Structural Studies of Galphaq and Its Complexes at the Cell Membrane

描述(申请人提供)：项目摘要。异三聚体G蛋白G？Q调节血小板活化、血压和心功能。虽然G？Q以其刺激磷脂酶CP(PLC|3)的能力而闻名，但它也与竞争性地抑制PLCP激活的G蛋白偶联受体激酶2(GRK2)以及调节细胞骨架结构的Rho鸟嘌呤核苷酸交换因子p63Rhogenf结合。我们最近证明，可以产生足够数量的功能性GaQ嵌合体来确定G？q-GRK2-GPY络合物的结构。在这个组装中，激活的异三聚体G蛋白的令人惊讶的排列表明RGS蛋白和受体也可以结合形成更高阶的信号复合体。目的1应用流式细胞仪结合分析、尺寸排斥层析和GGS-RGS和RGS-G？Q GRK2-GPY复合体的结晶学研究，研究RGS2等RGS蛋白与GRK2结合G？Q的能力。目的2研究在膜环境中，G？q-GRK2-GPV和RGS蛋白与完整受体或其胞质环相互作用时，激活的G？q在膜上的取向变化。G？Q嵌合体也为其他G？Q效应相互作用的结构分析打开了大门。为了启动这些工作，Aim 3试图通过定点突变、荧光偏振核苷酸交换分析和结构研究来确定G？Q介导的p63Rhogef激活的分子基础。关联性。通过我们对G？q、GRK2和p63Rhogef这两个独特的效应因子的研究，我们试图通过G？q来定义异源三聚体G蛋白信号的一般范式。我们还重点研究了G？q、GRK2、RGS2和p63Rhogef，因为它们都与心血管生理和疾病密切相关，期望这些蛋白质在活细胞中直接或间接地协调它们的活动并不是不合理的。GRK2和G？q/n对心脏的正常发育和功能是必不可少的，RGS2通过减弱G？q信号来调节血压，而p63Rhogef诱导心肌细胞的变化，这些变化是心肌肥大的特征。

DESCRIPTION (provided by applicant): Project Summary. The heterotrimeric G protein G?q regulates platelet activation, blood pressure and cardiac function. While G?q 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 G?q -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 G?q using a flow-cytometry binding assay, size-exclusion chromatography and crystallographic studies of Gaq-RGS and RGS- G?q GRK2-Gpy complexes. Aim 2 investigates changes in the orientation of activated G?q at the membrane upon effector binding as well as the interactions of G?q -GRK2-Gpv and RGS proteins with intact receptors or their cytoplasmic loops in a membrane environment. The G?q chimera also opens the door to the structural analysis of other G?q -effector interactions. To initiate these efforts, Aim 3 seeks to define the molecular basis for G?q -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 G?q, GRK2 and p63RhoGEF, we seek to define general paradigms for heterotrimeric G protein signaling through G?q. We have also focused on G?q, 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 G?q /n are essential for proper heart development and function, RGS2 regulates blood pressure via attenuation of G?q signaling, and p63RhoGEF induces changes in myocytes that are characteristic of cardiac hypertrophy.