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.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008168:Lung; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D013873:Thionucleotides / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism*
D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D010727:Phosphoric Diester Hydrolases; D013917:Thrombin; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase