Inhibition of insulin secretion by galanin is pertussis toxin (PTX) sensitive, suggesting the activation of one or more heterotrimeric (α, β, γ) G-proteins (Gi/Go). Multiple effectors, including the K+ATP and L-type Ca2+ channels, adenylyl cyclase, and an as yet unidentified system at a site close to exocytosis, are modulated by galanin. Therefore, it is necessary to delineate the particular G-proteins activated by the galanin receptor as a first step to understanding its net cellular response. During specific conditions, cholera toxin (CTX) can ADP-ribosylate the αi/αo-subunits of the PTX-sensitive substrates but only during receptor/G-protein interaction. Therefore, we used CTX-catalyzed A DP ribosylation to identify galanin receptor-associated G-protein α-subunits in RINmSF cells. Galanin enhanced the A DP ribosylation of membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in two bands at 39,000 and 42,000 Mr This labeling was blocked in membranes prepared from PTX-treated cells, enhanced by Mg2+, and showed a biphasic dependence on exogenous guanine nucleotides. Identification of the CTX ADP-ribosylated G-proteins by immunoprecipitation with selective antisera indicate activation by the galanin receptor of αi1, and αi3, which have the same mobility on SDS-PAGE (42,000 Mr), and αi2 (39,000 Mr). These studies provide evidence for the activation of multiple G-proteins by receptors for galanin in RINmSF cells.

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