Molecular Mechanisms of RhoA-mediated Ca2+Sensitization in Vascular Smooth Muscle

描述（由申请方提供）：平滑肌（SM）细胞形成血管壁、气道、胃肠道和生殖道的主要部分。SM收缩性的病理学在高血压、脑血管和冠状动脉痉挛、勃起功能障碍、支气管哮喘和其他疾病中起关键作用。在给定的Ca 2+水平下，SM的收缩性受到蛋白质-蛋白质相互作用的复杂网络的关键调节，这可以通过小的GTdR RhoA增强收缩作用。改变这些相互作用的分子的设计可以提供一种更具体的治疗靶向RhoA信号传导的方式。众所周知，RhoA是一种普遍存在的分子开关，由许多不同的GEF（鸟嘌呤核苷酸交换因子）和GAP（GTP酶激活蛋白）控制，其要么用GTP装载RhoA（GEF），要么通过催化GTP水解为GDP（GAP）来下调RhoA。哪些GEF和哪些GAP在SM中是活跃的，以及它们如何有助于调节收缩性-尚不清楚。我们建议，以确定全球环境基金和GAP活动在SM，并剖析其运作的机制。这是一个令人兴奋的阶段，在我们正在进行的研究机制的分子和结构生物学的RhoA依赖性信号通路。在与SM生理学相关的GEFs中，有三个RGS RhoGEFs与Ga 12/13亚基相互作用，我们已经积累了大量的结构信息。我们目前，第一次，生化和功能数据牵连p63 RhoGEF/GEFT与Gaq 11连接到特定的G蛋白偶联受体相互作用。我们的qRT-PCR实验的有希望的结果确定了几个GAP和GEF新的SM，可能下调和上调RhoA分别和调制SM收缩性。我们还制定了一个新的假设，初步数据的支持，这假定负控制施加在RhoA的环核苷酸（cAMP）通过Rap 1的GEF，Epac和Rap 1（另一个GTAPs）激活RhoA特异性GAP，包括ARAP 3和RA-RhoGAP。我们将使用一个协同的，多学科的方法，桥梁分子生理学与结构生物学。我们将研究SM组织从正常和基因敲除小鼠，与实验设计，允许去耦的Ca 2+依赖的现象从RhoA依赖的调节。利用X射线晶体学、NMR、SAXs和DXMS，我们将剖析多结构域GEF和GAP在体外和体内调节的分子机制。我们的研究将解释控制SM收缩性的基本方面，这些知识可用于设计治疗高血压和哮喘等广泛疾病的新疗法。

DESCRIPTION (provided by applicant): Smooth muscle (SM) cells form the main part of the walls of blood vessels, the airways, the gastrointestinal and reproductive tracts. Pathology of SM contractility plays a key role in hypertension, cerebral and coronary vasospasm, erectile dysfunction, bronchial asthma, and other diseases. SM contractility at a given level of Ca2+ is critically modulated by a complex network of protein-protein interactions, which can enhance the contractile effect acting via a small GTPase RhoA. The design of molecules that would alter these interactions could provide a more specific way of therapeutic targeting of RhoA signaling. It is well understood that RhoA, a ubiquitous molecular switch, is controlled by many different GEFs (guanine nucleotide exchange factors) and GAPs (GTPase activating proteins), which either load RhoA with GTP (GEFs) or downregulate it by catalyzing the hydrolysis of GTP to GDP (GAPs). Which GEFs and which GAPs are active in SM, and how they contribute to the regulation of contractility - is not known. We propose to identify GEFs and GAPs active in SM, and to dissect the mechanisms by which they operate. This is an exciting stage in our ongoing studies of the mechanisms underlying the molecular and structural biology of the RhoA-dependent signaling pathways. Among the GEFs relevant to SM physiology are three RGS RhoGEFs, interacting with the Ga12/13 subunits, for which we have already accumulated a substantial amount of structural information. We present, for the first time, biochemical and functional data implicating p63RhoGEF/GEFT that interacts with Gaq11 linked to specific G-protein-coupled receptors. Promising results of our qRT-PCR experiments identify several GAPs and GEFs new to SM that may down and up regulate RhoA respectively and modulate SM contractility. We also formulate a new hypothesis, supported by preliminary data, which postulates that negative control is exerted on RhoA by cyclic nucleotides (cAMP) acting via the Rap1 GEF, Epac and Rap1 (another GTPase) to activate RhoA specific GAPs including ARAP3 and RA-RhoGAP. We will use a synergistic, multidisciplinary approach that bridges molecular physiology with structural biology. We will study SM tissues from normal and knock-out mice, with an experimental design that allows for the decoupling of the Ca2+-dependent phenomena from RhoA dependent regulation. Using X-ray crystallography, NMR, SAXs and DXMS, we will dissect the molecular mechanism by which the multidomain GEFs and GAPs are regulated in vitro and in vivo. Our research will explain fundamental aspects that control SM contractility and this knowledge may be used to design novel therapies for widespread diseases such as hypertension and asthma.