(-)U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide] induces internalization and down-regulation of the human, but not the rat, kappa-opioid receptor: structural basis for the differential regulation.

We showed previously that prolonged activation by (-)U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide] led to internalization and down-regulation of the human kappa opioid receptor (hkor), but not the rat kappa opioid receptor (rkor). Herein, we investigated structural determinants in the receptors underlying these differences using chimeric and mutant receptor constructs epitope tagged with FLAG and stably expressed in Chinese hamster ovary cells (CHO). The FLAG-hkor, but not the FLAG-rkor, underwent internalization and down-regulation after exposure to (-)U50,488H. Monensin did not have any effect on the intracellular receptor pool of the FLAG-rkor or rkor with or without (-)U50,488H treatment, indicating that the lack of (-)U50,488H-induced internalization is not due to rapid resurfacing of the rkor. Two chimeric receptors, FLAG-h/rkor and FLAG-r/hkor, were generated, in which the C-terminal domains of the hkor and the rkor were switched. The FLAG-r/hkor displayed significant (-)U50,488H-induced internalization and down-regulation, whereas the FLAG-h/rkor did not, indicating that the C-terminal domain contributes to the differences between the rkor and the hkor. To further characterize, we generated two mutants, FLAG-hkorS358N and FLAG-rkorN358S in which the locus 358 was exchanged. The FLAG-hkorS358N mutant displayed greatly reduced (-)U50,488H-induced internalization and no down-regulation compared with the FLAG-hkor, indicating that Ser358 in the hkor is critical for these processes. However, the FLAG-rkorN358S mutant was internalized, but not down-regulated, demonstrating that N358 prevents the rkor from being internalized, but it may not have a role in the lack of down-regulation of the rkor. In addition, the trafficking of the FLAG-rkorN358S mutant seems to be more complex than the rkor and the hkor.