ANP Receptor:Molecular approach of signaling mechanisms

说明(由申请人提供)：心钠素(ANP)是一种调节钠排泄、液体容量和血管松弛的降压激素，这些都是控制血压和血量的重要因素。参与心钠素调节作用的主要基因之一是鸟氨酰环化酶连接的心钠素受体(GC-A)，命名为NPRA，属于GC受体家族，其信号转导机制尚不清楚。NPRA由一个含有胞外心钠素结合区、一个跨膜序列、胞内蛋白激酶样同源结构域(Protein-KHD)和鸟苷酸环化酶催化结构域的多肽组成。据推测，ANP与胞外区的结合引起构象变化，将信号传递给环化酶催化活性中心。也有人提出，作为正变构效应的ATP可以与蛋白-KHD结合，从而增强ANP依赖的鸟苷酸环化酶的激活，然而，还需要进一步的研究来揭示真正的信号转导机制。这些研究的长期目标是阐明NPRA的结构-功能关系，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区域，以刺激GC活性和cGMP的产生，受体内化，转运，并最终在稳定表达重组NPRA的人胚胎肾脏293细胞中失活和降解。提出了四个具体的目标：1)研究调节ANP/ATP信号转导活性的机制，揭示定义ATP结合序列的一致性和NPRA激活的结构基序；2)描述绿色荧光蛋白标记的NPRA内化、运输和隔离的分子机制；3)研究ANP作用的分子机制，这些机制可以通过减少激活的受体数量(下调)或通过恒定受体的功能失活(脱敏)来调节。4)阐明ANP介导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作用的分子机制。建议的研究应提供对NPRA功能模式的全面评估，以直接在分子水平上阐明NPRA信号通路独特的分子、药理学和生理学意义。由此产生的知识应该为高血压和心血管疾病的控制和治疗产生新的治疗靶点和新的基因座

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