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: 8119010
• 负责人: Zygmunt S Derewenda
• 金额: $ 59.27万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8119010
• 关键词: Accounting; Actins; Agonist; Asthma; Binding; Biochemical; Biological; Biological Assay; Blood Vessels; Blood flow; Brain; Calcium; Cells; Cerebrovascular Spasm; Complex; Coronary Artery Vasospasm; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; 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; Health; Heart; Homeostasis; Human; Human Genome; Hydrolysis; Hypertension; In Vitro; Individual; Ions; Knockout Mice; Knowledge; Lead; Light; Link; Mediating; Messenger RNA; Methods; Molecular; Molecular Biology; Molecular Profiling; Monomeric GTP-Binding Proteins; Mus; Myosin Type II; Organ; Pathology; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Physiology; Play; Proteins; RHOA gene; 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; receptor; reproductive; research study; response; 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、脑和冠状血管痉挛、勃起功能障碍、支气管哮喘等疾病中起着关键作用。 SM 在给定 Ca2+ 水平下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 GTP 酶 RhoA 增强收缩效应。改变这些相互作用的分子设计可以提供一种更具体的 RhoA 信号传导靶向治疗方法。众所周知，RhoA 是一种普遍存在的分子开关，受许多不同的 GEF（鸟嘌呤核苷酸交换因子）和 GAP（GTP 酶激活蛋白）控制，它们要么用 GTP（GEF）加载 RhoA，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 中活跃，以及它们如何促进收缩性调节 - 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 生理学相关的 GEF 中，有 3 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。我们首次提出了涉及 p63RhoGEF/GEFT 与特定 G 蛋白偶联受体相关的 Gaq11 相互作用的生化和功能数据。我们的 qRT-PCR 实验的有希望的结果确定了 SM 新的几个 GAP 和 GEF，它们可能分别下调和上调 RhoA 并调节 SM 收缩性。我们还提出了一个得到初步数据支持的新假设，该假设假设环核苷酸 (cAMP) 通过 Rap1 GEF、Epac 和 Rap1（另一种 GTPase）对 RhoA 施加负控制，以激活 RhoA 特异性 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基因敲除小鼠的 SM 组织，实验设计可以将 Ca2+ 依赖性现象与 RhoA 依赖性调节分离。利用 X 射线晶体学、NMR、SAX 和 DXMS，我们将剖析多域 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针对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（分别损害心脏和大脑的血液流动）以及气道收缩引起的哮喘等疾病，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物。我们的研究结果可能转化为这些疾病的新疗法。