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. 1992 Jan;3(1):95–102. doi: 10.1091/mbc.3.1.95

Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis.

V Vouret-Craviari 1, E Van Obberghen-Schilling 1, U B Rasmussen 1, A Pavirani 1, J P Lecocq 1, J Pouysségur 1
PMCID: PMC275505  PMID: 1312881

Abstract

alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl to the presumed thrombin cleavage site of the human receptor have been shown to activate platelets as well as the thrombin receptor expressed in Xenopus oocytes. In the present study we have examined the effects of synthetic peptides corresponding to the same region of the hamster receptor (S-42-L-55) and shorter peptides (2-7 residues) on signal transducing systems in CCL39 cells. Our results indicate that hamster receptor peptides of greater than or equal to 5 residues effectively stimulate phospholipase C in CCL39 cells via the thrombin receptor and induce rapid desensitization of the response. The same peptides also inhibit adenylate cyclase in a pertussis toxin-sensitive manner. Although the peptides are potent agonists of serotonin release in platelets, unlike thrombin, by themselves they are not mitogenic. However, they potentiate DNA synthesis in cooperation with growth factors possessing tyrosine kinase receptors. Hence, we conclude that the potent mitogenic action of thrombin cannot be accounted for solely by the activation of the cloned receptor. We postulate the existence of an additional receptor activated by thrombin, which is required for its full mitogenic potential.

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Selected References

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  1. Ashkenazi A., Winslow J. W., Peralta E. G., Peterson G. L., Schimerlik M. I., Capon D. J., Ramachandran J. An M2 muscarinic receptor subtype coupled to both adenylyl cyclase and phosphoinositide turnover. Science. 1987 Oct 30;238(4827):672–675. doi: 10.1126/science.2823384. [DOI] [PubMed] [Google Scholar]
  2. Chambard J. C., Paris S., L'Allemain G., Pouysségur J. Two growth factor signalling pathways in fibroblasts distinguished by pertussis toxin. Nature. 1987 Apr 23;326(6115):800–803. doi: 10.1038/326800a0. [DOI] [PubMed] [Google Scholar]
  3. Davey M. G., Lüscher E. F. Actions of thrombin and other coagulant and proteolytic enzymes on blood platelets. Nature. 1967 Dec 2;216(5118):857–858. doi: 10.1038/216857a0. [DOI] [PubMed] [Google Scholar]
  4. Dohlman H. G., Thorner J., Caron M. G., Lefkowitz R. J. Model systems for the study of seven-transmembrane-segment receptors. Annu Rev Biochem. 1991;60:653–688. doi: 10.1146/annurev.bi.60.070191.003253. [DOI] [PubMed] [Google Scholar]
  5. Fargin A., Raymond J. R., Regan J. W., Cotecchia S., Lefkowitz R. J., Caron M. G. Effector coupling mechanisms of the cloned 5-HT1A receptor. J Biol Chem. 1989 Sep 5;264(25):14848–14852. [PubMed] [Google Scholar]
  6. Fenton J. W., 2nd Regulation of thrombin generation and functions. Semin Thromb Hemost. 1988 Jul;14(3):234–240. doi: 10.1055/s-2007-1002783. [DOI] [PubMed] [Google Scholar]
  7. Ferrell J. E., Jr, Martin G. S. Platelet tyrosine-specific protein phosphorylation is regulated by thrombin. Mol Cell Biol. 1988 Sep;8(9):3603–3610. doi: 10.1128/mcb.8.9.3603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Golden A., Brugge J. S. Thrombin treatment induces rapid changes in tyrosine phosphorylation in platelets. Proc Natl Acad Sci U S A. 1989 Feb;86(3):901–905. doi: 10.1073/pnas.86.3.901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Harlan J. M., Thompson P. J., Ross R. R., Bowen-Pope D. F. Alpha-thrombin induces release of platelet-derived growth factor-like molecule(s) by cultured human endothelial cells. J Cell Biol. 1986 Sep;103(3):1129–1133. doi: 10.1083/jcb.103.3.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Higashijima T., Uzu S., Nakajima T., Ross E. M. Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins). J Biol Chem. 1988 May 15;263(14):6491–6494. [PubMed] [Google Scholar]
  11. Kohno M., Pouysségur J. Alpha-thrombin-induced tyrosine phosphorylation of 43,000- and 41,000-Mr proteins is independent of cytoplasmic alkalinization in quiescent fibroblasts. Biochem J. 1986 Sep 1;238(2):451–457. doi: 10.1042/bj2380451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. L'Allemain G., Paris S., Magnaldo I., Pouysségur J. Alpha-thrombin-induced inositol phosphate formation in G0-arrested and cycling hamster lung fibroblasts: evidence for a protein kinase C-mediated desensitization response. J Cell Physiol. 1986 Nov;129(2):167–174. doi: 10.1002/jcp.1041290207. [DOI] [PubMed] [Google Scholar]
  13. Magnaldo I., Pouysségur J., Paris S. Thrombin exerts a dual effect on stimulated adenylate cyclase in hamster fibroblasts, an inhibition via a GTP-binding protein and a potentiation via activation of protein kinase C. Biochem J. 1988 Aug 1;253(3):711–719. doi: 10.1042/bj2530711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Moss M., Wiley H. S., Fenton J. W., 2nd, Cunningham D. D. Photoaffinity labeling of specific alpha-thrombin binding sites on Chinese hamster lung cells. J Biol Chem. 1983 Mar 25;258(6):3996–4002. [PubMed] [Google Scholar]
  15. Paris S., Magnaldo I., Pouysségur J. Homologous desensitization of thrombin-induced phosphoinositide breakdown in hamster lung fibroblasts. J Biol Chem. 1988 Aug 15;263(23):11250–11256. [PubMed] [Google Scholar]
  16. Paris S., Pouysségur J. Guanosine 5'-O-(3-thiotriphosphate) and guanosine 5'-O-(2-thiodiphosphate) activate G proteins and potentiate fibroblast growth factor-induced DNA synthesis in hamster fibroblasts. J Biol Chem. 1990 Jul 15;265(20):11567–11575. [PubMed] [Google Scholar]
  17. Pouysségur J., Chambard J. C., L'Allemain G., Magnaldo I., Seuwen K. Transmembrane signalling pathways initiating cell growth in fibroblasts. Philos Trans R Soc Lond B Biol Sci. 1988 Jul 26;320(1199):427–436. doi: 10.1098/rstb.1988.0086. [DOI] [PubMed] [Google Scholar]
  18. Pouysségur J., Seuwen K. Transmembrane receptors and intracellular pathways that control cell proliferation. Annu Rev Physiol. 1992;54:195–210. doi: 10.1146/annurev.ph.54.030192.001211. [DOI] [PubMed] [Google Scholar]
  19. Rasmussen U. B., Vouret-Craviari V., Jallat S., Schlesinger Y., Pagès G., Pavirani A., Lecocq J. P., Pouysségur J., Van Obberghen-Schilling E. cDNA cloning and expression of a hamster alpha-thrombin receptor coupled to Ca2+ mobilization. FEBS Lett. 1991 Aug 19;288(1-2):123–128. doi: 10.1016/0014-5793(91)81017-3. [DOI] [PubMed] [Google Scholar]
  20. Seuwen K., Kahan C., Hartmann T., Pouyssegur J. Strong and persistent activation of inositol lipid breakdown induces early mitogenic events but not Go to S phase progression in hamster fibroblasts. Comparison of thrombin and carbachol action in cells expressing M1 muscarinic acetylcholine receptors. J Biol Chem. 1990 Dec 25;265(36):22292–22299. [PubMed] [Google Scholar]
  21. Seuwen K., Lagarde A., Pouysségur J. Deregulation of hamster fibroblast proliferation by mutated ras oncogenes is not mediated by constitutive activation of phosphoinositide-specific phospholipase C. EMBO J. 1988 Jan;7(1):161–168. doi: 10.1002/j.1460-2075.1988.tb02796.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Seuwen K., Magnaldo I., Kobilka B. K., Caron M. G., Regan J. W., Lefkowitz R. J., Pouysségur J. Alpha 2-adrenergic agonists stimulate DNA synthesis in Chinese hamster lung fibroblasts transfected with a human alpha 2-adrenergic receptor gene. Cell Regul. 1990 May;1(6):445–451. doi: 10.1091/mbc.1.6.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Seuwen K., Magnaldo I., Pouysségur J. Serotonin stimulates DNA synthesis in fibroblasts acting through 5-HT1B receptors coupled to a Gi-protein. Nature. 1988 Sep 15;335(6187):254–256. doi: 10.1038/335254a0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Van Obberghen-Schilling E., Chambard J. C., Lory P., Nargeot J., Pouysségur J. Functional expression of Ca2(+)-mobilizing alpha-thrombin receptors in mRNA-injected Xenopus oocytes. FEBS Lett. 1990 Mar 26;262(2):330–334. doi: 10.1016/0014-5793(90)80221-4. [DOI] [PubMed] [Google Scholar]
  26. Van Obberghen-Schilling E., Chambard J. C., Paris S., L'Allemain G., Pouysségur J. alpha-Thrombin-induced early mitogenic signalling events and G0 to S-phase transition of fibroblasts require continual external stimulation. EMBO J. 1985 Nov;4(11):2927–2932. doi: 10.1002/j.1460-2075.1985.tb04025.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Van Obberghen-Schilling E., Pouysségur J. Affinity labeling of high-affinity alpha-thrombin binding sites on the surface of hamster fibroblasts. Biochim Biophys Acta. 1985 Dec 12;847(3):335–343. doi: 10.1016/0167-4889(85)90039-4. [DOI] [PubMed] [Google Scholar]
  28. Van Obberghen-Schilling E., Vouret-Craviari V., Haslam R. J., Chambard J. C., Pouysségur J. Cloning, functional expression and role in cell growth regulation of a hamster 5-HT2 receptor subtype. Mol Endocrinol. 1991 Jul;5(7):881–889. doi: 10.1210/mend-5-7-881. [DOI] [PubMed] [Google Scholar]
  29. Veillette A., Bookman M. A., Horak E. M., Bolen J. B. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell. 1988 Oct 21;55(2):301–308. doi: 10.1016/0092-8674(88)90053-0. [DOI] [PubMed] [Google Scholar]
  30. 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]

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