Browsing by Subject "Opioid receptor"

Now showing 1 - 3 of 3
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    Agonist-dependent mechanism of Mu-opioid receptor desensitization.
    (2009-12) Chu, Ji
    Desensitization of the μ-opioid receptor (MOR) has been implicated as an important regulatory process in the development of tolerance to opiates. Desensitization of G-protein coupled receptor (GPCR) is thought to involve receptor phosphorylation and subsequent recruitment of βArrestins (βArrs). However, the roles of receptor phosphorylation and βArr in morphine-induced MOR desensitization remain to be demonstrated; this may result from the insensitivity of the methods used to study receptor function. Using MOR-induced intracellular Ca2+ ([Ca2+]i) release to monitor receptor activation, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) induced MOR desensitization in a receptor phosphorylation- and βArr-dependent manner. DAMGOinduced desensitization was blunted in HEK293 cells expressing the MORS375A mutant and was eliminated in MEF cells isolated from βArr2 knockout mice expressing the wild type MOR. However, although morphine induced a more rapid desensitization of [Ca2+]i release than DAMGO did and could induce the phosphorylation of the Ser375 residue of MOR, morphine-induced desensitization was not influenced by mutating MOR phosphorylation sites or in MEF cells lacking βArr1 and 2. In contrast, morphine induced MOR desensitization via protein kinase C (PKC). By using subtype-specific inhibitors, PKCε was shown to be the PKC subtype activated by morphine and the subtype responsible for morphine-induced desensitization. Meanwhile, DAMGO did not increase PKCε activity and DAMGO-induced MOR desensitization was not affected by a PKCε inhibitor. Among the various proteins within the receptor signaling complex, Gαi2 was phosphorylated by morphine-activated PKCε. Moreover, mutating v three putative PKC phosphorylation sites, Ser44, Ser144 and Ser302 on Gαi2 to Ala attenuated morphine-induced, but not DAMGO-induced desensitization. In addition, pretreatment with morphine desensitized cannabinoid receptor CB1 agonist WIN 55212-2-induced [Ca2+]i release, and this desensitization could be reversed by pretreating with a PKCε inhibitor or overexpressing of Gαi2 with the putative PKC phosphorylation sites mutated. Thus, depending on the agonist, activation of MOR could lead to heterologous desensitization and probable crosstalk between MOR and other Gαi-coupled receptors such as the CB1 receptor.
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    Alpha-2 adrenergic and opioid spinal analgesic synergy: utility and cellular mechanisms.
    (2010-06) Overland, Aaron C.
    Agonists acting at spinal α 2 -adrenergic receptors (α 2 AR) and opioid receptors (OR) produce analgesia through common intracellular signaling systems primarily mediated through inhibitory G proteins. Furthermore, co-activation of spinal α 2 AR and OR produces antinociceptive synergy. Synergistic analgesic interactions are important, as conventional opioid therapy is limited clinically due to the development of adverse side effects such as tolerance, dependence, abuse liability and opioid-induced hyperalgesia. Agonist combinations that interact synergistically may bypass these unwanted side effects by allowing decreases of analgesic dose and increasing the therapeutic index. Synergy between analgesic compounds has been shown experimentally and utilized clinically, yet the underlying cellular mechanisms mediating this phenomenon remain relatively unexplored. Elucidating the mechanisms underlying analgesic synergy may have broad clinical implications and may lead to the discovery of novel drug targets for pain management. The goal of this study was therefore to determine the cellular mechanisms mediating the synergistic interaction between agonists acting at two anatomically co-localized G protein-coupled receptors (GPCRs) in the spinal cord. In the first phase of these studies, we evaluated the ability of the selective delta-opioid receptor (DOP) agonist deltorphin II (DELT), the α 2 AR agonist clonidine (CLON) or their combination to inhibit nociceptive responses from mice in the tail flick test. We then examined the possible underlying signaling mechanisms involved through co-administration of inhibitors known to affect the above-mentioned receptor pair. Second, we looked at the ability of the DOP-selective agonist DELT, the α 2 AR agonist CLON, or their combination to inhibit calcitonin gene-related peptide (CGRP) release from spinal cord slices and spinal cord synaptosomes. Third, we determined the specific signaling mediator involved in α 2 AR/DOP analgesic synergy using genetically manipulated mice. We observed that the in vivo and in vitro synergistic interaction between agonists acting at α 2 AR/DOP is specifically mediated through activation of protein kinase C epsilon. These findings suggest that this particular enzyme could represent a novel pain therapy target.
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    Novel binding partners of Mu-opioid receptor and their regulatory roles.
    (2009-12) Ge, Xin
    G-protein-coupled receptors (GPCRs) represent a super-family of proteins in the human genome (>900), which include at least one third of current drug targets. Associated proteins of GPCRs consist of the down-stream signaling pathway, which convert the external stimulus to the final signal of cellular function change. Some useful methods have been used to explore the sophisticated network of GPCR-associated proteins, such as yeast-two hybridization and GST fusion protein pull-down assay. However, in both methods, only one or two domains of the receptor were used to construct a fusion protein for identifying scaffolding proteins. This cannot reflect the conditions in which an agonist/antagonist mediates the opioid receptors' conformational change that leads to protein recruitment. To identify the binding partners of MOR, a member of GPCR rhodopsin subfamily that clarified to be important in regulating drug tolerance and addiction, we purified MOR complexes from (His)6-tagged MOR stably expressing neuroblastma neuro2A (N2A) cells. Combine with Mass Spectrometry and LC MS/MS (Liquid chromatography-electrospray ionization tandem mass spectrometry), some novel MOR binding partners were found. Two of them are studied further about their important roles in regulating MOR functions. Ribophorin I (RPNI), a component of the oligosaccharidetransferase complex, could directly interact with MOR. RPNI can be shown to participate in MOR export by the intracellular retention of the receptor after siRNA knocking-down of endogenous RPNI. Over-expression of RPNI rescued the surface expression of the MOR 344KFCTR348 deletion mutant (C2) independent of calnexin. Furthermore, RPNI regulation of MOR trafficking is dependent on the glycosylation state of the receptor, as reflected in the inability of over-expression of RPNI to affect the trafficking of the N-glycosylation deficient mutants, or GPCR such as k-opioid receptor that has minimal glycosylation sites. Hence, this novel RPNI chaperone activity is a consequence of N-glycosylation-dependent direct interaction with MOR. G protein-regulated inducer of neurite outgrowth 1 (GRIN1) can influence MOR lipid raft location by tethering the receptor with the heterotrimeric G protein ?-subunit. GST fusion pull-down and receptor mutational analyses indicated the 267GSKEK271 sequence within the MOR 3rd intracellular loop was involved in interacting with the GRIN1 sequence distinct from that participated in the G? binding. The uncoupling of G? from MOR with PTX reduced the amount of GRIN1 co-immunoprecipitated with MOR while the amount of GRIN1 coimmunoprecipitated with G? was unchanged. Furthermore, over-expression of GRIN1 significantly enhanced the amount of MOR in lipid raft and the receptor signaling magnitude as measured by Src kinase activation. Such increase in MOR signaling was demonstrated further by determining the GRIN1-dependent neurite outgrowth. In contrast to minimal neurite outgrowth induced by etorphine in control cells, over-expression of GRIN1 and the increase in GRIN1-MOR interaction resulted in the increase in etorphine- and not-morphine-induced neurite outgrowth in the neuroblastoma N2A cell that was PTX sensitive. Knocking-down of endogenous GRIN1 by siRNA attenuated the agonist-induced neurite outgrowth. Disrupting lipid raft by M?CD also blocked neurite outgrowth. Hence, by serving as a tether between G? and MOR, GRIN1 stabilizes the receptor within the lipid rafts and potentiates the receptor signaling in the neurite outgrowth process.