Abstract
Ubiquitination of the human κ 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 β-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.
Footnotes
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This work was supported by National Institutes of Health grant DA17302.
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ABBREVIATIONS: 7TMR, seven transmembrane domain receptor; GRK, G protein-coupled receptor kinase; β2-AR, β2-adrenergic receptor; CHO cells, Chinese hamster ovary cells; CHO-FLAG-hKOR, CHO cell lines stably expressing FLAG-hKOR; CHO-FLAG-hKOR-10KR, CHO cell lines stably expressing FLAG-hKOR-10KR; CYLD, cylindromatosis tumor suppressor gene; DN, dominant-negative mutant; Dyn A, dynorphin A (1-17); ER, Endoplasmic reticulum; FLAG-hKOR, Flag-tagged human κ opioid receptor; hKOR, human κ opioid receptor; hKOR-10KR, a FLAG-hKOR mutant in which all 10 intracellular lysines were replaced with arginines; NK-1, neurokinin-1 receptor; PAR, protease-activated receptor; U50,488H, (-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny) cyclohexyl] benzeneacetamide; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; GTPγS, guanosine 5′-O-(3-thio)triphosphate; HA, hemagglutinin; HA-Ub, HA-ubiquitin; PBS, phosphate-buffered saline; UBPY, ubiquitin-specific protease Y.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received October 23, 2007.
- Accepted January 17, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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