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

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Abstract
Two different methods were used to study directly α-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-[su3H]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-γ-S. Of the two mitogens, α-thrombin and EGF, known to stimulate DNA synthesis in Chinese hamster fibroblasts, only α-thrombin is a potent activator of PIP2 breakdown in intact cells. Consistent with this observation, α-thrombin but not EGF potentiated GTP-γ-S-dependent phospholipase C activity in membrane preparations. These results strongly support the hypothesis that a GTP-binding protein couples α-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.