Abstract

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 (K_ir3) 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 K_ir3 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 K_ir3 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.