Differences in the ability of opioid drugs to promote regulated endocytosis of m-opioid receptors are related to their tendency to produce drug tolerance and dependence. Here we show that drugspecific differences in receptor internalization are determined by a conserved, 10-residue sequence in the receptor's carboxylterminal cytoplasmic tail. Diverse opioids induce receptor phosphorylation at serine (S)375, present in the middle of this sequence, but opioids differ markedly in their ability to drive higher-order phosphorylation on flanking residues [threonine (T)370, T376, and T379]. Multi-phosphorylation is required for the endocytosispromoting activity of this sequence and occurs both sequentially and hierarchically, with S375 representing the initiating site. Higherorder phosphorylation involving T370, T376, and T379 specifically requires GRK2/3 isoforms, and the same sequence controls opioid receptor internalization in neurons. These results reveal a biochemical mechanism differentiating the endocytic activity of opioid drugs.

This article is part of a themed section on Emerging Areas of Opioid Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.14/issuetoc.

Background and Purpose Homologous agonist‐induced phosphorylation of the μ‐opioid receptor (MOR) is initiated at the carboxyl‐terminal S375, followed by phosphorylation of T370, T376 and T379. In HEK293 cells, this sequential and hierarchical multi‐site phosphorylation is specifically mediated by G‐protein coupled receptor kinases 2 and 3. In the present study, we provide evidence for a selective and dose‐dependent phosphorylation of T370 after activation of PKC by phorbol esters. Experimental Approach We used a combination of phospho site‐specific antibodies, kinase inhibitors and siRNA knockdown screening to identify kinases that mediate agonist‐independent phosphorylation of the MOR in HEK293 cells. In addition, we show with phospho site‐specific antibodies were also used to study constitutive phosphorylation at S363 of MORs in mouse brain in vivo. Key Results Activation of PKC by phorbol esters or heterologous activation of substance P receptors co‐expressed with MORs in the same cell induced a selective and dose‐dependent phosphorylation of T370 that specifically requires the PKCα isoform. Inhibition of PKC activity did not compromise homologous agonist‐driven T370 phosphorylation. In addition, S363 was constitutively phosphorylated in both HEK293 cells and mouse brain in vivo. Constitutive S363 phosphorylation required ongoing PKC activity. When basal PKC activity was decreased, S363 was also a substrate for homologous agonist‐stimulated phosphorylation. Conclusions and Implications Our results have disclosed novel mechanisms of heterologous regulation of MOR phosphorylation by PKC. These findings represent a useful starting point for definitive experiments elucidating the exact contribution of PKC‐driven MOR phosphorylation to diminished MOR responsiveness in morphine tolerance and pathological pain.

Agonists of the nociceptin/orphanin FQ opioid peptide (NOP) receptor, a member of the opioid receptor family, are under active investigation as novel analgesics, but their modes of signaling are less well characterized than those of other members of the opioid receptor family. Therefore, we investigated whether different NOP receptor ligands showed differential signaling or functional selectivity at the NOP receptor. Using newly developed phosphosite-specific antibodies to the NOP receptor, we found that agonist-induced NOP receptor phosphorylation occurred primarily at four carboxyl-terminal serine (Ser) and threonine (Thr) residues, namely Ser346, Ser351, Thr362, and Ser363, and proceeded with a temporal hierarchy, with Ser346 as the first site of phosphorylation. G protein–coupled receptor kinases 2 and 3 (GRK2/3) cooperated during agonist-induced phosphorylation, which in turn facilitated NOP receptor desensitization and internalization. A comparison of structurally distinct NOP receptor agonists revealed dissociation in functional efficacies between G protein–dependent signaling and receptor phosphorylation. Furthermore, in NOP-eGFP and NOP-eYFP mice, NOP receptor agonists induced multisite phosphorylation and internalization in a dose-dependent and agonist-selective manner that could be blocked by specific antagonists. Our study provides new tools to study ligand-activated NOP receptor signaling in vitro and in vivo. Differential agonist-selective NOP receptor phosphorylation by chemically diverse NOP receptor agonists suggests that differential signaling by NOP receptor agonists may play a role in NOP receptor ligand pharmacology.

The efficiency of μ‐opioid receptor signalling is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. Here, we review and discuss recent progress in the generation and application of phosphosite‐specific μ‐opioid receptor antibodies, which have proved to be excellent tools for monitoring the spatial and temporal dynamics of receptor phosphorylation and dephosphorylation. Agonist‐induced phosphorylation of μ‐opioid receptors occurs at a conserved 10 residue sequence 370TREHPSTANT379 in the receptor's carboxyl‐terminal cytoplasmic tail. Diverse opioids induce receptor phosphorylation at S375, present in the middle of this sequence, but only high‐efficacy opioids have the ability to drive higher order phosphorylation on flanking residues (T370, T376 and T379). S375 is the initiating residue in a hierarchical phosphorylation cascade. In contrast, agonist‐independent heterologous μ‐opioid receptor phosphorylation occurs primarily at T370. The combination of phosphosite‐specific antibodies and siRNA knockdown screening also facilitated the identification of relevant kinases and phosphatases. In fact, morphine induces a selective S375 phosphorylation that is predominantly catalysed by GPCR kinase 5 (GRK5), whereas multisite phosphorylation induced by high‐efficacy opioids specifically requires GRK2/3. By contrast, T370 phosphorylation stimulated by phorbol esters or heterologous activation of Gq‐coupled receptors is mediated by PKCα. Rapid μ‐opioid receptor dephosphorylation occurs at or near the plasma membrane and is catalysed by protein phosphatase 1γ (PP1γ). These findings suggest that there are distinct phosphorylation motifs for homologous and heterologous regulation of μ‐opioid receptor phosphorylation. However, it remains to be seen to what extent different μ‐opioid receptor phosphorylation patterns contribute to the development of tolerance and dependence in vivo. Linked Articles This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2

The frequent overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of novel multireceptor somatostatin analogs. Although the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. Here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (T333) and 347 (T347), which enabled us to selectively detect either the T333-phosphorylated or the T347-phosphorylated form of sst5. We show that agonist-mediated phosphorylation occurs at T333, whereas T347 is constitutively phosphorylated in the absence of agonist. We further demonstrate that the multireceptor somatostatin analog pasireotide and the sst5-selective ligand L-817,818 but not octreotide or KE108 were able to promote a detectable T333 phosphorylation. Interestingly, BIM-23268 was the only sst5 agonist that was able to stimulate T333 phosphorylation to the same extent as natural somatostatin. Agonist-induced T333 phosphorylation was dose-dependent and selectively mediated by G protein-coupled receptor kinase 2. Similar to that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. However, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were rapidly completed within minutes. We also identify protein phosphatase 1γ as G protein-coupled receptor phosphatase for the sst5 receptor. Together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal T333. In addition, we identify G protein-coupled receptor kinase 2-mediated phosphorylation and protein phosphatase 1γ-mediated dephosphorylation of T333 as key regulators of rapid internalization and recycling of the human sst5 receptor.

Phosphorylation sites of KOPR following treatment with the selective agonist U50,488H were identified after affinity purification, SDS-PAGE, in-gel digestion with Glu-C and LC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated S356, T357, T363 and S369 in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pS356/pT357, pT363, and pS369, respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pT363, pS369 and pS356/pT357 antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. S369 phosphorylation affected T363 phosphorylation and vice versa and T363 or S369 phosphorylation was important for S356/T357 phosphorylation, revealing phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at S356/T357, T363 and S369 by immunoblotting. Using SILAC (stable isotope labeling by amino acids in cell culture) and LC-MS/MS, we found that compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at T363 and S369 with U/E ratio of 2.5 and 2, respectively. Both induced double phosphorylation at T363+S369 and T357+S369 with ratios of U/E=3.3 and 3.4, respectively. Only U50,488H induced triple phosphorylation at S356+T357+S369. An unphosphorylated KOPR(354–372) fragment containing all the phosphorylation sites was detected with a ratio of C/E/U =1/0.7/0.4, indicating that ~60% and ~30% of the mKOPR are phosphorylated following U50,488H and etorphine, respectively. Thus, KOPR internalization requires receptor phosphorylation above a certain threshold and higher-order KOPR phosphorylation may be disproportionally important.

the δ-opioid receptor (Dop) is an attractive pharmacological target due to its potent analgesic, anxiolytic and anti-depressant activity in chronic pain models. However, some but not all selective DOP agonists also produce severe adverse effects such as seizures. Thus, the development of novel agonists requires a profound understanding of their effects on DOP phosphorylation, post-activation signaling and dephosphorylation. Here we show that agonist-induced Dop phosphorylation at threonine 361 (T361) and serine 363 (S363) proceeds with a temporal hierarchy, with S363 as primary site of phosphorylation. This phosphorylation is mediated by G protein-coupled receptor kinases 2 and 3 (GRK2/3) followed by DOP endocytosis and desensitization. DOP dephosphorylation occurs within minutes and is predominantly mediated by protein phosphatases (PP) 1α and 1β. A comparison of structurally diverse Dop agonists and clinically used opioids demonstrated high correlation between G protein-dependent signaling efficacies and receptor internalization. In vivo, Dop agonists induce receptor phosphorylation in a dose-dependent and agonist-selective manner that could be blocked by naltrexone in Dop-eGfp mice. together, our studies provide novel tools and insights for ligandactivated Dop signaling in vitro and in vivo and suggest that DOP agonist efficacies may determine receptor post-activation signaling. The δ-opioid (DOP) receptor, as member of the opioid receptor family, was first discovered in 1975, based on the preference of [Leu]-enkephalin binding to receptors in mouse vas deferens, significantly later followed by the cloning of the single-copy gene OPRD for DOP receptor 1-3. The endogenous enkephalins ([Met]-enkephalin and [Leu]-enkephalin), and the frog skin peptides dermenkephalin and deltorphins I and II were identified as naturally-occurring ligands 4-6. Deltorphins have high DOP receptor selectivity, whereas enkephalins are moderately DOP receptor-selective 4. Through coupling to Gα i /Gα 0 proteins, DOP receptor activation leads to inhibition of cAMP production and voltage-gated calcium channels (N-and P/Q-type), as well as induction of β-arrestin signaling and activation of G protein-coupled inwardly rectifying potassium (GIRK) channels 7-10. In addition, signaling kinases such as ERK, c-Jun N-terminal kinase (JNK), src, Akt, p38 mitogen-activated protein kinase (p38 MAPK) or phospholipase C (PLC) and phospholipase A 2 (PLA2) are also activated by DOP receptors 11-17. DOP receptor mRNA and protein are widely expressed throughout the brain, spinal cord and dorsal root ganglia (DRG) 18-21. The DOP receptor is involved in the regulation of important physiological processes such as thermal and mechanical hyperalgesia, chronic inflammatory pain, anxiety and depression, migraine, locomotion, seizures, emotions, learning and memory, as well as addiction and tolerance development 22-26. DOP receptor is also involved in wound healing, neuronal, retinal and cardiovascular cytoprotection during hypoxia, as well as cardioprotection d...