Hansen C A, Schroering A G, Carey D J, Robishaw J D
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
J Cell Biol. 1994 Aug;126(3):811-9. doi: 10.1083/jcb.126.3.811.
Signal transducing heterotrimeric G proteins are responsible for coupling a large number of cell surface receptors to the appropriate effector(s). Of the three subunits, 16 alpha, 4 beta, and 5 gamma subunits have been characterized, indicating a potential for over 300 unique combinations of heterotrimeric G proteins. To begin deciphering the unique G protein combinations that couple specific receptors with effectors, we examined the subcellular localization of the gamma subunits. Using anti-peptide antibodies specific for each of the known gamma subunits, neonatal cardiac fibroblasts were screened by standard immunocytochemistry. The anti-gamma 5 subunit antibody yielded a highly distinctive pattern of intensely fluorescent regions near the periphery of the cell that tended to protrude into the cell in a fibrous pattern. Dual staining with anti-vinculin antibody showed co-localization of the gamma 5 subunit with vinculin. In addition, the gamma 5 subunit staining extended a short distance out from the vinculin pattern along the protruding stress fiber, as revealed by double staining with phalloidin. These data indicated that the gamma 5 subunit was localized to areas of focal adhesion. Dual staining of rat aortic smooth muscle cells and Schwann cells also indicated co-localization of the gamma 5 subunit and vinculin, suggesting that the association of the gamma 5 subunit with areas of focal adhesion was wide-spread.
信号转导异源三聚体G蛋白负责将大量细胞表面受体与合适的效应器偶联。在这三个亚基中，已鉴定出16种α亚基、4种β亚基和5种γ亚基，这表明异源三聚体G蛋白有超过300种独特组合的可能性。为了开始解读将特定受体与效应器偶联的独特G蛋白组合，我们研究了γ亚基的亚细胞定位。使用针对每种已知γ亚基的抗肽抗体，通过标准免疫细胞化学对新生心脏成纤维细胞进行筛选。抗γ5亚基抗体在细胞周边附近产生了高度独特的强烈荧光区域模式，这些区域倾向于以纤维模式向细胞内突出。用抗纽蛋白抗体进行双重染色显示γ5亚基与纽蛋白共定位。此外，如用鬼笔环肽双重染色所示，γ5亚基染色沿着突出的应力纤维从纽蛋白模式向外延伸一小段距离。这些数据表明γ5亚基定位于粘着斑区域。对大鼠主动脉平滑肌细胞和雪旺细胞的双重染色也表明γ5亚基与纽蛋白共定位，这表明γ5亚基与粘着斑区域的关联是广泛存在的。
