THE KAPPA-OPIOID RECEPTOR EXPRESSED ON THE MOUSE LYMPHOMA CELL-LINE R1.1 CONTAINS A SULFHYDRYL-GROUP AT THE BINDING-SITE

Studies were directed at determining whether the kappa-opioid receptor expressed on the mouse R1.1 thymoma cell line contained either a disulfide bond or a sulfhydryl group at the opioid binding site. The binding of the kappa-opioid receptor agonist [H-3](-)-bremazocine to R1.1 cell membranes was unchanged following treatment with the disulfide bond-reducing reagent dithiothreitol at concentrations up to 130 mM. However, treatment of membranes with the sulfhydryl-alkylating reagent N-ethylmaleimide, followed by extensive washing, reduced [H-3](-)-bremazocine binding by as much as 90%. Inhibition of [H-3](-)-bremazocine binding by N-ethylmaleimide was concentration- and time-dependent. When R1.1 cell membranes were treated with 1 mM N-ethylmaleimide for 10 min at 24-degrees-C, the B(max) value for [H-3](-)-bremazocine binding decreased by 50%, with no change in receptor affinity. N-Ethylmaleimide-induced reduction of [H-3](-)-bremazocine binding was attenuated by pretreatment of membranes with the kappa-selective opioids U50,488 and U69,593. The results indicate that a sulfhydryl group is present at or near the binding site on the kappa-opioid receptor expressed by the R1.1 thymoma cell line.