Localization of a heterotrimeric G protein gamma subunit to focal adhesions and associated stress fibers.

Abstract

Abstract. 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 o~, 4/~, and 5 3' 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 3' subunits. Using anti-peptide antibodies specific for each of the known 3, subunits, neonatal cardiac fibroblasts were screened by standard immunocytochemistry. The anti-3"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 3'5 subunit with vinculin. In addition, the 3's 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 3'5 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 3'5 subunit and vinculin, suggesting that the association of the 3"s subunit with areas of focal adhesion was widespread.H ETEROTRIMERIC G proteins are responsible for transmitting signals from a large variety of ligandbound receptors to specific effector(s), which, in turn, modulate the concentrations of intracellular signals, including cyclic-AMP, inositol 1,4,5-trisphosphate, diacylglycerol, and ions such as K ÷ and Ca 2+ (reviewed in references 15, 39). Given its interposition between receptor and effector, much of the fidelity of signal transmission must reside in the ot/sy subunit structure of the G protein. While it has generally been assumed that structural heterogeneity of the ot subunits provides the specificity for receptor-G protein and G protein-etfector interactions (reviewed in reference 46), a growing body of evidence indicates that a similar structural heterogeneity of the/5 and 3' subunits exists, and that this heterogeneity, particularly in the 3' subunits, contributes to the specificities of these interactions (24,25,35,37,45). While the functional significance of the individual /5 and 3' subunits identified thus far has yet to be elucidated, evidence indicates that/5~ subunits directly regulate the activities of several effectors. The rapidly expanding family of /5"r regulated effectors include the type II and IV adenylyl cyclase (11,22,48), phospholipase A2 (23), the/5 family of phospholipase (3,6,47), muscarinic and /5-adrenergic receptor kinases (16,36), and a plasma membrane Ca 2+

Analysis of G Protein βγ Dimer Formation in Live Cells Using Multicolor Bimolecular Fluorescence Complementation Demonstrates Preferences of β1for Particular γ Subunits

“…In addition, relationships between subcellular compartmentalization and association of particular ␤ and ␥ subunits can only be investigated using intact cells. ␥ 5 can associate with focal adhesions (Hansen et al, 1994), whereas ␥ 12 can associate with actin filaments (Ueda et al, 1997). Application of BiFC will enable determination of the ␤ subunits with which these ␥ subunits are associated and may demonstrate differential localization patterns depending on the associated ␤ subunit.…”

Mechanoregulation of gene expression in fibroblasts

“…G proteins are important cell surface receptors in mechanical signaling, the subunits of which are localized at the site of focal adhesions (Hansen et al, 1994). Co-localization of G proteins and integrins allows a single signal to activate two transmembrane receptors, indicating the indirect involvement of G proteins in integrin-mediated signaling (Lehoux and Tedgui, 2003).…”

The Expanding Roles of GβγSubunits in G Protein–Coupled Receptor Signaling and Drug Action

“…For example, Gg 12 was found to be associated with F-actin in Swiss 3T3 cells and C6 glioma cells. In contrast, Gg 5 seems to be associated with vinculin in the context of focal adhesions (Ueda et al, 1997) rather than directly associating with actin (Hansen et al, 1994).…”