G12 Requirement for Thrombin-stimulated Gene Expression and DNA Synthesis in 1321N1 Astrocytoma Cells

Abstract

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 G12 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 G12 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 o 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.

The G12 coupled thrombin receptor stimulates mitogenesis through the Shc SH2 domain

“…We have previously demonstrated that thrombin treatment induces Ras activation in 1321N1 cells and that the heterotrimeric G protein G 12 is required for thrombin-induced mitogenesis (LaMorte et al, 1993;Aragay et al, 1995). While expression of an activated form of Ga 12 can activate Ras , the mechanism of Ras activation by this PTX-insensitive G protein is unclear, particularly as the direct e ectors of Ga 12 are unknown.…”

“…We previously demonstrated that G 12 transduces thrombin's mitogenic signals in 1321N1 cells and that expression of the GTPase-de®cient Ga 12 Q229L results in Ras activation (Aragay et al, 1995;Collins et al, 1996). We hypothesized that Ga 12 may stimulate Ras through tyrosine kinase activation and Shc phosphorylation.…”

“…Ga 12 and thrombin both activate AP-1-mediated gene expression through a pathway that requires Ras (Aragay et al, 1995;LaMorte et al, 1993). To determine whether AP-1-dependent gene expression also depends upon the SH2 domain of Shc, 1321N1 cells were cotransfected with Ga 12 and either the SH2 domain of Shc or control vector.…”

“…Thrombin-stimulated mitogenesis occurs through the PTX-insensitive G protein G 12 in these cells (Aragay et al, 1995). Further, Ga 12 and thrombin activate similar signaling pathways.…”

“…Further, Ga 12 and thrombin activate similar signaling pathways. Expression of an activated form of Ga 12 increases Ras-GTP levels and AP-1 gene expression mediated by Ga 12 is Ras dependent (Aragay et al, 1995). The observation that Ga 12 can signal to Ras pathways may explain its mechanism of transformation in ®broblasts (Xu et al, 1993;Chan et al, 1993;Jiang et al, 1993;VoynoYasenetskaya et al, 1994); however, the direct e ectors of G 12 and mechanism by which Ga 12 activates Ras are unknown.…”