Structural and Functional Studies of Galphaq and Its Signaling Complexes

描述（由申请人提供）：异三聚体G蛋白Gq调节神经肌肉控制、血小板活化、血压和心功能，在高血压和心脏肥厚的发生中起核心作用。激活G ?q直接与对这些过程起重要作用的蛋白相互作用，包括效应酶磷脂酶C2 （PLC2）和p63RhoGEF，以及GTPase激活蛋白G蛋白信号蛋白2 （RGS2）。我们最近确定了无脊椎动物PLC2和G？q -p63RhoGEF-RhoA复合体，这让我们对G？RGS2-G1q复合体是RGS蛋白与G1q亚家族成员复合体的第一个结构。在Aim 1中，我们将确认G？q在活细胞和模式生物C. elegans中变构调节PLC2，并通过与G1q复合物的全长PLC2的功能研究、x射线晶体学和电镜分析来研究PLC23的c端调控区如何增强对G1q的催化和亲和力。在Aim 2中，我们通过溶液核磁共振确定了p63RhoGEF的自抑制基础结构，并评估了其结构在与G？Q，这将有助于充分描述其激活机制。在Aim 3中，我们将确定RGS2与G?螺旋结构域之间形成的独特相互作用的作用。q并测试它们对RGS2选择性调控G1q的贡献。总的来说，我们的实验有望揭示G？q激活其效应器，以及RGS2如何独特地进化以调节G？q。这些知识对于理解心脏和神经肌肉功能的基本过程以及开发治疗心血管疾病的新方法至关重要。

DESCRIPTION (provided by applicant): The heterotrimeric G protein Gq regulates neuromuscular control as well as platelet activation, blood pressure and cardiac function, and plays a central role in the development of hypertension and cardiac hypertrophy. Activated G?q directly interacts with proteins that have been shown to be important for these processes, including the effector enzymes phospholipase C2 (PLC2) and p63RhoGEF, and the GTPase activating protein regulator of G protein signaling protein 2 (RGS2). We recently determined structures of the autoinhibited catalytic core of invertebrate PLC2 and the G?q -p63RhoGEF-RhoA complex, which led to dramatic new insights into how G?q regulates effector activity, and of the RGS2-G1q complex, the first structure of an RGS protein in complex with a member of the G1q subfamily. In Aim 1, we will confirm our model for how G?q allosterically regulates PLC2 in living cells and in the model organism C. elegans, and investigate how the C-terminal regulatory region of PLC23 enhances catalysis and affinity for G1q using functional studies and X-ray crystallographic and electron microscopic analyses of full-length PLC2 in complex with G1q. In Aim 2, we determine the autoinhibited basal structure of p63RhoGEF by solution NMR and assess how its structure is perturbed upon complex formation with G?q which will help fully describe its mechanism of activation. In Aim 3, we will determine the role of unique interactions formed between RGS2 and the helical domain of G?q and test their contribution to the selective regulation of G1q by RGS2. Collectively, our experiments are expected to reveal the molecular basis for how G?q activates its effectors, and how RGS2 appears to have uniquely evolved to regulate G?q . This knowledge is essential for understanding fundamental processes that underlie cardiac and neuromuscular function, and for developing new approaches to treat cardiovascular disease. PUBLIC HEALTH RELEVANCE: The interactions of G?q with PLC2, GRK2, p63RhoGEF, and RGS2 are all strongly linked to cardiovascular physiology and/or disease in humans, and to neurotransmission at neuromuscular junctions in animal models. Our studies will shed significant light on the molecular processes that regulate cardiovascular physiology and potentially open new avenues for therapeutic intervention.