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.
Department of Medicine, University of California, San Diego, La Jolla 92093-0636.
V J
VJ
LaMorte
E D
ED
Kennedy
L R
LR
Collins
D
D
Goldstein
A T
AT
Harootunian
J H
JH
Brown
J R
JR
Feramisco
D001254:Astrocytoma; D002455:Cell Division / Q000187:drug effects*; D004261:DNA Replication / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism*; D011905:Genes, ras; D006133:Growth Substances / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D006133:Growth Substances; D010716:Phosphatidylinositols; D016755:Proto-Oncogene Proteins c-jun; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D013917:Thrombin; D019204:GTP-Binding Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)