Agonist-promoted Lys63-linked polyubiquitination of the human κ-opioid receptor is involved in receptor down-regulation

Ubiquitination of the human kappa opioid receptor ( hKOR) expressed in Chinese hamster ovary ( CHO) cells was observed in the presence of the proteasomal inhibitor N-benzoyloxycarbonyl ( Z)-Leu-Leu-leucinal ( MG132) and enhanced by the agonists (-)( trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny) cyclohexyl] benzeneacetamide (U50,488H) and dynorphin A ( Dyn A). The dominant-negative ( DN) mutants GRK2-K220R and beta-arrestin ( 319 - 418), but not dynamin I-K44A, reduced Dyn A-stimulated hKOR ubiquitination, and a phosphorylation-defective hKOR mutant ( hKOR-S358N) did not undergo Dyn A-stimulated ubiquitination, indicating that hKOR ubiquitination is enhanced by receptor phosphorylation but not by receptor internalization. A hKOR mutant ( hKOR-10 KR) in which all 10 intracellular Lys residues were changed to Arg showed greatly reduced basal and agonist-promoted receptor ubiquitination and substantially decreased Dyn A-induced receptor down-regulation, without changing ligand binding affinity, receptor-G protein coupling, or receptor internalization or desensitization. The ubiquitination sites were further determined to be the three Lys residues in the C-terminal domain. The K63R ubiquitin mutant decreased Dyn A-induced hKOR ubiquitination and down-regulation, but the K48R mutant did not. Expression of HN-CYLD, a DN mutant of deubiquitination enzyme cylindromatosis tumor suppressor gene ( CYLD) that breaks Lys63-linked polyubiquitin chain, increased Dyn A-induced hKOR ubiquitination and down-regulation. These results indicate that ubiquitinated hKOR after agonist treatment contains predominantly Lys63-linked polyubiquitin chains and ubiquitination of the hKOR involved in agonist-induced down-regulation.