3381/fm/frame Page 1 Wednesday, May 8, 2002 10:32 AM G Proteins: Techniques of Analysis Edited by David R. Manning, Ph.D. CRC Press Boca Raton London New York Washington, D.C. ©1999 CRC Press LLC 3381/fm/frame Page 2 Wednesday, May 8, 2002 10:32 AM Library of Congress Cataloging-in-Publication Data Mannning, David R. G proteins: techniques of analysis / [edited by] David R. Manning. p. cm. — (Methods in signal transduction) Includes bibliographical references and index. ISBN 0-8493-3381-4 (alk. paper) 1. G Proteins — Analysis. I. Manning, David R. II. Series. QP552.G16 G245 1999 572′.643 — dc21 DNLM/DLC for Library of Congress 98-49670 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. 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Visit the CRC Press Web site at www.crcpress.com © 1999 by CRC Press LLC No claim to original U.S. Government works International Standard Book Number 0-8493-3381-4 Library of Congress Card Number 98-49670 Printed in the United States of America 2 3 4 5 6 7 8 9 0 Printed on acid-free paper ©1999 CRC Press LLC 3381/fm/frame Page 3 Wednesday, May 8, 2002 10:32 AM Preface The field of heterotrimeric GTP-binding regulatory proteins (G proteins) has grown dramatically in the past decade. More than 20 G proteins have been identified, and all participate in both unique and shared functions relevant to signal transduction. G proteins are normally coupled to 7-transmembrane domain receptors, now esti- mated to number at least several hundred, which respond to hormones, neurotrans- mitters, autocoids, drugs, and sensory inputs such as light and odorants. The binding of an agonist to a receptor of this nature elicits an activation of one or more G proteins, which in turn regulates an appropriate set of second messenger systems. G proteins may well play additional roles in vesicle trafficking and protein docking. Incredibly, at a time when the interest in G proteins is at its highest, few existing reviews cover the methodologies basic to successful work in this field. This book is intended to help fill that void. G proteins are αβγ heterotrimers present at the inner surface of the plasma membrane and elsewhere in the cell. The identity of a G protein is usually equated with that of its α subunit. Currently recognized α subunits range in size from 40 to 46 kDa and, by virtue of primary structure, are the basis for classification of G proteins into four major families — the G, G, G, and G families. Members of the G family s i q 12 s (G and G ) stimulate adenylyl cyclases and dihydropyridine-sensitive Ca2+ channels s olf and inhibit cardiac Na+ channels. Members of the G family (G, G, G, G , and G) i i o t gus z variously inhibit adenylyl cyclases and voltage-sensitive Ca2+ channels, stimulate dif- ferent forms of K+ channels, and (through βγ) stimulate phosphoinositide-specific phospholipases C-β and phosphoinositide 3-kinase. Members of the G family (G, q q G , G , and G ) also (through α subunits) stimulate phospholipases C-β. G and 11 14 15/16 12 G interact with rho-specific guanine-nucleotide exchange factors and can regulate 13 Na+/H+ exchange, immediate early gene expression, and cell morphology. The activity of G proteins, and consequent status of target regulation, is tightly linked to the binding and hydrolysis of GTP. Upon binding to receptors at the cell surface, agonists promote the release of GDP from the G protein α subunit and thus an exchange for GTP present in the cytoplasm. Correlates of the exchange are an altered conformation of the α subunit and its dissociation from βγ. Regulation of effector activity can be achieved by the α subunit alone, the βγ heterodimer alone, or the α and βγ subunits working coordinately. The GTP on the α subunit is ©1999 CRC Press LLC 3381/fm/frame Page 4 Wednesday, May 8, 2002 10:32 AM eventually hydrolyzed to allow reversion of the subunit to an inactive, GDP-bound form that can reassociate with βγ. Factors that can accelerate the hydrolysis of GTP by the α subunit include certain effectors and a growing number of RGS (regulator of G protein signaling) proteins. G proteins are subject to a variety of covalent modifications. The α subunits of the G family are all N-myristoylated, and all α subunits but α are substrates for i t palmitoylation. Fatty-acid acylation is relevant to anchorage of the subunits to membranes and to interactions of the subunits with other proteins. βγ is anchored to membrane through isoprenylation of the γ subunit. The α subunits of G and G s t are substrates for ADP-ribosylation by cholera toxin, which inhibits hydrolysis of GTP directly and thus maintains the activated α subunit in an active conformation. The α subunits of G, G , and G are ADP-ribosylated by a pertussis toxin, which i o t inhibits the contact of these G proteins with receptors and thus agonist-promoted exchange of GDP for GTP. Several α subunits are substrates for phosphorylation by protein kinase C and tyrosine kinases. The first five chapters of this book deal with the expression and functional analysis of mammalian G protein subunits in cells of bacterial, insect, mammalian, and yeast origin. Escherichia coli and Sf9 insect cells are key sources of subunits for work in the areas of reconstitution, structural and biochemical analyses, and microinjection. Bacterial expression systems are amenable to large-scale production of recombinant subunits whose purification is relatively straightforward. Subunits expressed in Sf9 cells are extensively processed, and an elegant hexahistidine- tagging technique for rapid purification of appropriately modified α and βγ subunits is discussed. Expression of subunits in mammalian cells following transfection is perhaps the most widely used paradigm for functional analysis, and techniques of mutagenesis and analysis as applied to interactions of G proteins with effectors are described. Finally, the yeast two-hybrid system, which has been used extensively in the analysis of protein·protein interactions for monomeric G proteins, has recently been applied successfully to heterotrimeric G protein subunits. The use of the yeast two-hybrid system to identify novel interacting proteins and to define subunit·subunit interactions is described. The next four chapters cover the techniques of evaluating G protein regulation. A critical endeavor in this field is the deployment of antibodies to assess subunit expression and covalent modification within cells. The production of antibodies and their use for Western blots and immunoprecipitation therefore assumes a prominent place in any set of techniques. Procedures of measuring the basic parameters of GTP binding and hydrolysis are also quite important, as they provide an understanding of the activation and deactivation of G proteins at a biochemical level. Superimposed on this is the need to evaluate the regulation imposed by the growing array of RGS proteins. Fatty-acid acylation has also emerged as a critical determinant in G protein function. The role of fatty-acid acylation in anchorage is to some extent intuitive, but the regulation of palmitoylation by agonists provides a new dimension to schemes of activation and deactivation that has been only partly explored. The ADP-ribosy- lation catalyzed by cholera and pertussis toxins affords an easy means of radiola- beling selected sets of G proteins, and ADP-ribosylation catalyzed by pertussis toxin in particular remains the traditional method for exploring the utilization of G or G . i o ©1999 CRC Press LLC 3381/fm/frame Page 5 Wednesday, May 8, 2002 10:32 AM The remaining five chapters focus on techniques for mapping pathways estab- lished between receptors, G proteins, and effectors. These chapters address a critical need for defining how the cell sets up specific linkages among the three proteins, and determining how these linkages change from cell to cell and in response to regulatory pressures. Utilization of mutants of G and G resistant to pertussis toxin i o holds considerable potential in discriminating subtypes of this widely used family of G proteins. Microinjection of antibodies that disrupt interactions between recep- tors and G proteins represents a means of blocking pathways of transduction that can be analyzed in single cells. Expression of antisense molecules represents another technique for ablation, and can be extended from the intact cell to a transgenic animal. Coupling profiles for receptors and G proteins can be generated by an analysis of GTPγS/GDP exchange, which can also be used to explore concepts of efficacy. Resolving actions attributable to α subunits and those attributable to βγ heterodimers can be achieved by the deployment of molecules that sequester βγ. The ability to distinguish α and βγ is quite important, as the actions of the two types of subunits can differ radically. Each chapter is designed to provide a step-by-step description of how a given technique is performed. Each presents an overview of the technique, an account of the technique, and a section on commonly encountered problems. The overview is intended to provide useful background on the subject, together with references and any necessary theory. The Problems section covers troubleshooting and alternative methodologies. Each chapter concludes with examples of data obtained, ranges of values, and controls. ©1999 CRC Press LLC 3381/fm/frame Page 6 Wednesday, May 8, 2002 10:32 AM Contributors Alastair J. Barr, D. Phil. Zhiyong Cheng Department of Medicine Department of Physiology and Duke University Medical Center Biophysics Durham, NC School of Medicine, SUNY/Stony Brook Stony Brook, NY Catherine H. Berlot, M.D., Ph.D. Department of Cellular and Peter Chidiac, Ph.D. Molecular Physiology Department of Pharmacology Yale University School of Medicine University of Texas Southwestern New Haven, CT Medical Center Dallas, TX Gloria H. Biddlecome, Ph.D. Department of Pharmacology Gregory J. Della Rocca, Ph.D. University of Texas Southwestern Departments of Medicine and Medical Center Biochemistry Dallas, TX Howard Hughes Medical Institute Duke University Medical Center Durham, NC Patrick J. Casey, Ph.D. Departments of Biochemistry and James R. Feramisco, Ph.D. Pharmacology and Cancer Biology Departments of Medicine and Duke University Medical Center Pharmacology Durham, NC University of California at San Diego La Jolla, CA Catherine A. Chen Department of Pharmacology Jennifer L. Glick, Ph.D. University of Pennsylvania Department of Biochemistry School of Medicine Duke University Medical Center Philadelphia, PA Durham, NC ©1999 CRC Press LLC 3381/fm/frame Page 7 Wednesday, May 8, 2002 10:32 AM N. Gautam, Ph.D. Maurine E. Linder, Ph.D. Departments of Anesthesiology and Department of Cell Biology and Genetics Physiology Washington University Washington University School of Medicine School of Medicine St. Louis, MO St. Louis, MO Xunxian Liu, Ph.D. O.G. Kisselev, Ph.D. Department of Molecular Department of Pharmacology Pharmacology–HSC Washington University School of Medicine, School of Medicine SUNY/Stony Brook St. Louis, MO Stony Brook, NY Walter J. Koch, Ph.D. Craig C. Malbon, Ph.D. Department of Surgery Department of Molecular Howard Hughes Medical Institute Pharmacology–HSC Duke University Medical Center School of Medicine, Durham, NC SUNY/Stony Brook Stony Brook, NY Tohru Kozasa, M.D., Ph.D. Department of Pharmacology David R. Manning, Ph.D. University of Texas Southwestern Department of Pharmacology Medical Center University of Pennsylvania Dallas, TX School of Medicine Philadelphia, PA Vickie J. LaMorte, Ph.D. Beckman Laser Institute Thomas E. Meigs, Ph.D. University of California at Irvine Department of Pharmacology and Irvine, CA Cancer Biology Duke University Medical Center Robert J. Lefkowitz, M.D. Durham, NC Departments of Medicine and Biochemistry Judy L. Meinkoth, Ph.D. Howard Hughes Medical Institute Department of Pharmacology Duke University Medical Center University of Pennsylvania Durham, NC School of Medicine Philadelphia, PA Hui Ling Li Department of Molecular Albrecht Moritz, Ph.D. Pharmacology–HSC Department of Biological Chemistry School of Medicine, University of Michigan Medical School SUNY/Stony Brook Ann Arbor, MI Stony Brook, NY ©1999 CRC Press LLC 3381/fm/frame Page 8 Wednesday, May 8, 2002 10:32 AM Suchetana Mukhopadhyay, Ph.D. Hsien-yu Wang, Ph.D. Department of Pharmacology Department of Physiology and University of Texas Southwestern Biophysics Medical Center School of Medicine, Dallas, TX SUNY/Stony Brook Stony Brook, NY A.N. Pronin, Ph.D. Department of Anesthesiology Jun Wang, Ph.D. Washington University Department of Pharmacology School of Medicine University of Texas Southwestern St. Louis, MO Medical Center Dallas, TX Elliott M. Ross, Ph.D. Department of Pharmacology Philip B. Wedegaertner, Ph.D. University of Texas Southwestern Department of Microbiology and Medical Center Immunology Dallas, TX Thomas Jefferson University Philadelphia, PA Ronald Taussig, Ph.D. Department of Biological Chemistry Marilyn J. Woolkalis, Ph. D. University of Michigan Department of Physiology Medical School Thomas Jefferson University Ann Arbor, MI Philadelphia, PA Yaping Tu, Ph.D. K. Yan, Ph. D. Department of Pharmacology Department of Anesthesiology University of Texas Southwestern Washington University Medical Center School of Medicine Dallas, TX St. Louis, MO ©1999 CRC Press LLC 3381/fm/frame Page 9 Wednesday, May 8, 2002 10:32 AM Contents Chapter 1 Expression and Purification of G Protein α Subunits in Escherichia coli Maurine E. Linder Chapter 2 Purification of Recombinant G Protein α and βγ Subunits from Sf9 Cells Tohru Kozasa Chapter 3 Expression and Functional Analysis of G Protein α Subunits in Mammalian Cells Catherine H. Berlot Chapter 4 Identification of Regulators and Targets of G Protein α Subunits Using the Yeast Two-Hybrid System Jennifer L. Glick, Thomas E. Meigs, and Patrick J. Casey Chapter 5 Functional Analysis of G Protein βγ Subunits K. Yan, O.G. Kisselev, A.N. Pronin, and N. Gautam Chapter 6 Immunological Techniques of G Protein Analysis Catherine A. Chen and David R. Manning Chapter 7 GTPase-Activating Proteins (GAPs) for Heterotrimeric G Proteins Jun Wang, Yaping Tu, Suchetana Mukhopadhyay, Peter Chidiac, Gloria H. Biddlecome, and Elliott M. Ross ©1999 CRC Press LLC 3381/fm/frame Page 10 Wednesday, May 8, 2002 10:32 AM Chapter 8 Fatty Acid Acylation of G Protein α Subunits Philip B. Wedegaertner Chapter 9 ADP-Ribosylation of G Proteins with Cholera Toxin or Pertussis Toxin Marilyn J. Woolkalis Chapter 10 Analysis of Receptor-G Protein Coupling with Pertussis Toxin-Resistant Mutants of G and G i o Albrecht Moritz and Ronald Taussig Chapter 11 Microinjection of Antibodies Neutralizing G Protein Function Vickie J. LaMorte, James R. Feramisco, and Judy L. Meinkoth Chapter 12 Agonist-Promoted [35S]GTPγS-Binding as a Probe of Receptor·G Protein Communication in Reconstituted Sf9 Cells Alastair J. Barr and David R. Manning Chapter 13 Methods of Sequestering βγ in the Intact Cell Gregory J. Della Rocca, Robert J. Lefkowitz, and Walter J. Koch Chapter 14 In Vitro and In Vivo Analysis of Heterotrimeric G-Protein Signaling via DNA/RNA Antisense Strategies Hsien-yu Wang, Xunxian Liu, Zhiyong Cheng, Hui Ling Li, and Craig C. Malbon ©1999 CRC Press LLC