Association of the gamma12 subunit of G proteins with actin filaments.
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.
Ueda
H
H
Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Japan.
Saga
S
S
Shinohara
H
H
Morishita
R
R
Kato
K
K
Asano
T
T
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D003902:Detergents; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007150:Immunohistochemistry; D051379:Mice; D017830:Octoxynol; D011487:Protein Conformation; D015398:Signal Transduction; D012995:Solubility
D000199:Actins; D003902:Detergents; D017830:Octoxynol; D019204:GTP-Binding Proteins