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Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington
Homologous desensitization of the µ opioid receptor (µOR) can be resolved into distinct processes that include the uncoupling of the µOR from its G-protein effectors and internalization of cell surface receptors. Using electrophysiological recordings of µOR 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 µOR important for receptor uncoupling. Activation of µOR 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 µOR. A subsaturating concentration of DAMGO (100 nM) did not cause receptor internalization but markedly reduced the subsequent responsiveness of Kir3 by uncoupling µOR. µOR 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 µOR (Threonine 180) blocked agonist-dependent receptor uncoupling without affecting receptor internalization. These results suggest that GRK-dependent phosphorylation of µOR required threonine 180 for uncoupling but that a different GRK and arrestin-dependent mechanism controlled µOR internalization in AtT20 cells.
Address correspondence to: Dr. Charles Chavkin, Department of Pharmacology, Box 357280, University of Washington School of Medicine, Seattle, Washington 98195-7280. E-mail: cchavkin{at}u.washington.edu
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