Regulation of F-actin and endoplasmic reticulum organization by the trimeric G-protein Gi2 in rat hepatocytes. Implication for the activation of store-operated Ca2+ inflow.
The roles of the heterotrimeric G-protein, G(i2), in regulating the actin cytoskeleton and the activation of store-operated Ca(2+) channels in rat hepatocytes were investigated. Galpha(i2) was principally associated with the plasma membrane and microsomes. Both F-actin and Galpha(i2) were detected by Western blot analysis in a purified plasma membrane preparation, the supernatant and pellet obtained by treating the plasma membrane with Triton X-100, and after depolymerization and repolymerization of F-actin in the Triton X-100-insoluble pellet. Actin in the Triton X-100-soluble supernatant co-precipitated with Galpha(i2) using either anti-Galpha(i2) or anti-actin antibodies. The principally cortical location of F-actin in hepatocytes cultured for 0.5 h changed to a pericanalicular distribution over a further 3.5 h. Some Galpha(i2) co-localized with F-actin at the plasma membrane. Pretreatment with pertussis toxin ADP-ribosylated 70-80% of Galpha(i2) in the plasma membrane and microsomes, prevented the redistribution of F-actin, caused redistribution and fragmentation of the endoplasmic reticulum, and inhibited vasopressin-stimulated Ca(2+) inflow. It is concluded that (i) a significant portion of hepatocyte Galpha(i2) associates with, and regulates the arrangement of, cortical F-actin and the endoplasmic reticulum and (ii) either or both of these regulatory roles are likely to be required for normal vasopressin activation of Ca(2+) inflow.
Wang
Y J
YJ
Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, GPO Box 2100, Adelaide, South Australia 5001, Australia.
Gregory
R B
RB
Barritt
G J
GJ
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D017136:Ion Transport; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats
D000199:Actins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium