Structural and Functional Studies of Galphaq and Its Signaling Complexes

描述（由申请人提供）：异源三聚体G蛋白Gq调节神经肌肉控制以及血小板活化、血压和心脏功能，并在高血压和心脏肥大的发展中发挥核心作用。激活G？q直接与已显示对这些过程重要的蛋白质相互作用，包括效应酶磷脂酶C2（PLC 2）和p63 RhoGEF，以及G蛋白信号传导蛋白2（RGS 2）的GT3活化蛋白调节剂。我们最近确定了无脊椎动物PLC 2和G？q-p63 RhoGEF-RhoA复合物，这导致了戏剧性的新见解G？q调节效应子活性，并且RGS 2-G1 q复合物是与G1 q亚家族成员复合的RGS蛋白的第一结构。在目标1，我们将确认我们的模型如何G？q变构调节活细胞和模式生物C中的PLC 2。elegans，并探讨如何C-末端调控区的PLC 23增强催化和亲和力G1 q使用功能研究和X-射线晶体学和电子显微镜分析全长PLC 2与G1 q的复合物。在目标2中，我们确定了自抑制的基础结构的p63 RhoGEF溶液NMR和评估其结构是如何扰动后，复杂的形成与G？这将有助于充分描述其激活机制。在目的3中，我们将确定RGS 2和螺旋结构域的G？q，并测试它们对RGS 2选择性调节G1 q的贡献。总的来说，我们的实验有望揭示G？q激活其效应，以及如何RGS 2似乎有独特的进化来调节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.