Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8782-8786, October 1993 Biochemistry The disaggregation theory of signal transduction revisited: Further evidence that G proteins are multimeric and disaggregate to monomers when activated SALEEM JAHANGEER AND MARTIN RODBELL Signal Transduction Section, Laboratory of Ceilular and Molecular Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 Contributed by Martin Rodbell, May 27, 1993 ABSTRACT We have compared the sin on rates and those found in detergent extracts. Based on target on sucrose gradients of the heterotrhimeric GTP-binding regu- analysis, G proteins in the native membrane environment are latory (G) proteins G., Go, G,, and Gq etracted from rat brain thought to be higher-ordered structures, termed multimers, synaptoneurosomes with Lubrol and digtin. The individual that are converted to monomers by the combined actions of a and 3 subunits were monitored with specifi antisera. In all hormones and guanine nucleotides (4). Further support for cases, both subunits cosedimete dicatg that the subunits this hypothesis stems from recent findings that the major are likely complexed as heterotrimers. When extracted with heterotrimeric G proteins in rat brain synaptoneurosomes are Lubrol al of the G proteins s ted with rates of about 4.5 cross-linked in their native membrane environment, yielding S (condstent with heterotrimers) whereas diitonin extae very large structures compatible with their being multimeric 60% ofthe G proteins with peaksat 11 S; 40% pefleted aslager proteins (5). Different structures of G proteins and their structure. Dgtonin-extae G, was cross-linked by p-phe- products ofhormone and guanine nucleotide activation have nyle imaide, yielding structures too large to enter poly- also been obtained, depending on the types of detergents acrlamide gels. No cross-l of Lubrol-extracted G, oc- employed for extraction from membranes (6, 7). Extraction curred. Treatment of the membranes with g ne 5'-[r of G proteins from these membranes with octyl glucoside thioltriphosphate and Mg2+ yielded digitonin-extracted yields polydisperse structures that are suggestive of multi- structures with peak sedimentation values of 8.5 S-I.e., meric proteins. Glucagon activation of the G protein G, in comparable to that ofpurifid G. in digltonin and considerably hepatic membranes involves structures that sediment in octyl larger than the Lubrol-exracted 2S structures representing the glucoside extracts (6, 7). separated a and fty subunits formed by the actions of guano- Possibly because of the broad range of S values for G dne 5'-[rthioJtriphosphate. It is conduded thatthe multimeric proteins extracted by octyl glucoside, attempts to purify the structure of G proteins in brain membranes are at least multimeric forms of octyl glucoside-extracted G proteins pardally preserved in digitonin and that activation of these proved unsatisfactory. Moreover, the (-subunit antibodies structures in membranes yields monomers ofG proteins rather employed in those studies were subsequently found to be than the ted products (a and 1y complexes) ob- insensitive for detecting these proteins. Supplied with high- served in Lubrol. It is proposed that hormones and GTP affect titer antibodies to all of the G-protein subunits in synapto- the dymmic interplay between multimeric G proteins and neurosomes and prompted by earlier studies (8) showing that receptors in a fashion analogous to the actions of ATP on the G. extracted from reticulocyte membranes with digitonin dynamic interactions between myosin and actin filaments. yielded very large structures (>600 kDa) that are not asso- Sinal transduction is miated by activated monomers re- ciated with either receptor or adenylyl cyclase, we have kased from the multimers during the activation process. investigated the sedimentation rates of various types of G proteins (G., Gi, Go, and Gq) present in rat brain synapto- Great progress has been made in understanding the structure neurosomes by comparing Lubrol and digitonin as extracting andfunction ofGTP-binding regulatory (G) proteins and their agents. The results suggest that digitonin at least partially role in signal transduction. Much of this recent progress preserves the multimeric structures ofG proteins observed in stems from the powerful tools ofmolecular biology that have membranes. Moreover, the products ofactivation by guano- provided knowledge of the primary structure of their com- sine 5'-[y-thio]triphosphate (GTP[yS]) are monomers rather ponents and the fact that there is a growing family of these than dissociated a and (By subunits observed as products of proteins. Reconstitution studies with purified components activation in Lubrol. have provided major support for the theory that receptor activation causes exchange, in a catalytic fashion, of tightly MATERIALS AND METHODS bound GDP with GTP on the a subunits of heterotrimeric G proteins; the GTP-bound a subunit is then thought to disso- Materials. Goat anti-mouse IgG, goat anti-rabbit IgG, and ciate from the fry complexes leading ultimately to regulation peroxidase anti-peroxidase (rabbit) were purchased from of a variety of effector systems; turnover of the reactions Organon Teknika-Cappel. Rabbit polyclonal antisera 8129 occurs through hydrolysis of GTP to GDP (for review, see (GP35 specific) and 8132 (GP36 specific) (9) were generously ref. 1). Although these propositions have been widely ac- supplied by David Manning (University of Pennsylvania). cepted, some of the underlying assumptions have been chal- Rabbit polyclonal antisera W082 (Gaq specific) and X384 lenged (2) in part because of apparent discrepancies between (Gaq and Gall specific) (10) were kindly supplied by Paul the structures determined by target analysis of G-protein- Sternweis (University of Texas). Rabbit polyclonal antise- mediated systems in their native membrane environment (3) rum QL (Gaq and Gall specific) (11, 12) was supplied by Allen Spiegel (National Institute of Diabetes and Digestive The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: GTP[yS], guanosine 5'-[ythio]triphosphate; G pro- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tein, GTP-binding regulatory protein. 8782 Downloaded by guest on October 2, 2021 Biochemistry: Jahangeer and RodbeR Proc. Natl. Acad. Sci. USA 90 (1993) 8783 and Kidney Diseases, National Institutes ofHealth). Sources min, and the fractions were examined by SDS/PAGE and of rabbit polyclonal antisera specific for Gas, Gai, and Gao Western blot analysis. have been described (6). In all cases the antibodies were raised against selective peptide regions of the G-protein subunits. Mouse anti-a-tubulin monoclonal antibody was RESULTS purchased from Amersham, and peroxidase anti-peroxidase The sedimentation velocity measurements (S values) of G (mouse) was procured from Accurate Chemicals. proteins, monitored by immunoblot analysis of the fractions Detergents and Other Reagents. Lubrol PX [10% (wt/vol) obtained from sucrose gradients, are shown in Fig. 1. When aqueous solution] was from Pierce. An aqueous stock solu- extracted with Lubrol, the most extensively used detergent tion of digitonin [5% (wt/vol)] was prepared by boiling for purification of G proteins, all of the G-protein subunits followed by cooling to room temperature for 2-3 days and (Ga., Gai, Gaq, Ga., G(-1, and G,-2) displayed sedimenta- filtration through a 0.45-pm (pore size) filter to remove tion values with peaks in the range of 4.0-4.5 S, which insoluble material; the stock solution was then stored at room approximates that of heterotrimeric G proteins (15). In con- temperature. Working solutions of detergents were generally trast, digitonin extraction of all the G proteins invariably prepared freshjust before use. GTP[yS] was purchased from yielded structures that distributed in the soluble fractions as Boehringer Mannheim. All other reagents were ofthe highest structures of 10 to >12 S; an estimated 40%o ofthe G proteins quality available. pelleted at the bottom ofthe gradient tube (fraction 22). Note Preparation and Treatment of Synaptoneurosomes. Rat that both a subunits and 3(y) subunits gave similar sedimen- brain synaptoneurosomes were prepared (6) and stored as tation rates, indicating that the structures most likely contain aliquots in liquid nitrogen until use. Synaptoneurosomes (1 complexes of heterotrimeric G proteins. Identical results mg) were incubated at 30°C for 5 min in buffer A (20 mM were obtained using liver membranes and various cell lines Hepes-NaOH, pH 7.4/150 mM NaCl/2 mM MgSO4/1 mM extracted with digitonin (data not shown). EDTA/0.4 mM phenylmethylsulfonyl fluoride), without or An explanation for the large differences in hydrodynamic with an appropriate concentration ofGTP[yS] as indicated in properties of the G proteins in digitonin and Lubrol is that figure legends, in a total volume of 0.2 ml. Reactions were multimers are stabilized in digitonin but not in Lubrol. To test stopped by the addition ofdetergent stock solutions to obtain this possibility, the sucrose gradient fractions were treated a concentration of 1%. Extractions were carried out on ice for with 75 ,uM p-phenylenedimaleimide under conditions iden- 1 h. tical to those used for cross-linking native membrane forms Sucrose Density Gradient Fractionation. After detergent of G proteins; the products were subsequently examined by extraction on ice for 1 h, 0.2 ml of total reaction mixture was layered over a prechilled 5-20%6 (wt/wt) linear sucrose 40- A Lubrol l Digitonin gradient in buffer B (20 mM Hepes-NaOH, pH 7.4/150 mM ao NaCl/l mM EDTA/1 mM dithiothreitol) and either 1% cts m Lubrol or After at u 30'- 0.5% digitonin. centrifugation 50,000 rpm Oli E 15 h at in a about 22 fractions for 4°C Beckman SW60 rotor, a4cz (0.2 ml) were collected (fraction 22 being the pelleted mate- w Cu rial). Alternate fractions were then examined for a and ( i.-0 _ 2 subunits of G proteins by SDS/PAGE and Western blot ._ analysis (13, 14).