Hierarchical Phosphorylation of δ-Opioid Receptor Regulates Agonist-induced Receptor Desensitization and Internalization

Abstract

Treatment of HEK293 cells expressing the δ-opioid receptor with agonist [D-Pen 2,5 ]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e. Thr 358 and Ser 363 , were phosphorylated, with Ser 363 being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser 363 as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr 358 and Ser 363 , with Thr 358 being involved in the receptor internalization.Prolonged exposure to opioid drugs often produces tolerance, dependence, and addiction, and the molecular mechanisms underlying these phenomena are still poorly understood. Opioid receptors belong to the G protein-coupled receptor (GPCR)1 superfamily (1, 2). Therefore, one component of opioid tolerance is likely to be mediated by a phosphorylationdependent desensitization of the receptor. Phosphorylation of the prototypic β 2 -adrenergic receptor by protein kinases, including the G protein-coupled receptor kinases (GRKs), promotes the association with the receptor of inhibitory proteins called arrestins (3,4). This association uncouples the receptor from the G proteins and promotes targeting of activated receptors to clathrin-coated pits for subsequent internalization, thus blunting receptor signaling.Agonist-induced phosphorylation of opioid receptors has been demonstrated in different systems (5-10). Concrete demonstration of the phosphorylation of δ-opioid receptor (DOR) was first reported by Pei and colleagues (6). Although the sites of agonist-dependent phosphorylation have not yet been identified, truncation of the carboxyl tail (C-tail) of DOR showed that major phosphorylation sites are localized within this domain (11,12 Phosphorylation of DOR appears to be the mechanism for agonist-induced receptor desensitization (6,13,14). 5 ]enkephalin (DPDPE)-induced phosphorylation of DOR seems to involve one or more GRKs (6), and Ala substitution of the last four carboxylterminal Ser and Thr of the receptor impaired the GRK-and arrestin-mediated receptor desensitization (13). In Xenopus oocytes, co-expression of GRK3 and β-arrestin 2 resulted in an increased rate of agonist-induced homologous desensitization of DOR (13). However, truncation of the COOH-terminal 31 amino acids of DOR did not affect the agonist-induced desensitization of the receptor in CHO cells (15), unlike what Zhao and coll...

A Phosphorylation-regulated Brake Mechanism Controls the Initial Endocytosis of Opioid Receptors but Is Not Required for Post-endocytic Sorting to Lysosomes

“…However, because of the essentially complete blockade of DOR5A endocytosis, we are unable to exclude a possible additional role of the dephosphorylated tail in inhibiting later step(s) mediating trafficking of full-length (but not truncated) receptors to lysosomes. Such an additional role has been suggested previously in controlling post-endocytic trafficking of the full-length ␤ 2 -adrenergic receptor (30) and V2 vasopressin receptor (31,32), and is consistent with previous kinetic and mutational (22,28) studies of opioid receptor internalization and down-regulation. Future studies will be necessary to address this possibility and to elucidate the biochemical mechanism mediating phosphorylation-independent sorting of internalized opioid receptors to lysosomes.…”

“…The present results clarify the role of phosphorylation in controlling agonist-induced endocytic membrane trafficking of DOR, thus providing insight into differences in the conclusions of previous studies regarding whether or not phosphorylation is required for regulated endocytosis (10,21) and down-regulation of mutant opioid receptors (12,17,18,22,23,28). In particular, our results suggest that the distal portion of the carboxyl-terminal cytoplasmic domain of DOR is not required for regulated endocytosis but functions as an endocytic brake that is "released" by phosphorylation in order to allow endocytosis of full-length receptors to occur.…”

“…To examine whether phosphorylation was required for endocytosis of full-length receptors, we created a full-length mutant version of DOR in which all serine and threonine residues located in the carboxyl-terminal cytoplasmic tail were mutated to alanine (DOR5A mutant receptor). These include Thr-358 and Ser-363 shown to be primary sites of DOR phosphorylation (22,23). Consistent with these previous studies, the DOR5A full-length mutant receptor exhibited no detectable phosphorylation in intact cells, like the DOR344T truncated mutant receptor described previously (Fig.…”

Constitutive Internalization of the Leucine-rich G Protein-coupled Receptor-5 (LGR5) to the Trans-Golgi Network

“…However, from our data it appears that even if these sites are mutated, internalization still ensues but at a much slower rate. The requirement or redundancy of multiple residues regulating internalization has been observed for other receptors in which the most important residues serve some necessary binding or priming function (53). Interestingly, a putative "dileucine" motif, 867LV, resides just after those two Ser residues, whose phosphorylation could enhance the ability of this motif to interact with clathrin-coated pit adaptor proteins in an agonist-independent fashion (54, 55).…”

Adenosine A1 receptor agonist N6-cyclohexyl-adenosine induced phosphorylation of delta opioid receptor and desensitization of its signaling

“…Acute activation of A 1 R [5] and DOR [6] inhibit adenylyl cyclase activity via inhibitory G protein (G i ), and lead to a decrease in intracellular cAMP levels. However, upon prolonged exposure to their agonists, these receptors undergo desensitization which is indicated by the decreased ability to mediate agonistinduced inhibition of adenylyl cyclase [7][8][9][10][11] . Receptor desensitization was also measured by the activities of other downstream effectors stimulated by selective receptor agonists in various signaling pathway such as ERK1/2 and Akt/PKB [12] , which depends on not only the functional status of the receptor but also the signal transduction amplification between the receptor and the effectors [13] .…”