A novel regulatory mechanism for trimeric GTP-binding proteins in the membrane and secretory granule fractions of human and rodent β cells

Abstract

Recently we described roles for heterotrimeric and low-molecular-mass GTP-binding proteins in insulin release from normal rat islets. During these studies, we observed that a protein with an apparent molecular mass (37 kDa) similar to that of the β subunit of trimeric GTP-binding proteins underwent phosphorylation in each of five classes of insulin-secreting cells. Incubation of the β cell total membrane fraction or the isolated secretory granule fraction (but not the cytosolic fraction) with [γ-32P]ATP or [γ-32P]GTP resulted in the phosphorylation of this protein, which was selectively immunoprecipitated by an antiserum directed against the common β subunit of trimeric G-proteins. Disruption of the αβγ trimer (by pretreatment with either fluoroaluminate or guanosine 5ʹ-[γ-thio]triphosphate) prevented β subunit phosphorylation. Based on differential sensitivities to pH, heat and the histidine-selective reagent diethyl pyrocarbonate (and reversal of the latter by hydroxylamine), the phosphorylated amino acid was presumptively identified as histidine. Incubation of pure β subunit alone or in combination with the exogenous purified α subunit of transducin did not result in the phosphorylation of the β subunit, but addition of the islet cell membrane fraction did support this event, suggesting that membrane localization (or a membrane-associated factor) is required for β subunit phosphorylation. Incubation of phosphorylated β subunit with Gα·GDP accelerated the dephosphorylation of the β subunit, accompanied by the formation of Gα·GTP. Immunoblotting detected multiple α subunits (of Gi, Go and Gq) and at least one β subunit in the secretory granule fraction of normal rat islets and insulinoma cells. These data describe a potential alternative mechanism for the activation of GTP-binding proteins in β cells which contrasts with the classical receptor-agonist mechanism: Gβ undergoes transient phosphorylation at a histidine residue by a GTP-specific protein kinase; this phosphate, in turn, may be transferred via a classical Ping-Pong mechanism to Gα·GDP (inactive), yielding the active configuration Gα·GTP in secretory granules (a strategic location to modulate exocytosis).