Study of ANP Receptor: Gene Targeting and Expression

描述（由申请人提供）：心房利钠肽（ANP）与鸟苷酸环化酶/利钠肽受体-A（NPRA/GC-A）结合，并引起利钠、利尿、血管松弛和抗增殖反应，这些反应是控制血压和血容量的重要因素。然而，这些活动的分子基础和Nprl基因（编码NPRA）的功能表达和调控仍然不完全清楚。为了进一步了解Nprt基因的功能意义，我们将使用我们分离和测序的基因组克隆研究其转录调控，并使用我们在我们的设施中建立的基因靶向突变小鼠模型研究其生理功能。我们的基本假设是，Nprl基因的调节活性需要响应细胞内外信号的普遍存在的和组织特异性的转录因子之间的协同相互作用，并且在体内完整动物中Nprl基因表达的缺失使得无对抗的强大钠保留、血管收缩和增殖系统，而Nprl基因的过表达发挥基因剂量依赖性的作用，其是利钠的，血管舒张和抗增殖的性质。为了实现这一目标，我们将整合分子水平上的遗传信息，蛋白质水平上的生化信息，以及整个动物水平上的生理信息，从而形成垂直整合的分子生理策略。我们将利用分子遗传学技术的力量来回答细胞，生物化学和病理生理学问题，在完整的动物在体内和分离的培养细胞在体外，从而得出结论，是明确的和生理相关的。从上述研究中获得的信息将提供直接测试Nprl基因组调控元件的功效以及Nprl基因剂量和无效突变在ANP/cGMP介导的生物学应答中的影响的手段。总的来说，该项目预计将产生重要的新的见解的分子机制，NPRA依赖的血压调节，肾功能和心血管病理生理学一般。

DESCRIPTION (provided by applicant): Atrial natriuretic peptide (ANP), binds to guanylyl cyclase/natriuretic peptide receptor-A (NPRA/GC-A) and elicits natriuretic, diuretic, vasorelaxant, and antiproliferative responses, important factors in the control of blood pressure and blood volume. However, the molecular basis of these activities and the functional expression and regulation of Nprl gene (coding for NPRA) remain incompletely understood. To further understand the functional significance of Nprt gene, we will study its transcriptional regulation using genomic clones, which we have isolated and sequenced, and physiological function(s) using the gene-targeted mutant mouse models, which we have established at our facility. Our fundamental hypothesis is that the regulated activity of Nprl gene requires cooperative interactions among ubiquitous and tissue-specific transcription factors that respond to extra-and intracellular signals, and that the absence of Nprl gene expression in intact animals in vivo renders unopposed powerful sodium retaining, vasoconstrictive, and proliferative systems, whereas, overexpression of Nprl gene exerts a gene-dose-dependent action that is natriuretic, vasodilatory, and antiproliferative in nature. To accomplish the objective of this proposal, we will integrate genetic information at the molecular level, with biochemical information at protein level, and physiological information at the whole animal level, resulting in a vertically integrated molecular-physiological strategy. We will exploit the power of molecular genetic techniques to answer cellular, biochemical, and pathophysiological questions in intact animals in vivo and isolated cultured cells in vitro so as to arrive at conclusions that are definitive and physiologically relevant. The information obtained from the above lines of investigation will provide the means to test directly the efficacy of Nprl genomic regulatory elements and the impact of Nprl gene dosage and null mutation in ANP/cGMP-mediated biological responses. Overall, this project is expected to produce important new insights into the molecular mechanisms that underlie NPRA-dependent blood pressure regulation, kidney function, and cardiovascular pathophysiology in general.