Baffy G, Yang L, Raj S, Manning D R, Williamson J R
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104.
J Biol Chem. 1994 Mar 18;269(11):8483-7.
The specific involvement of G proteins in thrombin receptor-mediated Ca2+ mobilization and DNA synthesis has been studied in single Chinese hamster lung fibroblasts (CCL39 cells) activated by the hexapeptide SFLLRN. Immunoblots performed with antibodies directed against the COOH terminus of G protein alpha subunits revealed that alpha q, alpha i, and alpha o were each present in CCL39 cells. The Ca2+ response to SFLLRN was measured after microinjection of anti-alpha q or anti-alpha o antibodies, which produced a total blockade in 71 and 46% of cells, respectively. A partial inhibition of the SFLLRN-induced Ca2+ response was observed in the remaining cells. The lag time between exposure of the cells to SFLLRN and the onset of Ca2+ mobilization was significantly longer (20-24 s) in cells microinjected with anti-alpha q- or anti-alpha o-antibodies than in control cells microinjected with preimmune serum (9 +/- 1 s). Moreover, the peak height of the Ca2+ response to SFLLRN was decreased by 36 and 73%, respectively in cells microinjected with anti-alpha q or anti-alpha o antibodies. SFLLRN-induced DNA synthesis in growth-arrested CCL39 cells was also inhibited (44-78%) by prior microinjection of anti-alpha q or anti-alpha o antibodies. Anti-alpha 1 antibodies had no effect on the SFLLRN-induced Ca2+ response or on DNA synthesis. These results provide direct evidence that the thrombin receptor in CCL39 cells is coupled to two different types of G proteins, Gq and Go, both causing Ca2+ mobilization and mitogenesis.
在由六肽SFLLRN激活的单个中国仓鼠肺成纤维细胞（CCL39细胞）中，研究了G蛋白在凝血酶受体介导的Ca2+动员和DNA合成中的具体作用。用针对G蛋白α亚基COOH末端的抗体进行免疫印迹分析，结果显示CCL39细胞中存在αq、αi和αo。在显微注射抗αq或抗αo抗体后，测定对SFLLRN的Ca2+反应，结果分别在71%和46%的细胞中产生了完全阻断。在其余细胞中观察到SFLLRN诱导的Ca2+反应部分受到抑制。与注射免疫前血清的对照细胞（9±1秒）相比，注射抗αq或抗αo抗体的细胞在接触SFLLRN后到Ca2+动员开始之间的延迟时间明显更长（20 - 24秒）。此外，注射抗αq或抗αo抗体的细胞中，对SFLLRN的Ca2+反应峰值高度分别降低了36%和73%。预先显微注射抗αq或抗αo抗体也抑制了生长停滞的CCL39细胞中SFLLRN诱导的DNA合成（44% - 78%）。抗α1抗体对SFLLRN诱导的Ca2+反应或DNA合成没有影响。这些结果提供了直接证据，表明CCL39细胞中的凝血酶受体与两种不同类型的G蛋白Gq和Go偶联，二者均引起Ca2+动员和有丝分裂。
