Structural and kinetic investigation of the guanine nucleotide exchange factor Ric-8A and its interaction with G proteins (Gαq/Gαi)

项目摘要/摘要 异三聚体G蛋白参与许多生理过程的调节，是必不可少的 到多个细胞信号通路。G蛋白异源三聚体是通过α，β和γ亚基结合而形成的 与G-α亚基结合的二磷酸胍。规范的信号传递机制包括 G-α亚基上的GDPGTP的置换及随后的解离 由Gβγ-gtp形成的Gα二聚体复合体；然后Gβγ和Gα-gtp复合体起调节下游的作用 细胞中的效应器。G蛋白偶联受体(Gpcr)是研究最深入的一类蛋白质。 负责催化国内生产总值在Gα亚基上交换为GTP。然而，其他非受体鸟嘌呤 交换因子蛋白也存在，并且已被证明对G蛋白信号的调节是至关重要的。 抗胆碱酯酶-8A(Ric-8A)是一种蛋白，已被证明是一种全球环境基金和 GI、GQ和G12/13家族Gα亚基的分子伴侣。尽管RIC-8A充当全球环境基金以激活 两者都是GαQ/GαI，以前的研究已经证明Ric-8A与GαQ的亲和力不同 与GαI结合，并且Ric-8A催化的鸟嘌呤核苷酸反应的动力学与这些因素有关 两个子类型。此外，Ric-8A包含几个磷酸化位点，并且Ric-8A的磷酸化形式具有 全球环境基金针对Gα亚基的活性增强。Ric-8A的磷酸化效应已被证明是 GαQ与GαI不同。这项工作的目的1是系统地测量全球环境基金活性的动力学 用于GαQ/GαI的RIC-8A使用停流荧光光谱进行直接比较，并 测定Ric-8A磷酸化的效果。目的2阐明Ric-8A：GαQ络合物的结构 用X射线结晶学和低温电子显微镜观察了无GDP和结合GDP的形态。这些研究将 为GαQ/GαI提供了对RIC-8A全球环境基金活性的不同机制的新见解，并为第一次 时间，RIC-8A：GαQ复合体的原子分辨率模型。这项工作将增进我们对 Ric-8A在G蛋白信号转导中的作用 为未来针对细胞质G蛋白激活的疗法的开发奠定基础。

PROJECT SUMMARY/ABSTRACT Heterotrimeric G proteins are involved in the regulation of many physiological processes and are essential to multiple cell signaling pathways. G protein heterotrimers are formed through association of α, β, and γ subunits with guanidine diphosphate (GDP) bound to the Gα subunit. The canonical signaling mechanism involves displacement of GDP from the Gα subunit by guanidine triphosphate (GTP) and subsequent disassociation of the Gβγ dimeric complex from Gα-GTP; the Gβγ and Gα-GTP complexes then act to regulate downstream effectors in the cell. G protein coupled receptors (GPCRs) represent the most well-studied class of proteins responsible for catalyzing the exchange of GDP for GTP on the Gα subunit. However, other nonreceptor guanine exchange factor (GEF) proteins also exist and have been shown to be crucial to regulation of G protein signaling. Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) is a protein that has been shown to act as a GEF and molecular chaperone for Gα subunits of Gi, Gq and G12/13 families. Although Ric-8A acts as a GEF to activate both Gαq/Gαi, previous research has demonstrated that the affinity of Ric-8A is different for Gαq in comparison to Gαi, and that the kinetics of the Ric-8A-catalyzed guanine nucleotide reaction are unique in relation to these two subtypes. Further, Ric-8A contains several phosphorylation sites and the phosphorylated form of Ric-8A has enhanced GEF activity toward Gα subunits. The effect of phosphorylation of Ric-8A has been shown to be different for Gαq relative to Gαi. Aim 1 of this work is to systematically measure the kinetics of GEF activity of Ric-8A for Gαq/Gαi using stopped-flow fluorescence spectroscopy to allow for a direct comparison, and to determine the effect of Ric-8A phosphorylation. Aim 2 is to elucidate the structure of Ric-8A:Gαq complexes in the GDP-free and GDP-bound forms by X-ray crystallography and cryo-electron microscopy. These studies will provide new insight into the distinct mechanism of Ric-8A GEF activity for Gαq/Gαi, and provide, for the first time, an atomic-resolution model of the Ric-8A:Gαq complex. This work will enhance our understanding of the role of Ric-8A in G protein signaling and have broad implications in human health and disease by laying the foundation for development of future therapeutics that target cytoplasmic G protein activation.