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

描述(申请人提供)：血管壁、呼吸道、胃肠道和生殖道的主要部分是平滑肌细胞。SM收缩的病理在高血压、脑血管痉挛、冠脉痉挛、勃起功能障碍、支气管哮喘等疾病中起着关键作用。在给定的钙离子水平上，SM的收缩能力受到一个复杂的蛋白质-蛋白质相互作用网络的关键调节，该网络可以通过一个小的GTP酶RhoA来增强收缩效应。改变这些相互作用的分子的设计可以为RhoA信号的治疗靶向提供一种更具体的方式。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，它们要么将GTP(GEF)装载到RhoA中，要么通过催化GTP水解为GDP(GAP)来下调RhoA的表达。目前尚不清楚哪些GEF和哪些间隙在SM中起作用，以及它们如何有助于调节收缩能力。我们建议确定在SM中活跃的GEF和GAP，并剖析它们的运行机制。这是我们正在进行的RhoA依赖信号通路的分子和结构生物学基础机制研究中一个令人兴奋的阶段。在与SM生理相关的GEF中，有三个RGS RhoGEF与GA12/13亚基相互作用，我们已经积累了大量的结构信息。我们首次提供了与特定G蛋白偶联受体相关联的与Gaq11相互作用的p63Rhogenf/Geft的生化和功能数据。我们的qRT-PCR实验结果令人振奋，发现了SM新发现的几个缺口和GEF，它们可能分别下调和上调RhoA，并调节SM的收缩能力。我们还提出了一个新的假说，并得到了初步数据的支持，该假说假设通过环核苷酸(CAMP)作用于RhoA，从而激活RhoA特异的间隙，包括ARAP3和RA-RhoGAP。环核苷酸(CAMP)通过Rap1环境基金、EPAC和Rap1(另一种GTP酶)作用于RhoA。我们将使用一种协同、多学科的方法，将分子生理学与结构生物学联系起来。我们将研究正常和基因敲除小鼠的SM组织，其实验设计允许将钙依赖现象与RhoA依赖调节分离。利用X射线结晶学、核磁共振、SAXS和DXMS，我们将剖析多结构域GEF和GAP在体外和体内受到调控的分子机制。我们的研究将解释控制SM收缩能力的基本方面，这一知识可能被用来设计针对高血压和哮喘等广泛疾病的新疗法。与公共卫生相关：高血压、冠状动脉和脑血管痉挛等疾病分别损害了流向心脏和大脑的血液，以及由呼吸道收缩引起的哮喘，这些疾病都是由这些组织中平滑肌的异常收缩和松弛引起的。我们正在研究特定蛋白质在调节平滑肌对细胞内钙增加的反应中的作用，细胞内钙是收缩的主要刺激因素。我们的研究结果可能会转化为治疗这些疾病的新方法。

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. PUBLIC HEALTH RELEVANCE: Diseases like hypertension, coronary and cerebral vasospasm, which compromise blood flow to the heart and brain respectively, as well as asthma which is caused by constriction of the airways, are all caused by abnormal contraction and relaxation of smooth muscle in these tissues. We are studying the role of specific proteins in the regulation of smooth muscle's response to increases in intracellular calcium, which is the primary stimulus for contraction. The results of our research may translate into novel treatments for these diseases.