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-p roteins, increases insulin secretion from beta-cells. In this study, w e have examined the role of heterotrimeric G-proteins in mastoparan-in duced insulin secretion from the insulin-secreting beta-cell line beta -TC3. Mastoparan stimulated insulin secretion in a dose-dependent mann er from digitonin-permeabilized beta-TC3 cells, Active mastoparan anal ogues mastoparan 7, mastoparan 8, and mastoparan X also stimulated sec retion, Mastoparan 17, an inactive analogue of mastoparan, did not inc rease insulin secretion from permeabilized beta-TC3 cells, Mastoparan- induced insulin secretion from permeabilized beta-TC3 cells was inhibi ted 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 ins ulin secretory granules (ISG) were prepared by sucrose/nycodenz ultrac entrifugation, Western immunoblotting performed on beta-TC3 ho mogenat e and ISG demonstrated that G alpha(i) was dramatically enriched in IS G. Levels of G alpha(o) and G alpha(q) were comparable in homogenate a nd ISG. Mastoparan stimulated ISG GTPase activity in a pertussis toxin sensitive manner. Mastoparan 7 and mastoparan 8 also stimulated GTPas e activity in the ISG, while the inactive analogue mastoparan 17 had n o effect. Selective localization of G alpha(1) to ISG was confirmed wi th 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 sugges t that mastoparan may act through the heterotrimeric G-protein G alpha (i) located in the ISG of beta-cells to stimulate insulin secretion.