A novel regulatory mechanism for trimeric GTP-binding proteins in the membrane and secretory granule fractions of human and rodent beta cells.
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 beta subunit of trimeric GTP-binding proteins underwent phosphorylation in each of five classes of insulin-secreting cells. Incubation of the beta cell total membrane fraction or the isolated secretory granule fraction (but not the cytosolic fraction) with [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of this protein, which was selectively immunoprecipitated by an anti-serum directed against the common beta subunit of trimeric G-proteins. Disruption of the alpha beta gamma trimer (by pretreatment with either fluoroaluminate or guanosine 5'(-)[gamma-thio]triphosphate) prevented beta 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 beta subunit alone or in combination with the exogenous purified alpha subunit of transducin did not result in the phosphorylation of the beta 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 beta subunit phosphorylation. Incubation of phosphorylated beta subunit with G alpha.GDP accelerated the dephosphorylation of the beta subunit, accompanied by the formation of G alpha-GTP. Immunoblotting detected multiple alpha subunits (of Gi, G(o) and Gq) and at least one beta 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 beta cells which contrasts with the classical receptor-agonist mechanism: G beta 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 G alpha.GDP (inactive), yielding the active configuration G alpha.GTP in secretory granules (a strategic location to modulate exocytosis).
Department of Medicine, University of Wisconsin School of Medicine, Madison 53705, USA.
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A
Kowluru
S E
SE
Seavey
C J
CJ
Rhodes
S A
SA
Metz
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism* / Q000648:ultrastructure; D046911:Macromolecular Substances; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D014530:Uridine Diphosphate / Q000494:pharmacology
D007328:Insulin; D046911:Macromolecular Substances; D010750:Phosphoproteins; D006153:Guanosine Diphosphate; D014530:Uridine Diphosphate; D006160:Guanosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; D019204:GTP-Binding Proteins