Structural Studies of Galphaq and Its Complexes at the Cell Membrane

项目摘要。异三聚体G蛋白Gaq调节血小板活化、血压和 心脏功能。虽然GaQ以其刺激磷脂酶CP(PLC|3)的能力而闻名，但它也结合 到竞争性抑制PLCP激活的G蛋白偶联受体激酶2(GRK2)，以及到 P63Rhogef，一种调节细胞骨架结构的Rho鸟嘌呤核苷酸交换因子。我们最近 证明可以产生足够数量的功能性Gaq嵌合体以满足结构 Gaq-GRK2-GPY络合物的测定。活化的异三聚体G的惊人排列 这种组装中的蛋白质表明，RGS蛋白和受体也可以结合在一起形成甚至 更高阶的信号复合体。AIM 1检测RGS蛋白如RGS2与GRK2结合的能力 用流式细胞仪结合分析、尺寸排除层析和结晶学方法结合Gaq GaQ-RGS和RGS-GaQ-GRK2-GPY络合物的研究目标2调查了定位的变化 膜上激活的Gaq与效应器结合以及Gaq-GRK2-GPV和RGS的相互作用 在膜环境中具有完整受体或其细胞质环路的蛋白质。Gaq嵌合体也 为其他Gaq效应器交互的结构分析打开大门。为了启动这些努力，目标3 旨在确定Gaq通过定点定向激活p63Rhogef的分子基础 诱变、荧光偏振核苷酸交换分析和结构研究。 关联性。通过将我们的建议集中在Gaq的两个独特的效应器GRK2和p63Rhogef上，我们寻求 确定通过Gaq的异源三聚体G蛋白信号的一般范例。我们还将重点放在Gaq上， GRK2、RGS2和p63Rhogef因为它们都与心血管生理和疾病密切相关， 期待这些蛋白质直接或间接地协调它们的活动并不是不合理的， 在活细胞中。GRK2和Gaq/n对心脏的正常发育和功能是必不可少的，RGS2调节 血压通过衰减Gaq信号和p63Rhogef诱导心肌细胞的变化 以心脏营养不良为特征的。

Project Summary. The heterotrimeric G protein Gaq regulates platelet activation, blood pressure and cardiac function. While Gaq is best known for its ability to stimulate phospholipase Cp (PLC|3), it also binds to G protein-coupled receptor kinase 2 (GRK2), which competitively inhibits PLCp activation, and to p63RhoGEF, a Rho guanine nucleotide exchange factor that modulates cytoskeletal structure. We recently demonstrated that a functional Gaq chimera could be produced in amounts sufficient for the structure determination of the Gaq-GRK2-Gpy complex. The surprising arrangement of activated heterotrimeric G proteins in this assembly suggested that RGS proteins and receptors could also associate to form even higher order signaling complexes. Aim 1 examines the ability of RGS proteins such as RGS2 to bind GRK2- bound Gaq using a flow-cytometry binding assay, size-exclusion chromatography and crystallographic studies of Gaq-RGS and RGS-Gaq-GRK2-Gpy complexes. Aim 2 investigates changes in the orientation of activated Gaq at the membrane upon effector binding as well as the interactions of Gaq-GRK2-Gpv and RGS proteins with intact receptors or their cytoplasmic loops in a membrane environment. The Gaq chimera also opens the door to the structural analysis of other Gaq-effector interactions. To initiate these efforts, Aim 3 seeks to define the molecular basis for Gaq-mediated activation of p63RhoGEF through site-directed mutagenesis, fluorescence polarization nucleotide exchange assays and structural studies. Relevance. By focusing our proposal on two unique effectors of Gaq,GRK2 and p63RhoGEF, we seek to define general paradigms for heterotrimeric G protein signaling through Gaq. We have also focused onGaq, GRK2, RGS2 and p63RhoGEF because all are strongly linked to cardiovascular physiology and disease, and it is not unreasonable to expect that these proteins coordinate their activities, either directly or indirectly, in living cells. GRK2 and Gaq/n are essential for proper heart development and function, RGS2 regulates blood pressure via attenuation of Gaq signaling, and p63RhoGEF induces changes in myocytes that are characteristic of cardiachypertrophy.