # w # # N N # N

HUMAN dARX HUMAN PARK2 HUMAN G R K 5

HUMAN GRK6 HUMAN GRKl BOVINE RK

HRIIGRGGFG HRIIGRGGFG YRVLGKGGFG YRVLGKGGFG YRVLGKGGFG FRVLGRGGFG O--O-OOW

HUMAN

LIW

HUMAN tIARK2 HUMAN GRK5 HUMAN GRK6 HUMAN GRX4 BOVINE RK

HUMAN GRK6

HUMAN GRX4 BOVINE RK HUMAN UARK HUMAN PARK2 HUMAN GRKS HUMAN GRK6 HUMAN GRK4 BOVINE RK

KKRIKEIKQGE TLALNERIML KKRIKEIKQGE TLALNERIML KKRIKKRKGE SMALNEKQIL KKRIKKRKGE AMALNEKQIL KKRIKKRKGE AMALNEKRIL KKRLKKRKGY QGAMVEKKIL

-

000-0

0

SLVSTGDCPF SLVSTGDCPF EKV...NSQF EKV...NSRF EKV QSRF AKV...HSRF

...

0- 0- -0

-

0

IVCMSYAFHT IVCMTYAFHT WNLAYAYET WSUYAYET WSLAYAYET IVSLAYAFET

0 -0

-

00- 0

PDKLSFILDL PDKLCFILDL KDALCLVLTI KDALCLVLTL KDALCLVLTI KTDLCLVMTI 0-

-- -

MNGGDLHYHL MNGGDLHYHL MNGGDLKFHI MNGGOLKFHI MNGGDLKFHI MNGGDIRYHI 00000--4-

##II##NIN#N

.... SQHGVF

....SQHGVF YNM..GNPGF YHM..GQAGF YNL..GNPGF YNMEDNPGF C

# # + # # # # a # # # # # # t i # #i ## # t i t i n # # t# # # # : # # # ai # aa##n#### ###xaa###a

SEMMPXYAA SEKEMRFYAT EEERALFYIU PEIRIWYAA

PANILLDEHG PANILLDEHG PENlLLD3YG P5NILLDZYG PEV1LLD:IG PENVLLDDDG

HVRISDLGLA HARISDLGLA HIIISDLGLA H1IlSC:Z:A HlRlSCLGLA NVRISDLGLA

o-ooo-

-00000000

NRFWYRDLK NRFWYRDLK ENWYR3LX RCRIVYR3L% D E. O_ R A W Y L 4 ELCCGLPDLO RERIVYRDLK . QEPRAIFYTA QIVSGLEHLH QRNIIYRDLK

-w

EIILGLEHMH EIILGLEHVH ElLCGLEDLH LICCGLEDLH

-~

-

000

-

-00000 0

i i t w i ~ i +t # i w i n t i # t o i t i i u i HSPFRQHKTK DKH.EIDRHT LRlAVELPDS HSPFRQHKTK DKH.EIDRM LTVNVELPDT QSPFRGRKEK VKFZEV3PRV LETELVYSHK QSPFQQRKKK 1KRTEYERLV KEWEEYSES L ( P F I Y I Y F Y YI(YEBMORI .. .-.. KXDtEEYSEK RGPFRARGEK VENKELKQRV LEQAVTYPDK 00 0 0-

__

SFDELDTKGI SFDEEDTKGI OF..STVI(GV QF..STVKGV QF..SAVKGI AF..STVKGV 0

00-

HUMAN

UARKZ HUMAN GRKS HUMAN G R X 6

NRLEWRGEGE NRLEWRGEGE RPSQNNSKSS R..QDCCGNC

HUMAN @ARK HUMAN #ARK2

ANGL .NGL

HUMAN ,#ARK

GKMYAMKCLD GKMYAMKCLD GKMYACKRLE GKMYACKKLE GKHYACKKLQ GKLYACKKLN 00--0 0 0

KLLDSDQELY KLLDCDQELY NLDHTDDDFY ELEPTDQDFY YLDTMEDFY AFEKROTEFF 0

-

-

APQSLLTMZE SRQNLLTMEQ PSSKTSFNHH SDSEEELPTR

R&FPL.T:SE KMPL.V:SE SKFSTGSVS: QKFATGSVPI lUIFATGCVSI QEFASGTCPI 0

0

CDF..SKKKP HASVGTHGYM APEVLQKGVA CDF..SKKKP HASVGTHGYM APEVLQKGTA W ! ? E G D L . I RSSVGTVGYH APEVL.rCN3R VHV?EGOf.I KSRVGTV;YV APEW.KUL3 TE:?EZDR.V RZRVGTVGYM A?EW.NULK VELKAGQTKT KGYAGTPGFM APELL LGEE

-

-00 0-0 000.-

~~~~~~~~

00

-

MLLTKDMQI CLLCKCPAER HLLTKXPSKR ALLQKDPEKR ~~~

00

~~

-

LGCOEECME VKRHPFFiWn NFKRLIAGXL :G:RGSSAW WEHPLFKKL NFKRLGAGUL LGCRGEGIUG VKOHPVFK3I NFRRLEANW. LGFRDGSCDG LRTHPLFRDI SWRQLEAGML ~

0 00

~~

~

~

- - o

_____

289 289

283 283 284 286

YDSSADWFSL GCMLFKLLRG YDSSADWFSL GCMLFKLLRG

387 387

YSLSPCYWL1 '%C1IYPY!t6

381

G-:'"?YIA? YT'S?XWA: YTFSP3YWGL G: IYlVlOG YDFSVDYFAL GVTLYEMIRR

381 382 385

0

0 0.-

0 0

--

-0

i # i a # ~ n : n t a w t n i # : n ~ ~ t i i i i i#i #i i i t + # FSPELHSLLE GLLQRDVNRR LGCLGRGAQE VKESPFFRSL D W Q W L Q R Y PPPLIPPRGE VNAADAFDIG FSPELKSLLE GLLQRDVSXR LGCHGGGSQE VKEHSFFKGV DWQHVYLQKY PPPLIPPRGE VNAADAFDIG

FSEEAKSICK FSPQARSLCS FSEOAKSICR FSPASKDFCE

189 189

0

# i t # t i t i # tw # i t i # i i ## i # n t # i t # #a # # u + t n n # #

0

HUMAN dARK HUMAN BARK2 HUMAN GRK5

EVYGCRKRDT EVYGCRKADT EVCACQVRAT EVCACQVRAT EVCACQVRAT EVFACQMAT 00 -0 0

-

oooo-o-

188 188

3PYtVYDPRA EPPFKPDPQA EPPFCPDPHA TPPFVPDSRT 00

0

VYCKJVL2:I :YCKDVLC:L VYCKOVL3:E VYAKNIQDVG

-

-

186 486 481 481

482 485

0-

RWQQEVAEN F3TlliAETDR :.EARKKAKNK QLSHEEDYAL CKDCIMHGYM SKNGNPFLTC WQKRYFILtP

585

RWQQEVTEN YEAVNMTDK IE*RKRAIC4K QLGHEECYAL CKDCIMHGYM LKLSIPFLTQ WQRRYFYLF?

585 556 557

PYCNEMlETE PWQNEMYETE PUQNEDCLTM PWQEEMIETG 00 0

0

C......... C......... V......... V.........

..._.._.._ ...kKELNVF GPNGTLPP3L NR.IHPPEPP KKGLLORLFK ..__._..._ ...FQELNVf GLDGSVPPDL DWKGQPPAPP KKGLLQRLFS .......... ...PSEKEVE PKQC...... .......... .......... .......... ...FGDLNVW _ _ RPDGPEIPDDM KGVSGQEAAP SSKSGMCVLS

IQSVEETQIK ERKCLLLKIR GGKQFILQCD SDPELVQWKK ELRDAYREAQ QLVQRVPKMK NKPRSPWEL SKVPLVQRGS ILSVEETQIK DKKCILFRIK GGKQFVLQCE SDPEFVQWKK ELNETFKEAQ R L L W K F L NKPRSGTVEL PKPSLCHRNS INSNHVSSNS TGSS...... L.........

.......... .......... ..__..__.. .......... .......... .......... .......... .......... .......... ._.._._.__ .......... ........_...........

FIG. 1. Comparison of amino acid sequences of human PARK, PARK2, GRK5, GRK6, and GRK4 and bovine RK. The predicted sequences were aligned using the Pileup program [Wisconsin Genetics Computer Group (GCG)]. #, Residues that are part of the kinase catalytic domain as defined by Hanks and Quinn (1991); 0, identity among all six homologues; -, similar residues. For this analysis amino acids were deemed to be similar to other residues within six groups as follows: (a) S, T, and C; (b) D and E; (c) N and Q; (d) R, K, and H; (e) Y, W, and F; and (f) M, A, I, V, and L. The amino acids are numbered on the right-hand side of the sequence. The sequences for PARK, PARKB, GRK4, GRK5, RK, and GRK6 were obtained from the following sources: Benovic et al. (1991b), Parruti et al. (1993a), Ambrose et al. (19921, Kunapuli et al. (19931, Benovic and Gomez (1993). and Lorenz et al. (1991).

defined as described by Hanks and Quinn (199U.l With the exception of specific sequences in RK, PARK, and PARK2 (described below), neither the N- nor C-terminal regulatory .domains show significant

532 561

685 685 590 576

199


RK BARK

100

33.4(57.8)

33.566.1)

47.0(68.1)

47.266.5)

47.1(69.0)

100

83.7 (92.0)

36.8 (56.5)

37.0 (58.3)

38.6 (59.8)

100

37.0 (57.4)

37.8 (58.3)

36.3 (57.0)

100

68.7 (82.1) 67.6 (82.5)

BARK2 GRK4 GRK5 GRK6

100

70.1 (83.5)

100

FIG. 2. Comparison of amino acid homologies between the various GRKs. Amino acid sequences from human PARK, PARK2, GRK4, GRK5, and GRKG and bovine RK were compared in a pairwise fashion using the Gap program [Wisconsin Genetics Computer Group (GCG)]. The percentage of amino acid identity is given as well as the percentage of similarity (in parentheses). The references are as listed in the legend to Fig. 1.

homology to proteins in the database outside the GRK family. As mentioned above, the sequences of some GRK catalytic subdomains (I, 11, VI, VII, and VIII) are unique to the family. Most obvious in this regard is the substitution of leucine for phenylalanine in subdomain VII (DLG instead of DFG, which is found in all but two other protein kinases). The hallmark of the original members (RK and PARK) of the GRK family is their ability to specifically recognize and phosphorylate only the agonist (or lightbactivated form of the receptor. Indeed, all GRKs (except GRK4, which has yet to be heterologously expressed in a n active form), when expressed in COS-7 or Sf9 insect cells, specifically phosphorylate PzAR and/or rhodopsin in an agonist-dependent fashion (Lorenz et al., 1991; Benovic and Gomez, 1993; Kim et al., 1993b; Kunapuli and Benovic, 1993). B. TISSUELOCALIZATION l h o members of the GRK family appear to be expressed in a limited number of tissues (Table I). RK is predominantly expressed in the retina, where it localizes to both rod and cones by immunofluorescence, and in the pineal body to a lesser extent (Somers and Klein, 1984; Palczewski et al., 1993). While GRK4 was cloned from a brain library, its transcript is not easily detectable by Northern analysis in any tissue except testis (Ambrose et al., 1992). In contrast, PARK, PARKB, GRK5, and GRKG are ubiquitous. The patterns of tissue expression for PARK, PARKB, and GRKG are similar, with each homologue being most abundant in the brain, skeletal muscle, and hema-

TABLE I MOLECULAR PROPERTIES OF THE GRKsa Parameter

RK

PARK

PARK2

GRK4

Polypeptide mol. wt. Amino acids N-terminal Catalytic C-terminal Receptor sub&ratese

62,933

79,463

79,803

561 186 266 109 Rhodopsin, P2AR Acidic

688 190 263 235 P2AR, rhodopsin, m2 mAChR, m3 mAChR, SPR, azAAR, azcAR Acidic

532 186 264 826

Peptide substrates Tissue distribution

689 190 263 236 P2AR, rhodopsin, m2 mAChR, m3 mAChR, SPR, aZAAR, azcAR Acidic

Retina > pineal body

Ubiquitous, brain, hematopoietic, skeletal muscle

Testis

+++

+

Ubiquitous, brain, hematopoietic, olfactory, skeletal muscle

Autophosphorylation CovaIent modifications

Farnesylation

?

?

?

?

* brain

GRK5

GRK6

67,786

66,074

590 185 263 142 P A R , m2 mAChR, rhodopsin

576 185 263 128 P2AR, m2 mAChR, rhodopsin

Neutral > acidicd

Neutral > acidicd

Ubiquitous heart, lung, placenta, hematopoietic, retina

Ubiquitous, brain, hematopoietic, skeletal muscle

+

?

+++

9

9

9

+ Palmitoylation

Activators

Polycations

Inhibitors Sangivamycin Heparin sensitivity ? Chromosomal localizationf mRNA size (kb) 3.land 5.8

By-subunits, PS, PA, PG, PI, PE

py-subunits, PS, PA, PG, PI, PEe

?

Polyanions, PIP,

Polyanions, PIP,

? ?

llq13

22qll

4

8

+

Polycations, PC, PE, DAG, fatty acids Polyanions

Polycations

4p16.3

10q24-qter

2.5

3

5q35 13pter-q21 2.4 and 3

+

+++

Polyanions

++

aP2AR, p,-Adrenergic receptor; mAChR, muscarinic cholinergic receptor; SPR, substance P receptor; a,,AR, a,,-adrenergic receptor that maps to human chromosome 10; IX,~AR,a,,-adrenergic receptor that maps to human chromosome 2; PS, phosphatidylserine; PA, phosphatidic acid; PG, phosphatidylglycerol; PI, phosphatidylinositol; PE, phosphatidylethanolamine;PC, phosphatidylcholine; DAG, diacylglycerol; PIP,, phosphatidylinositol-4,5-bisphosphate. bPolymerase chain reaction analysis has suggested the presence of a n insert (the sequence of which has yet to be reported) in the C-terminal domain (Inglese et al., 1993). CReceptors that can act as phosphoacceptors in a light- or agonist-dependent fashion in uitro (irrespective of efficacy) or implicated as substrates from in viuo experiments. dAll peptides contained three arginine residues at the N terminus, which aids in the isolation of peptides on phosphocellulose paper. Peptides differed in that nearest neighbors of the phosphoacceptor serine were either neutral (alanine) or acidic (glutamic acid, aspartic acid, and phosphoserine). .The effects of phospholipids on PARK2 are presumed based on the similarity between @ARKand PARK2 i n the region thought to be involved in phospholipid binding. fSee the work of Benovic et al. (1991b), Calabrese et al. (1994b), Ambrose et al. (1992), Bullrich et al. (1995), and Haribabu and Snyderman (1993).

202


topoietic cells (Benovic et al., 1989, 1991a; Chuang et al., 1992; Benovic and Gomez, 1993; Parruti et al., 1993a). The tissues in which GRK5 is most prevalent are the heart, placenta, lung, retina, and hematopoietic cells (Kunapuli and Benovic, 1993; Premont et al., 1994; Sallese et al., 1994). Olfactory tissue expresses single isoforms of several proteins involved in signal transduction, that is, Golfand type I11 adenylyl cyclase. In line with this observation, PARK2 is apparently the only GRK expressed in olfactory epithelium (Dawson et al., 1993; Schleicher et al., 1993). With the generation of specific antibodies against each homologue it will be possible to quantitatively assess the relative amounts of each GRK in a given tissue or cell line. In situ hybridization and immunocytochemistry have been used to investigate PARK and PARK2 localization in the brain (Arriza et al., 1992). Both PARK and PARK2 are found extensively in many, but not all, regions of the brain. The patterns of abundance of the two proteins are similar but distinguishable. PARK is more abundant in most regions, but PARK2 was preferentially expressed in some. The expression of PARK and PARK2 does not precisely parallel the localization of the P2AR, consistent with the observation that other GPRs are substrates for these enzymes in uitro (see below). Among cell types in the brain, PARK and PARK2 are found predominantly in neurons. Both proteins are found in cell bodies, postsynaptic densities, and presynaptic axon terminals. C. MEMBRANE LOCALIZATION Although GRKs phosphorylate disk or plasma membrane receptors, RK and PARK are found in the soluble fraction following disruption of rod outer segments or cells, respectively. Upon agonist stimulation PARK activity has been reported to translocate from the cytosol to the membrane (Strasser et al., 1986; Mayor et al., 1987; Chuang et al., 1992). However, receptor activation alone may be insufficient to direct membrane translocation, since the GRKs appear to utilize distinct mechanisms to specify their subcellular localization. The C-terminal domain of GRKs plays a n important role in the membrane association of these kinases. The sequence of rhodopsin kinase terminates with -CVLS, which follows the consensus “CAAX box” (where C is cysteine, A is a small aliphatic residue, and X is a n uncharged amino acid), directing farnesylation (C15 isoprenylation) and carboxymethylation of the resulting farnesylated cysteine (Gibbs, 1991). Indeed, RK is farnesylated (Anant and Fung, 1992; Inglese et al., 1992a). Isoprenylation is neces-


203

sary for the membrane localization of one class of yeast mating pheromones; members of the ras superfamily, including some small G proteins involved in vesicular trafficking; the y-subunits of heterotrimeric G proteins; and the a-subunit of cGMP phosphodiesterase. Similar to small G proteins, but unlike the Ras oncoprotein, RK is not constitutively associated with the membrane and probably cycles on and off the membrane. In an in uitro assay RK associates with rod outer segments only in the presence of light (Inglese et al., 1992a,b). A mutant RK that lacks the isoprene moiety fails to translocate to the membrane upon light activation and also has diminished kinase activity. Conversely, a mutant that is modified with the more lipophilic C20 geranylgeranyl group is associated with rod outer segments independent of light activation and retains full kinase activity. Thus, isoprenylation not only directs RK to the membrane, but probably orients the kinase for optimal phosphorylation of its substrate. Unlike other GRKs, the ability of PARK and PARK2 to phosphorylate P2AR, M, muscarinic cholinergic receptor (m2 mAChR) and rhodopsin is significantly enhanced by brain Ply-subunits (Haga and Haga, 1990, 1992; Pitcher et al., 199213) and the Ply binding site of PARK has been mapped to residues 546-670 (Koch et al., 1993). This domain overlaps a recently described -100-amino acid domain (residues 553-656 of PARK), called the pleckstrin homology (PHI domain, found in cytoskeletal proteins as well as proteins involved in various signal transduction pathways (Musacchio et al., 1993; Gibson et al., 1994). While PH domains from several proteins are capable of binding Ply subunits, it is the C-terminal half of the PH domain and residues distal to the end of the recognized PH domain that are involved in the P/y interaction (Touhara et al., 1994). It has independently been proposed that residues 639-670 of PARK form a three-stranded coiled coil with polypeptide strands from the P- and y-subunits (Simonds et al., 1993), and this suggestion is consistent with the presence of a C-terminal a-helix, determined from the crystal and solution structures of PH domains (Macias et al., 1994; Yoon et al., 1994). It should be noted, however, that full-length PARK binds to ply-subunits with a Kd of -30 nM (Kim et al., 1993a), which is at least 10-fold tighter than the IC,,s (determined by phosphorylation inhibition or translocation inhibition assays) of fusion proteins bearing the entire C-terminal domain of PARK interacting with Ply-subunits (Koch et al., 1993; Touhara et al., 1994). Thus, it is possible that residues in the N-terminal and/or catalytic domain of PARK contribute to its high-affinity binding to p l y-subunits. The C-terminal domains of PARK and PARK2 are -120 amino acids

204


longer than that of RK (Fig. l),and truncation of the PH domain of PARK t o produce a RK-sized kinase abolishes Ply-inducible activity and even lowers the basal activity (Koch et al., 1993). Engineering a site for the addition of a geranylgeranyl group to the truncated PARK restores kinase activity up to -50% of the level observed with fulllength PARK in the presence of Ply-subunits. Since the Ply-subunits are constitutively associated with the membrane by virtue of geranylgeranylation of the y-subunit, Ply binding to PARK likely plays the same role in membrane localization as isoprenylation does for RK. Some PH domains also bind the membrane lipid phosphatidylinositol4,5-bisphosphate (PIP,), and residues in the N terminus of the PH domain are involved in this interaction (Harlan et al., 1994). In fact, recent studies have demonstrated that PARK can bind directly t o phospholipid vesicles containing either PIP, or phosphatidylserine (DebBurman et al., 199513). Interestingly, PIP, inhibits PARK activity severalfold, while PS, phosphatidylethanolamine,phosphatidylinositol, phosphatidic acid, and phosphatidylglycerol (but not phosphatidylcholine) all activate PARK -2-fold. Thus, these lipids may play a role in PARK localization and regulation. When cells from tissues or cells in culture are lysed in low or physiological salt concentrations by disruption utilizing sheer force, most of the immunoreactive PARK is found in the soluble fraction. In contrast, when cells are gently lysed in the presence of osmotic support, a substantial amount of PARK activity (as measured by the ability to phosphorylate rhodopsin) is associated with the membrane fraction (GarciaHiguera et al., 1994).In fact, sucrose gradient fractionation and immunoelectron microscopy studies indicate that 39-50% of the PARK is associated with the endoplasmic reticulum, 14-18% is associated with the plasma membrane, and 31-43% is associated with the cytoplasm. Furthermore, PARK can bind to microsomal membranes in a reversible and saturable manner. While the interaction of PARK with intracellular organelles is intriguing, the function of this association is currently unknown. Residues located in the C-terminal domain of GRK5 undergo autophosphorylation, which can be stimulated by the presence of various lipids, including phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, myristic acid, and palmitic acid (Kunapuli et al., 1994a). Direct binding of GRK5 to phosphatidylcholine vesicles can be blocked by a fusion protein containing the last 102 amino acids of the kinase, implicating this domain in lipid binding. Unlike RK, which undergoes light-dependent association with rod outer segments in an in uitro translocation assay, GRK5 associates with rod outer segment membranes in the dark or light in the absence or presence of ATP (Premont


205

et al., 1994). Thus, it seems possible that in the cell GRK5 is constitutively localized at the membrane. GRKG is covalently modified by palmitoylation of one or more cysteine residues located 12-15 amino acids from the C terminus of the protein at positions 561, 562, and 565 (Stoffel et al., 1994). When GRKG is overexpressed in Sf9 insect cells, all of the palmitate-conjugated protein is localized to the membrane fraction, while the majority of GRKG remains soluble. It is not clear whether the presence of cytosolic GRKG is a result of saturation of the acylation machinery and whether a greater percentage of GRKG may be palmitoylated when the protein is expressed at endogenous levels. Thus, it is not yet clear in normal cells whether GRKG is constitutively associated with the membrane or whether its membrane localization is modified by receptor activation.

D. REGULATION One of the fascinating features of GRKs is their ability to specifically recognize the light- or agonist-activated form of the receptor. Rhodopsin is phosphorylated by RK at as many as seven sites located within a 10-amino-acid stretch of the C terminus of rhodopsin (Wilden et al., 1986). However, since the C-terminal tail of rhodopsin can serve as a substrate for proteases in the absence of light, it is not likely that activation recognition simply involves exposure of the substrate serines and threonines to the kinase. Instead, since activated rhodopsin promotes the phosphorylation of free peptides by RK, intracellular domains of the receptor must alter the kinase, thereby converting it from an inactive to an active enzyme (Fowles et al., 1988). A form of rhodopsin that lacks the C-terminal tail (G32Q-Rho)is sufficient to activate RK, suggesting that the substrate peptide is not involved in kinase activation (Palczewski et al., 1991b). Proteolytic clipping of the third intracellular loop abrogates activation, implicating this loop in RK activation, analogous t o the requirement of this loop for GT activation. Stimulation of peptide phosphorylation by light-activated G329Rho (G329-Rho") is a result of increasing the V, as well as lowering the K,. Peptides from the first, second, and third loops and the C-terminal tail had previously been shown, by stimulation or inhibition of kinase activity, to interact with RK (Palczewski et al., 1988, 1989a; Kelleher and Johnson, 1990). Furthermore, the wasp venom peptide mastoparan, which has been shown to mimic the receptor activation of G proteins, also stimulates peptide phosphorylation by RK (Palczewski et al., 1991b). An antipeptide antibody that recognizes residues 17-34 of RK blocks the ability of RK to phosphorylate rhodopsin. Since this

206


antibody has no effect on the catalytic activity, as judged by the ability of RK to phosphorylate exogenous peptides in the presence of antibody, these experiments suggest that the N terminus of RK may be a site of activation by rhodopsin (Palczewski et al., 1993). Light-activated rhodopsin (Rho”) and agonist-treated P2AR and m2 mAChR each activate PARK to phosphorylate exogenous peptides (Chen et al., 1993; K. Haga et al., 1994). The EC5,s for p2AR and rhodopsin are 12 nit4 and -1 FM,respectively, suggesting that PARK interacts with P2AR with a much higher affinity than it interacts with Rho, consistent with kinetic studies (Benovic et al., 1987b).Residues in the first intracellular loop and the N-terminal portion of the third intracellular loop of P2AR are likely candidates for involvement in kinase activation, since peptides encompassing these regions are potent inhibitors of receptor phosphorylation by PARK (Benovic et al., 1990). Peptides derived from the second and third loops and C-terminal tail of the m2 mAChR as well as mastoparan also stimulate PARK to phosphorylate the m2 mAChR and a substrate consisting of a portion of the third intracellular loop of m2 mAChR linked to glutathione-S-transferase (GST) (GST-3I-m2AChR) (K.Haga et al., 1994).Some peptides, especially those that lack acidic residues, are extremely poor substrates for PARK (Benovic et al., 1990; Onorato et al., 1991). However, activation by Rho* can increase the catalytic efficiency (VmaX/Km) of a poor substrate (RRRASAAASAA) as much as 200-fold (Chen et al., 1993). By comparison, the catalytic efficiency of a “good” substrate is increased only -10-fold. Thus, the acidic nature of the substrate is not essential for catalysis, and instead, it is possible that, once activated, the kinase may phosphorylate any serine or threonine in close proximity to the substrate binding pocket. G protein Ply-subunits increase the initial rate of PARK (and PARK21 phosphorylation of Rho, P,AR and muscarinic acetylcholine receptors 10-fold (Haga and Haga, 1992; Pitcher et al., 1992b; Kim et aZ., 1993a,b). Brain Ply-subunits are more effective than transducin P l y (Pitcher et al., 1992b), and the effect is observed with Ply-subunits derived from purified G,, Gi, and Go (Haga and Haga, 1992). As described above, because ply-subunits are membrane associated due to the isoprenylation of the y-subunit, interaction of PARK with the PI y-subunits may serve as a mechanism for membrane association of the kinase. Allowing PARK to diffuse in two, rather than three, dimensions to find the receptor in the membrane can account for a dramatic increase in initial rates of phosphorylation. However, Pl y-subunits do have a small but detectable capacity to increase the phosphorylation of exogenous peptides (Kim et al., 1993a). Furthermore, the stimulation

-


207

of PARK activity by activated receptor together with Ply-subunits is greater than the sum of the stimulation by either of these agents alone. This synergy has been demonstrated in five assay systems: direct binding of PARK to Ply and P2AR; agonist-treated P2AR and P l y stimulation of peptide phosphorylation; G329-Rho*and P l y stimulation of peptide phosphorylation; mastoparan and P l y stimulation of m2 mAChR phosphorylation; and mastoparan and P l y stimulation of GST-3I-m2AChR (Kim et al., 1993a; K. Haga et al., 1994). Therefore, it is clear that a ternary complex consisting of PARK, P l y , and activated receptor is formed, and the simultaneous interaction of PARK with receptor and Ply-subunits generates the maximally active kinase. RK and GRK5 undergo intramolecular autophosphorylation at multiple sites, while autophosphorylation of PARK, PARKB, and GRK6 is substoichiometric (Kelleher and Johnson, 1990; Buczylko et al., 1991; Kunapuli et al., 1994a; Loudon and Benovic, 1994). Autophosphorylation of RK occurs rapidly to yield three phosphates per mole and more slowly to achieve the maximal phosphorylation of four phosphates per mole (Kelleher and Johnson, 1990; Buczylko et al., 1991). The serines and threonine at positions 21, 488, and 489 have been identified as phosphoacceptors (Palczewski et al., 1992). Phosphorylated and unphosphorylated RKs do not differ appreciably in their ability to bind to (Buczylko et al., 1991) or utilize light-activated rhodopsin as substrate (Kelleher and Johnson, 1990). However, phosphorylated RK binds only weakly to phosphorylated and light-activated rhodopsin (Rho*-PI,while unphosphorylated RK demonstrates significant binding to Rho*-P (Buczylko et al., 1991). Based on these results, it has been suggested that autophosphorylation of RK may aid the dissociation of the kinase from the receptor following its phosphorylation. Phosphorylation of receptors by GRK5 requires prior autophosphorylation of S484 andlor T485, since substitution of alanine a t these two positions abolishes autophosphorylation as well as Rho* and &AR* phosphorylation (Kunapuli et al., 1994a). Interestingly, autophosphorylation is stimulated by several types of lipids, but not specifically by the activated receptor (Kunapuli et al., 1994a; Premont et al., 1994). Unphosphorylated GRK5 can bind to lipid, since the autophosphorylation-defective mutant effectively competes with the wild-type protein for binding to lipid vesicles. Light-activated rhodopsin stimulates the phosphorylation of exogenous peptides by GRK5, although the level of activation is low relative to Rho* stimulation of PARK. Although GRK5 phosphorylation of rhodopsin is strictly light dependent, significant phosphorylation of P2AR occurs in the presence of antagonist (Premont et al., 1994). Phospholipid-stimulated autophos-

208


phorylation of GRK5 may be analogous to P l y binding to PARK in that it provides a means of membrane localization as well as sufficient activation to allow phosphorylation of an antagonist-occupied receptor. While palmitoylation certainly plays a role in the membrane localization of GRKG, the lipid may also be required for receptor activation of the kinase. This is a prediction based on the assumption that the GRKG that has been characterized to date was purified from Sf9 insect cells, in which the majority of the expressed kinase is not palmitoylated. Relative to GRK5 and PARK, GRKG displays a poor capacity to phosphorylate receptors but a comparable ability to phosphorylate nonreceptor substrates, such as casein and phosvitin (Loudon and Benovic, 1994). E. RECEPTORSPECIFICITY Upon stimulation of intact cells with various agonists, soluble “PARK” activity diminishes, while membrane-associated kinase activity increases. In S49 lymphoma cells isoproterenol (a P-agonist) and prostaglandin El (PGE,) treatment promote an -80% decrease in soluble PARK activity, while increasing the membrane kinase activity -10-fold (Strasser et al., 1986). Somatostatin induces -50% diminution in soluble PARK activity in S49 lymphoma cells (Mayor et al., 1987). In mononuclear leukocytes platelet-activating factor (PAF) decreases soluble PARK activity -3-fold, while increasing kinase activity in the particulate fraction over 2-fold (Chuang et al., 1992). These results suggest that P-agonists, PGE,, somatostatin, and PAF induce translocation of PARK from the cytosol to the plasma membrane and imply that PARK plays a role in the desensitization of the cognate receptors. If so, PARK would be involved in the regulation of at least three types of GPRs: those coupled to the stimulation of adenylyl cyclase (PAR and PGE,), stimulation of phospholipase C (PAF),and inhibition of adenylyl cyclase (somatostatin). Interestingly, in DDT,-MF2 smooth muscle cells, in which a P-agonist increases membrane PARK activity, an a,AR agonist did not alter PARK activity (Strasser et al., 1986). In view of the current understanding of GRKs, these results must be put in perspective. Since the Ply-subunits from Gs, Gi, and Go all stimulate PARK activity (Haga and Haga, 1992), agonist stimulation may induce membrane association of PARK irrespective of receptor interaction. Thus, PARK translocation may be necessary but insufficient for receptor phosphorylation. Furthermore, the assays used to measure PARK activity (phosphorylation of P2AR and Rho) do not distinguish between GRKs. Thus, the “PARK” activity in these studies


209

could also have included PARK2, GRK5, GRK6, and possibly other activities. Direct interaction of GRKs with receptors following agonist treatment may be necessary to infer specificity in uiuo. Several combinations of receptor and GRK preparations have been used to assess the specificity of GRKs in uitro (summarized in Table 11). With the exception of rhodopsin, native receptors in crude membranes do not exist in sumcient quantity to allow their detection by kinase assay due to the substantial “background” generated by other endogenous membrane kinases and their substrates. Therefore, receptor purification from endogenous sources was initially used to assess phosphorylation of the P2AR (Benovic et al., 19861, m2 mAChR (Kwatra et al., 19891, and a,*AR (Benovic et al., 1 9 8 7 ~by ) PARK. Alternatively, overexpression of receptors in Sf9 insect cells followed by affinity chromatography has provided ample amounts of specific receptors: P2AR (Kim et al., 1993b), m2 mAChR (Richardson et al., 1993), and substance P receptor (Kwatra et al., 1993). Likewise, PARK, pARK2, GRK5, and GRKG have all been overexpressed in and purified from Sf9 cells (Kim et al., 1993b; Kunapuli et al., 199413; Loudon and Benovic, 1994). Using in uitro assays with purified P,AR, m2 mAChR, and GRKs, PARK and PARK2 (in the presence of Ply-subunits) are the best kinases, as determined by initial rate and extent of phosphorylation. In fact, no significant differences between PARK and PARK2 have been observed in uitro. GRK5 has similar or slightly less activity than PARK and pARK2, while GRKG has significantly less activity using these receptors as substrates. It must be noted, however, that GRKG may not be fully activated in these assays, because it is likely that the majority of the preparation is not palmitoylated (Stoffel et al., 1994). Likewise, GRK5 may require co- or post-translational modification or a cofactor for full activation. Enrichment of a plasma membrane fraction by sucrose gradient centrifugation (Pei et al., 1994) and stripping total membranes with 4 M urea (DebBurman et al., 1995a) are techniques that have recently been used to demonstrate the agonist-dependent phosphorylation of receptors in membranes from Sf9 cells overexpressing GPRs. These procedures have obviated the need to purify and reconstitute receptors into lipid vesicles. Using urea-treated membranes as substrates, the m2 and m3 mAChRs are both much better substrates for PARK and PARK2 than they are for GRK5 and GRKG (DebBurman et al., 1995a). With sucrose gradient-purified membranes aZcAR is better substrate for PARK and PARK2 than it is for GRK5, while PzAR is good substrate for PARK, pARK2, and GRK5 (Pei et al., 1994). One feature of the m2 mAChR and CX,~AR in membranes that distinguishes them

TABLE I1 GRK RECEPTOR SPECIFICITY Assay

Kinase preparation

Receptor preparation

I n vitro phosphorylation

Purified

Purified

I n vitro phosphorylation

Purified

Sf9 membranes from cells overexpressing receptor

Crude soluble fraction from Sf9 cells overexpressing kinase In vivo desensitization Injection of RNA into Xenopus oocytes (Ca2+ mobilization) In vivo desensitization Overexpression of kinasedefective PARK mutant (CAMPproduction) in BEAS-2B cells GRK antibody inhibi- Endogenous tion of desensitization in permeabilized olfactory epithelium

Zn vitro phosphorylation

Receptor

GRK preference

Rhodopsin PZAR %*AR m2 mAChR PzAR azcAR m2 mAChR m3 mAChR PAR rhodopsin

PARK-PARK~ZGRK~>GRK~O PARK- PARKB?GRKS>GRKG PARK pARK-pARKS>GRK5>GRKG PARK=PARKBzGRKB PARK = PARK2 > GRK5 PARK PARK2 + GRKS, GRKG PARK = PARK2 + GRKS, GRKG PARK GRKB > GRKG PARK > GRKS > GRKG

Injection of RNA into Xenopus oocytes Endogenous

Thrombin receptor PzAR > PGE, receptor*

PARK2 > PARK > RK

Endogenous

Citralva (olfactory receptor)

Purified

i=

OIt is likely that the majority of the GRKG used in these experiments was not palmitoylated, potentially resulting in lower levels of receptor phosphorylation (see text). *The “PGE, (prostaglandin E,) receptor” appears to be the EP2 prostanoid receptor (J. Regan, personal communication).


211

from purified and reconstituted receptors is that the stoichiometry of phosphorylation by PARK in uitro is similar to the observed level of receptor phosphorylation in whole cells following agonist treatment (Pei et al., 1994; DebBurman et al., 1995a). In contrast, purified and reconstituted receptors incorporate -2-fold more phosphate (relative to the in uiuo level) when assayed in uitro. By virtue of its preferential expression in olfactory epithelium relative to PARK, PARK2 appears to be specifically involved in the regulation of odorant receptors (Dawson et al., 1993; Schleicher et al., 1993). When added to permeabilized olfactory cilia, PARK2, but not PARK, antibodies block desensitization of odorant receptors coupled to the production of CAMP.In isolated olfactory cilia the simultaneous addition of a mixture of odorants results in the dose-dependent translocation of PARK2 immunoreactivity from the soluble to the particulate fraction (Boekhoff et al., 1994). Further, a GST fusion protein encoding the C terminus of PARK2, when added to permeabilized cilia, prolongs the odorant-induced stimulation of cAMP and blocks the phosphorylation of putative membrane receptors. These results suggest that upon odorant stimulation, PARK2 translocates to the ciliary membrane where receptors or other membrane proteins are phosphorylated, leading to the rapid diminution of cAMP production. Since Protein kinase A is also required for desensitization of the odorant response (Schleicher et al., 1993), the details of these events may be a permutation of the current models for desensitization of rhodopsin by RK and arrestin and PzAR by PARK and parr. PARK2 also appears to regulate the thrombin receptor in an in uiuo reconstitution system (Ishii et al., 1994). When RNA for the thrombin receptor is coinjected into Xenopus oocytes along with RNA encoding PARK, PARK2, or RK, only the PARK2 RNA significantly diminished thrombin receptor signaling, even though lysates prepared from oocytes injected with each of the GRKs could aptly phosphorylate P2AR or rhodopsin. Injection of mutated forms of PARK2 RNA, including deletions of subdomains I and I1 or site-directed mutagenesis of subdomain VII of PARK2, leaves thrombin receptor signaling unaltered. Likewise, substitution of serine and threonine residues in the C-terminal tail of the receptor with alanine residues allows the receptor to respond to thrombin but yields a receptor that is refractory to coinjection of PARK2 RNA. These results strongly implicate PARK2 in the regulation of the thrombin receptor, albeit in a n environment where this receptor is not naturally expressed. Another approach to assessing the in viuo specificity of GRKs is the use of dominant-negative mutations. An invariant lysine in subdo-

212


main I1 of the kinase family is required for phosphoryl transfer, and therefore mutation of this residue results in a kinase-deficient protein. Substitution of this invariant lysine (K220 in PARK) abolishes the kinase activity of PARK. Since the regulatory N- and C-terminal domains of PARK remain intact, this kinase-deficient mutant can effectively compete with the wild-type kinase for phosphorylation of activated P2AR in uitro (Kong et al., 1994). When this mutant is stably overexpressed in BE AS-2B cells [a bronchial epithelial cell line endogenously expressing P2AR and PGE, receptors that undergo homologous desensitization (Penn et al., 1994)], short-term desensitization of the PzAR is mitigated, allowing CAMPto accumulate 2- to 2.5-fold over wild-type cell levels (Kong et al., 1994). These results are the strongest evidence to date that PARK regulates the P2AR in uiuo. Since the desensitization of PGE, receptors is unaffected by the kinase-deficient mutant, PARK demonstrates a preference toward desensitization of the P2AR. However, it is possible that the overexpression of the kinase mutant also hampers the activities of other endogenous kinases. Therefore, it remains to be determined whether kinase-deficient versions of PARK2, GRK5, or GRKG demonstrate similar or unique specificities.

F. DISTINGUISHING FEATURESOF MEMBERS OF THE GRK FAMILY Several characteristics of members of the GRK family allow their distinction (Table I). By sequence homology PARK and PARK2 are members of a subfamily, while RK, GRK4, GRK5, and GRKG belong to a second subfamily. PARK and PARK2 bind to and are activated by G protein Ply-subunits, while the other GRKs are insensitive to Pi y-subunits (Haga and Haga, 1992; Pitcher et al., 1992b; Kim et al., 1993a; Kunapuli and Benovic, 1993; Loudon and Benovic, 1994). In contrast, the catalytic activities of RK (Palczewski et al., 1989a), GRK5 (Kunapuli et al., 1994b), and GRKG (Loudon and Benovic, 1994) are stimulated by the polycations spermine and spermidine, while PARK and PARK2 are insensitive to these agents. RK (Anant and Fung, 1992; Inglese et al., 1992b) and GRKG (Stoffel et al., 1994) are covalently modified by farnesylation and palmitoylation, respectively. Other characteristics do not seem t o correlate with membership in either subfamily. The sensitivity of GRKs to the polyanion heparin varies over at least three orders of magnitude, with the rank order of sensitivity being GRK5 > GRKG > PARK, PARK2 > RK (Palczewski et al., 1989a; Kim et al., 1993b; Kunapuli et al., 199413; Loudon and Benovic, 1994). PARK and RK phosphorylate acidic peptides in uitro (Benovic et al., 1990; Onorato et al., 19911, while GRK5 and GRKG prefer neutral


2 13

or basic peptides (Kunapuli et al., 1994b; Loudon and Benovic, 1994). Finally, autophosphorylation of RK and GRK5 occurs a t 2-3 mol of phosphate per mole of kinase, while autophosphorylation of PARK, pARK2, and GRK6 is substoichiometric (Palczewski et al., 1992; Kunapuli et al., 1994a). 111. ARRESTINS A. CLONING Visual arrestin was initially identified as a major protein that redistributed (along with RK) from the cytoplasm to the disk membrane following light activation of rod outer segments (Kuhn, 1978). Molecular cloning of visual arrestin utilized monoclonal and polyclonal antibodies directed against bovine arrestin to probe a bovine retinal cDNA expression library (Shinohara et al., 1987; Yamaki et al., 1987). Interestingly, arrestin cDNAs were represented in almost 1%of the plaques in the library. Evidence for a n arrestin homologue was initially suggested during the course of PARK purification, when it was found that crude preparations of the kinase could adequately uncouple P2AR from G,, while purified PARK did so to a much lesser extent (Benovic et al., 1987a). Moreover, purified retinal arrestin could increase the efficiency of P,AR/Gs uncoupling by PARK, demonstrating that a n arrestin could indeed specifically interact with the PARK-phosphorylated &AR. Using the visual arrestin cDNA as a probe under conditions of low stringency, a molecular search for a homologue resulted in the identification of a bovine brain protein, parr, which could uncouple P2AR/Gs with 20-fold greater efficacy than retinal arrestin (Lohse et al., 1990). Subsequently, the cDNAs for retinal arrestin alone, parr alone, or a mixture of retinal arrestin and parr were used as probes to isolate a third arrestin homologue. This homologue is variously called hTHY-ARRX (from human thyroid) (Rapoport et al., 1992), parr2 (from rat brain) (Attramadal et al., 19921, and arr3 (from bovine brain) (Sterne-Marr et al., 1993). A fourth homologue was isolated by preparing a retinal-enriched cDNA library (by depleting “generic” sequences with fibroblast cDNA) and using sequence analysis to screen for arrestin homologs. Because this homologue maps to the human X chromosome, it is called X-arrestin (Murakami et al., 1993). This fourth homologue (also called C-arrestin, cone arrestin, or CAR) was also identified in a PCR screen for arrestins utilizing a sense primer designed from the sequence LKHEDTN, which is conserved in all ar-

214


restins, and a vector-derived antisense primer (Craft et al., 1994). For the purpose of this chapter, we refer to the 48-kDa originally identified arrestin as visual or retinal arrestin (arr) (even though the fourth arrestin probably plays a role in the visual system as well); the first arrestin, which was shown to preferentially uncouple the P,AR/G, interaction, as parr (or arr2); the third arrestin homologue as arr3 (because this seems less inappropriate than hTHY-ARRX or parr2); and the fourth arrestin as arr4. The members of the arrestin family are very similar, with the residues corresponding to 16-349 of human retinal arrestin being 45% identical and 70% similar among all four homologues (Figs. 3 and 4). Each arrestin is most similar to parr with the identity varying from 56.5% to 78.3% and the similarity varying from 74.4% to 88.5%. Among the arrestins there are four variable regions that represent islands of disparity, designated V-I, V-11, V-I11 and V-IV (Fig. 3). In relation to retinal arrestin numbering, these include the N terminus to residue 15 (V-I),residues 97-104 (V-111, residues 364-372 (V-1111, and residue 385 to the C terminus (V-IV).While the C-terminal 30 amino acids of each arrestin are very acidic, there are three regions that are enriched in basic residues: the N terminus through residue 33 (B-I), residues 156- 180 (B-IT), and residues 237-242 (B-111). Straddling the V-IV region of retinal arrestin is the sequence ARHNLKDAGEA, which bears significant homology to the C terminus of the a-subunit of transducin (IKENLKDCGLF). Since this region of transducin and other G protein a-subunits has been implicated in receptor binding (for a review see Conklin and Bourne, 19931, it has been speculated that this region of arrestin might play a similar role. However, this homology is much weaker when arr is compared to other G protein a-subunits and when the other arrestins are compared to transducin. Furthermore, binding studies with truncation mutants of arr have not invoked a role for this region of arr in receptor binding (see below). Nevertheless, this homology is intriguing.

FIG. 3. Comparison of amino acid sequences of bovine parr and human arr3, arr4, and arr. The predicted sequences were aligned using the Pileup program [Wisconsin Genetics Computer Group (GCG)]. 0, Identity among all four homologues; -, similar residues. Similarity was determined as in the legend to Fig. 1. V-I, V-11,V-I11 and V-IV indicate regions of greatest variability between the four arrestins. B-I, B-11, and B-I11 designate three regions that are highly enriched in basic residues. The amino acids are numbered on the right-hand side of the sequence. The sequences were obtained from the following sources: Lohse et al. (1990), Rapoport et al. (1992), Murakami et al. (19931, and Yamaki et al. (1988).

215


P I

********** ***** v-1

bovine parr human a r r 3 human arrd human a r r

.......MGD K.GTRVFKKA SPNGKLTVYL GKRDFVDHID LVEPVDGWL ....... MGE KPGTRVFKKS SPNCKLTVYL GKRDFVDHLD KVDPVDGVVL .......... ..MSKVFKKT SSNGKLSIYL GKRDFVDHVD TVEPIDGWL MAASGKTSKS EPNHVIFKKI SRDKSVTIYL GNRDYIDHVS WQPVDGWL -0oo

--a0 0

0

oo--co-

42 43 38 50

00-00000

*** bovine parr human a r r 3 human a r r 4 human a r r

VDPEYLKERR VYVTLTCAFR YGREDLDVLG LTFRKDLFVA NVQSFPPAPE 92 VDPDYLKDRK VFVTLTCAFR YGREDLDVLG LSFRKDLFIA TYQAFPPVPN 93 VDPEYLKCRK LFVMLTCAFR YGRDDLEVIG LTFWLYVQ TLQWPAESS 88 VDPDLVKGKK VYVTLTCAFR YGQEDVDVIG LTFRRDLYFS RVQVYPPVGA 100

ooo- -0 --

--0 ooo333 00

-o--o-o

0-03-00--

0

0

v-11 DKK.PLTRLQ ERLIKXLGEH AYPFTFEIPP NLPCSVTLQP GPEDTGKACG PPR.PPTRLQ DRLLRKLGQH AHPFFFTIPQ NLPCSVTLQP GPEDTGKACG SPQGALTVLQ ERLLHKLGDN A Y P F T W NLPCSVTLQP GPEDAGKPCG AS..TPTKLQ ESLLKKLGSN TYPFLLTFPD YLPCSVMLQP APQDSGKSCG

I*****

bovine parr human a r r 3 human a r r 4 human a r r

000-0--oOo

bovine parr human a r r 3 human a r r 4 human arr

0-

0

0-

0

- 0

-o-o-oooo--0o

0---

- 0 0

0---ooo-m--00

0-0

0-000

.DDDIWEDF ARQFUKGMKD DKEEEEDGTG SPRLNDR TDDDIVFEDF ARLRLKGMKD DDYDDQLC.. SSEDIVIEEF TRKGEEESQK AVEAEGDEGS QDANLVFEEF ARHNLKDAGE AEEGKRDKND ADE....

....... .......

-00-0

0

288 289 285 298

338 334 335 346

0

................... ................

******** ********** ******* bovine W r r human a r r 3 human a r r 4 human a r r

00

v-111 * * ********** ********** ********** ASSDVAVELP FTLMHPKPKE E....PPHRE VPEHETPVDT NLIELDTN.. DVSVELP FVLMHPKPHD HIPLPRPQSA APETDVPVDT NLIEFDTNYA TASDVGVELP LVLIHPKPSH EA........ A TSSEVATEVP FRLMHPQPED PA........ KESI -0

238 239 235 248

-00-0

LDGKLKHEDT NLASSTLLRE GANREILGII VSYKVKVKLV VSRGGLLGDL LDGKLKHEDT NLASSTIVKE GANKEVLGIL VSYRVKVKLV VSRGG..... LDGKLKHEDT NLASSTIIRP GMDKELLGIL VSYKVRVNLM VSCGGILGDL LDGKIKHEDT NLASSTIIKE GIDRTVLGIL VSYQIKVKLT VS..GFLGEL

...

188 189 185 198

0000-

00--00000

ADICLFNTAQ YKCPVAMEEA DDTVAPSSTF CKVYTLTPFL ANNREKRGLA ADICLFSTAQ YKCPVAQLEQ DDQVSPSSTF CKVYTITPLL SDNREKRGLA TDWLYSLDK YTKTVFIQEF TETVAANSSF SQSFAVTPIL AASCQKRGLA ANWLYSSDY YVKPVAMEEA QEKVPPNSTL TKTLTLLPLL ANNRERRGIA

o300-00000---

bovine parr human a r r 3 human a r r l human a r r

0-

-oooo-oo

0 0---0-

-0-

bovine parr human arr3 human a r r 4 human a r r

00000000000000

8-111 QFLMSDKPLH LEASLDKEIY YHGEPISVNV HVTNNTNKTV KKIKISVRQY HFLMSDRSLH LEASLDKELY YHGEPLNVNV HVTNNSTKTV KKIKVSVRQY RFLLSAQPLQ LQAiQCXUWH YHGEPISVNV SINNCTNKVI KKIKISVDQI QFFMSDKPLH LAVSLNREIY FHGEPIPVTV TVTNNTEKTV KKIKACVEQV 0-0

bovine p a r r human a r r 3 human a r r 4 human a r r

-

8-11 VDYEVKAFCA ENL...EEKI HKRNSVRLVI RKVQYAPERP GPQPTAETTR VDFEIWCA KSL...EEKS HKRNSVRLVI RKVQFAPEKP GPQPSAETTP IDFEVKSFCA ENP...EETV SKRDYVRLW WQFAPPEA GPGPSAQTIR VDFEVKAFAT DSTDAEEDKI PKKSSVRYLI RSVQHAPLEM GPQPRAEATW -0-0-- 0

bovine parr human a r r 3 human a r r d human a r r

00

141 142 138 148

382 381 382 372

V-N 418 409 388 405

216


Darr arr

100

parr arr3 am4

&

57.9 (76.4) 56.5(74.3) 50.5(70.6)

100

78.3(88.5) 59.7(77.5) 100

56.5 (74.4) 100

FIG.4. Comparison of amino acid homologies between the various arrestins. Amino acid sequences from human arr, arr3, and arr4 and bovine parr were compared in a pairwise fashion using the Gap program [Wisconsin Genetics Computer Group (GCG)I. The percentage of amino acid identity is given as well as the percentage of similarity (in parentheses). The references are as listed in the legend to Fig. 3.

B. POLYPEPTIDE VARIANTS Three of the four arrestin homologues are expressed as polypeptide variants. Bovine retinal arrestin is expressed in three forms: the originally described 404-residue form; a 370-amino-acid form, called p44 (recently isolated from high-salt extracts of rod outer segment membranes), in which the last 35 residues of the 404-amino-acid form are replaced by a single alanine (Palczewski et al., 1994; Smith et al., 1994); and a 396-residue form that lacks the eight homologous amino acids encoded by exon 13 of the human gene (residues 338-345) (Yamaki et al., 1990; Parruti et al., 199313). The 404-residue form is - 10-fold more abundant than the 370-residue form in bovine retina (Palczewski et al., 1994). In addition to its expression in the retina, “visual” arrestin is present at much lower levels in the cerebellum and in leukocytes (Parruti et al., 1993b). While the retinal and cerebellar forms of visual arrestin have 404 amino acids, a small proportion of the leukocyte visual arrestin is the 396-residue form. Parr exists as two polypeptide variants: the initially described 418-amino-acid “long” form (ParrL) and a 410-amino-acid “short” form (ParrS) that lacks residues 334-341, which correspond to the analogous residues deleted in the 396-residue form of visual arrestin (Parruti et al., 1993b; SterneMarr et al., 1993). In general, ParrL is the predominant form in the brain, that is, cortex, cerebellum, striatum, pineal body, retina, and heart, but not in the pituitary gland; ParrS is the major form in peripheral tissues, that is, spleen, kidney, lung, liver, and pituitary, but not in the heart (Sterne-Marr et al., 1993). The majority of the arr3 is expressed as a 409-amino-acid protein, but some tissues, including brain, pituitary gland, pineal body, spleen, and lung, express a small amount of a 420-residue polypeptide that contains an insert of 11amino acids following residue 361 (Sterne-Marr et al., 1993).Unlike the three other


217

arrestin homologues, both forms of arr3 lack the eight amino acids which are present in arr4 and in the long forms of arr and parr. AND SUBCELLULAR LOCALIZATION C. TISSUE,CELLULAR,

By Northern analysis both arr (Lohse et al., 1990) and arr4 (Murakami et al., 1993) are expressed most abundantly in the retina and the pineal body (summarized in Table 111). However, the sensitivity of PCR analysis has enabled the detection of much lower levels of arr and arr4 in various other tissues. Thus, in addition to the expression of arr in the brain and in leukocytes, arr and p44 have been detected in heart, kidney, lung, and skeletal muscle when large amounts of poly(A)+ RNA are used for reverse transcription preceding PCR (Smith et al., 1994). Arr4 is found in the pituitary gland and the cerebral cortex when purified mRNA is used as the original template for PCR (Craft et al., 1994). From the combination of Northern, PCR, and immunoblotting analyses, parr and arr3 appear to be fairly ubiquitously expressed, with highest levels of expression in the brain, spleen, and prostate (Parruti et al., 1993b; Sterne-Marr et al., 1993).When the two polypeptides are compared directly in tissues using a monoclonal antibody that recognizes an epitope found in all arrestins, parr appears to be expressed at significantly higher levels than arr3 (Sterne-Marr et al., 1993). However, in olfactory epithelium, arr3 is the predominant arrestin isoform (Dawson et al., 1993). Further, arr3 is expressed at higher levels than parr in several cell lines (R. Sterne-Marr, unpublished observations). Immunocytochemistry, immunoelectron microscopy, and in situ hybridization have been used to study the cellular and subcellular distributions of members of the arrestin family. In dark-adapted retinas antibodies specific for the 404-residue visual arrestin label the inner and outer segments of the rod cells, somata, and synaptic layer (Smith et al., 1994). Upon light activation the signal from the inner segments, somata, and synapses decreases, while the outer segment signal is intensified. Interestingly, p44 labeling is found exclusively in the outer segments (consistent with its membrane localization) in dark-adapted retinas, and the C-terminal epitope is apparently masked upon light activation (Smith et al., 1994).Two groups have used in situ hybridization with nucleic acid probes to analyze the cellular localization of arr4 in the retina. Unfortunately, disparate observations resulted. Craft et al. (1994) detected specific labeling of cone cells, while Murakami et al. (1993) found a much broader distribution of arr4 in the rod inner and outer segments as well as the inner plexiform layer. Since the exact

TABLE I11 MOLECULAR PROPERTIES OF THE ARRESTINS Parameter

Pam

arr ~~

arr3

arr4

~~

Polypeptide variants (amino acids) Tissue distribution

404, 396, 370

418.410

420. 409

388

Retina > pineal body

Ubiquitous, brain, hematopoietic, prostate

Retina (Cone?)

mRNA size (kb) Chromosomal localization" Receptor affinity (K,) (nM) Rhodopsinb PzARC m2 mAChR. Uncoupling efficacy Receptor binding preference

1.5 2q37

7.5, 4.1, 1.3 llq13

Ubiquitous, brain, hematopoietic, prostate, olfactory cilia 1.7-2.4 17~13

30-50 2.1 7.2 Rhodopsin P pzAR Rhodopsin % P,AR m2 mAChR

0.14 0.48 PzAR rhodopsin m2 mAChR > pzAR rhodopsin

-

3

*

3

-

0.33 0.35 PzAR rhodopsin PzAR m2 mAChR rhodopsin

-

1.35 Xcen-q2l

?

2

? ? -

aSee the work of Calabrese et al. (1994a,b) and Murakami et al. (1993). bDetermined by stabilization of metarhodopsin I1 (Schleicher et al., 1989). CDetermined by Scatchard analysis using radiolabeled arrestins (Gurevich et al., 1993b, 1995).


219

sequence of the probe used in the former case is not clear, it is not possible to rationalize this disparity at this time. Immunocytochemistry was also used to compare parr and arr3 localization in the rat brain (Attramadal et al., 1992).Although the relative level of reactivity of each antibody is distinct, parr and arr3 antibodies label the cortex, olfactory bulb, hippocampus, cerebellum, and other neuronal structures. The neuronal labeling pattern roughly correlates with the reactivity levels detected by immunoblotting of lysates derived from various brain structures. Arr3 antibodies light up pyramidal cells of the cortex, while parr staining in the cortex is more diffuse. At the subcellular level both antibodies react with neuronal structures, most notably postsynaptic specializations as well as nonsynaptic plasma membrane, Golgi, and multivesicular bodies. Thus, while parr and arr3 are present in the soluble fraction following cell lysis, ultrastructural analyses show an association with membrane structures.

D. MECHANISM OF RETINAL ARRESTIN BINDING TO RHODOPSIN Studies to date addressing the mechanism of action of members of the arrestin family have focused primarily on the interaction of retinal arr with rhodopsin using a variety of techniques, including spectral studies, uncoupling assays, and direct binding assays (Wilden et al., 1986; Schleicher et al., 1989; Palczewski et al., 1991a,c; Gurevich and Benovic, 1992). Arr is a very abundant soluble rod outer segment protein that was discovered by its ability to bind to light-activated (Kuhn, 1978) and phosphorylated (Kuhn et al., 1984) disk membranes, inactivating the visual transduction cascade by effectively competing with transducin for the activated receptor (Wilden et al., 1986) and preventing receptor dephosphorylation (Palczewski et al., 1989b). When rhodopsin absorbs a photon, 11-&-retinal rapidly isomerizes, yielding an equilibrium between the spectrally distinguishable MI and MII. Interaction of transducin with MI1 not only initiates the visual cascade, but drives the equilibrium toward the MI1 tautomer (sometimes called the formation of “extra-MII”)(Stryer, 1986).Upon light activation arr also enhances the formation of MII, and this stabilization requires prephosphorylation of rhodopsin. Kinetic analysis indicates that the binding of arr to rhodopsin proceeds with a large Arrhenius activation energy, suggesting that arr undergoes a conformational change upon binding phosphorylated and light-activated rhodopsin (Schleicher et al., 1989). This conformational change is also detected as an increase in the sensitivity of arr to limited proteolysis (Palczewski et al., 1991a). The putative conformational change does not disrupt the secondary

220


structure of arr (as measured by circular dichroism), suggesting that rhodopsin-arr interactions replace intramolecular arrestin interactions (Palczewski et al., 1991~). Various truncated forms of arr have been used to investigate the binding-induced conformational change in arr and the interactions of arr with the light-activated and phosphorylated form of rhodopsin, leading to the assignment of functional domains of retinal arrestin. These studies take advantage of the ability to prepare the various functional forms of rhodopsin in uitro. Rod outer segment membranes purified from dark-adapted bovine retinas and stripped of arrestin and RK by urea treatment contain predominantly rhodopsin (>go% of the total protein). In the presence of light, rhodopsin can be phosphorylated to varying extents using purified RK or PARK (both phosphorylate the same C-terminal tail residues). After washing to remove the kinase, rhodopsin is again dark-adapted and regenerated with unbleached chromophore to produce “dark phosphorylated rhodopsin” (Rho-P). Upon light activation rhodopsin and Rho-P assume their active conformations, light-activated rhodopsin (Rho*) and light-activated phosphorylated (Rho*-P)rhodopsin, respectively. Full-length visual arrestin binds to Rho*-P with high affinity (-30-50 nM) and selectivity, since binding to Rho*-P is 10- to 12-fold higher than binding to Rho* or Rho-P (Bennett and Sitaramayya, 1988; Schleigher et al., 1989; Gurevich and Benovic, 1992). It should be emphasized that wild-type arrestin can clearly detect Rho* and Rho-P, since binding to these forms is greater than binding to Rho. Thus, selectivity refers to the ability of arr to preferentially bind to the light-activated and phosphorylated form of rhodopsin. Since the C-terminal 20% of each arrestin represents the most variable region among family members and contains some homology to G protein a-subunits, it has been suggested that this portion of the molecule might determine the specificity of binding to different receptors and therefore be directly involved in receptor binding. While arr is 100-fold (Lohse et al., 1992) more effective than parr in uncoupling GT-Rho*-P interaction, substitution of the last 59 residues of visual arrestin with the corresponding 78 amino acids of parr has little affect on its ability to bind Rho*-P (Gurevich et al., 1995). While a role in binding to the receptor cannot be ruled out, truncation mutagenesis studies suggest that the C terminus of arrestin plays a regulatory role, perhaps by interacting with other portions of arrestin, such as the N-terminal domain. Proteolytic fragments (residues 3-354 and other similar products) of arr not only bind Rho*-P, but do so with higher affinity than does the full-length arrestin (Palczewski et al., 1991a).In


221

vitro translation of functionally active arr has allowed a rapid systematic study of this phenomenon using various-length arr molecules. A form of arr that lacks the last 13 residues retains most of its binding to Rho*-P while also maintaining its selectivity. In contrast, a molecule that contains the first 365 (A366-404) residues can bind Rho*-P fairly well, but has lower selectivity: it binds Rho* and Rho-P to a greater extent than does full-length arrestin (Gurevich and Benovic, 1992). Therefore, the C terminus apparently maintains the rigidity of arr, preventing it from making strong contacts with rhodopsin unless the receptor is in the light-activated and phosphorylated form. Interestingly, the A366-404 mutant is similar in structure to the naturally occurring p44, which contains the first 369 residues and terminates with an alanine. Analogous to the 365-deletion mutant, p44 binds to Rho*-P, Rho*, and Rho-P equally well. However, using an uncoupling assay (an in uitro system measuring the ability of an arr to block transducin-mediated phosphodiesterase activity), p44 is more than 30-fold more potent than arr in competing with transducin for Rho*, while p44 is unable to compete with transducin for Rho*-P (Palczewski et al., 1994). Since p44 binds more avidly to the membrane than arr, this suggests that p44 may quench the transducin-mediated signal independently of phosphorylation. Further, since p44 binds Rho*-P but is not effective in inactivating the visual transduction cascade, this experiment underscores the importance of using a functional assay to measure arrestin activities. The product of the Drosophila arrl gene is quite similar in length to p44, yet mutations in this gene do not significantly alter visual transduction or desensitization (Dolph et al., 1992). Indirect evidence suggests that the regulatory role of the C terminus of arr is mediated by its interaction with the N terminus. Retinal arrestin is sensitive to certain polyanions, such as heparin and dextran sulfate, but not polyglutamic or polyaspartic acids (Palczewski et al., 1991). Not only does heparin prevent binding of arrestin to Rh*-P, as measured by direct binding (Gurevich et al., 1994) and extra MI1 stabilization studies (Palczewski et al., 1991), but it also mimics the phosphorylated receptor by inducing the conformational change in arr (Palczewski et al., 1991). Furthermore, heparin can also mimic the arr C terminus, since low levels confer selectivity to the A366-404 arr that otherwise lacks selectivity (Gurevich et al., 1994). The heparin binding site in arr maps in the amino half of the molecule, since a proteolytic fragment of arrestin containing residues 3-205 binds tightly to heparin-Sepharose (Palczewski et al., 1991) and a truncation mutant that terminates at residue 191 is exquisitely sensitive to

222


heparin (IC50,

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