Received for publication October 24, 2007.
Revised January 17, 2008.
Accepted for publication January 17, 2008.

Agonist-promoted Lys 63-linked polyubiquitination of the human kappa opioid receptor is involved in receptor down-regulation

Abstract

Ubiquitination of the human opioid receptor (hKOR) expressed in CHO cells was observed in the presence of the proteasomal inhibitor MG132 and enhanced by the agonists 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 ten 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 the deubiquitinating enzyme CYLD that breaks Lys 63-linked polyubiquitin chain, increased Dyn A-induced hKOR ubiquitination and down-regulation. These results indicate that ubiquitinated hKOR following agonist treatment contains predominantly Lys63-linked polyubiquitin chains and ubiquitination of the hKOR involved in agonist-induced down-regulation.

Key words: Opioid, GRKs, barrestins, Receptor degradation