1、G Protein SignalingM E T H O D S I N M O L E C U L A R B I O L O G YJohn M. Walker, SERIES EDITOR260. Mobile Genetic Elements: Protocols and Genomic Applica- tions edited by Wolfgang J. Miller and Pierre Capy, 2004259. Receptor Signal Transduction Protocols, Second Edition,edited by Gary B. Willars

2、and R. A. John Challiss, 2004258. Gene Expression Profiling: Methods and Protocols, edited by Richard A. Shimkets, 2004257. mRNA Processing and Metabolism: Methods and Proto- cols, edited by Daniel R. Schoenberg, 2004256. Bacterial Artifical Chromosomes, Volume 2: Functional Studies, edited by Shayi

3、ng Zhao and Marvin Stodolsky, 2004255. Bacterial Artifical Chromosomes, Volume 1: Library Con- struction, Physical Map , and Sequencing, edited by Shaying Zhao and Marvin Stodolsky, 2004254. Germ Cell Protocols, Volume 2: Molecular Embryo Analy- sis, Live Imaging, Transgenesis, and Cloning, edited b

4、y Heide Schatten, 2004253. Germ Cell Protocols, Volume 1: Sperm and Oocyte Analy- sis, edited by Heide Schatten, 2004252. Ribozymes and siRNA Protocols, Second Edition, edited by Mouldy Sioud, 2004251. HPLC of Peptides and Proteins: Methods and Protocols,edited by Marie-Isabel Aguilar, 2004250. MAP

5、Kinase Signaling Protocols, edited by Rony Seger, 2004249. Cytokine Protocols, edited by Marc De Ley, 2004235.E coli Plasmid Vectors: Methods and Applications, edited byNicola Casali and Andrew Preston, 2003p53 Protocols, edited by Sumitra Deb and Swati Palit Deb, 2003 Protein Kinase C Protocols, ed

6、ited by Alexandra C. Newton, 2003Protein Misfolding and Disease: Principles and Protocols, edited by Peter Bross and Niels Gregersen, 2003Directed Evolution Library Creation: Methods and Proto- cols, edited by Frances H. Arnold and George Georgiou, 2003 Directed Enzyme Evolution: Screening and Selec

7、tion Meth- ods, edited by Frances H. Arnold and George Georgiou, 2003 Lenti Gene Engineering Protocols, edited by Maurizio Federico, 2003Membrane Protein Protocols: Expression, Purification, and Characterization, edited by Barry S. Selinsky, 2003 Membrane Transporters: Methods and Protocols, edited

8、by Qing Yan, 2003PCR Protocols, Second Edition, edited by John M. S. Bartlett and David Stirling, 2003Inflammation Protocols, edited by Paul G. Winyard and Derek A. Willoughby, 2003Functional Genomics: Methods and Protocols, edited byMichael J. Brownstein and Arkady Khodursky, 2003Tumor Suppressor G

9、enes: Volume 2: Regulation, Function, and Medicinal Applications, edited by Wafik S. El-Deiry, 2003 Tumor Suppressor Genes: Volume 1: Pathways and Isola- tion Strategies, edited by Wafik S. El-Deiry, 2003 Generation of cDNA Libraries: Methods and Protocols, edited by Shao-Yao Ying, 2003Cancer Cytoge

10、netics: Methods and Protocols, edited by John Swansbury, 2003Cardiac Cell and Gene Transfer: Principles, Protocols, and Applications, edited by Joseph M. Metzger, 2003Cancer Cell Signaling: Methods and Protocols, edited byDavid M. Terrian, 2003Neurogenetics: Methods and Protocols, edited by Nicholas

11、T. Potter, 2003PCR Detection of Microbial Pathogens: Methods and Protocols, edited by Konrad Sachse and Joachim Frey, 2003 Cytokines and Colony Stimulating Factors: Methods and Protocols, edited by Dieter Körholz and Wieland Kiess, 2003 Supergen Protocols, edited by Teresa Krakauer, 2003 Capill

12、ary Electrophoresis of Carbohydrates, edited by Pierre Thibault and Susumu Honda, 2003Single Nucleotide Polymorphisms: Methods and Protocols,edited by Pui-Yan Kwok, 2003Protein Sequencing Protocols, Second Edition, edited byBryan John Smith, 2003MHC Protocols, edited by Stephen H. Powis and Robert W

13、. Vaughan, 2003Transgenic Mouse Methods and Protocols, edited by Mar- ten Hofker and Jan van Deursen, 200348.body Engineering: Methods and Protocols, edited byBenny K. C. Lo, 2004222.247. Drosophila Cytogenetics Protocols, edited by Daryl S. Henderson

14、, 2004246. Gene Delivery to Mammalian Cells: Volume 2: Viral Gene Transfer Techniques, edited by William C. Heiser, 2004245. Gene Delivery to Mammalian Cells: Volume 1: Nonviral Gene Transfer Techniques, edited by William C. Heiser, 2004244. Protein Purification Protocols, Second Edition, edited byP

15、aul Cutler, 200418.243. Chiral Separations: Methods and Protocols, edited byGerald Gübitz and Martin G. Schmid, 2004242. Atomic Force Microscopy: Methods and Applications,edited by Pier Carlo Braga and Davide Ricci, 2004241. Cell Cycle Checkpoint Control Protocols, edited by Howard

16、B. Lieberman, 2004240. Mammalian Artificial Chromosomes: Methods and Proto- cols, edited by Vittorio Sgaramella and Sandro Eridani, 2003239. Cell Migration in Inflammation and Immu : Methods and Protocols, edited by Daniele DAmbrosio and Francesco Sinigaglia, 2003238. Biopolymer Methods in Tissue En

17、gineering, edited byAnthony P. Hollander and Paul V. Hatton, 2003237. G Protein Signaling: Methods and Protocols, edited by AlanV. Smrcka, 2003236. Plant Functional Genomics: Methods and Protocols, edited by Erich Grotewold, 200311.210.209.G Protein SignalingMethods and Prot

18、ocolsEdited byAlan V. SmrckaDepartment of Pharmacology and Physiology, University of Rochester School of Medicine, Rochester, NYHumana PressTotowa, New JerseyM E T H O D S I N M O L E C U L A R B I O L O G Y© 2004 Humana Press Inc.999 Riverview Drive, Suite 208 Totowa, New Jersey 07512. No part

19、 of this book may be reproduced, stored in a retrieval system, or transmitted in any form or byany means, electronic, mechanical, photocopying, microfil, recording, or otherwise without written permission fromthe Publisher. Methods in Molecular BiologyTM is a trademark of The Humana Press Inc.All pa

20、pers, comments, opinions, the views of the publisher.s, or recommendatoins are those of the author(s), and do not necessarily reflectpaper. ¥This publication is printed on acid-ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials.Production Editor: R

21、obin B. Weisberg. Cover design by Patricia F. Cleary.Cover illustrations: Background: Immunocytochemical localization of HA-tagged thyrotropin releasing hormone receptor in HEK293 cells. Image provided by Patricia Hinkle. Inset: Figure 1 from Chapter 20.For additional copies, pricing for bulk purcha

22、ses, and/or information about other Humana titles, contact Humana at the aboveaddress or at any of the following numbers: Tel.: visit our Website:; Fax:;: humana; orPhotocopy Authorization Policy:Photocopy Authorization Policy: Authorization to photocopy items for internal oral use, or the internal

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24、has been arranged and is acceptable to the Humana Press. The fee code for users of the Tranional Reporting Service is 1-58829-137-5/04 $25.00.Printed in the United States of America. 10 9 8 7 6 5 4 3 2 11-59259-430-1 (e-book)Library of Congress Cataloging in Publication DataG protein signaling : met

25、hods and protocols / edited by Alan V. Smrcka.p. ; cm. (Methods in molecular biology ; v. 237) Includes bibliographical references and index.ISBN 1-58829-137-5 (alk. paper) ISSN 1064-37451. G proteinsLaboratory manuals. 2. Cellular signal transductionLaboratory manuals. I. Smrcka, Alan, V. II. Serie

26、s.QP552.G16G185 2003 572'.643dc212003042323PrefaceSince the initial discovery of the G protein-coupled receptor system that regulates cyclicAMP production, the G protein field has rapidly expanded. Cell surface receptors that couple to heterotrimeric G proteins, the G protein- coupled receptors

27、(GPCRs), number in the hundreds and bind to a wide diver- sity of ligands including, biogenic amines (e.g., adrenaline), lipid derivatives (e.g., lysophosphatidic acid), peptides (e.g., opioid peptides), proteins (e.g., thyroid-stimulating hormone), and odorants to name a few. The GPCR system is fou

28、nd throughout biology in such simple organisms as yeast and in such more complex organisms as Dictyostelium discoideum (slime mold), Caenor- habditis elegans (nematode worm), and of course in humans. GPCRs and their associated G protein systems are the subject of intense academic research and becaus

29、e of their involvement in a human biology and disease, the pharmaceu- tical industry has large research initiatives dedicated to the study of GPCRs. By some estimates, more than 50% of the pharmaceuticals on the market are targeted at GPCRs.The G protein/G protein-coupled receptor system consists of

30、 a receptor (GPCR), a heterotrimeric G protein consisting of a, b, and g subunits, and an effector. G protein effector molecules, such as enzymes or ion channels, respond to activa- tion by the G protein to generate second messengers or changes in membrane potential that lead to alterations in cell

31、physiology. Superimposed on this classical G protein framework are the recently discovered regulators of G protein signaling (RGS proteins) that provide an additional level of G protein regulation, but whose physiological functions remain undefined. The heterotrimeric G proteins themselves form a di

32、verse family of subunits with more than 20 a subunits, 6 b subunits and 13 g subunits. There are many effectors, each of which may be repre- sented by multiple isoforms. Analysis of such a complex system requires meth- ods that can tackle each of these molecules and the interactions between them in

33、biochemical and cell biological contexts. The variety of strategies that are employed range from studies with purified components to studies in cell bio- logical and whole animal systems. In this volume we have gathered together many of the methodologies that are used to study mechanisms of G protei

34、n and G protein coupled receptor function and the roles of G protein subunits in cell biology and disease. Many of the chapters cover topics that use a variety of G protein methodologies, so within single chapters multiple specific methodolo-gies are described in. In some cases not only is the metho

35、d itselfdescribed, but the principles that underlie the experimental design are also out-vviPrefacelined in ways that are not described in other publications. And of course in eachchapter there is a Notes section dealing with criticals that are often notincluded in formal publications owing to space

36、 limitations and other issues.G Protein Signaling: Methods and Protocols has been divided into sevenparts somewhat arbitrarily, but based on teral types of systems beingused. The first part deals with purification of G proteins and effector enzymes from heterologous expressions systems. To study G p

37、roteins and their interac- tions with receptors and targets it is often necessary to purify the proteins prior to analysis. The second part of the volume has protocols for assays of theinteractions between these purified G proteins and effector enzymes. Alternate strategies outlined in Part III are

38、for the study of G protein interactions with effectors in intact cells, either with the endogenous components or with expressed components. Each of these approaches has advantages and disad- vantages with regard to data interpretation and analysis. The fourth part is con- cerned with various assays

39、of G protein coupled receptor structure, function, and localization. The fifth part has protocols for studying the physiological roles for endogenous G proteins, either in cell culture systems or in whole animals, using approaches that inhibit these endogenous systems. Regulators of G protein signal

40、ing (RGS) proteins are a novel class of proteins that regulate the activity of heterotrimeric G proteins. Part VI describes methods for study- ing lipid and phosphate modifications of these proteins. Finally, green fluores- cent proteins and their derivatives have been used to study the localization

41、 and interactions of many proteins in cells. In Part VII, the specific application of such green fluorescent proteins to G protein signaling systems are described.The methods outlined in G Protein Signaling: Methods and Protocols should be of interest to scientists studying the physiological roles o

42、f G protein sys- tems, the signal transduction systems associated with these G protein systems, as well as the molecular nature of the G proteins themselves. The volume con- tains complementary biochemical, molecular biological, and cell biological approaches to addressing specific questions so that

43、 they can be studied from multiple perspectives and adopted by different types of laboratories.Alan V. SmrckaContentsPrefacevContributorsxiPART I. PURIFICATION OF G PROTEIN SUBUNITS AND G PROTEIN EFFECTORS1Purification of Recombinant G Protein a Subunits from Escherichia coliWendy K. Greentree and M

44、aurine E. Linder3Purification of G Protein Subunits from Sf9 Insect Cells Using Hexahistidine-Tagged a and bg SubunitsTohru Kozasa21Expression and Purification of Soluble Adenylyl Cyclase fromEschericia coliJeff A. Beeler and Wei-Jen Tang39Purification of Phospholipase C b and Phospholipase C efrom

45、Sf9 CellsMousumi Ghosh, Huan Wang, Grant G. Kelley,and Alan V. Smrcka55234PART II. ASSAYS FOR G PROTEIN-DEPENDENT EFFECTOR ACTIVATION IN VITRO5Assay for G Protein-Dependent Activation of Phospholipase C bUsing Purified Protein ComponentsMousumi Ghosh and Alan V. Smrcka67Assays of Recombinant Adenyly

46、l Cyclases Expressed in Sf9 CellsRonald Taussig776PART III. ASSAYS FOR G PROTEIN-COUPLED RECEPTOR-MEDIATED AND G PROTEIN SUBUNIT-MEDIATED EFFECTOR ACTIVATION IN INTACT CELLS7Measurement of G Protein-Coupled Receptor-Stimulated Phospholipase D Activity in Intact CellsStephanie J. Walker and H. Alex B

47、rown89Analysis of G Protein-Mediated Activation of Phospholipase C in Cultured CellsBo Liu and Dianqing Wu998viiviiiPART IV. RECEPTOR MECHANISMS, ACTIVITY, AND LOCALIZATIONContents9Intensive Mutational Analysis of G Protein-Coupled Receptors in YeastAndjelka C´ elic´ , Sara M. Connelly, Ne

48、gin P. Martin,and Mark E. Dumont105Green Fluorescent Protein-Tagged b-Arrestin Translocation as a Measure of G Protein-Coupled Receptor ActivationStephen S. G. Ferguson and Marc G. Caron121Detection of G Protein-Coupled Receptors by Immunofluorescence MicroscopyPatricia M. Hinkle and John A. Puskas1

49、27Assay of G Protein-Coupled Receptor Activation of G Proteins in Native Cell Membranes Using 35SGTPgS BindingJean M. Bidlack and Amy L. Parkhill135Analysis of the Coupling of G12/13 to G Protein-Coupled Receptors Using a Luciferase Reporter AssayBo Liu and Dianqing WART V. DISRUPTION O

50、F ENDOGENOUS G PROTEIN-SIGNALING SYSTEMS TO UNDERSTAND THE ROLE OF G PROTEIN SUBUNITS IN INTACT SYSTEMS14 Selective Inhibition of G Protein-Mediated Pathways Using RGS DomainsTohru Kozasa and Richard D. Ye15315 Ribozymes as Tools for Suppression of G Protein g SubunitsJanet D. Robishaw, Zheng-Guo, a

51、nd Qin Wang16916 In Vivo Adenoviral-Mediated Gene Transfer of the bARKct to Study the Role of Gbg in Arterial RestenosisGuido Iaccarino and Walter J. Koch181PART VI. POSTTRANSLATIONAL MODIFICATIONS OF REGULATORS OF G PROTEIN SIGNALING17 Analysis of RGS Protein PalmitoylationLeah S. Bernstein, Maurin

52、e E. Linder, and John R. Hepler19518 Methods for Measuring RGS Protein Phosphorylation by G Protein-Regulated KinasesSusanne Hollinger and John R. Hepler205ixPART VII. USE OF GFP-TAGGED G PROTEINS AND EFFECTORS IN INTACT CELLSContents19 The Use of Green Fluorescent Proteins to View Association Betwe

53、en Phospholipase Cb and G Protein Subunits in CellsSuzanne Scarlata and Louisa Dowal22320 Cellular Localization of GFP-Tagged a SubunitsThomas R. Hynes, Thomas E. Hughes,and Catherine H. Berlot233Index247ContributorsJEFF A. BEELER Ben May Institute for Cancer Research and Committee on Neurobiology,

54、University of Chicago, Chicago, ILCATHERINE H. BERLOT Weis Center for Research, Geisinger Clinic, Danville, PALEAH S. BERNSTEIN Department of Pharmacology, Emory University School of Medicine, Atlanta, GAJEAN M. BIDLACK Department of Pharmacology and Physiology, University of Rochester School of Med

55、icine, Rochester, NYH. ALEX BROWN Department of Pharmacology and Institute of Chemical Biology, Vanderbilt University Medical Center, Nashville, TNMARC G. CARON Howard Hughes Medical Institute, Duke University Medical Center, Durham, NCANDJELKA C´ ELIC´ Department of Biochemistry and Bioph

56、ysics, Universityof Rochester School of Medicine, Rochester, NYSARA M. CONNELLY Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NYLOUISA DOWAL Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, NYMARK E

57、. DUMONT Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NYSTEPHEN S. G. FERGUSON The John P. Robarts Institute and Departments of Physiology, Pharmacology and Toxicology, and Medicine, University of Western Ontario, London, Ontario, CanadaMOUSUMI GHOSH Department of Pharmacology and Physiology, University of Rochester School of Medicine, Rochester, NYWENDY K. GREENTREE Department of Cell Biology and Physiology, Washington University, St. Louis, MOZHENG-GUO Department of Anesthesiology, Wayne State University,Detroit Medical Center, Detroit,