THE SEROTONIN RECEPTORS
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THE SEROTONIN RECEPTORS
T H E R E C E P T O R S
KIM A. NEVE, SERIES EDITOR
The Serotonin Receptors: From Molecular Pharmacologyto Human Therapeutics, EDITED BY Bryan L. Roth, 2006
The Adrenergic Receptors: In the 21st Century, EDITED BY
Dianne M. Perez, 2005
The Melanocortin Receptors, EDITED BY Roger D. Cone, 2000
The GABA Receptors, Second Edition, EDITED BY S. J. Ennaand Norman G. Bowery, 1997
The Ionotropic Glutamate Receptors, EDITED BY Daniel T.Monaghan and Robert Wenthold, 1997
The Dopamine Receptors, EDITED BY Kim A. Neve and RachaelL. Neve, 1997
The Metabotropic Glutamate Receptors, EDITED BY P. JeffreyConn and Jitendra Patel, 1994
The Tachykinin Receptors, EDITED BY Stephen H. Buck, 1994
The Beta-Adrenergic Receptors, EDITED BY John P. Perkins, 1991
Adenosine and Adenosine Receptors, EDITED BY MichaelWilliams, 1990
The Muscarinic Receptors, EDITED BY Joan Heller Brown, 1989
The Serotonin Receptors, EDITED BY Elaine Sanders-Bush, 1988
The Alpha-2 Adrenergic Receptors, EDITED BY Lee Limbird, 1988
The Opiate Receptors, EDITED BY Gavril W. Pasternak, 1988
The Alpha-1 Adrenergic Receptors, EDITED
BY Robert R. Ruffolo, Jr., 1987
The GABA Receptors, EDITED BY S. J. Enna, 1983
The SerotoninReceptors
From Molecular Pharmacologyto Human Therapeutics
Edited by
Bryan L. Roth, MD, PhD
Department of Biochemistry
Case Western Reserve University–School of Medicine
Cleveland, OH
© 2006 Humana Press Inc.999 Riverview Drive, Suite 208Totowa, New Jersey 07512
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Cover design by Patricia F. Cleary
Cover illustration: Figure 3 from Chapter 9, “Cellular and Subcellular Localization of SerotoninReceptors in the Central Nervous System,” by L. Descarries, V. Cornea-Hébert, and M. Riad
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Library of Congress Cataloging-in-Publication DataThe serotonin receptors : from molecular pharmacology to human therapeutics / edited by
Bryan L. Roth.p. cm. — (The receptors)
Includes bibliographical references and index.ISBN 1-58829-568-0 (alk. paper)1. Serotonin—Receptors. 2. Serotonin—Physiological effect. 3. Serotoninergic mechanisms. I. Roth, Bryan L. II. Series.QP801.S4.S474 2006612.8’042—dc22 2005029055
Preface
It has been nearly 20 years since the last Humana Press book devoted toserotonin (5-hydroxytryptamine; 5-HT) receptors has appeared. Since then, thefield of 5-HT receptors has undergone a revolution due to the discovery of manyadditional 5-HT receptors. Although 5-HT was chemically elucidated in 1948 byPage and colleagues (Rapport et al., 1948) and 5-HT receptors initially classifiedin 1957 (Gaddum and Picarelli, 1957), the complexity of 5-HT pharmacologywas not fully appreciated until the late 1970s and early 1980s when many puta-tive 5-HT receptors were identified by radioligand binding studies (e.g., 5-HT1A,5-HT2, 5-HT1E and so on) (Leysen et al., 1979; Hamon et al., 1980; Peroutkaet al., 1981; Leonhardt et al., 1989). The first 5-HT receptors were cloned in 1988(Fargin et al., 1988; Julius et al., 1988) and the discovery of 14 distinct human5-HT receptors since then ushered in the era of 5-HT receptor molecular biology(Kroeze et al., 2003). The cloning and sequencing of 5-HT receptors has alsorevealed the presence of post-transcriptionally modified mRNA species (RNAediting) (Burns et al., 1997) as well as naturally occurring mutations and theirrelations to various diseases (e.g., single nucleotide polymorphisms; SNPs)(Arranz et al., 1995).
The identification of the amino acid sequences of 5-HT receptors has allowedus to predict how 5-HT and related agonists bind to and activate 5-HT receptors(Shapiro et al., 2000; Shapiro et al., 2002). The hope has been that this informa-tion will lead, eventually, to the development of novel, subtype-selective 5-HTreceptor agonists and antagonists (Kroeze et al., 2002).
The first several chapters of The Serotonin Receptors: From Molecular Phar-macology to Human Therapeutics are aimed at reviewing our knowledge of themolecular and structural biology of 5-HT receptors, followed by our current under-standing of 5-HT receptor pharmacology. The elucidation of the sequences of 5-HT receptors has also facilitated the development of highly selective tools formapping the distribution of 5-HT receptors. These tools include selective 5-HTreceptor antibodies and hybridization probes. The use of these biochemical probeshas revealed an unexpected complexity in both the cellular and subcellular distri-bution of 5-HT receptors.
The next few chapters describe the anatomical, cellular, and subcellular dis-tribution of 5-HT receptors. Because of the plethora of receptors and receptorsubtypes, however, it has been exceedingly difficult to identify the physiologicalrole of various 5-HT receptors using pharmacological tools. A powerful approach
v
to elucidating the physiological role of 5-HT receptors was to use mice in which5-HT receptors were deleted (e.g., knockout mice); the first 5-HT receptor knock-outs were reported in 1994 (Saudou et al., 1994) and, since then, nearly all 5-HTreceptors have been “knocked-out”—typically with novel phenotypes (Tecott etal., 1995; Brunner et al., 1999).
The final chapters review our understanding the physiological role(s) of 5-HTreceptors based mainly on studies performed in genetically engineered mice.This book represents our collective attempts to provide the reader with a “snap-shot” of the 5-HT receptor field circa 2006. The scope of the book is vast,proceeding from the genomic to the therapeutic. Because it is unlikely that anyreader will devote the time to reading the entire book cover-to-cover, each chap-ter has been designed to represent a complete review of the particular field. Thus,each chapter begins with a short introduction to 5-HT receptors and then pro-ceeds to review the particular subfield in depth. Not surprisingly, therefore, theenterprising reader will find some overlap between various introductory sec-tions.
AcknowledgmentsI would like to especially thank Mr. Jon Evans who has tirelessly collected,
edited, and collated the finished chapters and who has done most of the “legwork” associated with this book. Without Jon’s devotion to this task, the bookwould never have been completed. Any omissions and errors are my sole respon-sibility. I would also like to thank my wife Judith and my daughter Rachel fortheir warmth and understanding during the gestation of this book. Lastly, I dedi-cate this book to “beings throughout the ten directions—hands palm-to-palm.”
Bryan L. Roth, MD, PhD
ReferencesArranz M, Collier D, Sodhi M, Ball D, Roberts G, Price J, Sham P, and Kerwin R.
Association between clozapine response and allelic variation in 5-HT2A receptor gene.Lancet 1995;346:281–282.
Brunner D, Buhot MC, Hen R, and Hofer M. Anxiety, motor activation, and maternal-infant interactions in 5HT1B knockout mice. Behav Neurosci 1999;113:587–601.
Burns CM, Chu H, Rueter SM, Hutchinson LK, Canton H, Sanders-Bush E, andEmeson RB. Regulation of serotonin-2C receptor G-protein coupling by RNA editing[see comments]. Nature 1997;387:303–308.
Fargin A, Raymond JR, Regan JW, Cotecchia S, Lefkowitz RJ, and Caron MG. Thegenomic clone G-21 which resembles a beta-adrenergic receptor sequence encodes the5-HT1A receptor. Nature 1988;335:358–360.
Gaddum JH and Picarelli ZP. Two kinds of tryptamine receptors. Br J Pharmacol1957;12:323–328.
vi Preface
Hamon M, Nelson DL, Herbet A. and Glowinski J. Multiple receptors for serotoninin the rat brain. Adv Biochem Psychopharmacol 1980;21:223–233.
Julius D, MacDermott AB, Axel R, and Jessell TM. Molecular characterization of afunctional cDNA encoding the serotonin 1c receptor. Science 1988;241:558–564.
Kroeze WK, Kristiansen K, and Roth BL. Molecular biology of serotonin receptorsstructure and function at the molecular level. Curr Top Med Chem 2002;2:507–528.
Kroeze WK, Sheffler DJ, and Roth BL. G-protein-coupled receptors at a glance. J CellSci 2002;116:4867-9.
Leonhardt S, Herrick-Davis K, and Titeler M. Detection of a novel serotonin receptorsubtype (5-HT1E) in human brain: interaction with a GTP-binding protein. J Neurochem1989;53:465–471.
Leysen JE, Gommeren W, Laduron PM, et al. Distinction between dopaminergic andserotonergic components of neuroleptic binding sites in limbic brain areas. BiochemPharmacol 1979;28:447–448.
Peroutka SJ, Lebovitz RM, and Snyder SH. Two distinct serotonin receptors withdistinct physiological functions. Science 1981;212:827-829.
Rapport MM, Green AA, and Page IH. Crystalline serotonin. Science 1948;108:329.Saudou F, Amara DA, Dierich A, et al. Enhanced aggressive behavior in mice lacking
5-HT1B receptor. Science 1994;265:1875–1878.Shapiro DA, Kristiansen K, Kroeze WK, and Roth BL. Differential modes of agonist
binding to 5-hydroxytryptamine(2A) serotonin receptors revealed by mutation andmolecular modeling of conserved residues in transmembrane region 5. Mol Pharmacol2000;58:877–886.
Shapiro DA, Kristiansen K, Weiner DM, Kroeze WK, and Roth BL. Evidence for amodel of agonist-induced activation of 5-HT2A serotonin receptors which involves thedisruption of a strong ionic interaction between helices 3 and 6. J Biol Chem 2002;18:18.
Tecott LH, Sun LM, Akana SF, et al. Eating disorder and epilepsy in mice lacking 5-HT2c serotonin receptors [see comments]. Nature 1995;374:542–546.
Preface vii
Contents
ix
Preface............................................................................................................... v
Contributors ..................................................................................................... xi
Color Plate .................................................................................................... xvii
1 Molecular Biology and Genomic Organizationof G Protein–Coupled Serotonin Receptors
Wesley K. Kroeze and Bryan L. Roth .................................................... 1
2 Structure and Function Reveal Insights in the Pharmacologyof 5-HT Receptor Subtypes
Richard B. Westkaemper and Bryan L. Roth....................................... 39
3 Polymorphic and Posttranscriptional Modifications of 5-HTReceptor Structure: Functional and Pathological Implications
Marilyn A. Davies, Chiao-ying Chang, and Bryan L. Roth ................ 59
4 Strategies for the Development of Selective Serotonergic Agents
Richard A. Glennon.............................................................................. 91
5 5-HT Receptor Signal Transduction Pathways
John R. Raymond, Justin H. Turner, Andrew K. Gelasco,Henry B. Ayiku, Sonya D. Coaxum, John M. Arthur,and Maria N. Garnovskaya ......................................................... 143
6 Agonist-Directed Trafficking of 5-HT Receptor-Mediated SignalTransduction
Kelly A. Berg and William P. Clarke ................................................ 207
7 Identification of 5-HT2 and 5-HT4 Receptor-InteractingProteins: A Proteomic Approach
Joël Bockaert, Carine Bécamel, Lara Joubert, Sophie Gavarini,Aline Dumuis, and Philippe Marin ............................................. 237
x Contents
8 5-HT Receptor-Associated Proteins (FRAPs): Relevance forTargeting, Trafficking, and Signal Transduction
Zongqi Xia, Douglas J. Sheffler, and Bryan L. Roth ........................ 257
9 Cellular and Subcellular Localization of Serotonin Receptorsin the Central Nervous System
Laurent Descarries, Virginia Cornea-Hébert,and Mustapha Riad ...................................................................... 277
10 Chemical Neuroanatomy of 5-HT Receptor Subtypes in theMammalian Brain
Guadalupe Mengod, M. Teresa Vilaró, Roser Cortés,Juan F. López-Giménez, Andreu Raurich,and José M. Palacios ................................................................... 319
11 Modulation of the Neuronal Activity and NeurotransmitterRelease by 5-HT1A and 5-HT1B/1D Receptors
Pau Celada, Albert Adell, and Francesc Artigas ....................... 365
12 Electrophysiology of 5-HT2A Receptors and Relevance forHallucinogen and Atypical Antipsychotic Drug Actions
Evelyn K. Lambe and George K. Aghajanian ................................... 403
13 The Emergence of 5-HT2B Receptors as Targets to Avoidin Designing and Refining Pharmaceuticals
Vincent Setola and Bryan L. Roth ..................................................... 419
14 The 5-HT3 Receptor
Andrew J. Thompson, Li Zhang, and Sarah C. R. Lummis ............... 439
15 5-HT3 and 5-HT4 Receptors as Targets for Drug Discoveryfor Dementia
Frank Lezoualc’h and Magali Berthouze .......................................... 459
16 Electrophysiological Properties of G s-Coupled 5-HTReceptors (5-HT4, 5-HT6, 5-HT7)
Rodrigo Andrade ................................................................................ 481
17 5-HT6 Receptors as Targets for the Treatment of CognitiveDeficits in Schizophrenia
Rudy Schreiber, Andrew Sleight, and Marie Woolley....................... 495
Contents xi
18 5-HT7 Receptors as Favorable Pharmacological Targets forDrug Discovery
Peter B. Hedlund and J. Gregor Sutcliffe ......................................... 517
19 Serotonin System Gene Knockouts:A Story of Mice With Implications for Man
Miles Berger and Laurence H. Tecott ............................................... 537
20 Effects of Serotonin-Related Gene Deletion on Measuresof Anxiety, Depression, and Neurotransmission
Anita J. Bechtholt and Irwin Lucki .................................................... 577
Index .............................................................................................................607
Contributors
xiii
ALBERT ADELL • Department of Neurochemistry, Institut d’ InvestigacionsBiomèdiques de Barcelona (CSIC), IDIBAPS, 08036 Barcelona, Spain
GEORGE K. AGHAJANIAN • Departments of Psychiatry and Pharmacology, YaleUniversity School of Medicine, New Haven, CT, USA
RODRIGO ANDRADE • Department of Psychiatry and Behavioral Neuroscience,Wayne State University School of Medicine, Detroit, MI, USA
JOHN M. ARTHUR • Division of Nephrology, Department of Medicine, MedicalUniversity of South Carolina; and the Medical and Research Services of theRalph H. Johnson Veterans Affairs Medical Center, Charleston, SC, USA
FRANCESC ARTIGAS • Department of Neurochemistry, Institut d’ InvestigacionsBiomèdiques de Barcelona (CSIC), IDIBAPS, 08036 Barcelona, Spain
HENRY B. AYIKU • Division of Nephrology, Department of Medicine, MedicalUniversity of South Carolina; and the Medical and Research Services of theRalph H. Johnson Veterans Affairs Medical Center, Charleston, SC, USA
CARINE BÉCAMEL • Institut de Génomique Fonctionnelle, UMR CNRS 5203,France
ANITA J. BECHTHOLT • Department of Psychiatry, University of Pennsylvania,Philadelphia, PA, USA
KELLY A. BERG • Department of Pharmacology, University of Texas HealthScience Center, San Antonio, TX, USA
MILES BERGER • Medical Scientist Training Program, Biomedical SciencesGraduate Program, and Department of Psychiatry, the University of California,San Francisco, CA
MAGALI BERTHOUZE • Signalisation et Physiopathogie Cardiaque, Inserm U-769,IFR-141, Faculté de Pharmacie, Université Paris XI, F-92296 Châtenay-Malabry, France
JOËL BOCKAERT • Institut de Génomique Fonctionnelle, UMR CNRS 5203,France
PAU CELADA • Department of Neurochemistry, Institut d’ InvestigacionsBiomèdiques de Barcelona (CSIC), IDIBAPS, 08036 Barcelona, Spain
CHIAO-YING CHANG • Department of Biochemistry, School of Medicine, CaseWestern Reserve University, Cleveland, OH, USA
xiv Contributors
WILLIAM P. CLARKE • Department of Pharmacology, University of TexasHealth Science Center, San Antonio, TX, USA
SONYA D. COAXUM • Division of Nephrology, Department of Medicine, MedicalUniversity of South Carolina; and the Medical and Research Services of theRalph H. Johnson Veterans Affairs Medical Center, Charleston, SC, USA
VIRGINIA CORNEA-HÉBERT • Departments of Pathology and Cell Biology andof Physiology, Faculty of Medicine, Université de Montréal, Montréal(QC), Canada
ROSER CORTÉS • Department of Neurochemistry, Institut d’InvestigacionesBiomédiques de Barcelona, CSIC, IDIBAPS, 08036 Barcelona, Spain
MARILYN A. DAVIES • Department of Biochemistry, School of Medicine, CaseWestern Reserve University, Cleveland, OH, USA
LAURENT DESCARRIES • Departments of Pathology and Cell Biology and ofPhysiology, Faculty of Medicine, Université de Montréal, Montréal (QC),Canada
ALINE DUMUIS • Institut de Génomique Fonctionnelle, UMR CNRS 5203, France
MARIA N. GARNOVSKAYA • Division of Nephrology, Department of Medicine,Medical University of South Carolina; and the Medical and Research Servicesof the Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC, USA
SOPHIE GAVARINI • Institut de Génomique Fonctionnelle, UMR CNRS 5203,France
ANDREW K. GELASCO • Division of Nephrology, Department of Medicine,Medical University of South Carolina; and the Medical and ResearchServices of the Ralph H. Johnson Veterans Affairs Medical Center, Charleston,SC, USA
RICHARD A. GLENNON • Department of Medicinal Chemistry, School ofPharmacy, Virginia Commonwealth University, Richmond, VA, USA
PETER B. HEDLUND • Department of Molecular Biology, The Scripps ResearchInstitute, La Jolla, CA, USA
LARA JOUBERT • Institut de Génomique Fonctionnelle, UMR CNRS 5203, France
WESLEY KROEZE • Department of Biochemistry, School of Medicine, CaseWestern Reserve University, Cleveland, OH, USA
EVELYN K. LAMBE • Departments of Psychiatry and Pharmacology, Yale UniversitySchool of Medicine, New Haven, CT, USA
FRANK LEZOUALC’H • Signalisation et Physiopathogie Cardiaque, Inserm U-769,IFR-141, Faculté de Pharmacie, Université Paris XI, F-92296 Châtenay-Malabry, France
JUAN F. LÓPEZ-GIMÉNEZ • Department of Biochemistry and Molecular Biology,University of Glasgow, Glasgow UK
IRWIN LUCKI • Departments of Psychiatry and Pharmacology, University ofPennsylvania, Philadelphia, PA, USA
SARAH C. R. LUMMIS • Department of Biochemistry, University of Cambridge,Cambridge, UK
PHILIPPE MARIN • Institut de Génomique Fonctionnelle, UMR CNRS 5203,France
GUADALUPE MENGOD • Department of Neurochemistry, Institut d’InvestigacionesBiomédiques de Barcelona, CSIC, IDIBAPS, 08036 Barcelona, Spain
JOSÉ M. PALACIOS • Parc Cientific de Barcelona, Universitat de Barcelona,08028 Barcelona, Spain
ANDREU RAURICH • Department of Neurochemistry, Institut d’InvestigacionesBiomédiques de Barcelona, CSIC, IDIBAPS, 08036 Barcelona, Spain
JOHN R. RAYMOND • Division of Nephrology, Department of Medicine, MedicalUniversity of South Carolina; and the Medical and Research Services of theRalph H. Johnson Veterans Affairs Medical Center, Charleston, SC, USA
MUSTAPHA RIAD • Departments of Pathology and Cell Biology and of Physiology,Faculty of Medicine, Université de Montréal, Montréal (QC), Canada
BRYAN L. ROTH • Department of Biochemistry, Neuroscience, and Psychiatry,School of Medicine, Case Western Reserve University, Cleveland, OH, USA
RUDY SCHREIBER • Pharmacology, Discovery Research, Sepracor, Marlborough,MA, USA
VINCENT SETOLA • Department of Biochemistry, School of Medicine, CaseWestern Reserve University, Cleveland, OH, USA
DOUGLAS J. SHEFFLER • Department of Biochemistry, School of Medicine,Case Western Reserve University, Cleveland, OH, USA
ANDREW SLEIGHT • PRBD-N, F. Hoffmann La-Roche, Basel, Switzerland
J. GREGOR SUTCLIFFE • Department of Molecular Biology, The Scripps ResearchInstitute, La Jolla, CA, USA
LAURENCE H. TECOTT • Department of Psychiatry, the University of California,San Francisco, CA, USA
ANDREW J. THOMPSON • Department of Biochemistry, University of Cambridge,Cambridge, UK
JUSTIN H. TURNER • Division of Nephrology, Department of Medicine, MedicalUniversity of South Carolina; and the Medical and Research Services of theRalph H. Johnson Veterans Affairs Medical Center, Charleston, SC, USA
Contributors xv
M. TERESA VILARÓ • Department of Neurochemistry, Institut d’InvestigacionesBiomédiques de Barcelona, CSIC, IDIBAPS, 08036 Barcelona, Spain
RICHARD B. WESTKAEMPER • Department of Medicinal Chemistry, School ofPharmacy, Virginia Commonwealth University, Richmond, VA, USA
MARIE WOOLLEY • Psychiatry CEDD, GlaxoSmithKline plc, Harlow, Essex, UK
ZONGQI XIA • Department of Biochemistry, School of Medicine, Case WesternReserve University, Cleveland, OH, USA
LI ZHANG • Laboratory of Integrative Neuroscience, National Institute onAlcohol Abuse and Alcoholism, National Institutes of Health, Bethesda,MD, USA
xvi Contributors
Color Plate
The following illustrations appear in color in the insert that follows page 240.
Chapter 2, Fig. 2, p. 46:A 5-HT2A-receptor model constructed from the rhodopsin crystal structure.
Chapter 8, Fig. 1, p. 260:An overview of yeast two-hybrid screens for FRAPs.
Chapter 8, Fig. 2, p. 261:An overview of phage display as a technique to identify FRAPs.
Chapter 13, Fig. 1, p. 426:Three-dimensional representation of VHD-associated and non-VHD-associated drug affinity for a battery of GPCRs, ligand-gated ion channels,and biogenic amine transporters.
Chapter 13, Fig. 2, p. 430:Mitogenic signal transduction from 5-HT2B receptors.
xvii
1
1
Molecular Biology and Genomic Organizationof G Protein–Coupled Serotonin Receptors
Wesley K. Kroeze and Bryan L. Roth
Summary
Among animals with nervous systems, serotonin (5-hydroxytryptamine;5-HT) is a ubiquitous neurotransmitter, and numerous classes and sub-classes of G protein–coupled 5-HT receptors have evolved to transduceextracellular 5-HT signals to the intracellular milieu. In this chapter, wesummarize naturally occurring variation in serotonin receptor sequences.These sequences vary by species and by class and subclass and are fur-ther modified from their canonical sequences by RNA editing, alterna-tive splicing, and the existence of single-nucleotide polymorphisms. Bythe presence of 5-HT receptors in such relatively simple organisms asCaenorhabditis elegans, it can be inferred that serotonergic signaling as ameans of intracellular communication arose fairly early in evolutionaryhistory. The multiple subclasses of 5-HT receptors and the various meansto further modify receptor sequences, such as splicing and editing, pre-sumably point to a biological requirement for very delicate “fine-tuning”of serotonergic signaling. How this fine-tuning is accomplished is likely tooccupy and intrigue biologists for many years.
Key Words: Serotonin; 5-hydroxytryptamine; receptor; sequence; database.
1. Introduction
The G protein–coupled serotonin (5-hydroxytryptamine; 5-HT) receptors aretypical group A rhodopsin-like G protein–coupled receptors (GPCRs) in thatthey are predicted to possess seven transmembrane spanning helices, threeintracellular and three extracellular loops, an extracellular amino-terminus, and
From: The Receptors: The Serotonin Receptors: From Molecular Pharmacology to Human Therapeutics
Edited by: B. L. Roth © Humana Press Inc., Totowa, NJ
an intracellular carboxy-terminus. The true structures of these receptors remainunknown, although the crystallization of the bovine rhodopsin receptor (1) pro-vides promise for the solution of the structures of the G protein–coupled 5-HTreceptors in the near future. Functionally, the transmembrane regions serve tobind ligands, especially the endogenous ligand serotonin, the intracellulardomains couple these receptors to various intracellular functions, and for themost part, the extracellular domains have uncertain functional roles (reviewedin ref. 2). The endogenous ligand is the neurotransmitter serotonin, and thepresence of serotonin and its receptors in a variety of invertebrates argues fora relatively early evolutionary origin of these receptors. Since our most recentreview on this topic (2), several genome sequencing projects have been com-pleted or nearly completed, and new 5-HT receptors have continued to beadded to the public databases. In addition, new insights have been gained on therelationship among sequence, structure, and function on many fronts. In thischapter, we will summarize aspects of protein and nucleic acid sequence vari-ation among the GPCRs, with emphasis on newer findings.
In mammals, there are six classes of G protein–coupled 5-HT receptors,namely 5-HT1, 5-HT2, 5-HT4, 5-HT5, 5-HT6, and 5-HT7. These classes arefurther subdivided as follows. The 5-HT1 receptor class contains the 5-HT1A,5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F receptor subclasses. The 5-HT2 receptorclass contains the 5-HT2A, 5-HT2B, and 5-HT2C receptors. The 5-HT5 receptorclass contains the 5-HT5A, and 5-HT5B receptor subclasses. For the most part,the 5-HT4, 5-HT6, and 5-HT7 classes do not contain subclasses of receptorsper se, and sequence diversity in these classes is provided mainly by alternativemRNA splicing, which will be the subject of a subsequent chapter. The orga-nization of classes and subclasses of receptors in mammals is not completelyconserved, even among other vertebrates, as, for example, the pufferfishTetraodon apparently has two subclasses of 5-HT7 receptors.
Additional sequence diversity is provided by RNA editing in some sub-classes of 5-HT receptors, and numerous single-nucleotide polymorphisms(SNPs) and splice variants are known to exist in many subclasses of thesereceptors. Editing of 5-HT receptors will be reviewed in this chapter, and SNPsand splice variants will be reviewed in subsequent chapters.
In addition to the 5-HT receptors identified from mammals, many 5-HTreceptors have also been cloned from nonmammalian vertebrates and fromseveral invertebrate species. Study of these 5-HT receptors should provide addi-tional information on the nature of the residues essential for binding of at leastthe natural ligand, serotonin, to 5-HT receptors and might provide insight intohow these receptors evolved. For example, residues that are completely con-served among all 5-HT receptors are likely to have essential roles in the func-tion of the receptor, whether that be ligand binding, interaction with signaling
2 Kroeze and Roth
or scaffolding proteins, or maintenance of the three-dimensional structure of theprotein. Residues that are conserved among one or a few subclasses of 5-HTreceptors might, for example, contribute to the subtype selectivity of certainmedications or explain coupling to restricted subsets of Gα-subunits. Thewealth of data now available regarding naturally occurring receptor sequencessuggests that methods such as the “evolutionary trace” method of the Lichtargegroup (4) could also be very useful in further unraveling the relationshipsamong sequence, structure, and function in these proteins.
Notwithstanding the progress that has been made in the sequencing of vari-ous genomes and cloning of new receptors, much remains to be done to gain afull appreciation of the natural variation in sequence of the 5-HT receptors, evenin relatively frequently studied animal species. For example, of the 13 likelyG protein–coupled 5-HT receptors in mammalian genomes, only 2 have beendescribed from rabbits, 5 from pigs and dogs, 7 from guinea pigs, and 3 fromhamsters. Table 1 lists the G protein–coupled 5-HT receptors known to date.
2. Database Annotation of 5-HT Receptors
With ongoing efforts in various genome sequencing projects, as well as thecloning of individual genes, 5-HT receptors are continually being added to thepublic databases. Although these additions to the databases provide a richness ofnew information for the investigator, a word of caution is in order. Functionalannotation is now being provided by automated or semiautomated algorithms;thus, sequences can be annotated as 5-HT receptors without pharmacologic orfunctional data. Automated annotation of new sequences is generally accuratewhen there are very clear homologies with previously known receptors, as withnewly cloned mammalian receptors (Table 1). However, when the databasesequences of newly cloned or sequenced, putative 5-HT receptors diverge signif-icantly from previously known 5-HT receptor sequences, as with some of thenewly discovered invertebrate and nonmammalian vertebrate receptors (Table 1),the annotations should be considered to be tentative until pharmacologic orfunctional data are obtained.
In addition to automated functional annotation of putative 5-HT receptorsequences, determination of open reading frames (ORFs), start codons, andintron/exon boundaries from genomic sequences is now also automated. Theabsence of expert human curation in such automated annotation can potentiallylead to the presence of incorrect sequence data in the databases that has arisenfrom misidentification of ORFs, start codons, and intron/exon boundaries in raw genomic sequences. For example, the sequences of several chimpanzee 5-HT receptors have recently been added to the GenBank databases, butthe sequences given for these receptors indicate that one or several of theseven transmembrane (TM) helices are not present in the sequence, although
Molecular Biology and Genomic Organization 3
4 Kroeze and Roth
Table 1G Protein–Coupled 5-HT Receptors Known to Date
Probablesecond Accession
Receptor Species messengera number Ref.
Invertebrates5-HTB1 Aplysia sea slug PI Q16950 485-HTB2 Aplysia sea slug PI Q16951 485-HT7-Ae Aedes mosquito AAG49292 495-HT7-ANO Anopheles mosquito XP–313129 Database only5-HT-DRO Drosophila fruit fly +AC AAA28305 505-HT-PLAN1 Dugesia planarian BAA22404 515-HT-PLAN4 Dugesia planarian BAA22403 515-HT7-He Helisoma snail AAQ84306 Database only5-HT-Ser7 C. elegans nematode AAB04582 Database only5-HT-DRO2 Drosophila fruit fly CAA57429 525-HT2A-ANO Anopheles mosquito XP–307953 Database only5-HT-Ser1 C. elegans nematode AAC15827 395-HT-ASC Ascaris nematode PI AAC78396 53, 545-HT-LYM2 Lymnaea snail PI AAC16969 555-HT-LOB Panulirus lobster PI AAS57919 565-HT-DRO1A Drosophila fruit fly +AC CAA77570 575-HT1A-ANO Anopheles mosquito EAA04158 Database only5-HT-HEL Heliothis moth −AC CAA64863 585-HT-BUT Papilio butterfly BAD72868 595-HT-DRO1B Drosophila fruit fly −AC CAA77571 575-HT-TICK Boophilus tick −AC AAQ89933 605-HT-BEE Apis honeybee XP–393915 Database only5-HT-BOM Bombyx moth −AC CAA64862 585-HT1B-ANO Anopheles mosquito XP–317820 Database only5-HT-LYM Lymnaea snail AAA29290 615-HT-1He1 Helisoma snail AAQ95277 Database only5-HT-AP1 Aplysia sea slug −AC AAM46088 625-HT-AP2 Aplysia sea slug −AC AAC28786 635-HT-Ser4 C. elegans nematode −AC NP–497452 645-HT-HAEM Haemonchus nematode AAO45883 655-HT-BARN Balanus barnacle BAA12013 665-HT-SH Metapenaeus shrimp AAS05316 Database only5-HT-Ser2 C. elegans nematode −AC NP–741936 675-HT-Ser3 C. elegans nematode NP–491954 Database only5-HT C. elegans nematode NP–508238 Database only
Molecular Biology and Genomic Organization 5
(continued)
Table 1 (continued)
Probablesecond Accession
Receptor Species messengera number Ref.
Vertebrates5-HT1A-α Fugu fish −AC CAA65175 685-HT1A-β Fugu fish −AC CAA65176 685-HT1A-α Tetraodon fish −AC CAF93441 Database only5-HT1A-β Tetraodon fish −AC CAF91711 Database only5-HT1A Tilapia fish −AC AAP83427 Database only5-HT1A Chicken −AC XP–429136 Database only5-HT1A Human −AC NP–000515 69, 705-HT1A Rat −AC NP–036717 715-HT1A Gorilla −AC BAA94490 Database only5-HT1A Orangutan −AC BAA94491 Database only5-HT1A Mouse −AC NP–032334 725-HT1A Chimpanzee −AC BAA94489 Database only5-HT1A Fox −AC AAP12466 735-HT1A Dog −AC AAN08044 745-HT1A Frog −AC CAA69208 755-HT1B Chimpanzee −AC BAA94456 Database only5-HT1B Human −AC AAH69065 76–845-HT1B Gorilla −AC BAA94457 Database only5-HT1B Dog −AC AAP12469 73, 855-HT1B Fox −AC AAP12468 735-HT1B Pig −AC NP–999463 865-HT1B Mouse −AC NP–034612 875-HT1B Rat −AC NP–071561 885-HT1B Rabbit −AC CAA90531 895-HT1B Golden hamster −AC AAK25827 Database only5-HT1B Mole rat −AC AAB82748 Database only5-HT1B Chinese hamster −AC CAA60175 Database only5-HT1B Guinea pig −AC AAB58500 905-HT1B Opossum −AC AAA17567 915-HT1B Tetraodon fish −AC CAF89927 Database only5-HT1B Chicken −AC XP–419875 Database only5-HT1D Chimpanzee −AC XP–524604 Database only5-HT1D Human −AC NP–000855 76, 925-HT1D Pig −AC NP–999323 935-HT1D Rabbit −AC CAA90530 895-HT1D Guinea pig −AC CAA64210 945-HT1D Rat −AC NP–036984 79, 88
6 Kroeze and Roth
5-HT1D Mouse −AC NP–032335 82, 955-HT1D Dog −AC CAA32207 96–985-HT1D Fugu fish −AC CAA58745 685-HT1D Tilapia fish −AC AAP83428 Database only5-HT1E Chimpanzee −AC XP–527443 Database only5-HT1E Human −AC NP–000856 81, 84, 995-HT1E Gorilla −AC BAA94460 Database only5-HT1E Orangutan −AC BAA94461 Database only5-HT1E Guinea pig −AC AAR05654 1005-HT1F Chimpanzee −AC XP–526246 Database only5-HT1F Human −AC NP–000857 101, 1025-HT1F Gorilla −AC BAA90455 Database only5-HT1F Orangutan −AC BAA90456 Database only5-HT1F Mouse −AC NP–032336 1035-HT1F Pig −AC NP–999266 1045-HT1F Guinea pig −AC AAB58496 1055-HT1F Rat −AC NP–068629 1015-HT1F Chicken −AC XP–425535 Database only5-HT1F Tetraodon fish −AC CAG09227 Database only5-HT2A Cow PI NP–001001157 Database only5-HT2A Dog PI NP–001005869 855-HT2A Pig PI NP–999382 106, 1075-HT2A Human PI NP–000612 108–1135-HT2A Rhesus PI AAB34691 1065-HT2A Orangutan PI CAH93260 Database only5-HT2A Rat PI NP–058950 108, 1145-HT2A Chinese hamster PI CAA37800 1155-HT2A Mouse PI NP–766400 1165-HT2A Chicken PI XP–425628 Database only5-HT2A Chimpanzee PI XP–522752 Database only5-HT2B Human PI NP–000858 117, 1185-HT2B Mouse PI NP–032337 1195-HT2B Rat PI NP–058946 1205-HT2B Frog PI CAD71264 1215-HT2B Tetraodon Fish PI CAC85912 1225-HT2C Chicken PI XP–426265 Database only5-HT2C Dog PI NP–001006649 85
Table 1 (continued)
Probablesecond Accession
Receptor Species messengera number Ref.
(continued)
Molecular Biology and Genomic Organization 7
Table 1 (continued)
Probablesecond Accession
Receptor Species messengera number Ref.
5-HT2C Rat PI NP–036897 1235-HT2C Human PI NP–000859 109, 1245-HT2C Mouse PI NP–032338 125, 1265-HT2C Chimpanzee PI XP–529113 Database only5-HT4 Pig +AC NP–001001267 1075-HT4 Guinea pig +AC CAA73912 Database only5-HT4 Human +AC CAC22248 107, 127–1315-HT4 Mouse +AC CAA70775 128, 129, 1325-HT4 Rat +AC NP–036985 37, 107, 1295-HT4 Chicken +AC XP–414481 Database only5-HT4 Tetraodon fish +AC CAF95370 Database only5-HT4 Chimpanzee +AC XP–518024 Database only5-HT5A Guinea pig CAD59057 1335-HT5A Rat NP–037280 1345-HT5A Human NP–076917 1355-HT5A Mouse NP–032340 136, 1375-HT5A Chicken XP–425970 Database only5-HT5A Zebrafish NP–001007122 Database only5-HT5A Tetraodon fish CAG04298 Database only5-HT5A Chimpanzee XP–519477 Database only5-HT5B Mouse NP–034613 1365-HT5B Rat XP–341112 134, 1385-HT6 Human +AC NP–000862 107, 1395-HT6 Chimpanzee +AC XP–524584 Database only5-HT6 Rat +AC NP–077341 140, 1415-HT6 Mouse +AC NP–067333 1425-HT7 Guinea pig +AC AAA83015 1435-HT7 Rat +AC NP–075227 144–1485-HT7 Pig +AC NP–999250 1495-HT7 Human +AC NP–000863 144, 145, 1505-HT7 Chimpanzee +AC XP–521556 Database only5-HT7 Dog +AC CAF31500 Database only5-HT7 Mouse +AC NP–032341 1515-HT7 Chicken +AC XP–420880 Database only5-HT7 Tetraodon fish +AC CAG05134 Database only5-HT7x Tetraodon fish +AC CAG09680 Database only
a+AC = stimulates adenylate cyclase activity; −AC = inhibits forskolin-stimulatedadenylate cyclase activity; PI = stimulates phosphatidylinositol (PI) hydrolysis.
8 Kroeze and Roth
obviously, for these receptors to be functional, all seven TM helices must bepresent. Thus, for example, the chimpanzee 5-HT2A receptor (accession numberXP–522752) has an approx 50-residue insertion in transmembrane helix 4(TM4) and a deletion that removes most of TM6 and TM7, although the distalend of the C-terminus is present in the database sequence (Fig. 1). Similarly,the first 35 residues of the chimpanzee 5-HT2C receptor (accession numberXP–529113) as given in the database bear no resemblance to the human 5-HT2C
receptor, and the “true” 5-HT2C receptor sequence of the chimpanzee protein aslisted in the database begins about halfway through TM4. In the chimpanzee5-HT4 receptor sequence (accession number XP–518024), the “Y” of the highlyconserved DRY motif at the intracellular end of TM3, the i2 intracellular loop,and TM4 are missing from the database sequence. The chimpanzee 5-HT5A
Fig. 1. Alignment of the putative chimpanzee 5-HT2A receptor sequence (accessionnumber XP–522752) with the human 5-HT2A receptor sequence (accession numberNP–000612), illustrating an example of differences between the receptor sequences ofthe two species probably caused by faulty database annotation.
Molecular Biology and Genomic Organization 9Molecular Biology and Genomic Organization 9
receptor sequence (accession number XP–519477) is incomplete and contains arun of “X”s that preclude alignment with the other known 5-HT receptors; addi-tionally, although both the N-terminus and C-terminus are present in thesequence, only the N-terminus is similar to known 5-HT receptors, and theC-terminal sequence, following the run of “X”s, bears no resemblance to anyknown 5-HT receptor by BLAST search. Although we have presented the chim-panzee 5-HT6 receptor sequence (accession number XP–524584) in the align-ment of all vertebrate 5-HT receptors shown in Fig. 2, the amino-terminus of thesequence in the database is likely to be incorrect because it is much longer in thechimpanzee sequence than in any other known 5-HT receptor sequence. A pos-sible explanation for this is that the DNA sequence for this region is very GCrich, which may have made DNA sequencing difficult, and, thus, that an error inthe reading of this sequence may have resulted in the abnormally long amino-terminus. Obviously, many of these chimpanzee sequences as listed wouldrepresent nonfunctional receptors. Therefore, it remains an open question(1) whether these represent aberrant splicing isoforms among the chimpanzee5-HT receptors that do not exist in humans, (2) whether chimpanzee 5-HTreceptors are truly different than those from other mammals, or (3) whetherfurther investigation of the genomic sequences with expert curation will reveal5-HT receptor sequences more like those from other primate or mammalianspecies. Of these three possibilities, the third seems most likely.
The chicken 5-HT2C receptor sequence in the database (accession numberXP–426265) is 1337 residues long, as compared to 458 residues for the humanreceptor (accession number NP–000859). Much of this “extra” sequence residesin the amino-terminus, which in the chicken sequence has the highly conserved“GN” motif in TM1 at positions 459–460, as opposed to positions 70–71 in thehuman 5-HT2C receptor sequence. In the chicken sequence, the e1 extracellularloop is also very long and the highly conserved “DRY,” motif is given as “DRC,”although many other features of 5-HT2C receptors are retained. It is possible that5-HT2C receptors of birds are in fact very different from the 5-HT2C receptors ofother species, as the current sequence data imply, or that the chicken 5-HT2C
receptor sequence in the database is incorrect. Which of these alternatives isfound to be true awaits further research.
As can be seen from the alignment shown in Fig. 2, the sequence given in thedatabase for the Tetraodon 5-HT4 receptor (accession number CAF95370) isunusually short, beginning with a methionine in TM1. This “ATG” is unlikely tobe the true “start” codon for this receptor because this results in part of TM1being absent, but examination of the genomic sequence shows a “TAG” stopcodon immediately upstream of the “ATG” methionine codon. Whether this is
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