Guanine Nucleotides Stimulate Soluble Phosphoinositide-specific ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1986 by The American Society

Vol. 261, No.35, Issue of December 15, pp. 16553-16558,1986 Printed in U.S.A.

ofBiological Chemists, Inc.

Guanine Nucleotides Stimulate Soluble Phosphoinositide-specific Phospholipase C in the Absence of Membranes* (Received for publication, July 10, 1986)

Hans Deckmyn, Shi-Ming Tu, and Philip W. Majerus From the Division of Hematology-Oncology, Departments of Internal Medicine and Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110

The effect of guanine nucleotides on platelet and calf blowfly salivary glands (12), porcine coronary artery (13), and brain cytosolic phospholipase C was examined in the rat liver (14). The extensively characterized phospholipase C absence of membranes or detergents in an assay using enzymes from seminal vesicles (15), brain (16), platelets (17), labeledlipid vesicles. Guaninenucleotidesstimulate liver (18), and heart (19) are soluble enzymes that do not hydrolysis of [3H]phosphatidylinositol 4,5-bisphos- show preferential hydrolysis of polyphosphoinositides (20phate([3H]PtdIns-4,5-P2)catalyzed bothbyenzyme 24). Thus, it has been presumed that there are two types of from humanplatelets and by partially purified enzyme phospholipase C enzymes. The first type are soluble enzymes from calf brain. Guanosine 5’-0-(3-thiotriphosphate) hydrolyzing all three phosphoinositides, and thereseem to be (GTP+) was the most potent guanine nucleotide with several such enzymes (15,19,23,25,26). Thesecond type are a half-maximal stimulation at 1-10 PM, followed by guanosine 5’-(fl,r-imido)triphosphate> GTP > GDP = membrane-bound enzyme(s) coupled to receptors. These enguanosine 5’-0-(2-thiodiphosphate).Guanosine 5‘-0- zymes are polyphosphoinositide-specific and activated by (2-thiodiphosphate) was able to reverse the GTPrS- membrane guanine nucleotide-binding protein(s). We now report that the soluble phospholipase C enzymes mediated stimulation.NaF also stimulated phospholipase C activity, further implying a role for a guanine from human platelets and also from calf brain are activated nucleotide-binding protein.In the presenceof GTPrS, by guanine nucleotides. This activation is calcium ion-dethe enzyme cleaved PtdIns-4,5-P2at higher pH values, pendent, relatively specific for polyphosphoinositides, and and the need for calcium ions was reduced 100-fold. independent of any membrane components. The stimulation of PtdIns-4,5-PZhydrolysis by GTPrS rangedfrom 2 to 25-fold undervariousconditions, EXPERIMENTALPROCEDURES [SH]phosphatidylinositol was whereashydrolysisof MateriaC+“3H]PtdIns-4,5-P~,1 [3H]PtdIns, and [cx-~’P]NADwere only slightly affected by guanine nucleotides. We pro- obtained from New England Nuclear. [32P]PtdInswas isolated as pose that a soluble guanine nucleotide-dependent pro- described previously (20) from HSD mouse fibrosarcoma cells (27) tein activates phospholipase C to hydrolyze its initial labeled for 12 h with 500 @Ci/ml [32P]-O-phosphoricacid (New substrate in the sequenceof phosphoinositide-derived England Nuclear). The specific activity of the isolated [32P]PtdIns messenger generation. was 200-800 cpm/pmol. ATP, CTP,UTP,GTP, GDP, GDPPS, Gpp(NH)p, insoluble NAD-glycohydrolase (porcine brain), dithiothreitol, NAD, and bovine serum albumin (fraction V) were purchased from Sigma. GTP-yS was from Boehringer Mannheim, pertussis toxin Phosphoinositide breakdown by phospholipase C is the from List Biological Laboratories (Campbell, CAI, and NaF from Allied Chemical (New York, NY). initial reaction in the liberation of the phosphoinositidePreparation of Platelet Supernatant and Membranes-Human derived messenger molecules which include the various ino- platelets from normal donors were isolated as described (28). They sitolphosphates, diglycerides, and icosanoids (1, 2). The were suspended in 10 mM Hepes, 160 m M sucrose, pH 7.5, in a final breakdown of phosphoinositides rapidly follows the binding concentration of 15 X lo9 platelets/ml. After 3 X 10 s sonication a t of specific agonists to cell surface receptors. Recently, numer- 100 watts (Biosonik IV, Bronwill, Rochester, NY) on ice, the crude ous studies have suggested that a guanine nucleotide-binding sonicate was centrifuged at 50,000 X g for 30 min. In some cases this supernatant was centrifuged for an additional 60 min at 200,000 X g protein may be involved in the activation of phospholipase C, to remove any residual membranes. Similar results were obtained in analogy to the guanine nucleotide-binding protein-medi- with both preparations. The supernatantfraction was usedas a source ated transduction of extracellular signals from receptors to of phospholipase C. The membrane pellet was washed twice with the adenylate cyclase (3). Phospholipase C activity, stimulated same buffer by sonication and centrifugation and finally resuspended either by specific agonists or by guanine nucleotides, has been in the original volume. Preparation of Single Bilrryer Vesicles from Total Platelet Lipidsmeasured using membranes from a variety of cells containing Platelet membrane lipids were obtained by extracting 1 ml of memendogenously radiolabeledphosphoinositides (4-10). Guanine branes (see above) with 5 mlof chloroform/methanol (1:l) for 30 nucleotide-dependent phospholipase C activity has also been min, followed by phase separation by addition of 1.5 ml of 2 M KC1 demonstrated using exogenously added phosphoinositides and containing 0.1 M EDTA. The lower phase was dried under a stream membranes from feline sarcoma virus transformed cells (11), of nitrogen, redissolved in 1 ml of chloroform, and kept at -20 “C. Lipids, 80 &I, plus 500,000 cpm [3H]PtdIns (3 &i/nmol), or [3H] *This work was supported by Grants HLBI 14147 (Specialized Center for Research inThrombosis),HLBI 16634, and Training I The abbreviations used are: PtdIns-4,5-P2, phosphatidylinositol Grant T32 HLBI 07088 from the National Institutes of Health, a 4,5-bisphosphate; PtdIns, phosphatidyIinositoI; Ins-1,4,5-P3, inositol NorthAtlantic Treaty Organization Research Fellowship, and a 1,4,5-trisphosphate; G-protein, guanine nucleotide-binding protein; Fullbright Award (to H. D.). The costs of publication of this article GTPrS, guanosine 5’-@(3-thiotriphosphate); Gpp(NH)p,guanosine were defrayed in part by the payment of page charges. This article 5’-(@,+y-imido)triphosphate;GDPPS, guanosine 5‘-0-(2-thiodiphosmust therefore be hereby marked “aduertisement” in accordance with phate); Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid 18 U.S.C. Section 1734 solelyto indicate this fact. EGTA, [ethyIenebis(oxyethylenenitrilo)]tetraacetic acid.

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Nucleotides Guanine Stimulate

mg:=

Phospholipase C

PtdIns-4,5-Pz (3 pCi/nmol), or [32P]PtdIns(0.1-0.4 pCi/nmol) or a mixture were added to 2-ml ampules. After drying under nitrogen, 2 ml of 50 mM Hepes, pH 7.0,lOO mM NaCl were added, and thesample was vortexed. Nitrogen gas wasled over the solution for 10 min, after which the vial was sealed tightly. The sample was sonicated using an ultrasonic cleaner (Cole Parmer, Model 8849) for 2 h. Vesicles were CJ 1200 i 400 800 stored at room temperature and used within 48 h. Preparation of Phospholipase C-Phospholipase CI and CII from ram seminal vesicles were isolated as described previously (15, 20). Phospholipase C from calf brain was purified by a modification of the previous procedure. Details of this purification will be reported elsewhere. ‘0 20 40 Assay of Phospholipase C Actiuity-Reaction mixtures contained 100 plof 0.1 M Tris maleate buffer, pH 6.0, 50 pi vesicles (-lo4 cpm), Proten. pg varying amounts of phospholipase C, and stimuli in 200 p1 final FIG. 1. Effect of GTPrS on phospholipase C activity. Phosvolume. Where indicated, free Ca2+ levels were maintained using pholipase C activity was measured using [3H]PtdIns-4,5-P2 as a Ca*+/EGTA buffers with either 1 or 10 mM EGTA, as calculated substrate. Activity was measured as a functionof platelet supernatant using Freecal (a program for the Macintosh Computer kindly provided protein concentration. Assays were done in the absence (0)or presby Dr. Lawrence Brass, University of Pennsylvania). Reactions were ence (0)of 100 p~ GTPyS at pH6.0. Results are expressed as total started by the addition of either vesicles or of phospholipase C, water-soluble radioactivity and arerepresentative of two experiments. followed by mixing and incubation at 37 “C for 10 min unless other- (See “Experimental Procedures” for other details.) wise stated. Reactions were terminated by adding 1ml of chloroform/ methanol/HCl (100:1000.6, v/v), vortexing, and phase separation 1 N HCl, 5 mM EGTA. The phases were further with 0.3mlof separated by centrifugation, and a 400-pl portion of the upper aqueous phase was removed for radioactivity measurement in a liquid scintillation counter. Results are expressed as total radioactivity in the aqueous phase. Treatment with Pertussis Toxin-Preactivated A protomer of pertussis toxin was obtained by incubation of the toxin for 10 min at 30 “C in 0.1 M potassium phosphate, pH 7.5, containing 10 mM dithiothreitol(29). To detect an effect of pertussis toxin on the phospholipase C activation, platelet supernatant (100 pg of protein) wasmixed with either 2 or 15 pg of preactivated A protomer of pertussis toxin, in the presence of 1 mM NAD in i;Imphate buffer, in a total volume of 100 pl for 60 min a t 30 “C. 150 pl of Hz0 was added and 30 p1 of this was immediately used in a phospholipase C assay. Controls were run in the absence of pertussis toxin. TIME(min) For the radioactive labeling of the pertussis toxin substrate, platelet FIG. 2. Time course of phospholipase C activity. Phospholisupernatant was first depleted of endogenous NAD by incubation at 30 “C for 30 min with insoluble NAD-glycohydrolase (30),followed pase C activity was measured using [3H]PtdIns-4,5-P2as a substrate. by centrifugation at 12,000 X g for 5 min. 50 p1 (100 pg of protein) of Activity was measured as a function of time using 8 (A) or 24 (0)pg this, or 20 pg of partially purified brain phospholipase C, or platelet of platelet supernatant protein in the absence (closedsymbols) or membranes (45 pg of protein) was mixedwith 10 pl of 0.1 M potassium presence (open symbols) of 100 p~ GTPyS at pH 6.0. Results are expressed as total water-soluble radioactivity and are representative phosphate buffer containing 10-15 pCi of [a-”PJNAD, and 15 pg of preactivated pertussis toxin A protomer. After 60 min a t 30 “C, the of five experiments. (See “Experimental Procedures” for other dereaction was stopped by addition of 0.5 ml of 15%trichloroacetic acid tails.) for the platelet supernatant, 0.5 ml of 15% trichloroacetic acid, and 20 pg of serum albumin for brain phospholipase C, or 0.5 ml of cold 0.1 M Tris-HC1 buffer, pH 6.0, and centrifugation at 50,000 X g, 30 min for the platelet membranes. Trichloroacetic acid precipitates were washed twice with 15% trichloroacetic acid and thentwice with diethyl ether. Precipitates and membrane pellets were dissolved in 4% sodium dodecyl sulfate, 10% P-mercaptoethanol by heating a t 100 “C for 2 min, and analyzed by polyacrylamide gel electrophoresis (31). The gel was then autoradiographed a t -80 “C for 24 h using XAR-5 x-ray films (Kodak). Other Methods-Protein was measured using the Bio-Rad protein 011 I& lob0 assay with bovine serum albumin as a standard.

*

i

Ib

[ guonine nucleotide ].JIM

RESULTS

Our experiments were designed to test thehypothesis that the soluble phospholipase C enzyme in platelets and brain might be stimulated by a guanine nucleotide-binding protein and thus be similar to the membrane-bound polyphosphoinositide-specific phospholipase C activity. We used unilamellar vesicles of total platelet lipids as substitutes for membranes to exclude all membrane proteinand detergents while still retaining the natural lipid composition. The lipids were tracer-labeled with tritiated phosphoinositides, which did not change the lipid composition of the vesicles since the added labeled lipids were never more than 1%of the mass of the endogenous phosphoinositides (18 nmol of PtdIns, 1.4 nmol of PtdIns-4,5-P2/1O9 platelets (32)). However, due tothe

FIG. 3. Effect of guanine nucleotides on phospholipase C activity. Phospholipase C activity was measured using [3H]PtdIns4,5-P2 as a substrate. Assayswere done at pH 5.5 with 12 pgof platelet supernatant protein and the following additions: 0, GTPyS; 0, Gpp(NH)p; 0, GTP; A, GDP; A, GDPPS. Results are expressed as total water-soluble radioactivity and are representative of two experiments. (See “Experimental Procedures” for other details.)

different mass of the endogenous phosphoinositides, the specific activity of the labeled lipids varied such that similar radioactivity indicated a 12-fold higher mass of PtdIns-derivedproducts as compared to PtdIns-4,5-P2-derivedproducts. Previous studies have shown that vesicles containing cell lipids are poor substrates for phospholipase C , mainly because of the high concentration of the inhibitory phospholipid phos-

Guanine Nucleotides Stimulate Phospholipase C 2000

a 400

[GDPBq, P M

FIG. 4. Inhibitionby GDPBS of the GTPyS-stimulatedphospholipase C activity. Phospholipase C activity was measured using [3H]PtdIns-4,5-P2assubstrate. Assaysweredonewith 24 pg of platelet supernatant proteinat pH 6.0. Results are expressedas the difference between total water-soluble radioactivity obtained in the presence of GDPj3S alone uersus GDPj3S plus either 10 (0)or 100 (0)p~ GTP-ySand are representative of twoexperiments.(See ”Experimental Procedures” for other details.)

FIG. 5. Stimulation of phospholipase C activity with NaF. Phospholipase C activity, using [3H]PtdIns-4,5-P2a substrate, as was measured as a function of increasing NaF or NaCl concentration. Assays were done at pH5.5using 16 pg of platelet supernatant protein. Resultsare expressed as total water-soluble radioactivity and are representativeof three experiments. (See “Experimental Procedures” for other details.)

t

3200

OH

FIG. 6. Effect of pH on phospholipase C activity. Phospholipase C activity was measured using [3H]PtdIns( a ) and [3H]PtdIns4,5-P2 (b) as substrates. Assays were done with 24 pg of platelet supernatant protein in the absence (0)or presence (0)of 100 p~ GTPrS. Results are expressed as total water-soluble radioactivity and are representativeof four experiments. (See “Experimental Procedures” for other details.) phatidylcholine (33, 34). Also, the phosphoinositide concentration (0.2-2 p M ) in our vesicle assays is far below saturating concentrations of the lipid substrates (20). However, we were able to measure phospholipase C activity using either labeled PtdIns orPtdIns-4,5-P2 as substrate. The resultsareex-

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pressed as water-soluble radioactivity. In two experiments we analyzed the products of phospholipase C reactions by high performance liquid chromatography (35) and found inositol 1-phosphate and cyclic inositol 1:2-phosphate formed from PtdIns andfrom PtdIns-4,5-P2 mainly Ins-1,4,5-P3and cyclic Ins-1:2,4,5-P3and lesser amounts of cyclic and noncyclic diand monophosphates. We saw no obvious change in the proportion of cyclic versw noncyclic products in reactions stimulated by guanine nucleotides as described below. Platelet Phospholipase C-When the nonhydrolyzable GTP analogue GTPyS was added to a mixture of platelet supernatant andmembrane-derived lipid vesicles labeled with 13H] PtdIns-4,5-P2, there was a 10-fold increase in phospholipase C activity, as shown in Fig. 1.This activity was dependent on the amount of enzyme added and was linear for 10 min, as shown in Fig. 2. The GTP+ activation of phospholipase C was immediate at high concentrations as prior incubation of enzyme with 100 p~ GTPyS did not further increase phospholipase C activity. The effect was dependent on the concentration of GTPyS with half-maximal activation between 1 and 10 p ~The . stimulation is dependent on the concentration of several guanine nucleotides, with GTPyS being the most potent followed by Gpp(NH)p, GTP, GDP, and GDPPS, as shown in Fig.3. ATP, CTP, and UTP, furthermore, were 32-, 16-, and &fold less potent thanGTP, respectively. GDPPS inhibited the stimulatory effect of GTPyS (Fig. 4). Sodium fluoride also stimulated the formation of water-soluble breakdown products of [3H]PtdIns-4,5-P2 by phospholipase C as shown in Fig. 5, whereas NaCl was without effect. Half-maximal stimulation was obtained with 200 p~ NaF. Phospholipase C as assayed in this system using [3H] PtdIns-4,5-P2 as substrate has a sharp pH optimum around 5, as shown in Fig. 6b. Above pH 6, there is little basal activity. However, in the presence of GTPyS, phospholipase C activity is increased, especially at higher pH values. GTPyS stimulated PtdIns breakdown only at pH 5 (Fig. 6a). The effect of varying calcium ion concentration on phospholipase C activity in the presence or absence of GTPyS is shown in Fig. 7. In these experiments the lipid vesicles were labeled with both [3H]PtdIns-4,5-P2 and [32P]PtdIns in order to determine the effects of guanine nucleotide on both substrates simultaneously. As noted above, 100 PM GTPyS has only a modest effect on phospholipase C activity toward PtdIns. Similarly, there is little effect of GTPyS on the Ca2+ dependence of the reaction. In contrast, GTPyS reduced the Ca2+concentration needed for hydrolysis of PtdIns-4,5-P2 by approximately 100-fold. Thus, this soluble phospholipase C enzyme has properties similar to those reported previously for membrane-bound enzyme. That is, it is stimulated by GTP-yS and at low Ca2+ concentrationsshows relative preference for the polyphosphoinositide substrate. Calf Brain Phospholipase C-We have purified a phospholipase C from calf brain which is most likely of the phospholipase CII type based on our previous work (15). Using enzyme purified 70-fold over a crude calf brain supernatant fraction, we again found a pH-dependent stimulation of activity by GTP+ when using [3H]PtdIns-4,5-P2 as substrate asshown in Fig. 8. We have not yet been able to separate a guanine nucleotide-binding protein from phospholipase C in a “reconstitution assay,” therefore, the possibility of a direct stimulation of phospholipase C by guanine nucleotides cannot be excluded. However,we do not believe that thestimulation by guanine nucleotide is due to a direct effect on phospholipase C, since brain phospholipase C purified (1000-fold) to homogeneity and ram seminal vesicle phospholipase CI and CII were not stimulated by GTP+.

Guanine Nucleotides Stimulate Phospholipase C

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FIG.7. Effect of free Ca2*concentration on phospholipase C activity. Phospholipase C activity was measured using double-labeled vesicles with [3zP] PtdIns ( a ) and [3H]PtdIns-4,5-Pz (b) as substrates. Assays were done a t pH 6.0 for 10 min in the absence (0)or presence (0)of 100 p~ GTPyS using 24pg of platelet supernatant protein. Results are expressed as total water-soluble radioactivity and are representative of four experiments. (See “Experimental Procedures” for other details.)

PH

FIG.8. Effect of pH on partially purified calf brain phospholipase C. Phospholipase C activity was measured using [‘HI PtdIns-4,5-P2 as substrate. Assays were done in the absence (0)or presence (0)of 100 p~ GTP-yS. Results are expressed as total watersoluble radioactivity. (See “Experimental Procedures” for other details.)

Pertussin Toxin Treatment-ADP-ribosylation of platelet membranes, supernatant, and brainphospholipase C by pertussis toxin in the presence of [“PINAD disclosed a 41-kDa substrate in platelet membrane and supernatant (Fig. 9), but not in the partially purified brain phospholipase C. Pertussis toxin treatment had no influence on the basal or GTP-ySstimulated breakdown of PtdIns-4,5-P2 by platelet supernatant phospholipase C.

41 K

DISCUSSION

The activation of phosphatidylinositol-specific phospholipase C is one of the early steps leading to cellular activation, resulting in the formation of second messengers such as Ins1,4,5-P3,cyclic Ins-1:2,4,5-P3, and diacylglycerol,which induce Ca2+ release and proteinkinaseCactivation (1, 36, 37). Guanine nucleotides have been shown to play a role in the activation of phospholipase C in a manner proposed to be analogous to their action on adenylate cyclase, whereupon receptor occupancy, and in thepresence of GTP, a G-protein dissociates into itsa- and &-subunits (38,39). The a-subunit stimulates (G.)(40) or inhibits (Gi) (41,42) adenylate cyclase. This effect is terminated by the GTPase activity intrinsic to the G-protein (43). Thus, stable GTP analogues have a more pronounced effect. In our system, we also found stimulation of the soluble phospholipase Cactivity by guanine nucleotides, with the

FIG. 9. ADP-ribosylation of the 41-kDa protein in platelet supernatant and membranes by the A protomer of pertussis toxin. Platelet supernatant fraction (lanes A and B ) and membrane fraction (lanes C and D) were incubated with [m3*P]NAD in the presence (lanes A and C ) or absence (lanes B and D )of the preactivated A protomer of pertussis toxin as described under “Experimental Procedures.” The autoradiographic patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, representative of two experiments, are shown.

stable GTP analogues, GTP-yS and Gpp(NH)p,more potent thanGTP itself, and much more potentthanGDPand GDPBS, which actually reversed the stimulatory effect of GTP-yS. AlF; is known to be a potent modulator of Gi, G., and transdu ‘n (see 39, 40, 42, 44), and NaF furthermore stimulates ph sphoinositide turnover in hepatocytes (45) and in

7

Phospholipase C

Nucleotides Guanine Stimulate platelets.' We found a lO-fold increase of platelet supernatant phospholipase Cactivity by 500 W M NaF, adding further evidence for the involvement of a G-protein in the phospholipase C stimulation. Guanine nucleotides stimulated phospholipase C activity greater toward PtdJns-4,5-P2 as compared to PtdIns. In the presence of GTP+, the enzyme cleaved PtdIns-4,5-Pz at higher pH values, and the need for free Ca2+was reduced. At low cellular Ca'+ levels, GTP-dependent stimulation of phospholipase C using PtdIns-4,5-Pz as a substrate forms Ins1,4,5-P3and cyclic Ins-1:2,4,5-P3, whichcan thenrelease Ca2+ from intracellular stores (35, 47). Elevated Ca2+levels result in numerous cellular reactions, including increased phospholipase C activity on the more abundant PtdIns to form large amounts of diacylglycerol, the natural stimulator of protein kinase C. We found no directeffect of guanine nucleotides on phospholipase C. This implies the need for a guanine nucleotide-binding protein to mediate guanine nucleotide-dependent stimulation. A 41-kDa pertussis toxin substratewas detected in our platelet supernatant preparation, which might be Gi (48). However, no such substrate was detected in the partially purified brain phospholipase C. Therefore, the nature of the proposed guanine nucleotide-binding protein coupled to phosphoinositide turnover is at present unknown. Our results imply that guanine nucleotide-regulated proteins may be cytosolic as well as membrane-bound. That Gproteins aresoluble in some cases was suggested by the recent finding of a cytosolic pertussis toxin substrate in mast cells (49). We also find a soluble substrate for pertussis toxin in platelets. Sternweis (50) has recently shown that the dissociated a-subunits of brain, GBand Go (a GTP-binding protein of unknown function), are soluble without detergents. Furthermore, Rodbell (51)demonstrated that guanine nucleotidebinding proteins can translocate from one membrane to another. Additionally, Bhat et al. (52) have shown that a variety of tissuescontaina soluble guanine nucleotide-dependent activity that can reconstitute adenylate cyclase activity in cyc- membranes, and Lynch et al. (46) have shown a-subunits of G, released from liver plasma membranes following cholera toxin activation. Taken together, these studies show that Gproteins or subunits thereof can be soluble. One could then speculate that the released G-proteins might bind to soluble phospholipase C, leading to its association with the membrane. Our study indicates that soluble phospholipase C enzymes have properties similar to those reported previously for membrane-bound enzyme. It remains to be shown whether the membrane-bound phospholipase C is the same enzyme or enzymes as found in cytosol. Thenature of the guanine nucleotide-binding protein that stimulates phospholipase C also requires further study. Acknowledgments-We thank A1 Gilman and Susie Mumby for assistance and advice on guanine nucleotide-binding proteins. We also thank The0 Ross, Susan Kaiser, and Cecil Buchanan for their help, and Teresa Bross, Vinay Bansal, Tom Connolly, and Roger Inhorn for helpful discussions.

4.

5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

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