Molecular basis for GPCR signaling fine-tuning in neurons

意义：G蛋白偶联受体（GPCR）激活异源三聚体G蛋白， 细胞中最重要的信号轴之一。因为GPCR是非常常见的目标， 治疗药物，其调节机制具有高度的生物医学重要性。虽然 已知许多细胞质因子在GPCR介导的活化后调节G蛋白的活性，它们 作为一个尚未开发的治疗干预机会，仍然被大大低估。我的目标是 描述了一种新的G蛋白胞质调节因子，其通过调节 神经传递，并已被证明是相关的神经系统疾病的背景下，包括 慢性疼痛和癫痫。目前对这些疾病的治疗包括在疼痛的情况下使用成瘾性阿片类药物， 或者是一个试错式的癫痫药物寻求过程，仍然有大约1/3的患者 无效的治疗。为此，在本提案中，我将通过这部小说来研究分子机制 调节子控制GPCR-G蛋白神经元信号传导。阐明了这一分子机制 生理上重要的G蛋白调节剂是开发新型靶向药物的第一步。 治疗由GPCR信号失调引起的疾病。 背景：在筛选与Gαi亚基结合的G蛋白激活剂的过程中，我的申办者的 一个实验室发现了一种通过独特的新机制调节G蛋白的蛋白质。我们创造了一个术语 “矛盾的G蛋白调节因子”（PGR）来表达它上调某些G蛋白的调节 同时下调其他G蛋白效应子的调节。其他人发现 这种“PGR”的缺失改变了外周神经系统神经元中的GPCR信号，并导致慢性炎症。 痛苦最近，我的赞助商的实验室发现，PGR基因敲除小鼠也有增加癫痫发作 易感性尽管其明确的生物医学重要性，这种G蛋白的分子机制， 调节器的运作，以及它是否调节大脑神经元的神经传递是完全未知的。 目的概要：基于令人信服的初步数据，我提出PGR调节Gαi和 Gβγ依赖性信号传导而不直接影响G蛋白酶活性（即，核苷酸结合 和/或水解），并且这种新机制微调了脑神经元中的GPCR信号传导。在AIM#1中， 剖析PGR如何调节重组系统中的G蛋白信号传导（体外和基于细胞的），而 在AIM#2中，我将通过结合质谱和诱变来描述它如何与Gαi物理结合。 在AIM#3中，我将通过使用初级PCR来表征内源性PGR如何调节神经元GPCR信号传导。 来自野生型和KO小鼠的神经元培养物。我的目标的实现将导致 一个以前未表征的GPCR信号调节机制的解剖与重要的 这类药物对正常神经功能和神经系统疾病（如慢性疼痛和癫痫）的影响。

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.