Abstract
Using subtype-specific antisera, we were able to identify the recently described alpha subunits of G12 and G13 in platelet membranes as 43-kDa proteins. Activation of the thromboxane A2 and the thrombin receptors in platelet membranes led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha 12 and alpha 13, indicating that both receptors couple to G12 and G13. In addition, both activated receptors were demonstrated to couple to one or more members of the Gq family. In the absence of receptor agonists, incorporation of [alpha-32P]GTP azidoanilide into alpha 12 and alpha 13 was low over a long time period (up to 45 min) due to an obviously low basal nucleotide exchange rate, whereas an agonist-stimulated photolabeling of alpha 12 and alpha 13 could be observed after 4-8 min and reached a maximum after 30-45 min. Effective activation of G12 and G13 via the thromboxane A2 and the thrombin receptors was not dependent on the presence of GDP. Our results provide evidence that G12 and G13 play a functional role in transmembrane signal transduction and suggest that both proteins are involved in pathways leading to platelet activation.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Baldassare J. J., Tarver A. P., Henderson P. A., Mackin W. M., Sahagan B., Fisher G. J. Reconstitution of thromboxane A2 receptor-stimulated phosphoinositide hydrolysis in isolated platelet membranes: involvement of phosphoinositide-specific phospholipase C-beta and GTP-binding protein Gq. Biochem J. 1993 Apr 1;291(Pt 1):235–240. doi: 10.1042/bj2910235. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Banga H. S., Walker R. K., Winberry L. K., Rittenhouse S. E. Platelet adenylate cyclase and phospholipase C are affected differentially by ADP-ribosylation. Effects on thrombin-mediated responses. Biochem J. 1988 May 15;252(1):297–300. doi: 10.1042/bj2520297. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Banno Y., Nakashima T., Kumada T., Ebisawa K., Nonomura Y., Nozawa Y. Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes. J Biol Chem. 1992 Apr 5;267(10):6488–6494. [PubMed] [Google Scholar]
- Berstein G., Blank J. L., Smrcka A. V., Higashijima T., Sternweis P. C., Exton J. H., Ross E. M. Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-beta 1. J Biol Chem. 1992 Apr 25;267(12):8081–8088. [PubMed] [Google Scholar]
- Birnbaumer L. Receptor-to-effector signaling through G proteins: roles for beta gamma dimers as well as alpha subunits. Cell. 1992 Dec 24;71(7):1069–1072. doi: 10.1016/s0092-8674(05)80056-x. [DOI] [PubMed] [Google Scholar]
- Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
- Camps M., Carozzi A., Schnabel P., Scheer A., Parker P. J., Gierschik P. Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits. Nature. 1992 Dec 17;360(6405):684–686. doi: 10.1038/360684a0. [DOI] [PubMed] [Google Scholar]
- Casey P. J., Fong H. K., Simon M. I., Gilman A. G. Gz, a guanine nucleotide-binding protein with unique biochemical properties. J Biol Chem. 1990 Feb 5;265(4):2383–2390. [PubMed] [Google Scholar]
- Florio V. A., Sternweis P. C. Mechanisms of muscarinic receptor action on Go in reconstituted phospholipid vesicles. J Biol Chem. 1989 Mar 5;264(7):3909–3915. [PubMed] [Google Scholar]
- Grandt R., Aktories K., Jakobs K. H. Evidence for two GTPases activated by thrombin in membranes of human platelets. Biochem J. 1986 Aug 1;237(3):669–674. doi: 10.1042/bj2370669. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gutowski S., Smrcka A., Nowak L., Wu D. G., Simon M., Sternweis P. C. Antibodies to the alpha q subfamily of guanine nucleotide-binding regulatory protein alpha subunits attenuate activation of phosphatidylinositol 4,5-bisphosphate hydrolysis by hormones. J Biol Chem. 1991 Oct 25;266(30):20519–20524. [PubMed] [Google Scholar]
- Hepler J. R., Gilman A. G. G proteins. Trends Biochem Sci. 1992 Oct;17(10):383–387. doi: 10.1016/0968-0004(92)90005-t. [DOI] [PubMed] [Google Scholar]
- Hinsch K. D., Tychowiecka I., Gausepohl H., Frank R., Rosenthal W., Schultz G. Tissue distribution of beta 1- and beta 2-subunits of regulatory guanine nucleotide-binding proteins. Biochim Biophys Acta. 1989 Sep 4;1013(1):60–67. doi: 10.1016/0167-4889(89)90128-6. [DOI] [PubMed] [Google Scholar]
- Hirata M., Hayashi Y., Ushikubi F., Yokota Y., Kageyama R., Nakanishi S., Narumiya S. Cloning and expression of cDNA for a human thromboxane A2 receptor. Nature. 1991 Feb 14;349(6310):617–620. doi: 10.1038/349617a0. [DOI] [PubMed] [Google Scholar]
- Houslay M. D., Bojanic D., Wilson A. Platelet activating factor and U44069 stimulate a GTPase activity in human platelets which is distinct from the guanine nucleotide regulatory proteins, Ns and Ni. Biochem J. 1986 Mar 15;234(3):737–740. doi: 10.1042/bj2340737. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hui K. Y., Jakubowski J. A., Wyss V. L., Angleton E. L. Minimal sequence requirement of thrombin receptor agonist peptide. Biochem Biophys Res Commun. 1992 Apr 30;184(2):790–796. doi: 10.1016/0006-291x(92)90659-9. [DOI] [PubMed] [Google Scholar]
- Jhon D. Y., Lee H. H., Park D., Lee C. W., Lee K. H., Yoo O. J., Rhee S. G. Cloning, sequencing, purification, and Gq-dependent activation of phospholipase C-beta 3. J Biol Chem. 1993 Mar 25;268(9):6654–6661. [PubMed] [Google Scholar]
- Katz A., Wu D., Simon M. I. Subunits beta gamma of heterotrimeric G protein activate beta 2 isoform of phospholipase C. Nature. 1992 Dec 17;360(6405):686–689. doi: 10.1038/360686a0. [DOI] [PubMed] [Google Scholar]
- Kucera G. L., Rittenhouse S. E. Human platelets form 3-phosphorylated phosphoinositides in response to alpha-thrombin, U46619, or GTP gamma S. J Biol Chem. 1990 Apr 5;265(10):5345–5348. [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Laugwitz K. L., Offermanns S., Spicher K., Schultz G. mu and delta opioid receptors differentially couple to G protein subtypes in membranes of human neuroblastoma SH-SY5Y cells. Neuron. 1993 Feb;10(2):233–242. doi: 10.1016/0896-6273(93)90314-h. [DOI] [PubMed] [Google Scholar]
- Lee C. H., Park D., Wu D., Rhee S. G., Simon M. I. Members of the Gq alpha subunit gene family activate phospholipase C beta isozymes. J Biol Chem. 1992 Aug 15;267(23):16044–16047. [PubMed] [Google Scholar]
- Manning D. R., Brass L. F. The role of GTP-binding proteins in platelet activation. Thromb Haemost. 1991 Oct 1;66(4):393–399. [PubMed] [Google Scholar]
- Milligan G., Mullaney I., Mitchell F. M. Immunological identification of the alpha subunit of G13, a novel guanine nucleotide binding protein. FEBS Lett. 1992 Feb 3;297(1-2):186–188. doi: 10.1016/0014-5793(92)80357-m. [DOI] [PubMed] [Google Scholar]
- Offermanns S., Schultz G., Rosenthal W. Evidence for opioid receptor-mediated activation of the G-proteins, Go and Gi2, in membranes of neuroblastoma x glioma (NG108-15) hybrid cells. J Biol Chem. 1991 Feb 25;266(6):3365–3368. [PubMed] [Google Scholar]
- Offermanns S., Schultz G., Rosenthal W. Identification of receptor-activated G proteins with photoreactive GTP analog, [alpha-32P]GTP azidoanilide. Methods Enzymol. 1991;195:286–301. doi: 10.1016/0076-6879(91)95174-i. [DOI] [PubMed] [Google Scholar]
- Offermanns S., Seifert R., Metzger J. W., Jung G., Lieberknecht A., Schmidt U., Schultz G. Lipopeptides are effective stimulators of tyrosine phosphorylation in human myeloid cells. Biochem J. 1992 Mar 1;282(Pt 2):551–557. doi: 10.1042/bj2820551. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Offermans S., Schäfer R., Hoffmann B., Bombien E., Spicher K., Hinsch K. D., Schultz G., Rosenthal W. Agonist-sensitive binding of a photoreactive GTP analog to a G-protein alpha-subunit in membranes of HL-60 cells. FEBS Lett. 1990 Jan 15;260(1):14–18. doi: 10.1016/0014-5793(90)80054-m. [DOI] [PubMed] [Google Scholar]
- Pang I. H., Sternweis P. C. Purification of unique alpha subunits of GTP-binding regulatory proteins (G proteins) by affinity chromatography with immobilized beta gamma subunits. J Biol Chem. 1990 Oct 25;265(30):18707–18712. [PubMed] [Google Scholar]
- Park D., Jhon D. Y., Lee C. W., Lee K. H., Rhee S. G. Activation of phospholipase C isozymes by G protein beta gamma subunits. J Biol Chem. 1993 Mar 5;268(7):4573–4576. [PubMed] [Google Scholar]
- Sage S. O., Rink T. J. The kinetics of changes in intracellular calcium concentration in fura-2-loaded human platelets. J Biol Chem. 1987 Dec 5;262(34):16364–16369. [PubMed] [Google Scholar]
- Shenker A., Goldsmith P., Unson C. G., Spiegel A. M. The G protein coupled to the thromboxane A2 receptor in human platelets is a member of the novel Gq family. J Biol Chem. 1991 May 15;266(14):9309–9313. [PubMed] [Google Scholar]
- Siffert W., Siffert G., Scheid P., Akkerman J. W. Na+/H+ exchange modulates Ca2+ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619. J Biol Chem. 1990 Jan 15;265(2):719–725. [PubMed] [Google Scholar]
- Simon M. I., Strathmann M. P., Gautam N. Diversity of G proteins in signal transduction. Science. 1991 May 10;252(5007):802–808. doi: 10.1126/science.1902986. [DOI] [PubMed] [Google Scholar]
- Simonds W. F., Goldsmith P. K., Codina J., Unson C. G., Spiegel A. M. Gi2 mediates alpha 2-adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with G alpha C-terminal antibodies. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7809–7813. doi: 10.1073/pnas.86.20.7809. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smrcka A. V., Hepler J. R., Brown K. O., Sternweis P. C. Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq. Science. 1991 Feb 15;251(4995):804–807. doi: 10.1126/science.1846707. [DOI] [PubMed] [Google Scholar]
- Spiegel A. M., Shenker A., Weinstein L. S. Receptor-effector coupling by G proteins: implications for normal and abnormal signal transduction. Endocr Rev. 1992 Aug;13(3):536–565. doi: 10.1210/edrv-13-3-536. [DOI] [PubMed] [Google Scholar]
- Sternweis P. C., Smrcka A. V. Regulation of phospholipase C by G proteins. Trends Biochem Sci. 1992 Dec;17(12):502–506. doi: 10.1016/0968-0004(92)90340-f. [DOI] [PubMed] [Google Scholar]
- Strathmann M. P., Simon M. I. G alpha 12 and G alpha 13 subunits define a fourth class of G protein alpha subunits. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5582–5586. doi: 10.1073/pnas.88.13.5582. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Takahara K., Murray R., FitzGerald G. A., Fitzgerald D. J. The response to thromboxane A2 analogues in human platelets. Discrimination of two binding sites linked to distinct effector systems. J Biol Chem. 1990 Apr 25;265(12):6836–6844. [PubMed] [Google Scholar]
- Theis J. G., Dellweg H., Perzborn E., Gross R. Binding characteristics of the new thromboxane A2/prostaglandin H2 receptor antagonist [3H]BAY U 3405 to washed human platelets and platelet membranes. Biochem Pharmacol. 1992 Aug 4;44(3):495–503. doi: 10.1016/0006-2952(92)90441-k. [DOI] [PubMed] [Google Scholar]
- Vu T. K., Hung D. T., Wheaton V. I., Coughlin S. R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991 Mar 22;64(6):1057–1068. doi: 10.1016/0092-8674(91)90261-v. [DOI] [PubMed] [Google Scholar]
- Wange R. L., Smrcka A. V., Sternweis P. C., Exton J. H. Photoaffinity labeling of two rat liver plasma membrane proteins with [32P]gamma-azidoanilido GTP in response to vasopressin. Immunologic identification as alpha subunits of the Gq class of G proteins. J Biol Chem. 1991 Jun 25;266(18):11409–11412. [PubMed] [Google Scholar]
- Wu D., Katz A., Lee C. H., Simon M. I. Activation of phospholipase C by alpha 1-adrenergic receptors is mediated by the alpha subunits of Gq family. J Biol Chem. 1992 Dec 25;267(36):25798–25802. [PubMed] [Google Scholar]
- Zavoico G. B., Cragoe E. J., Jr, Feinstein M. B. Regulation of intracellular pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+ exchange and its inhibition by amiloride analogs. J Biol Chem. 1986 Oct 5;261(28):13160–13167. [PubMed] [Google Scholar]