ANP Receptor:Molecular approach of signaling mechanisms

描述（由申请人提供）：心房利钠肽（ANP）是一种促尿激素，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。参与ANP调节作用的主要基因座之一是鸟苷酸环化酶连接的ANP受体（GC-A），命名为NPRA，其属于GC受体家族，其总体信号传导机制尚不清楚。NPRA由含有细胞外ANP结合结构域、单个跨膜序列、细胞内蛋白激酶样同源结构域（蛋白-KHD）和鸟苷酸环化酶催化结构域的单个多肽组成。据推测，ANP与胞外结构域的结合引起构象变化，将信号传递到环化酶催化活性中心。也有人提出，ATP，一个积极的变构效应，结合蛋白KHD，因此，增强ANP依赖的鸟苷酸环化酶的激活，然而，需要进一步的研究，以揭示信号转导的实际机制。本研究的长期目标是阐明NPRA的结构-功能关系，并了解ANP/ATP如何与受体相互作用，以及这种相互作用如何被转导到细胞内结构域，以刺激GC活性和cGMP产生，受体内化，运输，并最终在稳定表达重组NPRA的人胚肾293细胞中失活和降解。提出了四个具体目标：1）检测调节ANP/ATP信号转导活性的机制，并揭示定义ATP结合序列的身份和NPRA活化的结构基序，2）描绘用绿色荧光蛋白标记的NPRA的内化、运输和螯合的动态的分子机制，3）研究ANP作用的分子机制，其可以通过减少活性受体的数量在NPRA水平上调节。（下调）或通过功能失活（脱敏），和4）描绘ANP介导的NPRA在瞬时表达野生型和突变型受体的血管平滑肌和系膜细胞中的生理功能的分子机制。拟议的研究应提供NPRA功能模式的全面评估，以直接阐明NPRA信号通路在分子水平上的独特分子、药理学和生理学意义。由此产生的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

DESCRIPTION (provided by the applicant): Atrial natriuretic peptide (ANP) is a hypotensive hormone that regulates sodium excretion, fluid volume, and vasorelaxation, important factors in the control of blood pressure and blood volume. One of the principal loci involved in the regulatory action of ANP is the guanylyl cyclase-linked ANP receptor (GC-A) designated as NPRA that belongs to a family of GC-receptors for which collectively the signaling mechanisms are not well understood. NPRA consists of a single polypeptide containing an extracellular ANP-binding domain, a single transmembrane sequence, and intracellular protein kinase-like homology domain (protein-KHD) and guanylyl cyclase catalytic domain. It has been postulated that the binding of ANP to the extracellular domain causes a conformational change, transmitting the signal to cyclase catalytic active center. It has also been proposed that ATP, a positive allosteric effector, binds to the protein-KHD and, hence, augments ANP-dependent guanylyl cyclase activation, however, further studies are needed to reveal the actual mechanisms of signal transduction. The long-term goals of the proposed studies are to elucidate the structure-function relationship of NPRA and to understand how ANP/ATP interact with the receptor and how such interactions are transduced to the intracellular domain to stimulate GC activity and cGMP production, receptor internalization, trafficking, and ultimately inactivation and degradation in human embryonic kidney 293 cells stably expressing recombinant NPRA. Four specific aims are proposed: 1) examine the mechanisms that modulate ANP/ATP signal transduction activities and reveal the structural motifs defining the identity of ATP-binding sequence and activation of NPRA, 2) delineate the molecular mechanisms of the dynamics of internalization, trafficking, and sequestration of NPRA-tagged with green fluorescent protein, 3) examine the molecular mechanisms of ANP action that can be regulated at the level of NPRA by reducing the number of active receptors (down-regulation) or by functional inactivation (desensitization) of constant receptors, and 4) delineate the molecular mechanisms of ANP-mediated physiological functions of NPRA in vascular smooth muscle and mesangial cells transiently expressing wild-type and mutant receptors. The proposed studies should provide a comprehensive assessment of the mode of functioning of NPRA to directly elucidate the unique molecular, pharmacologic and physiologic implications of NPRA signaling pathways at the molecular level. The resulting knowledge should yield new therapeutic targets and novel loci for the control and treatment of hypertension and cardiovascular disorders