Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinases Block Internalization of δ-Opioid Receptors

Abstract

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.

Serine 363 of the δ-opioid receptor is crucial for adopting distinct pathways to activate ERK1/2 in response to stimulation with different ligands

“…Recently, agonist-specific desensitization of ERK signaling pathway has been reported for the OR (Eisinger et al, 2002;Hong et al, 2009). In addition, differential kinetic patterns of ERK activation by individual OR agonists have also been observed (Eisinger and Schulz, 2004;Audet et al, 2005). For example, the duration of ERK activated by morphine and etorphine is quite different.…”

“…For example, the duration of ERK activated by morphine and etorphine is quite different. ERK1/2 activated by etorphine is quite transient, whereas ERK1/2 activated by morphine is quite persistent (Eisinger and Schulz, 2004). Moreover, such different patterns of ERK activation induced by morphine and etorphine have been linked to their ability to differently desensitize and internalize OR (Eisinger and Schulz, 2004;Eisinger et al, 2002;Audet et al, 2005).…”

“…S2). It has also been reported that differential temporal patterns of ERK activation can be detected upon activation of the ORs by distinct agonists (Eisinger et al, 2002;Eisinger and Schulz, 2004). However, the role of Ser363 in the C-terminal for the OR adoption of distinct pathways to activate ERK remains to be elucidated.…”

“…An intriguing feature of OR-mediated ERK signaling is the differential desensitization properties following activation by distinct agonists (Eisinger et al, 2002;Eisinger and Schulz, 2004;Hong et al, 2009). To further confirm the distinct regulation of ERK1/2 activity by DPDPE and TIPP, we also examined the effects of either DPDPE or TIPP pretreatment on subsequent ERK1/2 activation by a second DPDPE stimulus, in CHO cells that stably coexpressed ORs.…”

Ligand stabilization of CXCR4/δ‐opioid receptor heterodimers reveals a mechanism for immune response regulation

“…Synthetic ligands such as [D-Pen2, D-Pen5]enkephalin (DPDPE), which have greater affinity and selectivity for the DOR, were thus generated and are used in pharmacological studies [25]. After ligand binding, DOR activates Ga i , which induces adenylate cyclase inhibition, inhibits voltage-sensitive Ca 2+ channels, activates PKC, promotes Ca 2+ release from internal stores, recruits membrane proteins such as Ras-G protein regulatory factor, and activates the ERK/MAPK transduction cascade [26][27][28][29].We show that simultaneous stimulation of CXCR4-and DOR-expressing cells by their respective ligands, CXCL12 and DPDPE, does not trigger function. We excluded differences in cell surface receptor expression and receptor internalization, as well as heterologous desensitization processes, as the cause of this effect.…”