A requirement for Ras protein function in thrombin-stimulated mitogenesis in astrocytoma cells

Thrombin stimulation of 1321N1 astrocytoma cells results in polyphosphoinositide hydrolysis, Ca2+ mobilization, AP-1-mediated transcriptional activation, and DNA replication. Thrombin stimulation also activates Ras as assessed by an increase in the proportion of Ras in a GTP bound state. We examined the functional requirement for endogenous Ras protein in mediating thrombin-induced responses. Microinjection of a dominant interfering mutant of H-Ras into 1321N1 cells inhibited DNA synthesis in response to thrombin as did microinjection of an inhibitory antibody to Ras. Stimulation of AP-1-mediated transcriptional activity was also reduced by the expression of interfering Ras mutants. However, neither the stimulation of polyphosphoinositide hydrolysis nor the mobilization of intracellular Ca2+ was dependent on endogenous Ras function. These observations indicate that thrombin stimulation of mitogenesis requires Ras protein function. Our data suggest that the G-protein-coupled thrombin receptor stimulates pathways, which in part are convergent with those stimulated by tyrosine kinase growth factor receptors.