THE HETEROTRIMERIC G-PROTEIN G(I) IS LOCALIZED TO THE INSULIN SECRETORY GRANULES OF BETA-CELLS AND IS INVOLVED IN INSULIN EXOCYTOSIS

Mastoparan, a tetradecapeptide found in wasp venom that stimulates G-proteins, increases insulin secretion from beta-cells. In this study, we have examined the role of heterotrimeric G-proteins in mastoparan-induced insulin secretion from the insulin-secreting beta-cell line beta-TC3. Mastoparan stimulated insulin secretion in a dose-dependent manner from digitonin-permeabilized beta-TC3 cells, Active mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan X also stimulated secretion, Mastoparan 17, an inactive analogue of mastoparan, did not increase insulin secretion from permeabilized beta-TC3 cells, Mastoparan-induced insulin secretion from permeabilized beta-TC3 cells was inhibited by pretreatment of the cells with pertussis toxin, suggesting that mastoparan-induced insulin secretion is mediated through a pertussis toxin sensitive G-protein present distally in exocytosis. Enriched insulin secretory granules (ISG) were prepared by sucrose/nycodenz ultracentrifugation, Western immunoblotting performed on beta-TC3 ho mogenate and ISG demonstrated that G alpha(i) was dramatically enriched in ISG. Levels of G alpha(o) and G alpha(q) were comparable in homogenate and ISG. Mastoparan stimulated ISG GTPase activity in a pertussis toxin sensitive manner. Mastoparan 7 and mastoparan 8 also stimulated GTPase activity in the ISG, while the inactive analogue mastoparan 17 had no effect. Selective localization of G alpha(1) to ISG was confirmed with electron microscopic immunocytochemistry in beta-TC3 cells and beta-cells from rat pancreas, In contrast to G alpha(o) and G alpha(q), G alpha(i) was clearly localized to the ISG, Together, these data suggest that mastoparan may act through the heterotrimeric G-protein G alpha(i) located in the ISG of beta-cells to stimulate insulin secretion.