G(12) REQUIREMENT FOR THROMBIN-STIMULATED GENE-EXPRESSION AND DNA-SYNTHESIS IN 1321N1 ASTROCYTOMA-CELLS
G(12) REQUIREMENT FOR THROMBIN-STIMULATED GENE-EXPRESSION AND DNA-SYNTHESIS IN 1321N1 ASTROCYTOMA-CELLS
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DOI：
10.1074/jbc.270.34.20073
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发表时间：
1995-08-25
影响因子：
4.8
通讯作者：
SIMON, MI
中科院分区：
文献类型：
--
作者：
ARAGAY, AM;COLLINS, LR;SIMON, MI
凝血酶刺激1321 N1星形细胞瘤细胞导致Ras依赖性AP-1介导的转录激活和DNA复制。与在大多数细胞系统中观察到的相反，在1321 N1细胞中，这些反应是百日咳毒素不敏感的。百日咳毒素不敏感的G蛋白G（12）与不同细胞系统中的细胞生长和转化有关。我们已经研究了这种蛋白在AP-1介导的转录激活和DNA合成在1321 N1细胞中的潜在作用。G α（12）的激活（GTP酶缺陷）突变体的瞬时表达增加了AP-1依赖性基因表达。这种反应被显性负性Ala-15 Ras蛋白的共表达抑制。为了确定百日咳毒素不敏感的G（12）蛋白是否参与凝血酶刺激的DNA合成，将针对G α（12）亚基C-末端序列的抑制性抗体显微注射到1321 N1细胞中。微量注射抗G α（12）导致凝血酶刺激的DNA合成的浓度依赖性抑制。相反，显微注射非免疫IgG或针对G α（0）C末端的抗体不会降低对凝血酶的促有丝分裂反应。此外，显微注射抗G α（12）抗体对成纤维细胞生长因子刺激的DNA合成没有影响。这些结果证明了G α（12）在人星形胶质细胞对凝血酶的促有丝分裂反应中的特定作用。
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.