Phospholipase C-PI Is a GTPase-Activating Protein for Gglll, Its Physiologic Regulator

Department of Pharmacology University of Texas Southwestern Medical Center Dallas, Texas 752359041 t Howard Hughes Medical Institute Department of Molecular Physiology and Biophysics Vanderbilt University School of Medicine Nashville, Tennessee 37232-2195 *Laboratory of Biochemistry National Heart, Lung and Blood Institute National Institutes of Health Bethesda, Maryland 20892 Summary Purified Ml muscarlnlc cholinergic receptor and G,,M were coreconstituted in llpid vesicles. Addition of puri- fled phospholipase C-p1 (PLC-pl) further stimulated the receptor-promoted steady-state GTPase activity of Gqlll up to PO-fold. Stimulation depended upon re- ceptor-medlated GTP-GDP exchange. Addition of PLCpl caused a rapid burst of hydrolysls of GWll- bound GTP that was at least IO-fold faster than In its absence. Thus, PLC-(31 stlmulates hydrolysis of GWI1- bound GTP and acts as a GTPase-activating protein (GAP) for its physiologic regulator, G,,,,,. GTPase- stimulating actlvlty was specific both for PLCpl and Gull. Such GAP activity by an effector coupled to a trimeric G protein can reconcile slow GTP hydrolysis by pure G protelns In vitro with fast physiologic deacti- vation of G protein-mediated signaling. Introduction G proteins convey information from cell surface receptors to intracellular effector proteins by following a controlled cycle of activation and deactivation. G proteins are acti- vated by binding GTP. They are deactivated when they hydrolyze bound GTP to GDP; GDP does not cause activa- tion. Hormone-liganded receptors promote the activation of G proteins by catalyzing the binding of GTP, both di- rectly and by accelerating the release of bound GDP. Re- ceptors have no effect on GTP hydrolysis, and the deacti- vation of G proteins by GTP hydrolysis has been widely assumed to be unregulated (for reviews see Bourne et al., 1990, 1991; Kaziro et al., 1991; Ross, 1989). The deactivation of monomeric, low molecular weight GTP-binding proteins, such as ras and rho, is promoted by GTPase-activating proteins (GAPS; Trahey and McCor- mick, 1987; reviewed by Bollag and McCormick, 1991; McCormick, 1989; Bourne et al., 1990). GAPS stimulate the hydrolysis of bound GTP and thus accelerate deactiva- tion about lOO-fold. Without GAPS, the GTPase activities of the small GTP-binding proteins are negligible. Because the GTP-binding a subunits of heterotrimeric G proteins are structurally similar and functionally homologous to the small GTP-binding proteins, the existence of GAPS for G proteins has been a subject for speculation. Some data suggest that there is a cellular mechanism to accelerate deactivation of G proteins. Hydrolysis of GTP bound to isolated G proteins is slow when measured in vitro. The tH for hydrolysis is 10-20 s most G proteins (Brandt and Ross, 1988; Higashijimaet al., 1987; reviewed in Gilman, 1987), 50 s for Gql, (a purified mixture of closely related G, and G,,; Berstein et al., 1992; Strathmann Simon, 1990), and >7 min for G, (Casey et al., 1990). For Gs-stimulated adenylylcyclase, the rate deactivation after hormonal stimulation (Cassel et al., 1977) agrees reasonably well with the measured rate of GTP hydrolysis by purified and reconstituted G, (Brandt and Ross, 1988). However, the rates of deactivation other G protein-regu- lated effecters are considerably faster than the rates at which their isolated G proteins hydrolyze bound GTP in vitro. For example, isolated transducin hydrolyzes bound GTP with a tH of about 15 s (Navon and Fung, 1984), but the deactivation of transducin-stimulated cGMP phospho- diesterase in rod outer segments takes less than 1 s (Dratz et al., 1987; Vuong and Chabre, 1990,199l; Arshavsky al., 1991). Similarly, isolated Gi hydrolyzes GTP as slowly as does purified transducin (Higashijima et al., 1987), but a Grregulated K+ channel in cardiac myocytes is deacti- vated about 100 times as fast upon removal of agonist (Breitwieser and Szabo, 1988). Such discrepancies be- tween slow GTP hydrolytic rates in vitro and rapid deacti- vation rates in cells argue that GAPS for G proteins must exist. proteins do appear to have the potential hydrolyze GTP rapidly. For example, G, that has been removed from detergent solution hydrolyzes bound GTP at least 20-fold faster than G, in detergent micelles or reconstituted into phospholipid vesicles (Brandt and Ross, 1985). It has been suggested that effector proteins such as adenylylcyclase, phospholipase C-81 (PLC-pl), or cyclic GMP phosphodiesterase may act as GAPS for the G pro- teins that regulate them. Direct evidence for this idea has been limited, though. In vitro studies of the GqrI-mediated activation of PLC-81 by the Ml muscarinic cholinergic re- ceptor suggested that PLC-51 may contribute to the deac- tivation of Gqlll (Berstein et al., 1992). Similarly, biphasic GTP hydrolysis by transducin in retinal rod outer segments appeared related to the presence of cyclic GMP phospho- diesterase because the rapid phase of hydrolysis was in- hibited by cyclic GMP and because the amount of rapid hydrolysis was roughly stoichiometric with the phosphodi- esterase (Arshavsky et al., 1991). Genetic evidence sug- gested that rasGAP binds to ras at a site that is needed to convey a signal to the still unknown ras effector (Adari et al., 1988; Cales et al., 1988). This finding prompted speculation that rasGAP is an effector protein for ras and, by extension, that other GAPS are effecters for ras-like monomeric GTP-binding proteins (Bollag and McCormick,