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）在细胞凋亡中起着重要作用。 高血压和心血管疾病的病理生理学。 获得 深入了解ANP/Npra信号通路的复杂性是关键 对于理解受体生物学和疾病状态的重要性 由异常的激素受体相互作用引起。 长期 本建议的目的是阐明在分子 NPRA的水平、性质和运作模式。 重组Npra 在缺乏内源性受体的转染细胞中表达， 一个有用的系统来实现这个目标。 从该领域获得的见解 将使用cDNA克隆作图和受体的生化分析 为了阐明那些可以通过缺失进一步分析的受体区域， 并在转染细胞中进行定点突变， 这些组件参与了NPRA的运作。 该提案有三个 主要的、密切相关的目标，所有这些目标都涉及以阿富汗国家警察为主要重点 受体作为一种方法，以获得更多的了解正常和 细胞和生理过程的异常控制。 PI希望 了解哪些结构成分参与配体结合、激活 蛋白激酶样结构域、环化酶活性位点和受体内吞作用， 下调和脱敏。 他会分析分子 受体序列中介导整体功能性 Npra的能力，对激素依赖性信号传导过程至关重要。 有三个具体的目的来研究这种结构/功能 关系：1）确定的分子调控机制， 蛋白激酶样结构域的Npra。 重点 将是为了验证一个假设，即 Gly 531-x1-Gly 533-x2-x3-x4 Gly 537（激酶结构域的残基影响激酶的活性） 受体的功能）。 此外，该建议认为， 还将研究含有内在蛋白激酶活性的蛋白质，2） 确定鸟苷酸环化酶催化结构域的分子决定簇 通过定点诱变获得。 目标是测试 环化酶催化位点的保守残基 （Glu 837、Lys 838、Lys 840、Pro 850、His 851和Val 853）对于蛋白质的合成是关键的。 激素结合后产生环GMP。 该地区发挥着 心钠素在血管平滑肌细胞生理反应中的重要作用 肌细胞和系膜细胞; 3）研究分子决定因素 介导Npra的结合后事件和代谢周转。 主要 这一目标的目的是检验保守残基 在蛋白激酶如鸟苷酸环化酶和Npra配体结合结构域中 确定内吞作用、下调和 与受体功能和细胞内信号相关的脱敏 使用转染的293细胞通过定点诱变进行转导。

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.