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Mol Pharmacol. 2004 Mar;65(3):528-37.

Distinct domains of the mu-opioid receptor control uncoupling and internalization.

Author information

1
Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195-7280, USA.

Abstract

Homologous desensitization of the micro opioid receptor (muOR) can be resolved into distinct processes that include the uncoupling of the muOR from its G-protein effectors and internalization of cell surface receptors. Using electrophysiological recordings of muOR activation of G-protein-coupled K+ channels (Kir3) in Xenopus laevis oocytes and AtT20 cells, confocal microscopy of receptor localization, and radioligand binding of cell surface receptors, we resolved these desensitization mechanisms to determine the domain of muOR important for receptor uncoupling. Activation of muOR by saturating concentrations of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), methadone, or fentanyl, but not morphine, produced robust internalization of a green fluorescent protein-tagged muOR. A subsaturating concentration of DAMGO (100 nM) did not cause receptor internalization but markedly reduced the subsequent responsiveness of Kir3 by uncoupling muOR. muOR desensitization in AtT20 cells was confirmed to be homologous, because desensitization by 100 nM DAMGO was blocked by dominant-negative forms of either G protein-coupled receptor kinase (GRK) or arrestin, and pretreatment with DAMGO did not affect the Kir3 response to somatostatin receptor activation. Alanine substitution of a single threonine in the second cytoplasmic loop of the muOR (Threonine 180) blocked agonist-dependent receptor uncoupling without affecting receptor internalization. These results suggest that GRK-dependent phosphorylation of muOR required threonine 180 for uncoupling but that a different GRK and arrestin-dependent mechanism controlled muOR internalization in AtT20 cells.

PMID:
14978231
DOI:
10.1124/mol.65.3.528
[Indexed for MEDLINE]

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