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

RhoA 介导的血管平滑肌 Ca2 敏化的分子机制

• 批准号: 8078690
• 负责人: Zygmunt S Derewenda
• 金额: $ 2.5万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8078690
• 关键词: Accounting; Actins; Agonist; Asthma; Binding; Biochemical; Biological; Biological Assay; Blood Vessels; Blood flow; Brain; Calcium; Cells; Cerebrovascular Spasm; Cerebrum; Complex; Coronary; Coronary Artery Vasospasm; Coupling; Cyclic AMP; Cyclic Nucleotides; Data; Disease; Down-Regulation; Equilibrium; Erectile dysfunction; Event; Experimental Designs; Family; G-Protein-Coupled Receptors; GTP Binding; GTPase-Activating Proteins; Genetic Transcription; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heart; Homeostasis; Human; Human Genome; Hydrolysis; Hypertension; In Vitro; Individual; Ions; Knockout Mice; Knowledge; Lead; Link; Mediating; Methods; Molecular; Molecular Profiling; Monomeric GTP-Binding Proteins; Mus; Myosin Type II; Organ; Pathology; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Physiology; Play; Proteins; Regulation; Relaxation; Research; Role; Signal Pathway; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Staging; Stimulus; Structure-Activity Relationship; Techniques; Thromboxane A2; Time; Tissues; Translating; Up-Regulation; Vascular Smooth Muscle; Vasoconstrictor Agents; Western Blotting; Widespread Disease; X-Ray Crystallography; blood pressure regulation; constriction; design; follow-up; gastrointestinal; in vivo; interdisciplinary approach; knock-down; man; member; novel; protein function; protein protein interaction; public health relevance; receptor; reproductive; research study; response; rho GTPase-activating protein; small hairpin RNA; structural biology; therapeutic target

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.

描述（由申请人提供）：平滑肌（SM）细胞构成血管壁、气道、胃肠道和生殖道的主要部分。SM收缩性的病理在高血压、脑和冠状血管痉挛、勃起功能障碍、支气管哮喘等疾病中起关键作用。在给定的Ca2+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严格调节，这可以通过小的GTPase RhoA增强收缩效应。改变这些相互作用的分子设计可以为RhoA信号的治疗靶向提供更具体的方法。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 （GTPase激活蛋白）控制，它们要么用GTP （GEFs）装载RhoA，要么通过催化GTP水解成GDP （gap）下调RhoA。在SM中，哪些gef和哪些gap是活跃的，以及它们如何对收缩性的调节起作用——目前尚不清楚。我们建议确定在SM中活跃的gef和gap，并剖析它们的运作机制。这是我们正在进行的rhoa依赖性信号通路的分子和结构生物学机制研究的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。我们首次提出了生化和功能数据，表明p63RhoGEF/GEFT与特异性g蛋白偶联受体连接的Gaq11相互作用。我们的qRT-PCR实验结果表明，SM中有几个新的gap和gef可能分别下调和上调RhoA并调节SM的收缩性。在初步数据的支持下，我们提出了一个新的假设，即环核苷酸（cAMP）通过Rap1 GEF、Epac和Rap1（另一种GTPase）对RhoA施加负性控制，激活RhoA特异性的gap，包括ARAP3和RA-RhoGAP。我们将使用一种协同的、多学科的方法，将分子生理学与结构生物学联系起来。我们将研究正常小鼠和敲除小鼠的SM组织，实验设计允许Ca2+依赖现象与RhoA依赖调节解耦。利用x射线晶体学、核磁共振、SAXs和DXMS，我们将剖析多域gef和gap在体外和体内调控的分子机制。我们的研究将解释控制SM收缩性的基本方面，这些知识可能用于设计治疗高血压和哮喘等广泛疾病的新疗法。公共卫生相关性：高血压、冠状动脉和脑血管痉挛等疾病，分别影响流向心脏和大脑的血液，以及由气道收缩引起的哮喘，都是由这些组织中的平滑肌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。我们的研究结果可能转化为治疗这些疾病的新方法。