Insulin activates G alpha il,2 protein in rat hepatoma (HTC) cell membranes.

Abstract

Insulin action is initiated by binding to its cognate receptor, which then triggers multiple cellular responses by activating different signaling pathways. There is evidence that insulin receptor signaling may involve G protein activation in different target cells. We have studied the activation of G proteins in rat hepatoma (HTC) cells. We found that insulin stimulated binding of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-35S) to plasma membrane proteins of HTC cells, in a dose-dependent manner. This effect was completely blocked by pertussis toxin treatment of the membranes, suggesting the involvement of G proteins of the G alpha i/G alpha o family. The expression of these G alpha proteins was checked by Western blotting. Next, we used blocking antibodies to sort out the specific G alpha protein activated by insulin stimulation. Anti-G alpha il,2 antibodies completely prevented insulin-stimulated GTP binding, whereas anti-G alpha o,i3 did not modify this effect of insulin on GTP binding. Moreover, we found physical association of the insulin receptor with G alpha il,2 by copurification studies. These results further support the involvement of a pertussis toxin-sensitive G protein in insulin receptor signaling and provides some evidence of specific association and activation of G alpha il,2 protein by insulin. These findings suggest that G alpha il,2 proteins might be involved in insulin action.