Characterization of galanin receptors in the insulin-secreting cell line Rin m 5F: evidence for coupling with a pertussis toxin-sensitive guanosine triphosphate regulatory protein.
The present work characterizes galanin receptors in the insulin-secreting pancreatic beta-cell line Rin m 5F and documents their regulation by guanine nucleotides. Binding of [125I]galanin to cell membranes was found to be temperature dependent, rapid, saturable, reversible, and highly peptide specific. Optimal steady state conditions were achieved after a 60-min incubation at 15 C. The concentration dependence of galanin binding determined by adding increasing concentrations of [125I]galanin indicated that galanin receptors were saturated at 2-3 nM peptide. Scatchard analysis revealed a single class of receptors, with a Kd of 0.3 nM and a binding capacity of 82 fmol/mg protein. Guanyl 5'-yl imidodiphosphate dramatically enhanced the dissociation of bound [125I]galanin. Some guanine nucleotides inhibited [125I]galanin binding to membranes with the following order of potency: guanyl 5'-yl imidodiphosphate greater than GTP = GDP. Other nucleotides had no effect. The effect of the guanine nucleotides was Mg2+ dependent, but Na+ independent, although Mg2+ ions alone (5 mM) slightly enhanced [125I]galanin binding, and Na+ ions alone (100 mM) induced a 60% decrease in the binding. Finally, overnight treatment of Rin m 5F cells with pertussis toxin (0.4 microgram/ml) dramatically reduced [125I]galanin binding to cell membranes. This was related to a 4-fold decrease in receptor affinity, with no change in binding capacity. In conclusion, for the first time evidence of the existence of galanin receptors on functional pancreatic beta-cells is presented. Also, other findings support the fact that galanin receptors are functionally associated with a pertussis toxin-sensitive GTP-binding protein mediating guanine nucleotide control of galanin binding.
Lagny-Pourmir
I
I
Unité de Recherches sur la Différenciation et la Neuroendocrinologie de Cellules Digestives, INSERM U178, Villejuif, France.
Amiranoff
B
B
Lorinet
A M
AM
Tatemoto
K
K
Laburthe
M
M
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D019004:Galanin; D006150:Guanine Nucleotides / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D037342:Pertussis Toxin; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D000067956:Adenylyl Cyclase Inhibitors; D006150:Guanine Nucleotides; D007328:Insulin; D010455:Peptides; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone; D010566:Virulence Factors, Bordetella; D006160:Guanosine Triphosphate; D019004:Galanin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases