Endocrinology, Vol 124, 2635-2641, Copyright © 1989 by Endocrine Society

ARTICLES

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

I Lagny-Pourmir, B Amiranoff, AM Lorinet, K Tatemoto and M Laburthe
Unite de Recherches sur la Differenciation et la Neuroendocrinologie de Cellules Digestives, INSERM U178, Villejuif, France.

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.

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