Agonist-Promoted Lys63-Linked Polyubiquitination of the Human -Opioid Receptor Is Involved in Receptor Down-Regulation

Ubiquitination of the human 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 -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. Effects of opioid drugs and endogenous peptides are mediated by opioid receptors. There are at least three types of opioid receptors: , , and . The opioid receptors, belonging to the rhodopsin subfamily of the seven transmembrane domain receptor (7TMR) family, are coupled to pertussis toxinsensitive Gi/Go proteins, and activation of their effectors include adenylate cyclase, potassium and calcium channels, and mitogen-activated protein kinase pathways (for review, see Law et al., 2000). Activation of the opioid receptor produces many effects, including analgesia, water diuresis, dysphoria, and antipruritic effects [see Liu-Chen (2004) for