Abstract

The agonist (−)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide [(−)U50,488H] caused desensitization of the human κ-opioid receptor (hkor) and Flag-tagged hkor (Flag-hkor) but not the rat κ-opioid receptor (rkor) and Flag-tagged rkor (Flag-rkor) stably expressed in CHO cells as assessed by guanosine 5′-O-(3-[³⁵S]thiotriphosphate) binding. In addition, (−)U50,488H stimulation enhanced phosphorylation of the Flag-hkor, but not Flag-rkor. (−)U50,488H-induced phosphorylation of the Flag-hkor was reduced by expression of the dominant negative mutant GRK2-K220R, demonstrating the involvement of G protein-coupled receptor kinases (GRKs). However, expression of GRK2 and arrestin-2 or GRK3 and arrestin-3 did not result in desensitization or phosphorylation of the Flag-rkor after (−)U50,488H pretreatment. To understand the molecular basis of the species differences, we constructed two Flag-tagged chimeric receptors, Flag-h/rkor and Flag-r/hkor, in which the C-terminal domains of Flag-hkor and Flag-rkor were switched. When stably expressed in CHO cells, Flag-r/hkor, but not Flag-h/rkor, was desensitized and phosphorylated after exposure to (−)U50,488H, indicating that the C-terminal domain plays a critical role in the differences. We then generated a Flag-hkor mutant, in which S358 was mutated to N (Flag-hkorS358N) and a Flag-rkor mutant, in which N358 was substituted with S (Flag-rkorN358S). Although Flag-hkorS358N was not phosphorylated or desensitized by (−)U50,488H stimulation, Flag-rkorN358S underwent (−)U50,488H-induced desensitization with slightly increased phosphorylation. These results indicate that there are differences in (−)U50,488H-induced desensitization and phosphorylation between the hkor and the rkor. In addition, the C-terminal domain plays a crucial role in these differences and the 358 locus contributes to the differences. Our findings suggest caution in extrapolating studies on κ-opioid receptor regulation from rats to humans.