Recent studies have suggested an association between heterotrimeric G proteins, which play a major role in transmembrane signal transduction, and intracellular components. We therefore examined the subcellular localization of isoforms of G protein gamma subunits in Swiss 3T3 and C6 glioma cells, mainly containing the gamma5 and gamma12 subunits. Immunocytochemical double staining with phalloidin showed co-localization of the gamma12 subunit with actin filaments (F-actin), while the gamma5 co-localized with vinculin, suggesting an association with focal adhesion. Pretreatment of cells with Triton X-100 eliminated the gamma5 but not the gamma12 staining. Co-localization of gamma12 and F-actin was preserved when F-actin was disorganized with cytochalasin D or reorganized using fetal calf serum. Large amounts of gamma12 were recovered in the vimentin- and tubulin-free F-actin-rich fraction prepared from crude cytoskeleton preparations by double depolymerization-repolymerization. Co-localization of Gi2alpha, beta and gamma12 in the F-actin-rich fraction suggested the existence of gamma12 as a betagamma or heterotrimeric complex. Furthermore, purified betagamma12 was found to associate with F-actin in vitro more tightly than betagamma5. These results strongly suggest that the gamma12 subunit associates with F-actin in cells. The observed differential distribution of gamma12 and gamma5 implies functional differences for the two gamma subunits.

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