ANP RECEPTOR--MOLECULAR APPROACH OF SIGNALING MECHANISMS

描述：(改编自应用程序)ANP与其 鸟苷酸环化酶-A受体(Npra)在细胞周期中起核心作用。 高血压和心血管疾病的病理生理学。收获 深入了解ANP/Npra信号通路的复杂性至关重要 了解受体生物学和疾病状态的重要性 由激素受体的异常相互作用引起。长期的 这一建议的目的是为了在分子水平上阐明 一级，全国人民代表大会的性质和运作模式。重组NprA 在缺乏内源性受体的转基因细胞中表达提供了 实现这一目标的有用系统。从该领域获得的见解 将使用cDNA克隆的作图和受体的生化分析 为了阐明哪些受体区域可以通过缺失进一步分析 并在转基因细胞中进行定点突变以了解什么结构 各组成部分参与NPRA的运作。这项建议有三个方面 重大的、密切相关的目标，所有这些目标都主要集中在ANP上 受体作为一种方法来增加对正常和 对细胞和生理过程的异常控制。公安部希望 了解配体结合、活化过程中涉及哪些结构成分 蛋白激酶样域、环化酶活性部位和受体内吞作用， 下调和脱敏。他将分析分子 受体序列中调节整体功能的决定因素 Npra的能力，对激素依赖的信号传递过程至关重要。 有三个具体目标来研究这种结构/功能 关系：1)确定细胞的分子调控机制 NprA蛋白激酶样域的定点突变。焦点 将检验这样一种假设，即基因突变 Gly531-x1-Gly533-x2-x3-x4Gly537(激酶结构域残基影响 受体的功能)。此外，有一种说法认为，受体 还将调查包含内在蛋白激酶活性，2)至 鸟苷酸环化酶催化区分子决定簇的测定 NPRA的定点突变。目标将是测试 假设环化酶催化部位的保守残基 (Glu837、Lys838、Lys840、Pro850、His851和Val853)对 激素结合后产生环状GMP。这个地区扮演着一个 心钠素在血管平滑肌生理反应中的关键作用 肌肉和系膜细胞，以及3)研究分子决定因素 调节NprA的结合后事件和代谢周转。主 这一目标的目的是检验这样一种假设，即保守残基 NprA的蛋白激酶样鸟苷酰环化酶及其配体结合域 确定内吞作用的分子机制，下调和 与受体功能和细胞内信号有关的脱敏 利用转基因293细胞进行定点突变转导。

DESCRIPTION: (Adapted from the application) Interaction of ANP with its guanylyl cyclase-A receptor (Npra) plays a central role in the pathophysiology of hypertension and cardiovascular disorders. Gaining insight into the intricacies of ANP/Npra signaling pathway is pivotal importance for understanding both receptor biology and the diseased state arising from the abnormal hormone receptor interplay. The long-term objective of this proposal is directed at elucidating at the molecular level, the nature and mode of functioning of Npra. The recombinant Npra expressed in transfected cells lacking the endogenous receptor provides the useful system to accomplish this goal. The insights gained from the domain mapping of cDNA clone and biochemical analysis of the receptor will be used to elucidate those receptor areas that can be analyzed further by deletion and site directed mutagenesis in transfected cells to learn what structural components are involved in the functioning of Npra. This proposal has three major, intimately linked goals, all involving the primary focus on the ANP receptor as an approach to gain increased understanding of the normal and abnormal control of cellular and physiological processes. The PI hopes to learn what structural components are involved in ligand binding, activation of protein kinase like domain, cyclase active site and receptor endocytosis, down-regulation and desensitization. He will analyze the molecular determinants in receptor sequence that mediate the overall functional ability of the Npra, critical for the hormone dependent signaling processes. There are three specific aims to investigate this structural/functional relationship: 1) To determine the molecular regulatory mechanisms of the protein kinase like domain of Npra by site directed mutagenesis. The focus will be to test the hypothesis that the mutations in the Gly531-x1-Gly533-x2-x3-x4Gly537 (residues of kinase domain affect the function of the receptors). In addition, the proposal that the receptor contains intrinsic protein kinase activity will also be investigated, 2) To determine the molecular determinants in guanylyl cyclase catalytic domain of Npra by site directed mutagenesis. The objective would be to test the hypothesis that the conserved residues in the cyclase catalytic site (Glu837, Lys838, Lys840, Pro850, His851 and Val853) are critical for the generation of cyclic GMP after hormone binding. This region plays a critical role in the physiological response of ANP in the vascular smooth muscle and mesangial cells, and 3) To study the molecular determinants mediating post-binding events and metabolic turnover of Npra. The main objective of this aim is to test the hypothesis that the conserved residues in protein kinase like guanylyl cyclase and ligand binding domains of Npra determine the molecular mechanism of endocytosis, down-regulation and desensitization relevant to receptor function and intracellular signal transduction by site directed mutagenesis using transfected 293 cells.