title: Mechanisms of agonist-induced down-regulation of the human kappa-opioid receptor: internalization is required for down-regulation.
abstract: Previously, we showed that the human kappa-opioid receptor (hkor) stably expressed in Chinese hamster ovary (CHO) cells underwent down-regulation after prolonged U50,488H treatment. In the present study, we determined the mechanisms underlying this process. U50, 488H caused a significant down-regulation of the hkor, although etorphine did not. Neither U50,488H nor etorphine caused down-regulation of the rat kappa-opioid receptor. Thus, similar to internalization, there are agonist and species differences in down-regulation of kappa-opioid receptors. Expression of the dominant negative mutants arrestin-2(319-418) or dynamin I-K44A significantly reduced U50,488H-induced down-regulation of the hkor. Coexpression of GRK2 or GRK2 and arrestin-2 permitted etorphine to induce down-regulation of the hkor, although expression of arrestin-2 or dynamin I alone did not. Expression of the dominant negative mutants rab5A-N133I or rab7-N125I blunted U50,488H-induced down-regulation. Pretreatment with lysosomal enzyme inhibitors [(2S, 3S)trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester or chloroquine] or proteasome inhibitors (proteasome inhibitor I, MG-132, or lactacystin) decreased the extent of U50,488H-induced down-regulation. A combination of chloroquine and proteasome inhibitor I abolished U50,488H-induced down-regulation. These results indicate that U50,488H-induced down-regulation of the hkor involves GRK-, arrestin-2-, dynamin-, rab5-, and rab7-dependent mechanisms and receptors seem to be trafficked to lysosomes and proteasomes for degradation. Thus, U50,488H-induced internalization and down-regulation of the hkor share initial common mechanisms. To the best of our knowledge, these results represent the first report on the involvement of both rab5 and rab7 in agonist-induced down-regulation of a G protein-coupled receptor. In addition, this study is among the first to show the involvement of proteasomes in agonist-induced down-regulation of a G protein-coupled receptor.
authors.affiliation: Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: J G
authors.initials: JG
authors.lastname: Li
authors.forename: J L
authors.initials: JL
authors.lastname: Benovic
authors.forename: L Y
authors.initials: LY
authors.lastname: Liu-Chen
mesh_terms: D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer / Q000494:pharmacology; D018712:Analgesics, Non-Narcotic / Q000494:pharmacology; D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000502:physiology; D016466:CHO Cells; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000502:physiology; D015536:Down-Regulation; D034284:Dynamin I; D034281:Dynamins; D005048:Etorphine / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000502:physiology; D006801:Humans; D008247:Lysosomes / Q000502:physiology; D009097:Multienzyme Complexes / Q000502:physiology; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology; D010873:Pinocytosis / Q000187:drug effects / Q000502:physiology; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D017473:Receptors, Opioid, kappa / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases; D020696:rab5 GTP-Binding Proteins / Q000502:physiology
chemical_list: D018712:Analgesics, Non-Narcotic; D000701:Analgesics, Opioid; D019390:Arrestins; D009097:Multienzyme Complexes; D010750:Phosphoproteins; D017473:Receptors, Opioid, kappa; D005048:Etorphine; D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D020696:rab5 GTP-Binding Proteins; D034281:Dynamins