Browsing by Subject "G protein-coupled receptor"

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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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    Agonist-selective signaling and MOR.
    (2009-12) Zheng, Hui
    Opioids are potent analgesics, but their application is limited by the development of tolerance (the increase in doses required to achieve the same effect) after chronic or repetitive usage. Because tolerance developed much more easily for the analgesia effect than for side effects (e.g. respiration depression), it is difficult to be overcome by simply increasing the doses of opioids. On the other hand, the identification of opioid receptors and endogenous agonists suggests the involvement of opioid pathways in the central nervous system. Thus exploring the mechanisms of tolerance development has been the focus of a vast number of laboratories for decades. Several hypotheses on tolerance development have been proposed. For example, because of the correlation between receptor internalization and tolerance development, receptor internalization has been considered as an inhibitor of tolerance. In addition, the involvement of δ-opioid receptor preproenkephalin, Ca2+/calmodulin-dependent protein kinase II, Protein Kinase C and β-arrestin2 has been suggested by knockout experiments. However, there is no universal explanation for tolerance development. The long-term goal of my studies is to understand the mechanism of tolerance development. Taking advantage of the observations that opioids have different abilities to induced tolerance and signaling events, I have proposed is that agonists induce different levels of tolerance by inducing different signaling events (agonist-selective signaling). Because of the inconsistency between the time courses of signaling cascades (usually seconds to hours) and the tolerance development (usually hours to days), It is suggested that the agonist-selective regulation on gene expression transduces the signals from the agonist-selective signaling to agonist-selective tolerance development. Hence, in my studies, the different abilities of agonists to initiate signaling events (ERK phosphorylation, receptor desensitization on intracellular calcium release) were compared. Then several determinants (receptor phosphorylation, cholesterol-rich lipid raft microdomain, receptor palmitoylation) for the agonist-selective signaling were identified. The final portion of my studies is to explore how agonist-selective regulation on gene expression (NeuroD and miR-190) results from the agonist-selective signaling and to determine whether the agonist-selective regulation on gene expression can contribute to the different levels of tolerance induced by agonists.