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

Insulin action is initiated by binding to its cognate receptor, which then triggers multiple cellular responses by activating different signaling path ways. There is evidence that insulin receptor signaling may involve G prote in activation in different target cells. We have studied the activation of G proteins in rat hepatoma (HTC) cells. We found that insulin stimulated bi nding of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S-35) to plasma mem brane proteins of HTC cells, in a dose-dependent manner. This effect was co mpletely blocked by pertussis toxin treatment of the membranes, suggesting the involvement of G proteins of the G alpha(i)/G alpha(o) family. The expr ession 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(i1,2) 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 associat ion of the insulin receptor with G alpha(i1,2) by copurification studies. T hese results further support the involvement of a pertussis toxin-sensitive G protein in insulin receptor signaling and provides some evidence of spec ific association and activation of G alpha(i1,2) protein by insulin. These findings suggest that G alpha(i1,2) proteins might be involved in insulin a ction.