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-γ-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 Gα i/Gα o family. The expression of these Gα proteins was checked by Western blotting. Next, we used blocking antibodies to sort out the specific Gα protein activated by insulin stimulation. Anti-Gα il,2 antibodies completely prevented insulin-stimulated GTP binding, whereas anti-Gα o,i3 did not modify this effect of insulin on GTP binding. Moreover, we found physical association of the insulin receptor with Gα i1,2 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 Gα i1,2 protein by insulin. These findings suggest that Gα i1,2 proteins might be involved in insulin action.
Keywords: Key words. Insulin; insulin receptor; G protein; insulin action; cellular signaling; hepatocytes.
Footnotes
Received 23 September 1998; received after revision 23 November 1998; accepted 25 November 1998