Modulating -Opioid Receptor Phosphorylation Switches Agonist-dependent Signaling as Reflected in PKC Activation and Dendritic Spine Stability

Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455-0217, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2011; 286(14):12724-33. DOI: 10.1074/jbc.M110.177089
Source: PubMed


A new role of G protein-coupled receptor (GPCR) phosphorylation was demonstrated in the current studies by using the μ-opioid receptor (OPRM1) as a model. Morphine induces a low level of receptor phosphorylation and uses the PKCε pathway to induce ERK phosphorylation and receptor desensitization, whereas etorphine, fentanyl, and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) induce extensive receptor phosphorylation and use the β-arrestin2 pathway. Blocking OPRM1 phosphorylation (by mutating Ser363, Thr370 and Ser375 to Ala) enabled etorphine, fentanyl, and DAMGO to use the PKCε pathway. This was not due to the decreased recruitment of β-arrestin2 to the receptor signaling complex, because these agonists were unable to use the PKCε pathway when β-arrestin2 was absent. In addition, overexpressing G protein-coupled receptor kinase 2 (GRK2) decreased the ability of morphine to activate PKCε, whereas overexpressing dominant-negative GRK2 enabled etorphine, fentanyl, and DAMGO to activate PKCε. Furthermore, by overexpressing wild-type OPRM1 and a phosphorylation-deficient mutant in primary cultures of hippocampal neurons, we demonstrated that receptor phosphorylation contributes to the differential effects of agonists on dendritic spine stability. Phosphorylation blockage made etorphine, fentanyl, and DAMGO function as morphine in the primary cultures. Therefore, agonist-dependent phosphorylation of GPCR regulates the activation of the PKC pathway and the subsequent responses.

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    • "The precise molecular mechanism by which this desensitization takes place depends on the opioid agonist used to activate the receptor. For example, highefficacy agonists such as DAMGO and Met-enkephalin induce MOR desensitization largely through a GPCR kinase (GRK)and arrestin-dependent mechanism, whereas lower-efficacy agonists such as the prototypical opioid agonist, morphine, induce desensitization largely through a PKC-dependent mechanism (Kovoor et al., 1998; Whistler and von Zastrow, 1998; Johnson et al., 2006; Feng et al., 2011; Grecksh et al., 2011; Zheng et al., 2011; Bailey et al., 2009; Levitt and Williams, 2012; Williams et al., 2013). "
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    ABSTRACT: The majority of studies examining desensitization of the μ-opioid receptor (MOPr) have examined those located at cell bodies. However, MOPrs are extensively expressed at nerve terminals throughout the mammalian nervous system. This study is designed to investigate agonist-induced MOPr desensitization at nerve terminals in the mouse ventral tegmental area (VTA). μ-opioid receptor function was measured in mature mouse brain slices containing the VTA using whole-cell patch-clamp electrophysiology. Presynaptic MOPr function was isolated from postsynaptic function and the functional selectivity, time-dependence and mechanisms of agonist-induced MOPr desensitization were examined. MOPrs located at GABAergic nerve terminals in the VTA were completely resistant to rapid desensitization induced by the high efficacy agonists DAMGO and met-enkephalin. MOPrs located postsynaptically on GABAergic cell bodies readily underwent rapid desensitization in response to DAMGO. However, after prolonged (>7 hour) treatment with met-enkephalin, profound homologous MOPr desensitization was observed. Morphine could induce rapid MOPr desensitization at nerve terminals when protein kinase C was activated. Agonist-induced MOPr desensitization in GABAergic neurons in the VTA is compartment-selective as well as agonist-selective. When MOPrs are located at cell bodies, higher-efficacy agonists induce greater levels of rapid desensitization than lower-efficacy agonists. However, the converse is true at nerve terminals where agonists that induce MOPr desensitization via PKC are capable of rapid agonist-induced desensitization while higher efficacy agonists are not. MOPr desensitization induced by higher efficacy agonists at nerve terminals only takes place after prolonged receptor activation.
    Full-text · Article · Jan 2014 · British Journal of Pharmacology
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    • "In locus ceruleus neurons, morphine-induced desensitization was shown to depend on PKC whereas DAMGO-induced desensitization is mediated by GRK2 and independent of PKC [20]. It is suggested that DAMGO- and morphine-bound MORs assume differential conformations that are targeted by different GRKs to give rise to various efficiencies in receptor internalization [16,21,22] and changes in the responses to PKC [23]. We also found that IK desensitization in hMOR expressing DRG neurons depends on opioid agonists. "
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    ABSTRACT: Phosphorylation sites in the C-terminus of mu-opioid receptors (MORs) are known to play critical roles in the receptor functions. Our understanding of their participation in opioid analgesia is mostly based on studies of opioid effects on mutant receptors expressed in in vitro preparations, including cell lines, isolated neurons and brain slices. The behavioral consequences of the mutation have not been fully explored due to the complexity in studies of mutant receptors in vivo. To facilitate the determination of the contribution of phosphorylation sites in MOR to opioid-induced analgesic behaviors, we expressed mutant and wild-type human MORs (hMORs) in sensory dorsal root ganglion (DRG) neurons, a major site for nociceptive (pain) signaling and determined morphine- and the full MOR agonist, DAMGO,-induced effects on heat-induced hyperalgesic behaviors and potassium current (IK) desensitization in these rats. A mutant hMOR DNA with the putative phosphorylation threonine site at position 394 replaced by an alanine (T394A), i.e., hMOR-T, or a plasmid containing wild type hMOR (as a positive control) was intrathecally delivered. The plasmid containing GFP or saline was used as the negative control. To limit the expression of exogenous DNA to neurons of DRGs, a neuron-specific promoter was included in the plasmid. Following plasmid injection, hMOR-T and hMOR were expressed in small and medium DRG neurons. Compared with saline or GFP rats, the analgesic potency of morphine was increased to a similar extent in hMOR-T and hMOR rats. Morphine induced minimum IK desensitization in both rat groups. In contrast, DAMGO increased analgesic potency and elicited IK desensitization to a significantly less extent in hMOR-T than in hMOR rats. The development and extent of acute and chronic tolerance induced by repeated morphine or DAMGO applications were not altered by the T394A mutation. These results indicate that phosphorylation of T394 plays a critical role in determining the potency of DAMGO-induced analgesia and IK desensitization, but has limited effect on morphine-induced responses. On the other hand, the mutation contributes minimally to both DAMGO- and morphine-induced behavioral tolerance. Furthermore, the study shows that plasmid gene delivery of mutant receptors to DRG neurons is a useful strategy to explore nociceptive behavioral consequences of the mutation.
    Full-text · Article · Dec 2013 · Molecular Pain
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    • "Recently, the extent of receptor phosphorylation was shown to regulate the agonist-specific activation of PKC (Zheng et al., 2011). The overexpression of GRK2 to increase morphineinduced receptor phosphorylation impairs its ability to activate PKC, whereas the blockade of receptor phosphorylation switches etorphine and DAMGO signaling from b-arrestin dependent to PKC dependent. "
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    ABSTRACT: Protein kinase C (PKC) activation plays an important role in morphine-induced μ-opioid receptor (OPRM1) desensitization and tolerance development. It was recently shown that receptor phosphorylation by G protein-coupled receptor kinase regulates agonist-dependent selective signaling and that inefficient phosphorylation of OPRM1 leads to PKCε activation and subsequent responses. Here, we demonstrate that such receptor phosphorylation and PKCε activation can be modulated by FK506 binding protein 12 (FKBP12). Using a yeast two-hybrid screen, FKBP12 was identified to specifically interact with OPRM1 at the Pro353 residue. In HEK293 cells expressing OPRM1, the association of FKBP12 with OPRM1 decreased the agonist-induced receptor phosphorylation at Ser(375). The morphine-induced PKCε activation and the recruitment of PKCε to the OPRM1 signaling complex were attenuated both by FKBP12 siRNA treatment and in cells expressing OPRM1 with a P353A mutation (OPRM1P353A), which leads to diminished activation of PKC-dependent extracellular signal-regulated kinases. Meanwhile, the over-expression of FKBP12 enabled etorphine to activate PKCε. Further analysis of the receptor complex demonstrated that morphine treatment enhanced the association of FKBP12 and calcineurin with the receptor. The blockade of the FKBP12 association with the receptor by the siRNA-mediated knockdown of endogenous FKBP12 or the mutation of Pro(353) to Ala resulted in a reduction in PKCε and calcineurin recruitment to the receptor signaling complex. The receptor-associated calcineurin modulates OPRM1 phosphorylation, as demonstrated by the ability of the calcineurin autoinhibitory peptide to increase the receptor phosphorylation. Thus, the association of FKBP12 with OPRM1 attenuates the phosphorylation of the receptor and triggers the recruitment and activation of PKCε.
    Preview · Article · Oct 2013 · Molecular pharmacology
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