Differential Regulation of Rho and Rac through Heterotrimeric G-proteins and Cyclic Nucleotides
2001; Elsevier BV; Volume: 276; Issue: 51 Linguagem: Inglês
10.1074/jbc.m104442200
ISSN1083-351X
AutoresMarie‐Pierre Gratacap, Bernard Payrastre, Bernhard Nieswandt, Stefan Offermanns,
Tópico(s)Cell Adhesion Molecules Research
ResumoPlatelets were used to study the activation of Rho and Rac through G-protein-coupled receptors and its regulation by cyclic nucleotides. The thromboxane A2(TXA2) mimetic U46619 rapidly activated both small GTPases independently of integrin αIIbβ3activation. U46619, which leads to the activation of G12/G13 and Gq did not induce Rac activation in Gαq-deficient platelets but was able to activate Rho, to stimulate actin polymerization and phosphatidylinositol 4,5-bisphosphate formation, and to induce shape change. Rac activation by U46619 in wild-type platelets could be blocked by chelation of intracellular Ca2+ and was partially sensitive to apyrase and AR-C69931MX, an antagonist of the Gi-coupled ADP receptor. Cyclic AMP, which completely blocks platelet function, inhibited the U46619-induced activation of Gq and G12/G13 as well as of Rac and Rho. In contrast, cGMP, which has no effect on platelet shape change blocked only activation of Gq and Rac. These data demonstrate that Rho and Rac are differentially regulated through heterotrimeric G-proteins. The G12/G13-mediated Rho activation is involved in the shape change response, whereas Rac is activated through Gq and is not required for shape change. Cyclic AMP and cGMP differentially interfere with U46619-induced Rho and Rac activation at least in part by selective effects on the regulation of individual G-proteins through the TXA2receptor. Platelets were used to study the activation of Rho and Rac through G-protein-coupled receptors and its regulation by cyclic nucleotides. The thromboxane A2(TXA2) mimetic U46619 rapidly activated both small GTPases independently of integrin αIIbβ3activation. U46619, which leads to the activation of G12/G13 and Gq did not induce Rac activation in Gαq-deficient platelets but was able to activate Rho, to stimulate actin polymerization and phosphatidylinositol 4,5-bisphosphate formation, and to induce shape change. Rac activation by U46619 in wild-type platelets could be blocked by chelation of intracellular Ca2+ and was partially sensitive to apyrase and AR-C69931MX, an antagonist of the Gi-coupled ADP receptor. Cyclic AMP, which completely blocks platelet function, inhibited the U46619-induced activation of Gq and G12/G13 as well as of Rac and Rho. In contrast, cGMP, which has no effect on platelet shape change blocked only activation of Gq and Rac. These data demonstrate that Rho and Rac are differentially regulated through heterotrimeric G-proteins. The G12/G13-mediated Rho activation is involved in the shape change response, whereas Rac is activated through Gq and is not required for shape change. Cyclic AMP and cGMP differentially interfere with U46619-induced Rho and Rac activation at least in part by selective effects on the regulation of individual G-proteins through the TXA2receptor. 5)P2, phosphatidylinositol 4,5-bisphosphate glycoprotein IIb/IIIa (integrin αIIbβ3) thromboxane A2 glutathione S-transferase phospholipase C 1,2-bis(2-aminophenoxy)ethane-N,N,N ′,N ′-tetraacetic acid tetrakis(acetoxymethyl ester) radioimmune precipitation buffer polyacrylamide gel electrophoresis 6-DCl-cBIMPS, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole-3′,5′-cyclic monophosphorothioate, Sp-isomer 8-(4-chlorophenylthio) guanosine-3′,5′-cyclic monophosphate The small GTPases Rho and Rac are central regulators of various cellular processes such as actin cytoskeleton dynamics, transcriptional regulation, cell cycle progression, and contractile processes (1Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1684) Google Scholar). They are activated by a variety of receptors, including those coupled to heterotrimeric G-proteins (2Kjoller L. Hall A. Exp. Cell Res. 1999; 253: 166-179Crossref PubMed Scopus (344) Google Scholar). Various heterotrimeric G-proteins have been involved in linking receptors to the regulation of Rho and Rac. The α-subunits of the G12 family of heterotrimeric G-proteins, Gα12 and Gα13, are able to activate Rho (3Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 4Sah V.P. Seasholtz T.M. Sagi S.A. Brown J.H. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Crossref PubMed Scopus (299) Google Scholar). Rho activation may be mediated by a group of Rho-specific guanine nucleotide exchange factors, which are able to interact with Gα12/Gα13 (5Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. 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G-protein-coupled receptor-mediated activation of Rac has been shown to be mediated by Gi-type G-proteins via a mechanism, which in some cases appears to involve G-protein βγ subunits and activation of phosphoinositide 3-kinase (11Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 12Belisle B. Abo A. J. Biol. Chem. 2000; 275: 26225-26232Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 13Geijsen N. van Delft S. Raaijmakers J.A. Lammers J.W. Collard J.G. Koenderman L. Coffer P.J. Blood. 1999; 94: 1121-1130Crossref PubMed Google Scholar, 14Ueda H. Morishita R. Yamauchi J. Itoh H. Kato K. Asano T. J. Biol. Chem. 2001; 276: 6846-6852Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Both Rho and Rac have been involved in early signaling processes underlying platelet activation. Platelets respond to various stimuli, which function through G-protein-coupled receptors with secretion of granule contents, aggregation, and a rapid change of their shape. Rac, which is rapidly activated after activation of the thrombin receptor PAR-1 (15Azim A.C. Barkalow K. Chou J. Hartwig J.H. Blood. 2000; 95: 959-964Crossref PubMed Google Scholar), has been suggested to mediate thrombin receptor-induced actin assembly via stimulation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)1production during platelets activation (16Hartwig J.H. Bokoch G.M. Carpenter C.L. Janmey P.A. Taylor L.A. Toker A. Stossel T.P. Cell. 1995; 82: 643-653Abstract Full Text PDF PubMed Scopus (613) Google Scholar, 17Tolias K.F. Hartwig J.H. Ishihara H. Shibasaki Y. Cantley L.C. Carpenter C.L. Curr. Biol. 2000; 10: 153-156Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), and evidence has been provided that Rho is involved in early processes underlying platelet activation by linking receptors to Rho-kinase and subsequent regulation of myosin light-chain phosphorylation (18Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (317) Google Scholar, 19Bauer M. Retzer M. Wilde J.I. Maschberger P. Essler M. Aepfelbacher M. Watson S.P. Siess W. Blood. 1999; 94: 1665-1672Crossref PubMed Google Scholar, 20Paul B.Z. Daniel J.L. Kunapuli S.P. J. Biol. Chem. 1999; 274: 28293-28300Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The mechanisms by which Rho and Rac become activated through G-protein-coupled receptors in platelets are unclear. Receptors activated by thrombin or thromboxane A2 (TXA2) couple to Gq and G12/G13. In addition, thrombin but not TXA2 is able to induce activation of Gi in platelet membrane fractions (18Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (317) Google Scholar, 21Offermanns S. Laugwitz K.L. Spicher K. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Crossref PubMed Scopus (398) Google Scholar). Platelets from Gαq-deficient mice have been instructive in delineating the initial mechanisms of platelet activation. TXA2 and thrombin are unable to induce phospholipase C activation as well as platelet aggregation and secretion in the absence of Gαq, whereas induction of platelet shape change through the activation of G12/G13 appears to be basically unaffected (18Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (317) Google Scholar,22Offermanns S. Toombs C.F. Hu Y.H. Simon M.I. Nature. 1997; 389: 183-186Crossref PubMed Scopus (501) Google Scholar). In this report we demonstrate by direct determination of Rho and Rac activation that both small GTPases become rapidly activated by TXA2 in an integrin αIIbβ3-independent manner. We used platelets from Gαq-deficient mice to show that Rho and Rac are differentially regulated. Rho activation occurs through G12/G13 and is involved in processes underlying platelet shape change, including PtdIns(4,5)P2 formation. In contrast, Rac activation is mediated by Gq and is not required for shape change. Cyclic nucleotides cAMP and cGMP differentially interfere with Rac and Rho activation in platelets at least in part by affecting receptor-mediated activation of Gq and G12/G13. U46619 was from Cayman Chemical (Ann Arbor, MI); thrombin, Sp-5,6-DCl-cBIMPS, and 8-pCPT-cGMP were from Biolog (Bremen, Germany). RO-31-8220, BAPTA-AM, A23187, and U-73122 were from Calbiochem. AR-C69931MX was a generous gift from Dr. J. Turner (ASTRA Charnwood, UK), whereas SR 121566A was obtained from Dr. P. Savi (Sanofi-Synthelabo, Toulouse, France). Anti-Rac monoclonal antibody was purchased from Upstate Biotechnology, Inc. Anti-RhoA monoclonal antibody was from Santa Cruz Biotechnology, Inc. Antisera against G-protein α-subunits have been described previously (21Offermanns S. Laugwitz K.L. Spicher K. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Crossref PubMed Scopus (398) Google Scholar, 23Laugwitz K.L. Spicher K. Schultz G. Offermanns S. Methods Enzymol. 1994; 237: 283-294Crossref PubMed Scopus (50) Google Scholar). All other reagents were obtained from Sigma Chemical Co. unless otherwise indicated. Y-27632 was kindly provided by Yoshitomi Pharmaceutical Industries, Ltd. (Saitama, Japan). Whole blood was collected from normal and Gαq-deficient mice (129/Sv × C57BL/6) anesthetized with pentobarbital by puncturing the inferior vena cava with syringes containing acid citrate dextrose (1/9 volume). The blood from 3–4 Gαq-deficient mice and wild-type mice was pooled for each platelet experiment. Blood was diluted with half the volume of Hepes-Tyrode's buffer (137 mm NaCl, 2 mm KCl, 12 mm NaHCO3, 0.3 mm NaH2PO4, 2 mmCaCl2, 1 mm MgCl2, 5.5 mm glucose, 5 mm Hepes, pH 7.3) containing 0.35% human serum albumin, and platelet-rich plasma was obtained by centrifugation for 8 min at 250 × g at 37 °C. Thereafter, prostacyclin at a final concentration of 500 nmwas added to the platelet-rich plasma, and platelets were pelleted twice by centrifugation at 1000 × g for 5 min at 37 °C. The platelet pellet was resuspended in Hepes-Tyrode's buffer at a density of 1 × 109 platelets per milliliter in the presence of 0.02 unit/ml of the ADP scavenger apyrase (adenosine-5′-triphosphate diphosphohydrolase) and incubated for 30 min at 37 °C. For inositol lipid analysis, platelets were labeled with 0.5 mCi/ml [32P]orthophosphate during 50 min in a phosphate-free washing buffer (pH 6.5) at 37 °C. 32P-Labeled platelets were then washed once in the same buffer and finally suspended at a final concentration of 1 × 109 cells/ml (pH 7.3). Optical aggregation experiments were conducted in a two-channel aggregometer (Chronolog). The amount of activated cellular Rho and Rac was determined by precipitation with a fusion protein consisting of GST and the Rho-binding domain of Rhotekin (GST-RBD) or the Rac-binding domain of PAK1 (PBD) as described (11Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 24Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1370) Google Scholar). Platelets were lysed in RIPA buffer (50 mm Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mm NaCl, 10 mm MgCl2, 10 μg/ml each of leupeptin and aprotinin, and 1 mmphenylmethylsulfonyl fluoride), and clarified cell lysates were incubated with GST-RBD or GST-PBD (20 μg of beads) at 4 °C for 45 min. The beads were washed four times with 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 150 mm NaCl, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 5 mm EGTA, and 5 μg/ml each of leupeptin and aprotinin. The bead pellet was finally suspended in 15 μl of Laemmli sample buffer. RhoA and Rac were separated by 12% SDS-PAGE and transferred to nitrocellulose membrane, and GTPases were detected using a specific monoclonal antibody against RhoA and Rac. SDS-PAGE of photolabeled proteins was performed on 12% (w/v) polyacrylamide gels. Photolabeled membrane proteins were visualized by autoradiography of the dried gels. Blotting of membrane proteins separated by SDS-PAGE, processing of immunoblots, and detection of immunoreactive proteins by chemiluminescence procedure (Amersham Pharmacia Biotech, Braunschweig, Germany) have been described previously (23Laugwitz K.L. Spicher K. Schultz G. Offermanns S. Methods Enzymol. 1994; 237: 283-294Crossref PubMed Scopus (50) Google Scholar). Reactions were stopped, and the cytoskeleton was immediately extracted by adding one volume of ice-cold twice-concentrated cytoskeleton buffer containing 100 mm Tris-HCl, pH 7.4, 20 mm EGTA, 2 mm Na3VO4, 4 μg/ml each of aprotinin and leupeptin, 2 mm phenylmethylsulfonyl fluoride, and 2% (v/v) Triton X-100 as described previously (25Grondin P. Plantavid M. Sultan C. Breton M. Mauco G. Chap H. J. Biol. Chem. 1991; 266: 15705-15709Abstract Full Text PDF PubMed Google Scholar, 26Guinebault C. Payrastre B. Racaud-Sultan C. Mazarguil H. Breton M. Mauco G. Plantavid M. Chap H. J. Cell Biol. 1995; 129: 831-842Crossref PubMed Scopus (224) Google Scholar). After 10 min at 4 °C, the cytoskeleton was pelleted by centrifugation (12,000 × g for 10 min at 4 °C), and a fraction of the supernatant was taken as a control. Pellets were washed once in cytoskeleton buffer with 0.5% Triton X-100 and once with the same buffer without Triton X-100. Cytoskeleton and a fraction of the post-spin supernatants as well as of the initial lysate were then immediately prepared for SDS-PAGE. Reactions were stopped by addition of chloroform/methanol (1/1, v/v) containing 0.4 nHCl, and lipids were immediately extracted following the modified procedure of Bligh and Dyer (27Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43301) Google Scholar, 28Gratacap M.P. Payrastre B. Viala C. Mauco G. Plantavid M. Chap H. J. Biol. Chem. 1998; 273: 24314-24321Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). PtdIns(4,5)P2 lipids were immediately deacylated by 20% methylamine and analyzed by high performance liquid chromatography on a Whatman Partisphere 5 SAX column (Whatman International Ltd., UK) as described previously (28Gratacap M.P. Payrastre B. Viala C. Mauco G. Plantavid M. Chap H. J. Biol. Chem. 1998; 273: 24314-24321Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Isolated platelets were preincubated under the indicated conditions. Thereafter, platelets were incubated in the absence or presence of U46619 (5 μm) for 5 s at 37 °C and then fixed for 10 min with 3% paraformaldehyde, 3.75% glutaraldehyde, 0.06 mm cacodylate buffer, and 3.4 mm CaCl2. The fixed platelets were suction-filtered onto Nucleopore polycarbonate filters (0.45 μm), which had been preincubated with 10 μg/ml polylysine. Filters were washed three times with 0.9% NaCl and dehydrated stepwise in aqueous ethanol. After exchange of ethanol for hexadimethyldisilazane, samples were air-dried and sputtered with gold. Scanning electron microscopy was carried out on a Zeiss-Gemini instrument using a beam voltage of 5 kV. After a preincubation of platelets for 20 min with or without Sp-5,6-DCl-cBIMPS (cAMP) (100 μm) or 8-pCPT-cGMP (cGMP) (1 mm), platelet membranes were prepared and photolabeled as described (21Offermanns S. Laugwitz K.L. Spicher K. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Crossref PubMed Scopus (398) Google Scholar). Briefly, cell membranes (50–100 μg of protein per assay tube) were incubated at 30 °C in a buffer containing 0.1 mm EDTA, 10 mmMgCl2, 30 mm NaCl, 1 mmbenzamidine, and 50 mm Hepes-NaOH (pH 7.4). After 3 min of preincubation in the absence and presence of receptor agonist, samples were incubated for another 15 min with 10–20 nm[α-32P]GTP azidoanilide (130 kBq per tube). [α-32P]GTP azidoanilide was synthesized and purified as described (29Offermanns S. Schultz G. Rosenthal W. Methods Enzymol. 1991; 195: 286-301Crossref PubMed Scopus (59) Google Scholar). Samples were washed, dissolved in labeling buffer, and irradiated as described (21Offermanns S. Laugwitz K.L. Spicher K. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 504-508Crossref PubMed Scopus (398) Google Scholar). Photolabeled membranes were pelleted, and proteins were predenatured in SDS. Solubilized membranes were preabsorbed with Protein A-Sepharose beads, and immunoprecipitation was performed as described (23Laugwitz K.L. Spicher K. Schultz G. Offermanns S. Methods Enzymol. 1994; 237: 283-294Crossref PubMed Scopus (50) Google Scholar). Human as well as wild-type and Gαq-deficient mouse platelets were incubated with the TXA2 mimeticU46619, and activation of Rho and Rac was investigated after different incubation times using the pull-down assays based on GST-Rho-binding domain or GST-Rac-binding domain fusion proteins (11Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 24Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1370) Google Scholar). Stimulation of human and mouse platelets by U46619 led to a very rapid and sustained activation of Rho and Rac, which reached a maximum a few seconds after addition of the stimulus (Fig.1, A and B). In contrast, no activation of Rac was observed in Gαq-deficient platelets stimulated by U46619 (Fig.1 B). However, in the absence of Gαq the TXA2 analogue still induced a Rho activation with a time course indistinguishable from that observed in wild-type platelets (Fig. 1 A). Quantification of precipitated Rho and of Rho in the lysates by densitometric analysis of immunoblots demonstrated that the amount of activated Rho in Gαq-deficient platelets exposed to U46619 was the same as that observed in wild-type platelets and amounted to about 10% of the total Rho (data not shown). These data indicate that the Gq-mediated pathway is not required for Rho activation in platelets. Gαq-deficient platelets do not show any aggregation and secretion in response to U46619 (22Offermanns S. Toombs C.F. Hu Y.H. Simon M.I. Nature. 1997; 389: 183-186Crossref PubMed Scopus (501) Google Scholar). Thus, the sustained Rho activation observed in Gαq-deficient platelets is obviously independent of integrin αIIbβ3activation and feedback effects of secreted stimuli. To test whether Rac activation was dependent on integrin αIIbβ3-mediated aggregation, we pretreated platelets with the GP IIb/IIIa antagonist SR 121566A (30Herault J.P. Peyrou V. Savi P. Bernat A. Herbert J.M. Thromb. Haemostasis. 1998; 79: 383-388Crossref PubMed Scopus (50) Google Scholar) as well as with the anti-GP IIb/IIIa antibody JON/A (31Nieswandt B. Bergmeier W. Rackebrandt K. Gessner J.E. Zirngibl H. Blood. 2000; 96: 2520-2527Crossref PubMed Google Scholar), which completely inhibited aggregation induced by U46619 (Fig.2 A). Rac activation by U46619in mouse platelets was not affected by pretreatment of platelets with GP IIb/IIIa blocking agents (Fig. 2 B). Similarly, blockade of integrin αIIbβ3 in human platelets by the peptide Arg-Gly-Asp-Ser (RGDS) had no effect on both Rho and Rac activation (Fig. 2 C). These data demonstrate that the rapid activation of Rho and Rac after platelet activation occurs independently of integrin outside-in-signaling. The lack of Rac activation in the absence of Gαqindicates that the Gq-mediated pathway is upstream of Rac. Rac activation by U46619 required an increase in the free cytosolic Ca2+ concentration, because it was strongly inhibited by pretreatment of platelets with the Ca2+ chelator BAPTA-AM and the phospholipase C inhibitor U-73122 but not by the protein kinase C inhibitor RO-31-8220 or the extracellular Ca2+ chelator EGTA (Fig. 3, A and B). To test whether an increase in the cytosolic calcium concentration would induce Rac activation, we incubated cells withA23187. Ionophore treatment resulted in a robust activation of Rac (Fig. 3 C). The dependence of Rac activation on Gq, PLC, and Ca2+ suggests that a direct pathway mediated by phospholipase Cβ is involved in Rac activation or that Rac activation follows the Gq-mediated release of mediators, which in turn may activate Gi-coupled receptors. ADP, which is an important mediator of some TXA2 effects, can activate various receptors, including the Gi-coupled P2Y12 receptor (32Hollopeter G. Jantzen H.-M. Vincent D. Li G. England L. Ramakrishnan V. Yang R.-B. Nurden P. Nurden A. Julius D. Conley P.B. Nature. 2001; 409: 202-207Crossref PubMed Scopus (1306) Google Scholar). To exclude a possible contribution of Gi activated through U46619-induced ADP release, we degraded ADP with apyrase or blocked the Gi-coupled P2Y12 receptor with AR-C69931MX. Apyrase and AR-C69931MX completely blocked ADP-induced aggregation (Fig. 3 D). Both apyrase and AR-C69931MX partially inhibited the U46619-induced Rac activation in wild-type platelets (Fig. 3 D) indicating that ADP and the Gi-coupled P2Y12 receptor are involved in the effect of U46619 on Rac activation. To test whether Gi activation alone is sufficient to lead to activation of Rac in platelets, we tested the effect of thrombin and ADP on GTP loading of Rac in Gαq-deficient platelets in which both agonists induce activation of G12/G13 and Gi (18Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (317) Google Scholar). Although thrombin and ADP and ADP+U46619 led to Rac activation in wild-type platelets (data not shown), no Rac activation could be observed in response to these stimuli in Gαq-deficient platelets (Fig. 3 E). This indicates that Gi activation alone is not sufficient for Rac activation and that a direct Gq/PLCβ-mediated mechanism as well as the Ca2+-dependent release of ADP and possibly other mediators are required for full Rac activation in platelets. Platelet activation is accompanied by rapid actin polymerization, a process that is regulated through the formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) (33Janmey P.A. Physiol. Rev. 1998; 78: 763-781Crossref PubMed Scopus (657) Google Scholar, 34Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Crossref PubMed Scopus (245) Google Scholar). The major PtdIns(4,5)P2-forming enzyme, phosphatidylinositol-4-phosphate 5-kinase, has been shown to be regulated by Rho and Rac (35Chong L.D. Traynor-Kaplan A. Bokoch G.M. Schwartz M.A. Cell. 1994; 79: 507-513Abstract Full Text PDF PubMed Scopus (594) Google Scholar, 36Tolias K.F. Cantley L.C. Carpenter C.L. J. Biol. Chem. 1995; 270: 17656-17659Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 37Ren X.D. Bokoch G.M. Traynor-Kaplan A. Jenkins G.H. Anderson R.A. Schwartz M.A. Mol. Biol. Cell. 1996; 7: 435-442Crossref PubMed Scopus (199) Google Scholar). Especially in platelets, evidence has been provided that Rac links receptors to PtdIns(4,5)P2formation and actin polymerization via stimulation of phosphatidylinositol-4-phosphate 5-kinase (16Hartwig J.H. Bokoch G.M. Carpenter C.L. Janmey P.A. Taylor L.A. Toker A. Stossel T.P. Cell. 1995; 82: 643-653Abstract Full Text PDF PubMed Scopus (613) Google Scholar, 17Tolias K.F. Hartwig J.H. Ishihara H. Shibasaki Y. Cantley L.C. Carpenter C.L. Curr. Biol. 2000; 10: 153-156Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Although a considerable portion of actin polymerization during platelet activation is mediated in an integrin αIIbβ3-dependent manner, some actin polymerization accompanies the protrusion of filopodia and lamellipodia during platelet shape change (18Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (317) Google Scholar, 38Wurzinger L.J. Adv. Anat. Embryol. Cell Biol. 1990; 120: 1-96Crossref PubMed Google Scholar, 39Fox J.E.B. Thromb. Haemostasis. 1993; 70: 884-893Crossref PubMed Scopus (204) Google Scholar). Accordingly, in Gαq-deficient platelets, which do not aggregate in response to TXA2 receptor activation, U46619 induced an increase in the Triton X-100-insoluble actin fraction (Fig.4, A and B). The latter was comparable to the effect observed in wild-type platelets treated with a GP IIb/IIIa blocking antibody (Fig. 4, A and B). To test whether there was still some receptor-dependent PtdIns(4,5)P2 formation in the absence of Gq-mediated Rac activation, we determined PtdIns(4,5)P2 levels in response to U46619 in wild-type and Gαq-deficient platelets. Although there was no Rac activation in Gαq-deficient platelets in response toU46619, a significant increase in PtdIns(4,5)P2 levels by about 30% could be observed shortly after addition of the TXA2 analogue, which was about half of that seen in wild-type mouse platelets (Fig. 4 C). At longer times of stimulation (3 min), the PtdIns(4,5)P2 levels decreased in wild-type platelets probably due to the activation of phospholipase C, whereas in Gαq-deficient platelets, which do not show any phospholipase C activation, PtdIns(4,5)P2 levels remained elevated. These data show that platelets can form PtdIns(4,5)P2 and undergo shape change in response toU46619 by a mechanism independent of Ca2+ and Rac activation. To test whether the Rho/Rho-kinase-mediated pathway is involved in the formation of PtdIns(4,5)P2 found in Gαq-deficient platelets, we preincubated wild-type platelets and platelets lacking Gαq with 10 μm of the Rho-kinase inhibitor Y-27632. Although PtdIns(4,5)P2 formation in wild-type platelets was partially reduced, the U46619-dependent formation of PtdIns(4,5)P2 in Gαq-deficient platelets was completely blocked by Y27632 (Fig. 4 D). This clearly suggests that PtdIns(4,5)P2 formation in platelets is under dual control through a pathway mediated by Gq/Rac and G12/13/Rho/Rho-kinase. The two main intracellular mediators of platelet inhibition, cAMP and cGMP strongly inhibit platelet aggregation but have been shown to differentially affect platelet shape change. While cAMP blocks the platelet shape change response, cGMP has no effect on shape change of human or mouse platelets (Fig. 5, Ref.18Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (317) Google Scholar). Preincubation of mouse platelets with prostacyclin which induces cAMP formation through the prostacyclin receptor or with the cAMP analogue Sp-5,6-DCl-cBIMPS strongly suppressed U46619-induced Rho activation in wild-type (Fig.6 A) and Gαq-deficient platelets (data not shown). In contrast, the cGMP analogue 8-pCPT-cGMP was without effect (Fig. 6 A). Activation of Rac by U46619 was, however, strongly inhibited by the cGMP analogue as well as by prostacyclin and the cAMP analogue. Very similar effects of cyclic nucleotides on Rho and Rac activation were observed in human platelets (Fig. 6 B). Thus, cAMP inhibits both Rac and Rho activation while cGMP blocks only receptor-mediated Rac activation.Figure 6Effect of cyclic nucleotides on RhoA and Rac activation. Platelets from wild-type mice (A) or human (B) were preincubated for 20 min with Sp-5,6-DCl-cBIMPS (cAMP) (100 μm) or 8-pCPT-cGMP (cGMP) (1 mm) or for 1 min with prostacyclin (PGI 2, 500 nm). Thereafter, stimulation was started by the addition of U46619 (1 μm). After 10 s, activation was stopped by addition of ice-cold 2× RIPA buffer. The amount of activated Rho and Rac was determined as described under "Experimental Procedures." Data are representative of three independent experiments. Shown are blots of the precipitates (Rho/Rac pull-down) as well as of 40 μl of the lysates (500 μl).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because Rho activation by U46619 appears to be mediated by G12/G13, whereas Rac involves Gq, we tested whether cAMP and cGMP interfere with TXA2receptor-mediated activation of Gq and/or G12/G13. Photolabeling of receptor-activated G-proteins in mouse platelet membranes and subsequent immunoprecipitation of individual G-protein α-subunits showed that, in wild-type mouse platelets, activated TXA2 receptors couple to Gq, G12, and G13 (18Klages B. Brandt U. Simon M.I. Schultz G. Offermanns S. J. Cell Biol. 1999; 144: 745-754Crossref PubMed Scopus (317) Google Scholar). Preincubation of human platel
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