G ProteinsTechniques of Analysis

320 Pages·1999·106.663 MB·\320

by  David R. Manning (Editor)

G PROTEINS
Technines
ûflnHttte

GPROTEINS
Techniques
or Analysis
Edited by
David R. Manning
Boca Raton London New York
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Library of Congress Cataloging-in-Publication Data
Mannning,DavidR.
Gproteins:techniquesofanalysis/ feditedbyJDavidR.Manning.
p. cm.- (Methodsinsignaltransduction)
Includesbibliographicalreferencesandindex.
ISBN0-8493-3381-4(alk.paper)
I. GProteins- Analysis. I.Manning,DavidR. II.Series.
QP552.Gl6G245 1999
572'.643- dc21
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гжя
CRC METHODS IN
SIGNAL TRANSIOCTION SERIES
Joseph Eichberg, Jr., Series Editor
SERIES PREFACE
In the past few years, the field of signal transduction has expanded at an
enormous rate and has become a major force in the biological and biomedical
sciences. Indeed, the importance of this area can hardly be exaggerated and
still continues to grow. The knowledge gained has vastly increased and, in
some instances, revolutionized our perceptions of the vast panoply of signaling
pathways and molecular mechanisms through which healthy cells respond to
extracellular and intracellular cues, and how these responses can malfunction
in a variety of disease states.
The increase in our understanding of signal transduction mechanisms
has relied upon the development and refinement of new and existing methods.
Successful investigations in this field now often require an integrated approach
that utilizes techniques drawn from molecular biology, cell biology, biochem­
istry, genetics, and immunology. The overall aim of this series is to bring
together the wealth of methodology now available for research in many aspects
of signal transduction. Since this is such a fast-moving field, emphasis will
be placed wherever possible on state-of-the-art techniques. Each volume is
assembled by one or more editors who are expert in their particular topic and
leaders in research relevant to it. Their guiding principle is to recruit authors
who will present detailed procedures and protocols in a critical yet reader-
friendly format that will be of practical value to a broad audience, including
students, seasoned investigators, and researchers who are new to the field.
Research of heterotrimeric GTP-binding proteins has vastly expanded
since the discovery of the first of these signaling molecules nearly 30 years
ago. Thus, although the field is well established, our knowledge in this area
is far from complete as we continue to encounter and appreciate the structural
and functional complexities of signaling pathway regulation by G proteins.
This volume presents cutting edge techniques that are already or should
shortly be finding wide application in G protein research. The first group of
methods involves the utilization of molecular genetics approaches for expression
and functional studies of these proteins. This is followed by descriptions of
diverse procedures to investigate the regulation of G proteins. Finally,
techniques for assessment of G protein interactions with other molecules in
the course of delineating signaling pathways are presented. Collectively, it
is the intent of the volume editor and authors to provide methodologies for
study of G proteins, which are of broad interest and practical benefit to
researchers in the field.
Joseph Eichberg, Ph.D.
Advisory Editor for the Series
CRC Press
METHODS IN THE LIFE SCIENCES
Gerald D. Fasman - Advisory Editor
Brandeis University
Series Overview
Methods in Biochemistry
John Hershey
Department of Biological Chemistry
University of California
Cellular and Molecular Neuropharmacology
Joan M. Lakoski
Department of Pharmacology
Penn State University
Research Methods for Inbred Laboratory Mice
John P Sundberg
The Jackson Laboratory
Bar Harbor, Maine
Methods in Neuroscience
Sidney A. Simon
Miguel Nicolelis
Department of Neurobiology
Duke University
Methods in Pharmacology
John H. McNeill
Professor and Dean
Faculty of Pharmaceutical Science
The University of British Columbia
Methods in Signal Transduction
Joseph Eichberg, Jr.
Department of Biochemical and Biophysical Sciences
University of Houston
Methods in Toxicology
Edward J. Massaro
Senior Research Scientist
National Health and Environmental Effects Research Laboratory
Research Triangle Park, North Carolina
CRC Press
METHODS IN THE LIFE SCIENCES
— METHODSINSIGNAL TRANSDUCTION
,
Joseph Eichberg Jr., Editor
The CRC Press Methods in the Life Sciences —Methods in Signal Transduction
Series provides the reader with state-of-the-art research methods that address
the cellular and molecular mechanisms of the neuropharmacology of brain
function in a clear and concise format.
Published Titles
Lipid Second Messengers, Suzanne G. Laychock and Ronald R Rubin
G Proteins Techniques of Analysis, David R. Manning
Signaling Through Cell Adhesion Molecules, Jun-Lin Guan
G Protein-Coupled Receptors, Gabriel Berstein and Tatsuya Haga
Calcium Signaling, James W. Putney
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 a subunit. Currently recognized a 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 Gs, Gj, Gq, and G12 families. Members of the Gs family
(Gs and Golf) stimulate adenylyl cyclases and dihydropyridine-sensitive Ca2+ channels
and inhibit cardiac Na+ channels. Members of the Gj family (Gj, G0, Gt, Ggus, and Gz)
variously inhibit adenylyl cyclases and voltage-sensitive Ca2+ channels, stimulate dif­
ferent forms of K+ channels, and (through βγ) stimulate phosphoinositide-specific
phospholipases C-ß and phosphoinositide 3-kinase. Members of the Gq family (Gq,
GH, G14, and G15/16) also (through a subunits) stimulate phospholipases C-ß. G12 and
G13 interact with rho-specific guanine-nucleotide exchange factors and can regulate
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 a subunit and thus
an exchange for GTP present in the cytoplasm. Correlates of the exchange are an
altered conformation of the a subunit and its dissociation from βγ. Regulation of
effector activity can be achieved by the a subunit alone, the βγ heterodimer alone,
or the a and βγ subunits working coordinately. The GTP on the a subunit is