ANP Receptor: Molecular approach of signaling mechanisms

描述（申请人提供）：心房钠素肽（ANP）是一种心脏激素，调节钠排泄、体液量和血管舒张，是控制血压和血容量的重要因素。ANP与其受体鸟酰环化酶- a /利钠肽受体- a （GC-A/NPRA）相互作用产生细胞内第二信使cGMP，在高血压和心血管疾病的病理生理中起核心作用。了解ANP/NPRA/cGMP信号通路的复杂性对于理解受体生物学和异常激素-受体相互作用引起的疾病状态至关重要。该建议的长期目标是在分子水平上阐明NPRA的性质和功能模式。从cDNA克隆的结构域定位和受体的生化分析中获得的见解将用于阐明这些受体区域，这些受体区域可以通过体外转染细胞的定点诱变和体内转基因小鼠模型进一步分析，以了解哪些结构成分参与了NPRA的功能。该建议有四个主要的、密切相关的目标，所有这些目标都涉及到将NPRA作为一种方法来获得对细胞和生理过程的正常和异常控制的更多理解。这些目标如下：1)通过位点定向诱变方法确定NPRA GC催化活性位点的分子决定因素，确定ANP/NPRA信号通路在体外和体内分别转染野生型和羧基末端突变受体的血管平滑肌和系膜细胞中的作用和生理功能；2)确定NPRA蛋白激酶样同源结构域（KHD）的细胞和分子调控机制，并研究ANP/ATP对NPRA磷酸化状态和信号转导活性的影响；3)在活细胞中实时荧光显微镜检测介导NPRA功能方面的分子决定因素，包括内化、转运、隔离和再循环的动态。4)在体外培养的VSMCs、MCs和HEK-293细胞和体内转基因小鼠中，描述介导NPRA脱敏和下调的结合后事件和代谢转换的分子决定因素。这些研究将在分子水平上对NPRA的功能模式进行全面评估，其中有关受体结构和信号传导的详细信息将为理解受体功能和调控提供基础。公共卫生相关性：高血压影响全世界10亿人，它是心血管疾病的一个潜在危险因素。这项研究将进一步加深我们对心脏激素调节血容量和血压稳态的机制的理解。最终，这些知识将为控制和治疗高血压和心血管疾病提供新的治疗靶点和新的基因位点。

DESCRIPTION (provided by applicant): Atrial natriuretic peptide (ANP) is a cardiac hormone that regulates sodium excretion, fluid volume, and vasorelaxation, important factors in the control of blood pressure and blood volume. Interaction of ANP with its receptor guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA) produces the intracellular second messenger cGMP, which plays a central role in the pathophysiology of hypertension and cardiovascular disorders. Gaining insight into the intricacies of ANP/NPRA/cGMP signaling pathways is of pivotal importance for understanding both receptor biology and the disease state arising from abnormal hormone-receptor interplay. The long-term objective of this proposal is directed at elucidating the nature and mode of functioning of NPRA at the molecular level. The insights gained from domain mapping of cDNA clones and biochemical analysis of the receptor will be used to elucidate those receptor areas that can be further analyzed by site- directed mutagenesis in transfected cells in vitro and transgenic mouse models in vivo to learn what structural components are involved in the functioning of NPRA. This proposal has four major, intimately linked goals, all involving a primary focus on NPRA as an approach to gain increased understanding of the normal and abnormal control of cellular and physiological processes. These goals are as follows: 1) delineate the molecular determinants in the GC catalytic active-site of NPRA by site-directed mutagenesis and determine the role of ANP/NPRA signaling and physiological function(s) in vascular smooth muscle and mesangial cells transfected with wild-type and carboxyl-terminal mutant receptors in vitro and transgenic mice in vivo, 2) determine the cellular and molecular regulatory mechanisms of the protein kinase-like homology domain (KHD) of NPRA and examine the effect of ANP/ATP on the phosphorylation state and signal transduction activities of NPRA, 3) examine the molecular determinants mediating the functional aspects of NPRA including the dynamics of internalization, trafficking, sequestration, and recycling by fluorescence microscopy in living cells in real time, and 4) delineate the molecular determinants mediating post-binding events and metabolic turnover including desensitization and down-regulation of NPRA in cultured VSMCs, MCs, and HEK-293 cells in vitro and transgenic mice in vivo. The proposed studies will delineate a comprehensive assessment of the mode of functioning of NPRA at the molecular level, in which detailed information about receptor structure and signaling will provide the basis for understanding receptor function and regulation. PUBLIC HEALTH RELEVANCE: Hypertension affects one billion people worldwide, and it is a potent risk factor for cardiovascular diseases. The proposed research should further our understanding of the mechanisms by which cardiac hormones regulate the blood volume and blood pressure homeostasis. Ultimately, this knowledge should yield new therapeutic targets and novel loci for the control and treatment of hypertension and cardiovascular diseases. PUBLIC HEALTH RELEVANCE: Information gained from the proposed studies will yield a more accurate assessment of the integrative role of guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA) in possible mechanisms of pathogenesis whereby malregulation of receptor-mediated cardiac hormones; atrial and brain natriuretic peptides (ANP, BNP) bioactivity could result in abnormalities of fluid volume regulation and blood pressure homeostasis. Ultimately, this knowledge should yield new therapeutic molecular targets for the diagnosis, control, and treatment of hypertension and cardiovascular diseases.