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.
    British Journal of Pharmacology 01/2014; 172(2). DOI:10.1111/bph.12605 · 4.84 Impact Factor
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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.
    Molecular Pain 12/2013; 9(1):63. DOI:10.1186/1744-8069-9-63 · 3.65 Impact Factor
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    • "Using microarray analysis, we determined the opioid-induced changes in the expression profiles of miRNAs in primary cultures of hippocampal neurons and in mice hippocampi (Zheng et al., 2010d). Two opioids, morphine and fentanyl, were used in our studies, because of their different characteristics in inducing receptor internalization, receptor phosphorylation, and receptor desensitization (Keith et al., 1996; Zhang et al., 1998; Chu et al., 2010; Zheng et al., 2011). The two opioids induced similar changes in the expression of miR-224, miR-331, and miR-365, but had agonist-selective effects on the expression of miR-20a, miR-184, miR-190, and miR-301 (Zheng et al., 2010d). "
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    ABSTRACT: Non-coding RNAs (ncRNAs), especially microRNAs, are reported to be involved in a variety of biological processes, including several processes related to drug addiction. It has been suggested that the biological functions of opioids, one typical type of addictive drugs, are regulated by ncRNAs. In the current review, we examine a variety of mechanisms through which ncRNAs could regulate μ-opioid receptor (OPRM1) activities and thereby contribute to the development of opioid addiction. Using miR-23b as an example, we present the possible ways in which ncRNA-mediated regulation of OPRM1 expression could impact opioid addiction. Using miR-190 as an example, we demonstrate the critical roles played by ncRNAs in the signal cascade from receptor to systemic responses, including the possible modulation of adult neurogenesis and in vivo contextual memory. After discussing the possible targets of ncRNAs involved in the development of opioid addiction, we summarize the mechanisms underlying the interaction between ncRNAs and opioid addiction and present suggestions for further study.
    Frontiers in Genetics 06/2012; 3:113. DOI:10.3389/fgene.2012.00113
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