LOCALIZATION OF A HETEROTRIMERIC G-PROTEIN GAMMA-SUBUNIT TO FOCAL ADHESIONS AND ASSOCIATED STRESS FIBERS

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 effecters, 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 cell that tended to protrude into the cell in a fibrous pattern. Dual staining with anti-vinculin anti-body 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 ys 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 ys subunit and vinculin, suggesting that the association of the ys subunit with areas of focal adhesion was widespread.