THE KAPPA-OPIOID RECEPTOR EXPRESSED ON THE MOUSE R1.1 THYMOMA CELL-LINE IS COUPLED TO ADENYLYL-CYCLASE THROUGH A PERTUSSIS-TOXIN-SENSITIVE GUANINE-NUCLEOTIDE-BINDING REGULATORY PROTEIN

The R1.1 mouse thymoma cell line expresses a high-affinity kappa opioid binding site. Opioid binding to this site is inhibited by guanine nucleotides, suggesting that the receptor is coupled to a guanine nucleotide-binding protein. Here, we present evidence that the kappa opioid binding site on R 1. 1 cell membranes is negatively coupled to adenylyl cyclase. The kappa-selective agonists (trans)-3,4-dichloro-N-methyl-N-[2-(l-pyrrolidinyl)-cy-clohexyl]benzeneacetamide methane-sulfonate hydrate [(-)-U50,488],(5alpha,7alpha,8beta)-(-)-N-methyl-N-(7-(l-pyrrolidinyl)-l-oxas-piro(4,5)dec-8-yl)benzeneacetamide (U69,593) and several dynorphin peptides inhibited basal and forskolin-stimulated cyclic AMP production by up to 40% in R1.1 cell membranes. The order of potency for the inhibition of adenylyl cyclase activity by opioid agonists correlated with their K(i) values for the inhibition of [H-3]U69,593 binding. Opioid-mediated inhibition of adenylyl cyclase activity was stereoselective, as (-)-U50,488 was more potent than the (+) isomer, and the inhibition was blocked by the kappa-selective antagonist nor-binaltorphimine. The opioid-mediated inhibition of adenylyl cyclase activity was also completely blocked by incubating R1.1 cells with Bordetella pertussis toxin (PTX). Incubation of R1.1 cell membranes with PTX and [adenylate-P-32]NAD+ resulted in the exclusive labeling of a 41-kDa protein, as determined by separating the membrane proteins under reducing conditions on a SDS polyacrylamide gel, followed by autoradiography. These results suggest that a PTX-sensitive inhibitory guanine nucleotide-binding protein mediates the link between the thymoma kappa opioid receptor and adenylyl cyclase.