title: ADP ribosylation by cholera toxin identifies three G-proteins that are activated by the galanin receptor. Studies with RINm5F cell membranes.
abstract: Inhibition of insulin secretion by galanin is pertussis toxin (PTX) sensitive, suggesting the activation of one or more heterotrimeric (alpha, beta, gamma) 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 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-associated G-protein alpha-subunits in RINm5F cells. Galanin enhanced the ADP ribosylation of membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in two bands at 39,000 and 42,000 M(r). 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 alpha i1 and alpha i3, which have the same mobility on SDS-PAGE (42,000 M(r)), and alpha i2 (39,000 M(r)). These studies provide evidence for the activation of multiple G-proteins by receptors for galanin in RINm5F cells.
authors.affiliation: Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
authors.forename: S L
authors.initials: SL
authors.lastname: Gillison
authors.forename: G W
authors.initials: GW
authors.lastname: Sharp
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000378:metabolism* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019004:Galanin; D007515:Islets of Langerhans; D046911:Macromolecular Substances; D008970:Molecular Weight; D009479:Neuropeptides / Q000494:pharmacology*; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D009479:Neuropeptides; D010455:Peptides; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone; D000246:Adenosine Diphosphate Ribose; D019004:Galanin; D002772:Cholera Toxin; D019204:GTP-Binding Proteins