G(12) REQUIREMENT FOR THROMBIN-STIMULATED GENE-EXPRESSION AND DNA-SYNTHESIS IN 1321N1 ASTROCYTOMA-CELLS

Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependent AP-1-mediated transcriptional activation and to DNA replication. In contrast to what has been observed in most cell systems, in 1321N1 cells these responses are pertussis toxin-insensitive. The pertussis toxin-insensitive G-protein G(12) has been implicated in cell growth and transformation in different cell systems. We have examined the potential role of this protein in AP-1-mediated transcriptional activation and DNA synthesis in 1321N1 cells. Transient expression of an activated (GTPase-deficient) mutant of G alpha(12) increased AP-1-dependent gene expression. This response was inhibited by co-expression of a dominant negative Ala-15 Ras protein. To determine whether the pertussis toxin-insensitive G(12) protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of G alpha(12) subunit was microinjected into 1321N1 cells. Microinjection of the anti-G alpha(12) resulted in a concentration-dependent inhibition of thrombin-stimulated DNA synthesis. In contrast, microinjection of nonimmune IgG or an antibody directed against the C terminus of G alpha(0) did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-G alpha(12) antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for G alpha(12) in the mitogenic response to thrombin in human astroglial cells.