Agonist-promoted Lys63-linked polyubiquitination of the human kappa-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.
Li
Jian-Guo
JG
Department of Pharmacology, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140. lliuche@temple.ed.
Haines
Dale S
DS
Liu-Chen
Lee-Yuan
LY
D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000074749:Deubiquitinating Enzyme CYLD; D004174:Diprenorphine / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009292:Narcotic Antagonists / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D025821:Polyubiquitin / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D017473:Receptors, Opioid, kappa / Q000819:agonists* / Q000737:chemistry / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects
D050505:Mutant Proteins; D009292:Narcotic Antagonists; D043562:Receptors, G-Protein-Coupled; D017473:Receptors, Opioid, kappa; D025521:Tumor Suppressor Proteins; D025821:Polyubiquitin; D004174:Diprenorphine; D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D001120:Arginine; C409984:CYLD protein, human; D000074749:Deubiquitinating Enzyme CYLD; D008239:Lysine