heterotrimeric g-proteins

Preface Heterotrimeric (a/33,) G proteins function as cell surface signal trans- ducers for a large number of hormones, neurotransmitters, and for autocrine and paracrine factors. It is now known that a hundred or so (not counting the olfactory) receptors are coupled to various effectors through G proteins. This large number of receptors couple to members of one of the four families of G proteins to transmit their signals. The specificity of the receptor-G protein interactions determines the transmission of the signal to different downstream pathways. As with all real life situations, the specificity of interactions between receptors and G proteins is varied and complex. Consequently, signals from a single receptor may be trans- mitted through several pathways simultaneously. Each of the four fami- lies of G proteins has many members. Cloning studies have indicated that there are twenty a, four/3, and six ~/subunits. It has been suggested that very large numbers of heterotrimeric G proteins with defined subunit compositions can be generated from these individual subunits. "Knock- out" experiments indicate that G proteins of defined subunit composition communicate signals from different receptors. However, at this time there is not sufficient general information to indicate that the functional identity of a G protein is defined by the molecular identity of all of its subunits. Consequently, in spite of the molecular heterogeneity of the/3 and ~/subunits, the different G proteins are still classified by the identity of their o~ subunits. This classification is useful because it indicates which intracellular messenger pathway is used. In this volume the nomenclature of G proteins is based on the molecular identity of the a subunit. Hence it should be noted that native purified G proteins may have a single molecu- lar species of ~ subunits, but multiple forms of/3 and ~/subunits. In the past decade there has been a substantial increase in our under- standing of signal-transducing G proteins. This advance has been brought about by a combination of molecular biological and biochemical tech- niques. Both approaches are represented in this book. Two types of chap- ters are included. The first presents techniques unique to the field of signal-transducing G proteins, the second general techniques that have been applied to the study of G-protein systems. Chapters of the latter type may be useful even to researchers who do not work on signal-transducing G proteins. The field of heterotrimeric G proteins has been covered in part in Volumes 109 and 195 of Methods in Enzymology. However, the subject has not been covered in a systematic fashion. Consequently, in planning XV xvi PREFACE this volume, an attempt was made to include all or at least most of the techniques in one volume. Thus techniques such as cholera and pertussis toxin labeling as well as some techniques for the purification of native G proteins are covered but by different authors. It should be noted that in G protein research many laboratories use different protocols for the same overall experiments. Since a single experimental protocol does not always work for the same G protein in different systems it can be useful to have more than one experimental procedure available to tackle the same ques- tion. Several, but not all subjects, are thus addressed by multiple labora- tories in this volume. I thank Lutz Birnbaumer, Henry Bourne, and Buzz Brown for their useful suggestions during the initial planning of this volume. I also thank Ms. Lina Mazzella for her valuable assistance. Last, but not least, I thank the authors for their contributions and for complying with my suggestions for change in a timely fashion. RAVI IYENGAR Contributors to Volume 237 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. JANMEET S. ANANT (40), Jules Stein Eye Institute, University of California at Los Angeles School of Medicine, Los Angeles, California 90024 ANNA M. ARAGAY (26), Department of Biol- ogy, California Institute of Technology, Pasadena, California 91125 YVES AUDIGIER (19), UMR 9925, Universitd Paul Sabatier, 31062 Toulouse Cedex, France ERIC A. BALCUEVA (39), Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822 JOgLLE BIGAY (11, 34), Institut de Pharma- cologie moldculaire et cellulaire, Unitd propre 411 du Centre National de la Re- cherche Scientifique, 06560 Valbonne, France LUTZ BIRNBAUMER (9, 29), Departments of Cell Biology, Medicine, Molecular Physi- ology, and Biophysics, and Division of Neurosciences, Baylor College of Medi- cine, Houston, Texas 77030 JONATHAN L. BLANK (14), Division of Basic Sciences, National Jewish Center for Im- munology and Respiratory Medicine, Denver, Colorado 80206 THOMAS BOUILLON (2), Department of Clin- ical Pharmacology, University of G6t- tingen, 37075 Gfttingen, Germany Jose. L. BOYER (15), Department of Phar- macology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 ALLAN BRADLEY (29), Institute for Molecu- lar Genetics and Howard Hughes Medi- cal Institute, Baylor College of Medicine, Houston, Texas 77030 DONNA J. CARTY (4, 6, 35), Department of Pharmacology, Mount Sinai School of Medicine, New York, New York 10029 RICHARD A. CERIONE (31), Department of Pharmacology, Cornell University, Ithaca, New York 14853 MARC CHABRE (1 l, 34), Institut de Pharma- cologie mol~culaire et cellulaire, Unit~ propre 411 du Centre National de la Re- cherche Scientifique, 06560 Valbonne, France JIANQIANG CHEN (35), Department of Phar- macology, Mount Sinai School of Medi- cine, New York, New York 10029 JUAN CODINA (9), Department of Cell Biol- ogy, Baylor College of Medicine, Hous- ton, Texas 77030 BRADLEY M. DENKER (18), Department of Medicine, Brigham and Women's Hospi- tal and Harvard Medical School, Boston, Massachusetts 02115 N. DHANASEKARAN (7), Department of Bio- chemistry and Fels Institute for Cancer and Molecular Biology, Temple Univer- sity School of Medicine, Philadelphia, Pennsylvania 19140 JANE DINGUS (36), Department of Cell and Molecular Pharmacology and Experimen- tal Therapeutics, Medical University of South Carolina, Charleston, South Caro- lina 29425 WILLIAM J. DUNN III (8), Departments of Medicinal Chemistry and Pharmacog- nosy, University of Illinois College of Pharmacy, Chicago, Illinois 60612 JOHN H. EXTON (14), Department of Molec- ular Physiology and Biophysics, Vander- bilt University School of Medicine, Nash- ville, Tennessee 37232 ROnERT A. FIGLER (17), Departments of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 ix X CONTRIBUTORS TO VOLUME 237 MICHAEL FORTE (33), Vollum Institute, Or- egon Health Sciences University, Port- land, Oregon 97201 BERNARD K. -K. FUNG (40), Jules Stein Eye Institute, University of California at Los Angeles School of Medicine, Los Angeles, California 90024 CHRISTINE GALLAGHER (37), Departments of Anesthesiology and Genetics, Wash- ington University School of Medicine, St. Louis, Missouri 63110 JAMES C. GARRISON (17), Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 NARASIMHAN GAUTAM (37, 38), Depart- ments of Anesthesiology and Genetics, Washington University School of Medi- cine, St. Louis, Missouri 63110 PABLO V. GEJMAN (24), Unit on Molecular Clinical Investigation, Clinical Neuro- genetics Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892 PETER GIERSCHIK (2), Department of Phar- macology and Toxicology, University of Ulm, 89069 Ulm, Germany ALFRED G. GILMAN (12, 16), Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 STEPHEN G. GRABER (17), Department of Pharmacology and Toxicology, West Vir- ginia University, Morgantown, West Vir- ginia 26505 DAGOBERTO GRENET (9), Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 HEIDI E. HAMM (32), Department of Physi- ology and Biophysics, University of Illi- nois at Chicago, Chicago, Illinois 60612 T. KENDALL HARDEN (15), Department of Pharmacology, University of North Caro- lina School of Medicine, Chapel Hill, North Carolina 27599 RANDY S. HAUN (5), Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 JOHN R. HEPLER (16), Department of Phar- macology, University of Texas South- western Medical Center, Dallas, Texas 75235 TSUTOMU HIGASHIJIMA I (3), Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 JOHN D. HILDEBRANDT (36), Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical Uni- versity of South Carolina, Charleston, South Carolina 29425 ATSUSHI INANOBE (10), Department of Life Science, Tokyo Institute of Technology, Yokohama, Kanagawa 227, Japan RAVI IYENGAR (4, 35), Department of Phar- macology, Mount Sinai School of Medi- cine, New York, New York 10029 KARL H. JAKOBS (1, 2), lnstitutfiir Pharma- kologie, Universitiitsklinikum Essen, 45122 Essen, Germany GARY L. JOHNSON (25), Division of Basic Sciences, National Jewish Center for Im- munology and Respiratory Medicine, Denver, Colorado 80206 TOSHIAKI KATADA (10), Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan CHRISTIANE KLEUSS (27), Bereich Moleku- larbiologie und Biolnformatik, Institut fiir Molekularbiologie und Biochemie, Freie Universitiit Berlin, D-14195 Berlin, Ger- many ICHIRO KOBAYASHI (10), Department of Life Science, Tokyo Institute of Technology, Yokohama, Kanagawa 227, Japan RUSSELL KOHNKEN (36), Molecular Geriat- rics, Libertyville, Illinois 60048 KENJI KONTANI (10), Department of Life Science, Tokyo Institute of Technology, Yokohama, Kanagawa 227, Japan Deceased. CONTRIBUTORS TO VOLUME 237 xi TOHRU KOZASA (16), Department of Phar- macology, University of Texas South- western Medical Center, Dallas, Texas 75235 KARL-LUDWIG LAUGWITZ (22), Institut fiir Pharmakologie, Freie Universitiit Berlin, 14195 Berlin, Germany ETHAN LEE (12), Department of Pharma- cology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 FANG-JEN SCOTT LEE (5), Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Insti- tutes of Health, Bethesda, Maryland 20892 WuN-CHEN LIN (40), Jules Stein Eye Insti- tute, University of California at Los Angeles School of Medicine, Los Angeles, California 90024 MAURINE E. LINDER (12, 20), Department of Cell Biology and Physiology, Washing- ton University School of Medicine, St. Louis, Missouri 63110 JOHN LYONS (23), Department of Drug Dis- covery, Onyx Pharmaceuticals, Rich- mond, California 94806 GRAEME MILHGAN (21), Molecular Phar- macology Group, Departments of Bio- chemistry and Pharmacology, University of Glasgow, G12 8QQ Glasgow, Scot- land, United Kingdom RICHARD M. MORTENSEN (28), Department of Medicine, Brigham and Women's Hos- pital and Harvard Medical School, Bos- ton, Massachusetts 02115 JOEL MOSS (5), Laboratory of Cellular Me- tabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 SUSANNE M. MUMBY (20), Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 EVA J. NEER (18), Department of Medicine, Brigham and Women's Hospital and Har- vard Medical School, Boston, Massachu- setts 02115 HIROSH1 NISH1NA (10), Department of Life Science, Tokyo Institute of Technology, Yokohama, Kanagawa 227, Japan STEFAN OFFERMANNS (22), Department of Biology, California Institute of Technol- ogy, Pasadena, California 91125 YOSHIHARU OHOKA (10), Department of Life Science, Tokyo Institute of Technol- ogy, Yokohama, Kanagawa 227, Japan OLIVIA C. ONG (40), Jules Stein Eye Insti- tute, University of California at Los Angeles School of Medicine, Los Angeles, California 90024 IoK-Hou PANG (13), Department of Glau- coma Research, Alcon Laboratories, Fort Worth, Texas 76134 S. Russ PRICE (5), Renal Division, Emory University Hospital, Atlanta, Georgia 30322 ALEXEY N. PRONIN (38), Departments of Anesthesiology and Genetics, Washing- ton University School of Medicine, St. Louis, Missouri 63110 FRANKLIN QUAN (33), Vollum Institute, Or- egon Health Sciences University, Port- land, Oregon 97201 HELEN M. RARICK (32), Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612 MARK M. RASENICK (8), Departments of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, Illi- nois 60612 FERNANDO RIBEIRO-NETo (9), Department of Pharmacology, Duke University Medi- cal Center, Durham, North Carolina 27710 JANET D. RontsnAw (39), Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822 ELLIOTT M. ROSS (3), Department of Phar- macology, University of Texas South- western Medical Center, Dallas, Texas 75235 UWE RUDOLPH (29), Institute of Pharma- cology, University of Zurich, CH-8057 Zurich, Switzerland xii CONTRIBUTORS TO VOLUME 237 ARNOLD E. RUOHO (7), Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706 MARUANE RUSSELL (25), Division of Basic Sciences, National Jewish Center for Im- munology and Respiratory Medicine, Denver, Colorado 80206 CARL J. SCHMIDT (18), Department of Med- icine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 GONTER SCHULTZ (22, 27), lnstitut far Pharmakologie, Freie Universitdt Berlin, D-14195 Berlin, Germany J. G. SEIDMAN (28), Department of Genet- ics, Harvard Medical School, Boston, Massachusetts 02115 MELVIN I. SIMON (26), Department of Biol- ogy, California Institute of Technology, Pasadena, California 91125 ALAN V. SMRCKA (13), Department of Phar- macology, University of Texas South- western Medical Center, Dallas, Texas 75235 B. EWA SNAAR-JAGALSKA (30), Cell Biology and Genetics Unit, Clusius Laboratory, Leiden University, 2333 AL Leiden, The Netherlands KARSTEN SPICHER (22), lnstitutfiir Pharma- kologie, Freie Universitiit Berlin, 14195 Berlin, Germany PAUL C. STERNWEIS (13), Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 KATSUNOBU TAKAHASHI (10), Department of Life Science, Tokyo Institute of Tech- nology, Yokohama, Kanagawa 227, Ja- pan MADHAVI TALLURI (8), Departments of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, Illi- nois 60612 THOMAS C. THOMAS (18), Alexion Pharma- ceuticals, Inc., New Haven, Connecticut 06511 Su-CHEN TSAI (5), Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 RlCHARD R. VAILLANCOURT (7), Division of Basic Sciences, Department of Pediat- rics, National Jewish Center for Immu- nology and Respiratory Medicine, Den- ver, Colorado 80206 PETER J. M. VAN HAASTERT (30), Depart- ment of Biochemistry, University of Gr6- ningen, 9747 AG Gr6ningen, The Nether- lands MARTHA VAUGHAN (5), Laboratory of Cel- lular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 GARY L. WALDO (15), Department of Phar- macology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 A. JOHN WATSON (26), Department of Biol- ogy, California Institute of Technology, Pasadena, California 91125 LEE S. WEINSTEIN (24), Molecular Patho- physiology Branch, National Institute of Diabetes and Digestive and Kidney Dis- eases, National Institutes of Health, Be- thesda, Maryland 20892 CATHERINE F. WELSH (5), Department of Cell Biology and Anatomy, University of Miami, Miami, Florida 33136 THOMAS WIELAND (1), Institutfiir Pharma- kologie, Universitiitsklinikum Essen, 45122 Essen, Germany MICHAEL D. WILCOX (36), Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical Uni- versity of South Carolina, Charleston, South Carolina 29425 CONTRIBUTORS TO VOLUME 237 xiii THOMAS M. WILKIE (26), Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 SIM WINITZ (25), Division of Basic Sci- ences, National Jewish Center for Immu- nology and Respiratory Medicine, Den- ver, Colorado 80206 BURGHARDT WITTIG (27), Bereich Moleku- larbiologie und Biolnformatik, Institut fiir Molekularbiologie und Biochemie, Freie Universitiit Berlin, D-14195 Berlin, Ger- many HARVEY K. YAMANE (40), Jules Stein Eye Institute, University of California at Los Angeles School of Medicine, Los Angeles, California 90024 [1] RECEPTOR-STIMULATED GTPyS BINDING BY G PROTEINS 3 [1] Measurement of Receptor-Stimulated Guanosine 5'-O-(y-Thio)triphosphate Binding by G Proteins By THOMAS WIELAND and KARL H. JAKOBS Introduction Many transmembrane signaling processes caused by extracellular hor- mones and neurotransmitters are mediated by receptors interacting with heterotrimeric (a/3y) guanine nucleotide-binding proteins (G proteins) attached to the inner face of the plasma membrane. Agonist-liganded receptors apparently initiate activation of G proteins by catalyzing the exchange of guano sine 5'-diphosphate (GDP) by guanosine 5'-triphosphate (GTP) bound to the a subunits.l,2 In membrane preparations and reconsti- tuted systems, this activation process is frequently monitored by studying agonist stimulation of high-affinity GTPase, an enzymatic activity of G-protein a subunits) However, the measurement of G-protein GTPase activity reflects steady-state kinetics of the overall G-protein activity cycle and not only the first step in the signal transduction cascade (i.e., the GDP/GTP exchange reaction). Furthermore, with regard to the molecular stoichiometry of receptor-G-protein interactions, only qualitative but not quantitative data can be obtained. To study the initial steps of G-protein activation by agonist-liganded receptors in a quantitative manner, the binding of radiolabeled GTP ana- logs, which are not hydrolyzed by the GTPase activity of G-protein a subunits, to G proteins is determined. Of these GTP analogs, guanosine 5'-O-(y-[35S]thio)triphosphate ([35S]GTPyS) is most frequently used. This nucleotide has a high affinity for all types of G proteins and is available with a relatively high specific radioactivity (1000-1400 Ci/mmol; physical half-life 87.4 days). Here we describe the measurement of receptor- induced binding of [35S]GTPyS to membranous and detergent-solubilized G proteins and how this method can be adapted to different G proteins for an optimal response to receptor stimulation. Materials The [3SS]GTPyS (1000-1400 Ci/mmol) is obtained from Du Pont New England Nuclear (Bad Homburg, Germany). The substance is delivered I A. G. Gilman, Annu. Rev. Biochem. 56, 615 (1987). 2 L. Birnbaumer, J. Abramovitz, and A. M. Brown, Biochim. Biophys. Acta 1031, 163 (1990). 3 D. Cassel and Z. Selinger, Biochim. Biophys. Acta 452, 538 (1976). Copyright @ 1994 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 237 All rights of reproduction in any form reserved. 4 Ga SUBUNITS [1] in a buffer containing 10 mM N-tris(hydroxymethyl)methylglycine- NaOH, pH 7.6, and l0 mM dithiothreitol (DTT). To minimize decomposi- tion, the solution is diluted 100-fold in this buffer and stored in aliquots at or below - 70 ° before use. If the reagent is not stored at these recom- mended conditions, and after repeated freezing and thawing, chemical decomposition is rather high. Unlabeled nucleotides and 3-[(3-cholamidopropyl)dimethylammonio]- 1-propane sulfonate (CHAPS) are from Boehringer Mannheim (Mann- heim, Germany). N-Ethylmaleimide, N-formylmethionyUeucylphenyl- alanine (fMet-Leu-Phe), isoproterenol, and carbachol are from Sigma (St. Louis, MO). Glass fiber filters (GF/C) are from Whatman (Clifton, NJ), and nitrocellulose filters (pore size 0.45 /xm) are from Schleicher and Schuell (Keene, NH). Membranes of various cells and tissues are prepared as previously described 4-7 and stored in aliquots at -70 °. Before use in the binding assay, the membranes are thawed, diluted with l0 mM triethanolamine hydrochloride, pH 7.4, containing 5 mM EDTA, centrifuged for I0-30 min at 30,000 g, and resuspended in 10 mM triethanolamine hydrochlo- ride, pH 7.4, at the appropriate membrane protein concentration. Equipment Incubator or water bath Filtration funnel with vacuum pump Cooled centrifuge (4 °, up to 30,000 g) for membrane preparation Ultracentrifuge with fixed-angle and swing-out rotors for preparation of membranes, solubilized proteins, and sucrose density gradient centrifugation Shaker to equilibrate the filters with the scintillation cocktail Liquid scintillation spectrometer Freezer (preferably -70 ° or lower) for storage of membranes and [35S]GTPyS Measurement of Agonist-Induced [35S]GTPTS Binding to G Proteins in Membranes The assay is performed in 3-ml plastic reaction tubes. The assay volume is 100/zl. The final concentrations of the reaction mixture constituents 4 p. Gierschik, M. Steisslinger, D. Sidiropoulos, E. Herrmann, and K. H. Jakobs, Eur. J. Biochem. 283, 97 (1989). 5 G. Hilt" and K. H. Jakobs, Eur. J. Pharmacol. 172, 155 (1989). 6 D. S. Papermaster and W. J. Dreyer, Biochemistry 13, 2438 (1974). 7 G. Puchwein, T. Pfeuffer, and E. J. M. Helmreich, J. Biol. Chem. 249, 3232 (1974). [1] RECEPTOR-STIMULATED GTP3,S BINDING BY G PROTEINS 5 are as follows: triethanolamine hydrochloride (pH 7.4), 50 mM; MgCI2, 5 raM; EDTA, 1 mM; DTT, 1 mM; NaCl, 0-150 mM; GDP, 0-100/zM; [35S]GTPyS, 0.3-0.5 nM (-50 nCi). The incubation temperature and mem- brane concentration as well as the concentrations of NaCl and GDP have to be adjusted to the individual cell type and the G-protein subtype acti- vated by the receptor under study. 1. The reaction mixture (40/zl) together with the receptor agonist or its diluent (10/zl) are thermally preequilibrated for 5 min at the desired reaction temperature. 2. The binding reaction is started by addition of the membrane suspen- sion (50/.d) and vortexing. 3. Samples are incubated for the appropriate incubation time, for ex- ample, 60 min at 30 °. 4. The incubation is terminated by the addition of 2.5 ml of an ice- cold washing buffer (50 mM Tris-HC1, pH 7.5, 5 mM MgCI2). 5. This mixture is passed through the filtration funnel. For systems containing only membrane-bound G proteins, Whatman GF/C glass fiber filters are used. In systems containing soluble G proteins (e.g., transducin), nitrocellulose filters are required. 6. The reaction tube is washed two times with 2.5 ml of the washing buffer, and this solution is also passed through the same filter. 7. The filter is additionally washed two times with 2.5 ml of the washing buffer and then dried at room temperature. 8. The dried filters are put into 5-ml counting vials and equilibrated with 4 ml of a scintillation cocktail for 20 rain at room temperature by moderate shaking. Any commercially available scintillation cocktail suit- able for counting of 35S can be used. Also a self-made cocktail consisting of 2 liters toluene, 1 liter Triton X-100, 15 g 2,5-diphenyloxazole, and 3 g 2,2'-p-phenylenebis(4-methyl-5-phenyloxazole) can be used. Application to Various Cell Types and Different G Proteins In membranes of various cell types, including human neutrophils: human platelets, 9 human leukemia cells (HL-60), ~°'1~ rat myometrium, ~2 8 R. Kupper, B. Dewald, K. H. Jakobs, M. Baggiolini, and P. Gierschik, Biochem. J. 282, 429 (1992). 9 C. Gachet, J P. Cazenave, P. Ohlmann, G. Hilf, T. Wieland, and K. H. Jakobs, Eur. J. Biochem. 2117, 259 (1992). l0 p. Gierschik, R. Moghtader, C. Straub, K. Dieterich, and K. H. Jakobs, Eur. J. Biochem. 197, 725 (1991). IIT. MI. Schepers, M. E. Brier, and K. R. McLeish, J. Biol. Chem. 267, 159 (1992). 12 C. Liebmann, M. Schnittler, M. Nawrath, and K. H. Jakobs, Eur. J. Pharmacol. 207, 67 (1991). [...]... higher sedimentation coefficient (about 6.4 S) contains essentially heterotrimeric G proteins, whereas 2n G Hill and K H Jakobs, Cellular Signalling 4, 787 (1992) [2] RECEPTOR-STIMULATED HYDROLYSISOF GTP 13 the second peak (sedimentation coefficient about 3.3 S) is mainly due to small molecular mass G proteins In fractions containing both heterotrimeric G proteins and receptor(s), agonist-induced binding... signal-transducing heterotrimeric guanine nucleotide-binding proteins (G proteins) is their cyclic movement through a series of activation and deactivation steps, which is characterized by changes in the nature of the bound guanine nucleotide and in the status of the subunit association Activation of G proteins is initiated by the release of guanosine 5'-diphosphate (GDP) from the heterotrimeric G protein,... stopped, and binding of [35S]GTP~/S to solubilized G proteins is analyzed by filtration through nitrocellulose filters as described above In fractions containing both muscarinic acetylcholine receptors and heterotrimeric G proteins, carbachol stimulates binding of [35S]GTP~/S to solubilized G proteins by about 2-fold (Fig 4B) Similar data have been obtained for fMet-Leu-Phe-stimulated binding of [3SS]GTP~/Sin... for results and a more detailed discussion of the influence of the guanine nucleotide level on the regulation of receptor-stimulated GTPase in membrane preparations) 58 p Gierschik and K H Jakobs, in "G-Proteins as Mediators of Cellular Signalling Processes" (M D Houslay and G Milligan, eds.), p 67 Wiley, Chichester, 1990 59 p Gierschik, K McLeish, and K H Jakobs, J Cardiovasc Pharmacol 12(Suppl 5), . Preface Heterotrimeric (a/33,) G proteins function as cell surface signal trans- ducers for a large number. there are twenty a, four/3, and six ~/subunits. It has been suggested that very large numbers of heterotrimeric G proteins with defined subunit compositions can be generated from these individual. be useful even to researchers who do not work on signal-transducing G proteins. The field of heterotrimeric G proteins has been covered in part in Volumes 109 and 195 of Methods in Enzymology.