Aragay, Anna M. and Collins, Lila R. and Post, Ginell R. and Watson, A. John and Feramisco, James R. and Brown, Joan Heller and Simon, Melvin I. (1995) G12 Requirement for Thrombin-stimulated Gene Expression and DNA Synthesis in 1321N1 Astrocytoma Cells. Journal of Biological Chemistry, 270 (34). pp. 20073-20077. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:ARAjbc95
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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 Gα12 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 Gα12 subunit was microinjected into 1321N1 cells. Microinjection of the anti-Gα12 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 Gα(o) did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-Gα12 antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for Gα12 in the mitogenic response to thrombin in human astroglial cells.
|Additional Information:||©1995 by The American Society for Biochemistry and Molecular Biology, Inc. (Received for publication, May 30, 1995; and in revised form, June 26, 1995) We thank David Goldstein and Carolan Buckmaster for expert technical assistance and Nadia Al-Alawi, Vickie LaMorte, and Vlad Slepak for helpful advice and discussions. This research was supported in part by National Institutes of Health Grants GM36927 (to J.H.B.) and GM34236 (to M.I.S.). The microinjection facilities were supported by National Institutes of Health Grants CA50528 and CA58689 (to J.R.F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. [L.R.C. was] [s]upported by National Institutes of Health Predoctoral Fellowship GM17277. [G.R.P. was] [s]upported by National Institutes of Health Postdoctoral Training Grant HL07444.|
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