Molecular basis for GPCR signaling fine-tuning in neurons

意义：G蛋白偶联受体(GPCRs)激活异源三聚体G蛋白，它们共同形成 细胞中发现的最重要的信号轴之一。因为GPCR是非常常见的目标 治疗药物，其调节机制具有很高的生物医学重要性。尽管它 已知有许多细胞质因子调节G蛋白在GPCR介导的激活后的活性，它们 作为一个尚未开发的治疗干预机会，目前仍未得到充分研究。我在这里的目标是 鉴定一种新的G蛋白胞质调节因子，它通过调节 神经传递，并已被证明与神经疾病的背景相关，包括 慢性疼痛和癫痫。目前这些疾病的治疗方法包括令人上瘾的阿片类药物，用于治疗疼痛， 或者是一种反复尝试的癫痫药物寻找过程，仍然有大约三分之一的患者患有 无效的治疗方法。为此，在这项建议中，我将通过这部小说来研究分子机制 调节因子调控GPCRG蛋白神经元信号转导。阐明其发生的分子机制 具有重要生理意义的G蛋白调节剂是开发新型靶向药物的第一步 治疗因gpcr信号失控引起的疾病。 背景：在筛选与GαI亚基结合的G蛋白激活剂的过程中，我的赞助人的 实验室发现了一种蛋白质，它通过一种独特而新颖的机制来调节G蛋白。我们创造了这个词 “矛盾G蛋白调节剂”(PGR)表达它上调某些G蛋白的调节 同时下调其他G蛋白效应器的调节。其他人已经找到了 这种“PGR”的缺失改变了周围神经系统神经元中的GPCR信号，并导致慢性 疼痛。最近，我的赞助人的实验室发现，PGR KO小鼠的癫痫发作也增加了 敏感度。尽管它具有明显的生物医学重要性，但这种G蛋白的分子机制 调节器起作用，它是否调节脑神经元的神经传递是完全未知的。 AIMS简介：基于令人信服的初步数据，我认为PGR调节GαI-和 Gβγ依赖的信号转导而不直接影响G蛋白的酶活性(即核苷酸结合 和/或水解)，并且这一新的机制微调了脑神经元中的GPCR信号。在目标1中，我将 剖析PGR如何在重组系统(体外和基于细胞)中调节G蛋白信号，而 在AIM#2中，我将结合质谱学和诱变来描述它是如何与GαI发生物理作用的。 在AIM#3中，我将描述内源性PGR如何通过使用初级的 野生型和KO小鼠神经元的培养。共同努力，我的目标的实现将导致 对以前未被描述的GPCR信号调节机制的剖析 对正常神经功能和神经疾病，如慢性疼痛和癫痫的后果。

SIGNIFICANCE: G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins, which together form one of the most important signaling axes found in the cell. Because GPCRs are very common targets for therapeutic drugs, the mechanisms that underlie their regulation are of high biomedical importance. Although it is known that many cytoplasmic factors regulate the activity of G proteins after GPCR-mediated activation, they remain greatly understudied as an untapped opportunity for therapeutic intervention. My goal here is to characterize a novel cytoplasmic regulator of G proteins that operates through modulation of neurotransmission, and has been shown to be relevant in the context of neurological disorders including chronic pain and epilepsy. Current treatments for these diseases include addictive opioids in the case of pain, or a trial-and-error drug seeking process for epilepsy that still leaves approximately 1/3 of patients with ineffective treatments. For this reason, in this proposal I will study the molecular mechanism by how this novel regulator controls GPCR-G protein neuronal signaling. Elucidating the molecular mechanisms of this physiologically important G protein regulator is a first step towards the development of novel targeted treatments for diseases that arise from dysregulated GPCR signaling. BACKGROUND: In the course of a screen for G protein activators that bind to Gαi subunits, my Sponsor's laboratory identified a protein that regulates G proteins via a unique and novel mechanism. We coined the term “paradoxical G protein regulator” (PGR) to convey that it upregulates the modulation of some G protein effectors while simultaneously downregulating the modulation of other G protein effectors. Others had found that loss of this “PGR” alters GPCR signaling in neurons of the peripheral nervous system and causes chronic pain. More recently my Sponsor's laboratory has found that PGR KO mice also have increased seizure susceptibility. Despite its clear biomedical importance, the molecular mechanisms by which this G protein regulator operates, and whether it modulates neurotransmission in brain neurons are completely unknown. SYNOPSIS OF AIMS: Based on compelling preliminary data, I propose that the PGR modulates both Gαi- and Gβγ-dependent signaling without directly affecting the G protein enzymatic activity (i.e., nucleotide binding and/or hydrolysis), and that this novel mechanism fine tunes GPCR signaling in brain neurons. In AIM#1 I will dissect how the PGR regulates G protein signaling in reconstituted systems (in vitro and cell-based), whereas in AIM#2 I will characterize how it engages physically Gαi by combining mass-spectrometry and mutagenesis. In AIM#3 I will characterize how the endogenous PGR regulates neuronal GPCR signaling by using primary cultures of neurons from wild-type and KO mice. Together, the achievement of my goals will lead to the dissection of a previously uncharacterized mechanism of regulation of GPCR signaling with important consequences in normal neural function and neurological disorders such as chronic pain and epilepsy.