Regulation of heterotrimeric G proteins by non-receptor activators

描述（由申请人提供）：Ric-8A和Ric-8B是异源三聚体G蛋白a亚基功能的正调节剂。我们最近定义了Ric-8蛋白对Ga亚基的细胞作用。Ric-8蛋白在生物合成Ga蛋白折叠过程中充当无核苷酸Ga构象的分子伴侣。在Ric-8基因缺失的细胞中，Ga亚基子集的细胞丰度降低了95%。Ric-8蛋白还表现出对Ga亚基的体外鸟嘌呤核苷酸交换刺激活性，并且我们现在认为这是源于Ric-8诱导对GDP具有降低的亲和力的部分折叠Ga状态的能力的体外现象。我们将发展我们的假设，即Ric-8蛋白是Ga亚基分子伴侣，并研究Ric-8蛋白与其他细胞伴侣包括TRiC/CCT（伴侣蛋白）复合物和HSC 70/90系统一起折叠单个Ga亚基的机制。我们有初步的证据表明，有复杂的规则和独特的伴侣要求个别Ga亚基。然后，我们将测试一个假设，即小鼠组织特异性Ric-8A基因缺失可以在遗传上抑制致癌G蛋白或过度活跃的GPCR驱动的癌症。将使用两种小鼠黑色素瘤模型，其中GNAQ/11-Q209 L（组成型活性）等位基因是转移性眼黑色素瘤的致癌驱动突变，并且来自黑色素细胞启动子的异位mGluR 5表达引起稳健的黑色素瘤。我们假设黑素细胞mGluR 5不适当地激活Gq/11以促进黑色素瘤。在Ric-8缺失将通过降低Gaq/11细胞丰度来抑制这些癌基因的作用的概念验证后，我们将进行筛选以鉴定Ric-8和G蛋白相互作用的小分子抑制剂。我们的目标是开发Ric-8抑制剂作为通过减少由Ric-8折叠的（突变）Ga亚基的丰度来间接阻断G蛋白疾病信号传导的手段。Ric-8抑制剂可以证明对致癌G蛋白疾病和/或作为现有GPCR疗法的增强疗法有效。我们还将进行一个小项目，研究Ric-8A通过磷酸化事件和14-3-3与Ric-8A结合的调节。

DESCRIPTION (provided by applicant): Ric-8A and Ric-8B are positive regulators of heterotrimeric G protein a subunit function. We have recently defined the cellular action of Ric-8 proteins towards Ga subunits. Ric-8 proteins act as molecular chaperones of nucleotide-free Ga conformations during biosynthetic Ga protein folding. In cells in which Ric-8 genes are deleted, cellular abundances of subsets of Ga subunits were reduced by 95%. Ric-8 proteins also exhibit in vitro guanine nucleotide exchange stimulatory activity towards Ga subunits, and we now believe this to be an in vitro phenomenon stemming from the capacity of Ric-8 to induce a partially folded Ga state that has reduced affinity for GDP. We will develop our hypothesis that Ric-8 proteins are Ga subunit molecular chaperones and investigate the mechanisms by which Ric-8 proteins work with other cellular chaperones including the TRiC/CCT (Chaperonin) complex and HSC70/90 systems to fold individual Ga subunits. We have preliminary evidence that there are complex rules and unique chaperone requirements of individual Ga subunits. We will then test a hypothesis that mouse tissue-specific Ric-8A gene deletion can genetically suppress oncogenic G protein or overactive GPCR-driven cancers. Two mouse melanoma models will be used in which GNAQ/11-Q209L (constitutively-active) alleles are the oncogenic driver mutations of metastatic ocular melanoma, and ectopic mGluR5 expression from a melanocyte promoter elicited robust melanoma. We hypothesize that melanocyte mGluR5 inappropriately activates Gq/11 to promote melanoma. Upon proof-of-concept that Ric-8 deletion will suppress the actions of these oncogenes by reducing Gaq/11 cellular abundances, we will perform screens to identify small molecule inhibitors of Ric-8 and G protein interactions. Our goal is to develop Ric-8 inhibitors as a means to indirectly block G protein disease signaling by reducing the abundance of (mutant) Ga subunits folded by Ric-8. Ric-8 inhibitors may prove efficacious against oncogenic G protein diseases and/or as augmentation therapies for existing GPCR therapeutics. We will also conduct a small project to investigate Ric-8A regulation by phosphorylation events and 14-3-3 binding to Ric-8A.