A REQUIREMENT FOR RAS PROTEIN FUNCTION IN THROMBIN-STIMULATED MITOGENESIS IN ASTROCYTOMA-CELLS

Thrombin stimulation of 1321N1 astrocytoma cells results in polyphosph oinositide hydrolysis, Ca2+ mobilization, AP-1-mediated transcriptiona l activation, and DNA replication. Thrombin stimulation also activates Ras as assessed by an increase in the proportion of Ras in a GTP boun d state. We examined the functional requirement for endogenous Ras pro tein in mediating thrombin-induced responses. Microinjection of a domi nant interfering mutant of H-Ras into 1321N1 cells inhibited DNA synth esis in response to thrombin as did microinjection of an inhibitory an tibody to Ras. Stimulation of AP-1-mediated transcriptional activity w as also reduced by the expression of interfering Ras mutants. However, neither the stimulation of polyphosphoinositide hydrolysis nor the mo bilization of intracellular Ca2+ was dependent on endogenous Ras funct ion. These observations indicate that thrombin stimulation of mitogene sis requires Ras protein function. Our data suggest that the G-protein -coupled thrombin receptor stimulates pathways, which in part are conv ergent with those stimulated by tyrosine kinase growth factor receptor s.