Galanin receptors in a hamster pancreatic beta-cell tumor: identification and molecular characterization.
High affinity binding sites for galanin are identified and characterized in membranes from a hamster pancreatic beta-cell tumor. Using the radioiodinated peptide [125I] galanin, interaction of the peptide with pancreatic membranes is shown to be saturable, reversible, and time, temperature, membrane protein concentration, pH, and ionic strength dependent. In optimized equilibrium conditions of binding (90 min at 10 C), native galanin competitively inhibits the binding of [125I]galanin in a dose-dependent manner (from 10(-11)-10(-8) M); half-maximal inhibition is induced by 1 nM peptide. Scatchard analysis indicates the existence of a single population of sites of high affinity (Kd = 1.5 nM) and low capacity (44 fmol/mg protein). The monophasic dissociation process confirms the homogeneity of galanin-binding sites. Galanin-binding sites are highly specific, since apart from native galanin, none of the numerous biologically active peptides tested competes with [125I] galanin for binding to pancreatic membranes. The cross-linking of [125I]galanin to beta-cell membranes is performed using the chemical bifunctional reagent ethylene glycol bis-(succinimidyl succinate). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in the presence or absence of dithiothreitol, one single band of 57,000 mol wt is observed, which may be corresponding to the [125I]galanin-receptor complex. Indeed, labeling of this 57,000 mol wt component is abolished only by native galanin but is unaffected by various other digestive peptides. Assuming one molecule of [125I]galanin is bound per molecule of protein, a 54,000 mol wt protein is identified as the pancreatic galanin receptor. In conclusion, our results indicate for the first time the identification of galanin receptors. Their presence in pancreatic beta-cells suggests a direct role of galanin in regulating endocrine beta-cell function.
D007516:Adenoma, Islet Cell / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003432:Cross-Linking Reagents; D019004:Galanin; D006863:Hydrogen-Ion Concentration; D007340:Insulinoma / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism; D007700:Kinetics; D008647:Mesocricetus; D009994:Osmolar Concentration; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D013388:Succinimides
D003432:Cross-Linking Reagents; D010455:Peptides; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone; D013388:Succinimides; C028722:ethylene glycolylbis(succinimidyl succinate); D019004:Galanin