The Heterotrimeric G-protein G Is Localized to the Insulin Secretory Granules of -Cells and Is Involved in Insulin Exocytosis (*)

Abstract

Mastoparan, a tetradecapeptide found in wasp venom that stimulates G-proteins, increases insulin secretion from β-cells. In this study, we have examined the role of heterotrimeric G-proteins in mastoparan-induced insulin secretion from the insulin-secreting β-cell line β-TC3. Mastoparan stimulated insulin secretion in a dose-dependent manner from digitonin-permeabilized β-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 β-TC3 cells. Mastoparan-induced insulin secretion from permeabilized β-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 β-TC3 homogenate and ISG demonstrated that Gαⁱ was dramatically enriched in ISG. Levels of Gα^o and Gα^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 Gαⁱ to ISG was confirmed with electron microscopic immunocytochemistry in β-TC3 cells and β-cells from rat pancreas. In contrast to Gα^o and Gα^q, Gαⁱ was clearly localized to the ISG. Together, these data suggest that mastoparan may act through the heterotrimeric G-protein Gαⁱ located in the ISG of β-cells to stimulate insulin secretion.