Mechanisms of agonist-induced down-regulation of the human kappa-opioid receptor: internalization is required for down-regulation

• Published in: Molecular pharmacology 2000-10, Vol.58 (4), p.795-801
• Main Authors: Li, J G, Benovic, J L, Liu-Chen, L Y
• Format: Article
• Language: English
• Subjects: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer - pharmacology; Analgesics, Non-Narcotic - pharmacology; Analgesics, Opioid - pharmacology; Animals; Arrestins - genetics; Arrestins - physiology; beta-Adrenergic Receptor Kinases; CHO Cells; Cricetinae; Cyclic AMP-Dependent Protein Kinases - genetics; Cyclic AMP-Dependent Protein Kinases - physiology; Cysteine Endopeptidases - physiology; Down-Regulation; Dynamin I; Dynamins; Etorphine - pharmacology; GTP Phosphohydrolases - genetics; GTP Phosphohydrolases - physiology; Humans; Lysosomes - physiology; Multienzyme Complexes - physiology; Mutation; Phosphoproteins - genetics; Phosphoproteins - physiology; Pinocytosis - drug effects; Pinocytosis - physiology; Proteasome Endopeptidase Complex; rab5 GTP-Binding Proteins - physiology; Rats; Receptors, Opioid, kappa - agonists; Receptors, Opioid, kappa - genetics; Receptors, Opioid, kappa - metabolism; Transfection
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.58.4.795

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.