A select set of opioid ligands induce up-regulation by promoting the maturation and stability of the rat kappa-opioid receptor in human embryonic kidney 293 cells.

Ligand-induced regulation of the rat kappa-opioid receptor (rKOR) was investigated in human embryonic kidney 293 cells stably expressing the FLAG-tagged rKOR. Incubation of rKOR cells with naltrexone for 24 h increased the B(max) >3-fold, with no change in the affinity of [(3)H]diprenorphine. Two immunoreactive receptor species were present in cell lysates: naltrexone treatment caused a >3-fold increase in the 52-kDa species while decreasing the level of the 42-kDa species. Dynorphin(1-13), U69,593 [(5alpha,7alpha,8beta)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4,5]dec-8-yl)benzeneacetamide], or salvinorin A [2S,4aR,6aR,7R,9S,10aS, 10bR)-9-(acetyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho[2,1c]pyran-7-carboxylic acid methyl ester] treatment did not alter the level of immunoreactive rKOR protein, whereas etorphine, cyclazocine, naloxone, and naloxone methiodide increased the 52-kDa and decreased the 42-kDa rKOR bands. Receptor up-regulation was associated with an increase in the number of cell surface receptors and a 2-fold increase in the E(max) for guanosine 5'-O-(3-[(35)S]thio)triphosphate binding. Glycosidase digestion indicated that the 52- and 42-kDa receptors contained complex and high-mannose N-glycans, respectively, Pulse-chase analysis and glycosidase digestion sensitivities suggested that the 42-kDa rKOR species was a precursor of the 52-kDa species. Naltrexone did not alter rKOR mRNA levels or translational efficiency, and rKOR up-regulation was not inhibited by cycloheximide. Brefeldin A caused accumulation of intracellular rKOR intermediates, and coincubation with naltrexone increased the levels of the brefeldin-induced species significantly. These results suggest that select opioid ligands up-regulate rKOR by enhancing the rate of receptor folding and maturation and by protecting the receptor from degradation, resulting in an increase in the number of rKOR binding sites, immunoreactive protein, and functional receptors.