G protein-coupled receptors PDF
Preview G protein-coupled receptors
G P -C ROTEIN OUPLED R ECEPTORS Edited by Tatsuya Haga Gabriel Berstein CRC Press Boca Raton New York London Tokyo Series: Methods in Signal Transduction Library of Congress Cataloging-in-Publication Data G protein-coupled receptors / Tatsuya Haga and Gabriel Berstein, editors. p. cm. -- (Signal transduction series) Includes bibliographical references and index. ISBN 0-8493-3384-9 (alk. paper) 1. G proteins--Receptors Laboratory manuals. I. Haga, Tatsuya. II. Berstein, Gabriel. III. Series. QP552.G16G17 1999 572'.6--DC21 99-26359 CIP This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage or retrieval system, without prior permission in writing from the publisher. The consent of CRC Press LLC does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from CRC Press LLC for such copying. Direct all inquiries to CRC Press LLC, 2000 Corporate Blvd., N.W., Boca Raton, Florida 33431. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are only used for identification and explanation, without intent to infringe. Cover: Illustrates a 3D model of muscarinic acetylcholine receptors M2 subtype; modified from the model calculated on the basis of the atomic structure of bacteriorhodopsin by Dr. G. Vriend. © 2000 by CRC Press LLC No claim to original U.S. Government works International Standard Book Number 0-8493-3384-9 Library of Congress Card Number 99-26359 Printed in the United States of America 1 2 3 4 5 6 7 8 9 0 Printed on acid-free paper Preface G proteins (heterotrimeric GTP-binding proteins) have been said to be “involved in everything from sex in yeast to cognition in humans.”* G protein-coupled receptors (GPCRs), which activate G proteins when bound to specific ligands, constitute one of the largest superfamilies of proteins. In Caenorhabditis elegans, an estimated 1049 genes which code for GPCRs are present among 19,099 genes (5.5%).** In the human genome, 252 GPCR genes were identified at the time when 15% of the genome was sequenced, suggesting that approximately 1700 GPCR genes may be present.*** Ligands of GPCRs are extremely diverse and include hormones, neu- rotransmitters, autacoids, cytokines, chemotactic factors, odorants, pheromones, and taste substances. Rhodopsin and cone pigments in retina, which respond to light, are also GPCRs, as are targets of many drugs including those for pain, hypertension, duodenal ulcer, schizophrenia, and asthma. An enormous variety of chemical species comprise GPCR ligands, among which are amines, amino acids, peptides, proteins, nucleosides, nucleotides, Ca2+ ions, and lipids, such as prostaglandins and leukot- rienes. In addition, 1000 distinct GPCR odorant receptors are thought to discriminate over 100,000 kinds of odorants, which have diverse chemical structures. This book covers current techniques used to study GPCRs, particularly methods related to studies that become possible by the molecular cloning of GPCRs. Most chapters include step by step protocols which are intended to be useful for new researchers as well as specialists in the field. The book consists of three sections: Section I Novel Ligands: Ligand Screening Systems and Orphan Receptors Section II Function and Regulation: Receptor-G Protein Coupling and Desensitization Section III 3D Structure: Large-Scale Expression and Electron- and X-Ray Crystal- lography Section I should be particularly useful for researchers who are interested in drug discovery. Basic theory of receptor-ligand interactions and different kinds of ligand screening systems are introduced. Historically, receptors and receptor subtypes were functionally identified by the action of specific ligands, exogenous agonists and antagonists. For example, muscarinic acetylcholine receptors were discriminated from nicotinic acetylcholine receptors by the actions of nicotine and muscarine and were further subdivided into M , M , and M subtypes through the use of specific 1 2 3 * Gilman, A.G., Science 266, 368, 1994. ** Bargman, C., Science 282, 2028, 1998. *** Henikoff, S. et al., Science 278, 609, 1997. ©2000 CRC Press LLC antagonists. Molecular cloning revealed the existence of two additional subtypes, M and M . A current challenge is to find specific ligands for the M and M receptor 4 5 4 5 subtypes. A similar requirement exists for almost all GPCRs. This kind of “reverse pharmacology” is more important for novel GPCRs, for which no specific ligands have been identified, and for “orphan receptors,” for which endogenous ligands have not been identified. Identification of subtype-specific ligands or an endogenous ligand for a given GPCR will be a key step in elucidating receptor functions and developing novel drugs. Chapter 1 provides fundamental knowledge and techniques concerning the interaction between GPCRs and iso- or allosteric ligands. Chapters 2 and 3 outline ligand screening systems that utilize yeast which express mammalian GPCRs and frog melanophores, respectively. Methods used to screen orphan recep- tors for endogenous ligands are described in Chapter 4. Section II is concerned with the interactions of GPCRs with G proteins and with GPCR desensitization. Interactions between GPCRs and G proteins can be analyzed using in vitro reconstitution of purified proteins in lipid membranes as presented in Chapter 5, or by means of expression of mutated proteins in cultured cells, as described in Chapter 6. These approaches have provided mechanistic insights into the initial biochemical events that are triggered by receptor activation, and they continue to be useful strategies in the characterization of novel receptors. Attenuation of receptor-mediated responses following prolonged exposure to agonists has long been known as desensitization. This is an active area of research because it is critical to understand how molecular mechanisms of desensitization control the duration and amplitude of receptor-mediated signals. Chapter 7 introduces general methods used to study desensitization of GPCRs and related phenomena of GPCR phospho- rylation. Chapter 8 discusses a specific form of desensitization, namely internaliza- tion of GPCRs. Chapter 9 describes the application of electrophysiology to studies of GPCR desensitization. Section III addresses the three-dimensional structure of GPCRs. The determi- nation of these membrane protein structures will unquestionably have a strong impact on our understanding of the interaction between GPCRs and G proteins and may also lead to theoretical prediction of effective ligands. Currently no three-dimen- sional structures of GPCRs are available, except a low resolution structure of rhodop- sin. The first difficulty in structural determination is that except rhodopsin, sufficient amounts of GPCRs cannot be readily attained. Large-scale expression and purifica- tion of GPCRs is therefore a prerequisite for structural studies. Chapter 10 presents a method for expression of functional GPCRs in E. coli. Chapter 11 extensively surveys and compares different expression systems for GPCRs, placing particular emphasis on expression in yeast. Examples for determination of the atomic structure of membrane proteins are described in Chapters 12 through 14. The structure of bacteriorhodopsin, which has seven transmembrane segments and is often assumed to be a model for GPCRs, has been elucidated using both electron crystallography (Chapter 12) and X-ray crystallography (Chapter 14). Principles and applications of two-dimensional crystallization and cubic phase crystallization are introduced in Chapters 12 and 14, respectively. Another example of the structural determination of a membrane-bound receptor both with and without its ligand-induced conforma- tional change is provided in Chapter 13. ©2000 CRC Press LLC Thus we believe that this book will be useful for those who wish to develop new drugs targeted to GPCRs, find endogenous ligands for orphan receptors, eluci- date the molecular mechanisms underlying the function and regulation of GPCRs, or determine the three-dimensional structure of GPCRs. Finally, we would like to sincerely thank each of the authors for taking time out of their busy schedules to write these excellent chapters. Tatsuya Haga Gabriel Berstein June 18, 1999 ©2000 CRC Press LLC Contributors Robert S. Ames James J. Foley SmithKline Beecham Pharmaceuticals SmithKline Beecham Pharmaceuticals King of Prussia, PA King of Prussia, PA Derk J. Bergsma Yoshinori Fujiyoshi SmithKline Beecham Pharmaceuticals Department of Biophysics King of Prussia, PA Faculty of Science Kyoto University Gloria H. Biddlecome Kyoto, Japan Howard Hughes Medical Institute Reinhard Grisshammer University of Iowa College of Medicine MRC Laboratory of Molecular Biology Iowa City, IA Cambridge, UK Nigel J.M. Birdsall John R. Hadcock Division of Physical Biochemistry Pfizer Central Research National Institute for Medical Research Division of Cardiovascular/Metabolic Mill Hill, London, UK Diseases Groton, CT Mark R. Boyett Department of Physiology Mac E. Hadley University of Leeds Departments of Cell Biology and Leeds, UK Anatomy College of Medicine Jon K. Chambers University of Arizona SmithKline Beecham Pharmaceuticals Tucson, AZ New Frontiers Science Park Harlow, Essex, UK Brian D. Hellmig SmithKline Beecham Pharmaceuticals Mark L. Chiu King of Prussia, PA Department of Chemistry Seton Hall University Jan Jakubik South Orange, NJ Laboratory of Bioorganic Chemistry NIDDK Jeffrey S. Culp National Institutes of Health SmithKline Beecham Pharmaceuticals Bethesda, MD King of Prussia, PA ©2000 CRC Press LLC Iftikhar A. Khan Michele Loewen Department of Physiology Department of Biology University of Leeds Massachusetts Institute of Technology Leeds, UK Cambridge, MA Evi Kostenis Dean E. McNulty Laboratory of Bioorganic Chemistry SmithKline Beecham Pharmaceuticals NIDDK King of Prussia, PA National Institutes of Health Bethesda, MD Dino Moras Laboratorie de Biologies Structurale Hitoshi Kurose Institut de Génétique et de Biologie Laboratory of Pharmacology and Moléculaire at Cellulaire Toxicology CNRS/INSERM/ULP Graduate School of Pharmaceutical Illkirch Cedex, France Sciences Alison I. Muir University of Tokyo Tokyo, Japan SmithKline Beecham Pharmaceuticals New Frontiers Science Park Jelveh Lameh Harlow, Essex, UK Molecular Research Institute Peter Nollert Palo Alto, CA University of California Departments of Biopharmaceutical San Francisco, CA Sciences and Pharmaceutical Chemistry University of California Janet E. Park San Francisco, CA SmithKline Beecham Pharmaceuticals New Frontiers Science Park Ehud M. Landau Harlow, Essex, UK Biozentrum University of Basel Mark H. Pausch Basel, Switzerland Wyeth-Ayerst Research Molecular Genetic Screen Design Group Sebastian Lazareno Princeton, NJ MCR Collaborative Centre 1-3 Burtonhole Lane J. Mark Quillan Mill Hill, London, UK Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry Kaspar P. Locher University of California Department of Microbiology San Francisco, CA Biozentrum University of Basel Bernard Rees Basel, Switzerland Laboratorie de Biologies Structurale present address: Institut de Génétique et de Biologie Division of Chemistry Moléculaire at Cellulaire California Institute of Technology CNRS/INSERM/ULP Pasadena, CA Illkirch Cedex, France ©2000 CRC Press LLC Helmut Reiländer Jeffrey M. Stadel Max Planck Institut für Biophysik SmithKline Beecham Pharmaceuticals Abt. Mol. Membranbiologie King of Prussia, PA Frankfurt/M, Germany Agnes Szmolenszky Christoph Reinhart Max Planck Institut für Biophysik Max Planck Institut für Biophysik Abt. Mol. Membranbiologie Abt. Mol. Membranbiologie Frankfurt/M, Germany Frankfurt/M, Germany Julie Tucker Jurg P. Rosenbusch MRC Laboratory of Molecular Biology Department of Microbiology Cambridge, UK Biozentrum present address: University of Basel Laboratory of Molecular Biophysics Basel, Switzerland Oxford, UK Henry M. Sarau Jürgen Wess SmithKline Beecham Pharmaceuticals Laboratory of Bioorganic Chemistry King of Prussia, PA NIDDK National Institute of Health National Institutes of Health Torsten Schöneberg Bethesda, MD Institut fur Pharmakologie Fachbereich Humanmedizin Christine Widmer Freie Universität Berlin Biozentrum Berlin, Germany University of Basel Basel, Switzerland Zhigang Shui Department of Physiology Shelagh Wilson University of Leeds SmithKline Beecham Pharmaceuticals Leeds, UK New Frontiers Science Park Harlow, Essex, UK J. Randall Slemmon SmithKline Beecham Pharmaceuticals Fu-Yue Zeng King of Prussia, PA Laboratory of Bioorganic Chemistry NIDDK National Institutes of Health Bethesda, MD Xiangyang Zhu Laboratory of Bioorganic Chemistry NIDDK National Institutes of Health Bethesda, MD ©2000 CRC Press LLC List of Protocols Chapter 1 1-1. Tissue Culture 1-2. Membrane Preparation 1-3. Protein Estimation and Final Membrane Preparation 1-4. Saturation Analysis with 3H-N-methylscopolamine 1-5. Allosteric Interaction with a Labeled and Unlabeled Ligand— the Affinity Ratio Plot 1-5a. The Affinity Ratio Plot, With Estimation of 3H-NMS Kd 1-5b. The Affinity Ratio Plot, With the Kd of 3H-NMS Already Known Chapter 2 2-1. Modified Rapid Protocol for Lithium Acetate Transformation of Yeast 2-2. Yeast Membrane Preparation 2-3. GTPg[35S] Binding to Yeast Membrane Preparations 2-4. Agar-Based Bioassay for GPCRs Expressed in Yeast 2-5. Liquid-Based Bioassays for GPCRs Expressed in Yeast Chapter 3 3-1. Preparation and Maintenance of Melanophore Cells 3-2. Photometric Reflectance Skin/Melanophore Bioassays 3-3. Photometric Absorbance Measurements from Multi-well Plates Using Cultured Melanophores 3-4. Synthesis of MUPLs 3-5. Gas-Phase Cleavage and Detoxification 3-6. Gel Assays Chapter 4 4-1. Transient Transfection of Orphan Receptors into HEK 293 Cells 4-2. Use of Cytosensor Microphysiometer to Screen Orphan Receptors 4-3. Ca2+ Mobilization Measurements Using Fluoskan Ascent Fluorescent Plate Reader (Labsystems) 4-4. Ca2+ Mobilization Measurements Using FLIPR (Fluorometric Imaging Plate Reader) 4-5. cAMP Determination Using Flashplates 4-6. Preparation of Peptide Libraries from Tissue Extracts 4-7. Purification and Identification of a Novel Peptide Ligand for the Orphan Receptor GPR10 ©2000 CRC Press LLC Chapter 5 5-1. Sf9 Cell Membrane Preparation 5-2. Purification of M Muscarinic Acetylcholine Receptor 1 5-3. Assay for Membrane-Bound M Muscarinic Receptor 1 5-4. Assay for Soluble M Muscarinic Receptor 1 5-5. Purification of G qa 5-6. Measurement of Protein-Bound GDP 5-7. Assay of PLC-b1 Activation by Purified G qa 5-8. Purification of G bg 5-9. Purification of PLC-b1 5-10. Assay of Purified PLC-b1 Activation by G qa 5-11. Preparation of Lipid Vesicles that Contain M 1 Muscarinic Receptor and G qa 5-12. Quantitation of M Muscarinic Receptor in Vesicles that 1 Contain [3H]-PIP 2 5-13. M Muscarinic Receptor-Stimulated [3H]PIP Hydrolysis 1 2 by PLC-b1 5-14. M Muscarinic Receptor-Stimulated [35S]-GTPgS 1 Binding to G qa 5-15. M Muscarinic Receptor-Stimulated [g32P]-GTP Hydrolysis 1 by G qa 5-16. Amido Black Protein Assay 5-17. Measurement of Phospholipid Concentration by Phosphorus Determination 5-18. Preparation and Calibration of Gel Filtration Column 5-19. Enzymatic Synthesis of [g32P]-GTP Chapter 6 6-1. A Universal PCR Mutagenesis Strategy 6-2. Cotransfection of COS-7 Cells with GPCRs and G Protein a Subunits 6-3. Inositol Phosphate Assay Using Transfected COS-7 Cells 6-4. ELISA to Measure Cell Surface Expression of GPCRs/GPCR Fragments (Example) 6-5. Sandwich ELISA to Measure Association of GPCR Fragments (Example) Chapter 7 7-1. Stable Transfection of CHO Cells 7-2. Transient Transfection of HEK 293 Cells 7-3. Ligation of cDNA With a Cosmid Vector 7-4. Co-transfection of Cosmid Vector and Adenovirus DNA into HEK 293 Cells 7-5. Determination of the Adenovirus Titer 7-6. Expression of Recombinant Protein by Adenovirus ©2000 CRC Press LLC
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