Structural Studies of Galphaq and Its Complexes at the Cell Membrane

项目摘要。异源三聚体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）的能力而闻名|（3）它也具有约束力 G蛋白偶联受体激酶2（GRK 2），其竞争性抑制PLCp活化，和 p63 RhoGEF，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 证明了功能性Gaq嵌合体可以以足够的量产生， Gaq-GRK 2-Gpy复合物的测定。激活的异源三聚体G 这种装配中的蛋白质表明，RGS蛋白和受体也可以联合形成， 高阶信号复合物目的1检测RGS蛋白如RGS 2结合GRK 2 - 1的能力。 使用流式细胞术结合测定、尺寸排阻色谱法和晶体学方法结合Gaq。 Gaq-RGS和RGS-Gaq-GRK 2-Gpy复合物的研究。目标2研究了 在效应物结合以及Gaq-GRK 2-Gpv和RGS的相互作用后， 在膜环境中具有完整受体或其胞质环的蛋白质。盖格奇美拉还 打开了大门，其他Gaq效应器相互作用的结构分析。为了开展这些工作，目标3 试图通过定点突变来确定Gaq介导的p63 RhoGEF激活的分子基础。 诱变、荧光偏振核苷酸交换测定和结构研究。 本案无关通过将我们的建议集中在Gaq的两个独特效应子GRK 2和p63 RhoGEF上，我们寻求 定义通过Gaq的异源三聚体G蛋白信号传导的一般范例。We have also也focused聚焦onGaq盖格， GRK 2、RGS 2和p63 RhoGEF，因为它们都与心血管生理学和疾病密切相关， 并且预期这些蛋白质直接或间接地协调它们的活性， 在活细胞中。GRK 2和Gaq/n对心脏正常发育和功能至关重要，RGS 2调节 血压通过减弱Gaq信号传导，p63 RhoGEF诱导心肌细胞的变化， 以心肌肥厚为特征。

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.