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

项目摘要/摘要 本研究的目的是确定G-α-z信号在阿片类药物药理调节中的作用。 为了产生各种各样的细胞反应，所有的G蛋白偶联受体(GPCRs)都连接到 异三聚体G蛋白作为受体和下游效应物之间的中介。 有16个不同的Gα亚基，5个Gβ亚基和12个Gγ亚基。因为G蛋白的数量 亚基，有许多可能的三聚体组合。G-α蛋白通过结合控制信号持续时间 GDP或GTP。阿片受体(ORs)主要与GαI/O类偶联，包括GαI1， Gαi2、Gαi3、GαOA、GαOB和Gαz。 先前的研究表明，缺乏Gαz的小鼠对吗啡的抗伤害性反应速度更快 宽容发展。敲除G蛋白信号调节因子RGSz1，增加Gαz 并增加MOR激动剂对小鼠的镇痛效果，延缓小鼠的发育。 吗啡耐受性。总体而言，通过Gαz增强信号可增加吗啡的止痛效果 并减少吗啡耐受性的发展。 我们发表的初步研究，使用生物发光共振能量转移(BRET)和HEK 转κ阿片受体(KOR)的293T细胞显示κ的不同效力和效果 当KOR通过不同的Gα亚基发出信号时，阿片激动剂。当韩国人发出信号通过 Gαz，阿片类激动剂，特别是部分激动剂，比 韩国人通过其他Gα亚基发出信号。我们的假设是µ阿片激动剂通过 耦合到Gαz的MOR将比MOR信号更有效，有时甚至更有效 通过其他GαI/O子单元。通过了解阿片类药物如何受到MOR信号的影响 通过Gαz与来自GαI/O家族的其他Gα亚基进行比较，可能设计出治疗方法 通过Gαz增加或减少MOR的信号，从而增加或减少效力和 微阿片类激动剂的疗效。将使用Bret检测来调查以下特定目标 受体激活和稳态条件。1)将测试Mu阿片类激动剂的疗效和效力 通过Gαz亚基发送MOR信号，并将结果与通过 其他Gα亚基。当MOR通过Gαz发出信号时，阿片类药物的简单分类为完全或部分 激动剂或拮抗剂可以改变。2)研究将确定RGSZ，即RGS20，对 激活和失活动力学，并确定RGSZ是否影响阿片类药物的疗效和效力 MOR通过Gαz发送信号。总的来说，这些实验将确定MOR信号是否通过 G-α-z改变MOR药理，如果增加或降低G-α-z活性是一个潜在的治疗靶点。

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.