ANP Receptor: Molecular approach of signaling mechanisms

描述（由申请人提供）：心房利钠肽（ANP）是一种心脏激素，可调节钠排泄、液体容量和血管舒张，是控制血压和血容量的重要因素。ANP与其受体鸟苷酸环化酶-A/利钠肽受体-A（GC-A/NPRA）相互作用产生细胞内第二信使cGMP，其在高血压和心血管疾病的病理生理学中起中心作用。深入了解ANP/NPRA/cGMP信号通路的复杂性对于理解受体生物学和异常的受体相互作用引起的疾病状态至关重要。本提案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那些受体区域，这些受体区域可以通过体外转染细胞和体内转基因小鼠模型中的定点诱变进一步分析，以了解NPRA的功能涉及哪些结构组分。该提案有四个主要的，密切相关的目标，所有涉及的主要重点是NPRA作为一种方法，以增加对细胞和生理过程的正常和异常控制的理解。这些目标如下：1）通过定点诱变描绘NPRA的GC催化活性位点中的分子决定簇，并在体外用野生型和羧基末端突变体受体转染的血管平滑肌和系膜细胞以及体内转基因小鼠中确定ANP/NPRA信号传导和生理功能的作用，2）确定NPRA的蛋白激酶样同源结构域（KHD）的细胞和分子调节机制，并检查ANP/ATP对NPRA的磷酸化状态和信号转导活性的影响，3）通过荧光显微镜在活细胞中真实的时间检查介导NPRA功能方面的分子决定簇，包括内化、运输、螯合和再循环的动力学，以及4）描述介导结合后事件和代谢周转的分子决定簇，包括体外培养的VSMC、MC和HEK-293细胞以及体内转基因小鼠中NPRA的脱敏和下调。拟议的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节提供基础。公共卫生相关性：高血压影响着全球10亿人，是心血管疾病的潜在危险因素。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，这些知识将产生新的治疗靶点和新的位点，用于控制和治疗高血压和心血管疾病。 公共卫生相关性：从拟定研究中获得的信息将更准确地评估鸟苷酸环化酶-A/利钠肽受体-A（GC-A/NPRA）在可能的发病机制中的综合作用，其中受体介导的心脏激素、心房和脑利钠肽（ANP，BNP）生物活性的失调可能导致液体容量调节和血压稳态异常。最终，这些知识将产生新的治疗分子靶点，用于高血压和心血管疾病的诊断、控制和治疗。

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.