Chronic opioid receptor (OR) activation by morphine causes distinct cellular adaptations responsible for the development of tolerance. The present study examines the effect of chronic morphine exposure on the ability of high-efficacy agonists to mediate ␦-OR (DOR) and -OR (MOR) uncoupling and internalization, two regulatory mechanisms contributing to rapid desensitization of OR function. Chronic morphine treatment (1 M; 72 hr) of DOR carrying neuroblastoma x glioma (NG108-15) hybrid cells, a prototypical model system frequently used to study cellular aspects of opioid tolerance, completely blocked the capacity of [D-Ala 2 , D-Leu 5 ]enkephalin (DADLE) and etorphine to desensitize opioid-stimulated [ 35 S]GTP␥S binding and to mediate DOR internalization. Similar findings were obtained on stably DOR-and MOR-transfected human embryonic kidney (HEK) 293 cells. Chronic morphine treatment also heterologously impaired agonist regulation of non-opioid G-proteincoupled receptors, such as the m 4 -muscarinic acetylcholine receptor and the brain-type cannabinoid receptor. As a possible underlying mechanism, we found that chronic morphine treatment completely blocked agonist-induced redistribution of ␤-arrestin1 in both NG108-15 and stably MOR-transfected HEK293 cells. Moreover, attenuation of ␤-arrestin1 function appears to depend on persistent stimulation of MAP kinase activity during the course of chronic morphine treatment, because coincubation of the cells together with the MAP kinase blocker PD98059 fully restored ␤-arrestin1 translocation and receptor internalization. These results demonstrate that chronic morphine treatment produces adaptational changes at the ␤-arrestin1 level, which in turn attenuates agonist-mediated desensitization and internalization of G-protein-coupled receptors.

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Translocation of G protein-coupled receptors (GPCRs) from the cell membrane to cytosol depends on the kind of ligand activating the receptor. This principle is clearly demonstrated for opioid receptors, because diverse opiate agonists rapidly induce receptor internalization, whereas morphine almost fails. We report here the impact of mitogen-activated protein (MAP) kinase isoforms extracellular signal-regulated kinase (ERK)1/2 on the internalization of ␦-opioid receptors (DORs) expressed in human embryonic kidney (HEK)293 cells. Receptor activation by etorphine turned out to transiently phosphorylate ERK/MAP kinases and bring about DOR internalization within 20 min. In contrast, prolonged exposure of HEK293 cells to morphine excited persistent phosphorylation of ERK/MAP kinases, and those cells failed to internalize the opioid receptor. When ERK/ MAP kinase phosphorylation was blocked by 2Ј-Amino-3Ј-methoxyflavone (PD98059), morphine gained the ability to strongly induce DOR endocytosis. The importance of activated MAP kinases for DOR internalization is further demonstrated by glutamate and paclitaxel because these substances induce phosphorylation of ERK1/2 and concomitantly prevent DOR sequestration by etorphine. In addition, receptor internalization by morphine was facilitated by inhibition of protein kinase C and opioid-mediated transactivation of epidermal growth factor receptor (EGFR), both activating ERK/MAP kinases by opioids. The mechanism affording DOR internalization by PD98059 may relate to arrestin, which uncouples GPCRs and thus triggers receptor internalization. Arrestin considerably translocates toward the cell membrane upon DOR activation by morphine in presence of the MAP kinase blocker, but it fails in the absence of PD98059. We conclude that ERK/MAP kinase activity prevents opioid receptor desensitization and sequestration by blocking arrestin 2 interaction with activated DORs.

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