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）细胞形成血管壁，气道，胃肠道和生殖区的主要部分。 SM收缩力的病理在高血压，脑血管痉挛，勃起功能障碍，支气管哮喘和其他疾病中起关键作用。通过复杂的蛋白质 - 蛋白质相互作用网络对给定水平的Ca2+水平的SM收缩性进行了严格的调节，该网络可以增强通过小的GTPase RhoA作用的收缩效应。将改变这些相互作用的分子的设计可以提供更具体的RhoA信号靶向方法。众所周知，RhoA是一种无处不在的分子开关，由许多不同的GEF（鸟嘌呤核苷酸交换因子）和GAPS（GTPase激活蛋白）控制，它们用GTP（GEFS）加载RhoA或通过将GTP的GTP水解为GDP（GDP）（GAPP）（GAPP）（GAPS）催化。尚不清楚哪些GEF和哪些差距在SM中有效，以及它们如何贡献对收缩性的调节。我们建议确定在SM中活跃的GEF和差距，并剖析其操作的机制。这是我们正在进行的对RhoA依赖性信号通路分子和结构生物学基础机制的持续研究中的一个令人兴奋的阶段。在与SM生理学相关的GEF中，有三个RGS Rhogefs与GA12/13亚基相互作用，我们已经积累了大量的结构信息。我们首次介绍了与与特定G蛋白偶联受体相互作用的p63rhogef/geft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新的几个差距和GEF，它们可能会分别调节RhoA并调节SM收缩性。我们还提出了一个新的假设，并得到了初步数据的支持，该假设假定通过RAP1 GEF，EPAC和RAP1（另一种GTPase）（另一个GTPase）作用的环状核苷酸（CAMP）在RhoA上施加了阴性对照，以激活RHOA特定的差距，包括ARAP3和RA-RHOGAP。我们将使用一种协同的多学科方法，该方法将分子生理学与结构生物学融为一体。我们将通过实验设计研究正常小鼠和敲除小鼠的SM组织，该设计允许将Ca2+依赖性现象与RhoA依赖性调节脱钩。使用X射线晶体学，NMR，SAXS和DXM，我们将剖析多个域GEF和间隙在体外和体内调节多域GEF和GAP的分子机制。我们的研究将解释控制SM收缩性和这些知识的基本方面，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，冠状动脉和大脑血管痉挛等疾病，分别损害了流向心脏和大脑的血液，以及由于气道收缩而引起的哮喘，都是由异常的肌力和平滑肌肉在这些组织中引起的。我们正在研究特定蛋白在调节平滑肌对细胞内钙增加的反应中的作用，这是收缩的主要刺激。我们的研究结果可能转化为这些疾病的新型治疗方法。