Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C.

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization.     

Sphingosine inhibits thyrotropin-releasing hormone binding to pituitary cells by a mechanism independent of protein kinase C

Sphingosine inhibited [3H]methylhistidine-thyrotropin-releasing hormone (MeTRH) binding to intact GH3 cells and to GH3 membranes. This inhibition was dependent on the concentration of sphingosine and on the ratio of sphingosine to cell number (or membrane protein) and was partly reversed by washing. In intact cells, the IC50 was 63 microM (1.8 X 10(6) cells/ml; 2 nM MeTRH), and 100 microM sphingosine was found, by Scatchard analysis, to increase the apparent dissociation constant (Kd) from 1.1 +/- 0.3 to 6.5 +/- 2.3 nM and to decrease the maximal binding capacity (Bmax) to 41 +/- 9.5% of control. Kinetic analysis showed that the major effect of sphingosine on Kd was due to a marked decrease in the apparent association rate constant for MeTRH from 2.5 +/- 0.4 X 10(5) M-1 s-1 to 0.10 +/- 0.015 X 10(5) M-1 s-1. At 100 microM, sterylamine was as effective as sphingosine in inhibiting MeTRH binding, whereas sphinganine was less effective, and psychosine and steroylsphingosine were without effect. The following observations show that sphingosine inhibition of MeTRH binding did not involve protein kinase C. The IC50 for sphingosine inhibition of MeTRH binding was the same in GH3 cells that had been incubated with 1 microM phorbol 12-myristate 13-acetate for 16 h, to "down-regulate" protein kinase C, as in control cells. Sphingosine inhibited MeTRH binding to membranes isolated from GH3 cells that contain very little protein kinase C activity. In GH3 membranes, 100 microM sphingosine increased the Kd for MeTRH from 3.4 +/- 0.1 to 13 +/- 3.1 nM but did not significantly decrease Bmax (12 +/- 5.0% of control, p greater than 0.05). And, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, an inhibitor of protein kinase C, failed to decrease MeTRH binding to intact GH3 cells or to membranes, and did not interfere with the effects of sphingosine. These data show that sphingosine and its analogs have complex actions to inhibit MeTRH binding to GH3 cells, at least some of which are independent of protein kinase C, and thereby demonstrate that sphingolipids cannot be used as specific inhibitors of protein kinase C.     

Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins.

The superoxide-generating NADPH oxidase system in phagocytes consists of at least membrane-associated cytochrome b558 and three cytosolic components named SOCI/NCF-3/sigma 1/C1, SOCII/NCF-1/p47-phox, and SO-CIII/NCF-2/p67-phox. p47-phox and p67-phox were isolated, and their primary structures were determined, but SOCI has not been well characterized. In the present study, we first purified SOCI to homogeneity from the cytosol fraction of the differentiated HL-60 cells. The purified SOCI was a small GTP-binding protein (G protein) with a M(r) of about 22,000. The guanosine 5'-(3-O-thio)triphosphate-bound form, but not the GDP-bound form, of this small G protein showed the SOCI activity. The partial amino acid sequence of SOCI thus far determined was identical to the amino acid sequence deduced from the cDNA encoding rac2 p21. None of the purified small G proteins, including Ki-ras p21, smg p21B/rap1B p21, rhoA p21, and rac1 p21, showed the SOCI activity. These results indicate that SOCI is a small G protein very similar, if not identical, to rac2 p21. The GDP/GTP exchange reaction of SOCI was stimulated and inhibited by stimulatory and inhibitory GDP/GTP exchange proteins for small G proteins, named smg GDS and rho GDI, respectively. The NADPH oxidase activity was also stimulated and inhibited by smg GDS and rho GDI, respectively. These results indicate that the superoxide-generating NADPH oxidase system is regulated by both smg GDS and rho GDI through rac2 p21 or the rac2-related small G protein in phagocytes.     

Properties of a novel GTP-binding protein which is associated with soluble phosphoinositides-specific phospholipase C

Recently we demonstrated the presence in calf thymocytes of a GTP-binding protein (G-protein) composed of three polypeptides, 54, 41, and 27 kDa, which was physically and functionally associated with a soluble phosphoinositides-specific phospholipase C (PI-phospholipase C). The properties of this G protein were further investigated with the following results. 1) In addition to the ability to bind [35S]guanosine-5'-[gamma-thio]triphosphate (GTP gamma S), the G-protein exhibited GTPase activity, which was enhanced by Mg2+, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, but inhibited by sodium cholate, GTP gamma S and F-.2) The 54-kDa polypeptide was ADP-ribosylated by pertussis toxin and also by endogenous membrane-bound ADP-ribosyltransferase, but none of these three polypeptides was ADP-ribosylated by cholera toxin. 3) The G-protein did not cross-react with either anti-rat brain alpha 1 (alpha-subunit of inhibitory G-protein, G1), alpha 0 (alpha-subunit of other G1-like G-protein, G0) or beta gamma antibodies. 4) Incubation of this G Protein with GTP gamma S caused dissociation of the three polypeptides. 5) The 27 kDa polypeptide showed GTP-binding activity and enhanced the phosphatidylinositol 4,5-bisphosphate hydrolysis by purified PI-phospholipase C. These results suggest that the PI-phospholipase C-associated G-protein in calf thymocytes may be a novel one and that it is involved in the regulation of PI-phospholipase C activity.     

Loss of receptor regulation by a phospholipase D1 mutant unresponsive to protein kinase C

Activation of phosphatidylcholine-specific phospholipase D (PLD) constitutes an important part of the cellular response to agonist signaling. PLD1 is stimulated in vitro in a direct and synergistic manner by protein kinase C (PKC), ADP-ribosylation factor (ARF) and Rho family members. However, the direct and specific role of each of these effectors in agonist-stimulated PLD activation is poorly understood. We have used transposon mutagenesis to generate a library of PLD1 alleles containing random pentapeptide insertions. Forty-five alleles were characterized to identify functionally important regions. Use of an allele unresponsive to PKC, but otherwise seemingly normal, to examine coupling of PLD1 to a subset of G-protein-coupled receptors demonstrates for the first time direct stimulation of PLD1 in vivo by PKC and reveals that this direct stimulation is unexpectedly critical for PLD1 activation.     

Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: possible involvement of an inhibitory GTP-binding protein

Cultured pituitary cells prelabeled with myo-[2-3H] inositol were permeabilized by ATP4-, exposed to guanine nucleotides and resealed by Mg2+. Addition of guanosine 5'-0-(3-thio triphosphate) (GTP gamma S) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5'-0-(2-thiodiphosphate) (GDP beta S) or by neomycin. Surprisingly, addition of GDP beta S (100-600 microM) on its own resulted in a dose-related increase in [3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP greater than GTP gamma S greater than ATP greater than CTP. The stimulatory effect of UTP, ATP and CTP, but not GTP gamma S or GDP beta S, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTP gamma S decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.     

Coupling of the alpha 1-adrenergic receptor to a guanine nucleotide-binding regulatory protein by a discrete domain distinct from its ligand recognition site

At rat hepatic membrane alpha 1-adrenergic receptors, the nonhydrolyzable GTP analogue p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding. This p[NH]ppG effect is consistent with the involvement of a guanine nucleotide-binding regulatory protein (G-protein) in alpha 1-adrenergic receptor signalling. Although readily apparent in membranes prepared to avoid retention of endogenous nucleotides and activation of Ca2+-sensitive proteinases (+pi), this p[NH]ppG effect is not observed in membranes prepared without proteinase inhibitors (-pi), or in -pi membranes treated with Ca2+ (-pi, +Ca2+). In these various membrane preparations, different Mr forms of the receptor are also identified by photoaffinity labeling with [125I]CP65526, an aryl azide analog of the alpha 1-selective antagonist, prazosin, followed by SDS-polyacrylamide gel electrophoresis and autoradiography. Whereas a predominant Mr = 80,000 subunit is identified in +pi membranes, in -pi membranes a proteolytic Mr = 59,000 fragment is also observed. In -pi, +Ca2+ membranes, only this latter peptide is detected. To evaluate the ability of each of these forms of the receptor to couple with a G-protein, the effect of p[NH]ppG on the agonist-inhibition of [125I]CP65526 labelling was determined by laser densitometry scanning and computer analysis. At the Mr = 80,000 subunit, p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding, even in -pi membranes. By contrast, agonist-binding at the Mr = 59,000 subunit is of low-affinity and was not affected by p[NH]ppG. These data indicate that the cleaved Mr = 59,000 fragment, while retaining hormone binding activity is unable to undergo G-protein coupling. Thus, the alpha 1-adrenergic receptor appears to contain a discrete domain necessary for G-protein coupling that is distinct from its ligand recognition site.     

Parathyroid hormone receptor internalization is independent of protein kinase A and phospholipase C activation

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) binding to their common receptor stimulates second messenger accumulation, receptor phosphorylation, and internalization. LLC-PK(1) cells expressing a green fluorescent protein-tagged PTH/PTHrP receptor show time- and dose-dependent receptor internalization. The internalized receptors colocalize with clathrin-coated pits. Internalization is stimulated by PTH analogs that bind to and activate the PTH/PTHrP receptor. Cell lines expressing a mutant protein kinase A regulatory subunit that is resistant to cAMP and/or a mutant receptor (DSEL mutant) that does not activate phospholipase C internalize their receptors normally. In addition, internalization of the wild-type receptor and the DSEL mutant is stimulated by the PTH analog [Gly(1),Arg(19)]hPTH-(1-28), which does not stimulate phospholipase C. Forskolin, IBMX, and the active phorbol ester, phorbol-12-myristate-13-acetate, did not promote receptor internalization or increase PTH-induced internalization. These data indicate that ligand-induced internalization of the PTH/PTHrP receptor requires both ligand binding and receptor activation but does not involve stimulation of adenylate cyclase/protein kinase A or phospholipase C/protein kinase C.     

Identification of a prostacyclin receptor coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mouse mastocytoma P-815 cells.

A stable analogue of prostacyclin, iloprost, specifically bound to 30,000 x g pellet (the membrane fraction) prepared from mouse mastocytoma P-815 cells. The binding was dependent on time, temperature and pH, and absolutely required a divalent cation. The equilibrium dissociation constant and the maximal concentration of the binding site as determined by Scatchard plot analysis were 10.4 nM and 1.12 pmol/mg of protein, respectively. The Hill coefficient was 1.0, indicating a single entity of binding site and no cooperativity. The binding site was highly specific for iloprost among PGs tested (iloprost much greater than PGE1 greater than carbacyclin greater than PGE2). In contrast, the membrane fraction had the binding site specific for PGE2 and PGE1, which was distinct from the prostacyclin receptor. The dissociation of bound [3H]iloprost from the membrane fraction was specifically enhanced by guanine nucleotides. Furthermore, iloprost dose-dependently enhanced the activity of adenylate cyclase in a GTP-dependent manner. These results indicate that a specific prostacyclin receptor is coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mastocytoma cells.     

The G alpha q and G alpha 11 proteins couple the thyrotropin-releasing hormone receptor to phospholipase C in GH3 rat pituitary cells.

Thyrotropin-releasing hormone stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in GH3 cell membranes. The stimulation of the phosphoinositide phospholipase C (PI/PLC) activity can be blocked by incubation of GH3 membranes with polyclonal antibodies directed against a peptide derived from the C-terminal region of G alpha q and G alpha 11. Antibodies directed against the C-terminal region of other G alpha-subunits had no detectable effect. The inhibition was specific since addition of the peptide that was used to prepare the antibody completely reversed the inhibition. Further evidence for the coupling of the TRH receptor to G alpha q or G alpha 11 comes from a reconstitution experiment in which human embryonic kidney cells were transiently transfected with cDNAs corresponding to the TRH receptor, G alpha q or G alpha 11. The PIP2 hydrolysis detected with membranes from cells that over-expressed the TRH receptor alone was low, however, co-expression with the G alpha q or G alpha 11 subunits produced a synergistic stimulation of PI-PLC activity. In contrast, co-expression of these alpha-subunits with the M2 muscarinic acetylcholine receptor induced a weak stimulation of PIP2 hydrolysis. The results presented here suggest that the TRH-dependent stimulation of PI-PLC in GH3 cells is mediated through the G-protein alpha-subunits, G alpha q and/or G alpha 11.     

Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5,800-fold from the cytosolic fraction of calf thymocytes. The purification was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTP gamma S-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTP gamma S-binding activities in the partially purified enzyme preparation could be separated by the column chromatography on Sephadex G-100 only in the presence of 1% sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTP gamma S (10 microM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTP gamma S. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.     

The formylpeptide chemoattractant receptor copurifies with a GTP-binding protein containing a distinct 40-kDa pertussis toxin substrate

Detergent extraction of plasma membranes from differentiated HL60 cells, specifically labeled with the chemoattractant, formyl-Nle-Leu-Phe-Nle-[125I-Tyr] Lys, resulted in the solubilization of a receptor-radioligand complex. GTP-binding activity coeluted with the radioligand when the sodium cholate extract was purified by chromatography on wheat germ agglutinin-Sepharose 6MB. A molecular size of approximately 59 A was estimated for the lectin-Sepharose-purified receptor complex by gel filtration chromatography on Ultrogel AcA 34. The isolated complex eluted from the gel filtration column exhibited an enhanced rate of ligand dissociation in response to GTP gamma S. Approximately 0.65 mol of pertussis toxin substrate/mol of receptor was estimated following partial purification of the receptor-ligand complex by sequential chromatography on wheat germ agglutinin-Sepharose, DEAE-Fractogel, and Ultrogel AcA 34. The pertussis toxin substrate which copurified with the receptor was compared with two distinct G proteins, containing alpha-subunits of 40 and 41 kDa, previously purified from HL60 cell plasma membranes. Approximately 86% of the pertussis toxin substrate identified in the receptor preparation consisted of the 40-kDa polypeptide. Differences in the peptide maps indicate that the predominant G protein which coelutes with the receptor is distinct from the purified G protein with an alpha-subunit of 41 kDa but homologous to the purified G protein with an alpha-subunit of 40 kDa.     

Functional interactions of stimulatory and inhibitory GDP/GTP exchange proteins and their common substrate small GTP-binding protein.

smg GDS and rho GDI are stimulatory and inhibitory GDP/GTP exchange proteins, respectively, for a group of ras p21-related small GTP-binding proteins (G proteins). rho p21 is a common substrate small G protein for both GDP/GTP exchange proteins. We examined here the functional interactions of these GDP/GTP exchange proteins with rho p21 as a substrate. smg GDS and rho GDI interacted with the GDP-bound form of rho p21 and thereby stimulated and inhibited, respectively, the dissociation of GDP. The inhibitory effect of rho GDI was much stronger than the stimulatory effect of smg GDS. The GDP-bound form of rho p21 formed a complex with rho GDI but not with smg GDS in their simultaneous presence. Since the content of smg GDS was generally less than that of rho GDI in cells, these results suggest that there is some mechanism to release the inhibitory action of rho GDI and to make rho p21 sensitive to the smg GDS action during the conversion of rhoA p21 from the GDP-bound inactive form to the GTP-bound active form in intact cells. On the other hand, rho p21 was previously shown to be ADP-ribosylated by bacterial ADP-ribosyltransferases, named C3 and EDIN, at Asn41 in the putative effector region of rho p21. This ADP-ribosylation was inhibited by rho GDI much more efficiently than by smg GDS. These results suggest that rho GDI may mask the putative effector region of rho p21 and thereby inhibit its interaction with the target protein even in the presence of smg GDS. Thus, both smg GDS and rho GDI are important to regulate the rho p21 activity and action in cooperation with each other.     

Purification and partial amino acid sequences of a phospholipase C-associated GTP-binding protein from calf thymocytes

We recently identified a phosphoinositide-specific phospholipase C (PI-PLC)-stimulating GTP-binding protein (G protein) in calf thymocyte cytosol (Wang, P., Toyoshima, S., & Osawa, T. (1987) J. Biochem. 102, 1275-1287; and (1988) 103, 137-142). In this study we completely purified a G protein whose properties are quite similar to the G protein mentioned above from the calf thymocyte membrane and determined partial amino acid sequences of it. The purification was achieved by first treating the membrane with GTP gamma S, followed by sequential column chromatographies on DEAE-Sepharose CL-6B, Sephacryl S-200, Mono Q, and Mono S. The G protein was purified in a GTP gamma S-binding form and assayed as to the radioactivity of the [35S]GTP gamma S-bound PI-PLC-associated G protein standard obtained from calf thymocyte cytosol. The purified G protein could stimulate the activity of a partially purified PI-PLC for phosphatidylinositol 4,5-bisphosphate hydrolysis. From approximately 5.6 g of membrane protein we obtained about 5 micrograms of a purified sample. The purified G protein showed a molecular weight of 21 kDa on SDS-PAGE and one of 25 kDa on gel filtration. The partial amino acid sequences were determined by treating the purified sample with lysylendopeptidase, purifying the resultant peptide fragments on a HPLC-reverse phase column and then sequencing the peptide fragments with a sequencer. Comparison of the obtained sequences with those of known lower molecular weight GTP-binding proteins suggested that, although structurally similar to rho gene products, this is a novel G protein.     

GTP gamma S-stimulated hydrolysis of phosphatidylinositol-4,5-bisphosphate soluble phospholipase C from human platelets requires soluble GTP-binding protein

GTP-binding activity was fractionated into two peaks (GI and GII) by chromatography on heparin-agarose. GTP-dependent PLC activity eluted as a single peak, which co-chromatographed with GTP-binding peak GII. Rechromatography of peak GII on heparin-agarose, in the presence of 0.5% sodium cholate, resulted in separation of PLC and GTP-binding activities, and loss of GTP-dependent PLC activity. Recombining fractions containing PLC and GTP-binding activities restored GTP-dependent PLC activity. A specific GTP-binding protein of 29,000 daltons was identified in peak GII by Western blotting of column fractions with [alpha-32P]GTP. These results demonstrate that the soluble phospholipase C from human platelets is regulated by GTP S-binding protein (G29).     

Protein kinase C impairs the coupling of the GTP-binding protein to LTB4 receptor in neutrophil.

In the present study, the mechanism of LTB4 receptor down regulation by protein kinase C (PKC) has been investigated using porcine neutrophil membranes. Pretreatment of intact porcine neutrophils with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 2 min prior to the preparation of plasma membrane, demonstrated a reduced binding sites (Bmax) for LTB4 without altering the receptor affinity (Kd). This effect of TPA on LTB4 receptor binding was found to be due to the activation of PKC as membrane treated with purified PKC (type III) produced the same effect. When membranes from neutrophils pretreated with TPA were exposed to non-hydrolyzable GTP analog, GTP-gamma S, or GMP-PNP, no further decrease in receptor Kd was observed, while the Bmax was reduced to the level observed in TPA treated samples. Treatment of isolated neutrophil membranes with purified PKC reduced the Bmax and blocked the effect of GTP analogs on the receptor affinity. These results suggest that, PKC interrupts the receptor binding to G-protein.     

Coupling of bradykinin receptors to phospholipase C in cultured fibroblasts is mediated by a G-protein

In cultured foreskin fibroblasts, bradykinin stimulates inositol phosphate generation, arachidonic acid release, and Na+/H+ exchange, with doses of 1-3 nM yielding half-maximal stimulation. Binding of 3H-bradykinin to these cells demonstrates a single receptor site with a Kd of 2.0 nM and a Bmax of 91 fmoles/mg protein. Bradykinin analogs of the B2 type inhibit this binding. GTP synergizes with bradykinin to stimulate phosphatidylinositol turnover in permeabilized fibroblasts and GTP-gamma-S decreases the Bmax of bradykinin binding to fibroblast membranes, indicating that a G-protein couples the receptor to phospholipase C. Pretreatment of fibroblasts with either cholera or pertussis toxin enhances bradykinin stimulation of inositol phosphate accumulation.     

Evidence for a GTP-binding protein coupling thrombin receptor to PIP2-phospholipase C in membranes of hamster fibroblasts

Two different methods were used to study directly alpha-thrombin modulation of polyphosphoinositide breakdown in membranes prepared from Chinese hamster lung (CHL) fibroblasts. In the first one we labelled the lipid pool by incubating the intact cells with myo-[3H]inositol prior to membrane isolation; in the other we used exogenous [3H]PIP2 with phosphatidylethanolamine (1:10) added as liposomes to freshly isolated membranes. A Ca2+-dependent PIP2 and PIP phospholipase C activity was characterized by measuring the rate of formation of inositol tris- and bisphosphate. Basal phospholipase C activity was stimulated up to 3-fold by GTP or GTP-gamma-S. Of the two mitogens, alpha-thrombin and EGF, known to stimulate DNA synthesis in Chinese hamster fibroblasts, only alpha-thrombin is a potent activator of PIP2 breakdown in intact cells. Consistent with this observation, alpha-thrombin but not EGF potentiated GTP-gamma-S-dependent phospholipase C activity in membrane preparations. These results strongly support the hypothesis that a GTP-binding protein couples alpha-thrombin receptor to PIP2 hydrolysis. Because both methods used to assay phospholipase C gave identical results, we conclude that the coupling is at the level of PIP2-phosphodiesterase activity.