ADP-RIBOSYLATION BY CHOLERA-TOXIN IDENTIFIES 3 G-PROTEINS THAT ARE ACTIVATED BY THE GALANIN RECEPTOR

Inhibition of insulin secretion by galanin is pertussis toxin (PTX) se nsitive, suggesting the activation of one or more heterotrimeric (alph a, beta, gamma), G-proteins (Gi/Go). Multiple effecters, including the K(+)ATP and L-type Ca2+ channels, adenylyl cyclase, and an as yet uni dentified system at a site close to exocytosis, are modulated by galan in. 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 alpha i/alpha o-subunits of the PTX-sensitive substrates but only during receptor/G-protein interaction. Therefore, we used CTX-catalyzed ADP ribosylation to identify galanin receptor-a ssociated G-protein alpha-subunits in RINm5F cells. Galanin enhanced t he ADP ribosylation of membrane proteins separated by sodium dodecyl s ulfate- polyacrylamide gel electrophoresis (SDS-PAGE) in two bands at 39,000 and 42,000 M(r). This labeling was blocked in membranes prepare d from PTX-treated cells, enhanced by Mg2+, and showed a biphasic depe ndence on exogenous guanine nucleotides. Identification of the CTX ADP -ribosylated G-proteins by immunoprecipitation with selective antisera indicate activation by the galanin receptor of alpha i(1), and alpha i(3), which have the same mobility on SDS-PAGE (42,000 M(r)), and alph a i(2) (39,000 M(r)). These studies provide evidence for the activatio n of multiple G-proteins by receptors for galanin in RINm5F cells.