G alpha Z subunit as a potential therapeutic target to modulate mu opioid receptor pharmacology

项目概要/摘要 本研究的目的是确定Gαz信号在调节阿片药理学中的作用。 为了产生各种各样的细胞反应，所有G蛋白偶联受体（GPCR）都偶联到 异源三聚体G蛋白作为受体和下游效应子之间的中间体。 有16种不同的Gα亚基，5种Gβ亚基和12种Gγ亚基。由于G蛋白的数量 亚基，存在许多可能的三聚体组合。Gα蛋白通过结合控制信号持续时间 GDP或GTP。阿片受体（OR）主要与Gαi/o类偶联，包括Gα i 1， Gαi2、Gαi3、GαoA、GαoB和Gαz。 先前的研究表明，缺乏Gαz的小鼠具有加快的吗啡抗伤害感受速率 宽容发展。敲除G蛋白信号调节因子（RGS）RGSz 1， 信号传导，并增加小鼠中莫尔激动剂的镇痛功效，并延迟 吗啡耐受总的来说，通过Gαz增强信号传导增加吗啡镇痛功效 并减少吗啡耐受性的发展。 我们初步发表的研究，使用生物发光共振能量转移（BRET）和HEK 用κ阿片受体（KOR）转染的293 T细胞显示κ阿片受体的不同效力和功效。 当KOR通过不同的Gα亚基传递信号时，当大韩民国发出信号 Gαz，阿片受体激动剂，特别是部分激动剂，更有力，有时更有效， KOR通过其他Gα亚基传递信号。我们的假设是，μ阿片激动剂通过 与Gαz耦合的莫尔将比当莫尔信号时更有效，有时更有效 通过其他Gαi/o亚基。通过了解阿片药理学如何受到莫尔信号的影响， 通过Gαz与Gαi/o家族的其他Gα亚基的比较， 通过Gαz增加或减少莫尔的信号传导，从而增加或降低效力， µ阿片激动剂的疗效。将使用BRET测定法研究以下特定目的，以测量 受体活化和稳态条件。1)将测试μ阿片激动剂的功效和效力 与通过Gαz亚基的莫尔信号传导进行比较，并将结果与通过Gαz亚基的信号传导进行比较。 其他Gα亚基。当莫尔通过Gαz进行信号传导时，阿片类药物完全或部分的简单分类 可以改变激动剂或拮抗剂。2)研究将确定RGSZ（即RGS 20）对 激活和失活动力学，并确定RGSZ是否影响阿片样物质的功效和效力， 莫尔通过Gαz发信号。总的来说，这些实验将确定莫尔信号是否通过 Gαz改变了莫尔药理学，如果增加或降低Gαz活性是一个潜在的治疗目标。

Project Summary/Abstract The goal of this study is to determine the role of Gαz signaling in modulating opioid pharmacology. To produce a wide variety of cellular responses, all G protein coupled receptors (GPCRs) couple to heterotrimeric G proteins to serve as the intermediaries between the receptor and downstream effectors. There are 16 different Gα subunits, five Gβ, and 12 Gγ subunits. Because of the number of G protein subunits, there are numerous possible trimer combinations. Gα proteins control signal duration by binding to either GDP or GTP. Opioid receptors (ORs) predominately couple to the Gαi/o class, comprised of Gαi1, Gαi2, Gαi3, GαoA, GαoB, and Gαz. Previous studies showed that mice lacking Gαz had an accelerated rate of morphine antinociceptive tolerance development. Knockout of the regulator of G protein signaling (RGS), RGSz1, increased Gαz signaling, and increased the analgesic efficacy of MOR agonists in mice and delayed the development of morphine tolerance. Collectively, enhancing signaling through Gαz increases morphine analgesic efficacy and reduces the development of morphine tolerance. Our preliminary published studies, using bioluminescence resonance energy transfer (BRET) and HEK 293T cells transfected with the κ opioid receptor (KOR), showed differential potency and efficacy of κ opioid agonists when the KOR signaled through different Gα subunits. When the KOR signaled through Gαz, opioid agonists, particularly partial agonists, were more potent and sometimes more efficacious than when the KOR signaled through other Gα subunits. Our hypothesis is that µ opioid agonists signaling through the MOR coupled to Gαz will be more potent and sometimes more efficacious than when the MOR signals through other Gαi/o subunits. By understanding how opioid pharmacology is affected by the MOR signaling through Gαz in comparison to other Gα subunits from the Gαi/o family, it may be possible to design therapeutics to increase or decrease signaling of the MOR through Gαz, thereby increasing or decreasing the potency and efficacy of µ opioid agonists. The following Specific Aims will be investigated using a BRET assay to measure receptor activation and steady-state conditions. 1) Mu opioid agonists will be tested for efficacy and potency with the MOR signaling through the Gαz subunit and results will be compared with signaling through other Gα subunits. When the MOR is signaling through Gαz, the simple classification of opioids as full or partial agonists or antagonists may be changed. 2) Studies will determine the effect of RGSZ, which is RGS20, on activation and deactivation kinetics and to determine if RGSZ affects opioid efficacy and potency when the MOR is signaling through Gαz. Collectively, these experiments will determine if MOR signaling through Gαz changes MOR pharmacology, and if increasing or decreasing Gαz activity is a potential therapeutic target.