Structural and Functional Studies of Galphaq and Its Signaling Complexes

描述(申请人提供)：异三聚体G蛋白GQ调节神经肌肉控制以及血小板激活、血压和心脏功能，并在高血压和心肌肥厚的发展中发挥核心作用。被激活的G？q直接与这些过程中重要的蛋白质相互作用，包括效应酶磷脂酶C2(PLC2)和p63Rhogef，以及G蛋白信号蛋白2的GTP酶激活蛋白调节因子(RGS2)。我们最近确定了无脊椎动物PLC2和G？q-p63Rhogef-RhoA复合体的自抑制催化核心的结构，这使得我们对G？q如何调节效应器活性以及RGS2-G1q复合体的结构有了新的见解，RGS2-G1q复合体是RGS蛋白与G1q亚家族成员形成的复合体的第一个结构。在目标1中，我们将确认我们的模型，即G？q如何以变构方式调节活细胞和模式生物秀丽线虫中的PLC2，并通过功能研究以及与G1q形成的全长PLC2的X射线结晶学和电子显微镜分析，研究PLC23的C端调控区如何增强对G1q的催化和亲和力。在目标2中，我们通过溶液核磁共振确定了p63Rhogef的自抑制基本结构，并评估了它的结构在与G？q形成络合物时是如何被扰动的，这将有助于全面描述它的激活机制。在目标3中，我们将确定RGS2与G？q螺旋结构域之间形成的独特相互作用的作用，并测试它们对RGS2选择性调节G1q的贡献。总而言之，我们的实验有望揭示G？Q如何激活其效应器的分子基础，以及RGS2似乎如何独特地进化来调节G？Q。这一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是必不可少的。 公共卫生相关性：G？Q与PLC2、GRK2、p63Rhogef和RGS2的相互作用都与人类的心血管生理和/或疾病密切相关，并与动物模型中神经肌肉接头的神经传递密切相关。我们的研究将对调节心血管生理学的分子过程提供重要的启示，并可能为治疗干预开辟新的途径。

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.