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Natriuretic peptide receptor: Structure and signaling

利钠肽受体：结构和信号传导

基本信息

批准号：
6873038
负责人：
KUNIO S MISONO
金额：
$ 29万
依托单位：
UNIVERSITY OF NEVADA RENO
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-07-01 至 2007-04-30
项目状态：
已结题

项目摘要

EXCEED THE SPACE PROVIDED. Atrial natriuretic peptide (ANP) plays a major role in the regulation and maintenance of the cardiovascular system and its aberrations may lead to hypertension, cardiac hypertrophy, congestive heart failure, and other cardiovascular diseases. The actions of ANP are mediated by cell membrane receptors coupled to guanylate cyclase (GCase). The goal of our research is to elucidate the mechanisms of ANP-receptor interaction and transmembrane signal transduction. The ANP receptor is a 130-kDa transmembrane protein containing an extracellular ANP-binding domain (BCD), a single transmembrane sequence, and an intracellular domain (ICD) consisting of a kinase-homologous regulatory domain and a guanylate cyclase catalytic domain. The mechanism of signal transduction by the ANP receptor remains largely unknown.It has been suggested that GCase activation by ANP may be mediated by ligand-induced receptor dimerization. However, the ANP receptor has been shown by us and others to dimerize in the absence of the hormone, indicating that dimerization alone is not sufficient for receptor activation. Thus, we hypothesize that ANP binding induces, in addition to dimerization, a conformational change in the extracellular domain which is transduced across the cell membrane to the intracellular region and causes activation of the GCase catalytic activity. Recently, we have found that ANP binding to its receptor is chloride concentration dependent. This finding suggest a possible chloride mediated feedback control of the ANP receptor playing a role in salt-fluid homeostasis. To understand the mechanisms of signaling and its control by chloride, we propose 1) to elucidate the mechanism of receptor-ANP interaction and the ANP-induced conformational changes responsible for signal transduction by a combination of site-directed mutagenesis and crystallographic determination of the structures of the BCD bound with ANP and with the inactiveanalog AP-I, 2) to elucidate the mechanism of chloride-control of ANP binding by characterizing the chloride-binding site by mutagenesis, analyzingchloride effects on the BCD conformation and dimerization by biophysical analyses, and determining the crystal structure of the BCD lacking chloride, and 3) to express and purify the ICD and to characterize its activities and molecular behavior to understand the mechanism of ICD action in ANP receptor signaling. These studies will provide us a more accurate understanding of the mechanisms of ANP-receptor interaction and signaling, chloride-control of the ANP receptor, and the mechanism of GCase activation in response to ANP binding signal. Such knowledge will give us a better understanding of the physiological actions of ANP that, in turn, will promote development of more reliable diagnostic methods and more effective therapies for the cardiovascular diseases. PERFORMANCE SITE ========================================Section End===========================================

超出所提供的空间。心房利钠肽（ANP）在心血管系统的调节和维持中起重要作用，其异常可导致高血压、心肌肥厚、充血性心力衰竭等心血管疾病。ANP的作用是通过与鸟苷酸环化酶（GCase）偶联的细胞膜受体介导的。本研究的目的是阐明心钠素-受体相互作用和跨膜信号转导的机制。ANP受体是一种130-kDa的跨膜蛋白，含有胞外ANP结合结构域（BCD）、单一跨膜序列和由激酶同源调节结构域和鸟苷酸环化酶催化结构域组成的胞内结构域（ICD）。ANP受体的信号转导机制尚不清楚，有研究认为ANP激活GCase可能是通过配体诱导的受体二聚化介导的。然而，我们和其他人已经证明，在没有激素的情况下，ANP受体会二聚化，这表明单独的二聚化不足以激活受体。因此，我们假设，ANP结合诱导，除了二聚化，在细胞外结构域的构象变化，这是跨细胞膜转导到细胞内区域，并导致激活的GCase催化活性。最近，我们发现心钠素与其受体的结合是氯离子浓度依赖性的。这一发现表明，可能的氯化物介导的反馈控制的ANP受体在盐液稳态中发挥作用。为了理解信号传导机制及其受氯离子的控制，我们提出：1）通过定点突变和晶体学测定与ANP和无活性类似物AP-Ⅰ结合的BCD的结构，阐明受体-ANP相互作用的机制和ANP诱导的信号传导构象变化，2）通过突变法确定氯离子结合位点，生物物理分析氯离子对BCD构象和二聚化的影响，确定无氯BCD的晶体结构，阐明氯离子控制ANP结合的机制; 3）表达、纯化ICD，并对其活性和分子行为进行表征，以了解ICD在ANP受体信号转导中的作用机制。这些研究将使我们更准确地了解ANP-受体相互作用和信号传导机制，ANP受体的氯控制，以及GCase响应ANP结合信号的机制。这些知识将使我们更好地了解ANP的生理作用，反过来，将促进更可靠的诊断方法和更有效的治疗心血管疾病的发展。性能现场=