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 [3H](-)-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 [3H](-)-bremazocine binding by as much as 90%. Inhibition of [3H](-)-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 Bmax value for [3H](-)-bremazocine binding decreased by 50%, with no change in receptor affinity. N-Ethylmaleimide-induced reduction of [3H](-)-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.
Joseph
D B
DB
Department of Pharmacology, University of Rochester, School of Medicine and Dentistry, NY 14642.
Bidlack
J M
JM
D000478:Alkylation; D000818:Animals; D001575:Benzomorphans / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D008223:Lymphoma / Q000378:metabolism*; D051379:Mice; D009294:Narcotics / Q000494:pharmacology; D017473:Receptors, Opioid, kappa / Q000737:chemistry* / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000032:analysis*; D014407:Tumor Cells, Cultured
D001575:Benzomorphans; D009294:Narcotics; D017473:Receptors, Opioid, kappa; D013438:Sulfhydryl Compounds; C027248:bremazocine; D005033:Ethylmaleimide; D004229:Dithiothreitol