Natriuretic peptide receptor: Structure and signaling

超出提供的空间。心钠素(ANP)在心血管系统的调节和维持中起重要作用，其异常可导致高血压、心肌肥厚、充血性心力衰竭等心血管疾病。ANP的作用是通过与鸟苷环化酶(GCase)偶联的细胞膜受体介导的。本研究的目的是阐明ANP-受体相互作用和跨膜信号转导的机制。ANP受体是一种130 kDa的跨膜蛋白，含有一个胞外ANP结合域(BCD)、一个单一的跨膜序列和一个胞内结构域(ICD)，ICD由一个激酶同源调节域和一个鸟苷环化酶催化结构域组成。ANP受体的信号转导机制目前尚不清楚，已有研究表明ANP激活GCase可能是通过配体诱导受体二聚化来实现的。然而，我们和其他人已经证明，在没有激素的情况下，ANP受体可以二聚化，这表明仅有二聚化是不足以激活受体的。因此，我们假设，除了二聚化外，ANP结合还诱导细胞外结构域的构象变化，该构象变化通过细胞膜传递到细胞内区域，并导致GCase催化活性的激活。最近，我们发现ANP与其受体的结合是氯离子浓度依赖的。这一发现表明，可能是氯介导的心钠素受体反馈控制在盐液平衡中发挥作用。为了了解氯离子调控信号的机制，我们提出：1)通过定点突变结合结合ANP和非活性类似物AP-1的BCD结构测定，阐明受体-ANP相互作用的机制和ANP诱导的构象变化引起的信号转导；2)通过突变表征氯结合部位，通过生物物理分析氯离子对BCD构象和二聚化的影响，以及测定无氯BCD的晶体结构，阐明氯离子控制ANP结合的机制。3)表达和纯化ICD，并鉴定其活性和分子行为，以了解ICD在ANP受体信号转导中的作用机制。这些研究将使我们更准确地了解ANP-受体相互作用和信号传递的机制，ANP受体的氯离子控制，以及GCase激活响应ANP结合信号的机制。这些知识将使我们更好地了解ANP的生理作用，从而促进心血管疾病更可靠的诊断方法和更有效的治疗方法的发展。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. Atrial natriuretic peptide (ANP) plays a major role in the regulation and maintenance of the cardiovascular system and its aberrations may lead to hypertension, cardiac hypertrophy, congestive heart failure, and other cardiovascular diseases. The actions of ANP are mediated by cell membrane receptors coupled to guanylate cyclase (GCase). The goal of our research is to elucidate the mechanisms of ANP-receptor interaction and transmembrane signal transduction. The ANP receptor is a 130-kDa transmembrane protein containing an extracellular ANP-binding domain (BCD), a single transmembrane sequence, and an intracellular domain (ICD) consisting of a kinase-homologous regulatory domain and a guanylate cyclase catalytic domain. The mechanism of signal transduction by the ANP receptor remains largely unknown.It has been suggested that GCase activation by ANP may be mediated by ligand-induced receptor dimerization. However, the ANP receptor has been shown by us and others to dimerize in the absence of the hormone, indicating that dimerization alone is not sufficient for receptor activation. Thus, we hypothesize that ANP binding induces, in addition to dimerization, a conformational change in the extracellular domain which is transduced across the cell membrane to the intracellular region and causes activation of the GCase catalytic activity. Recently, we have found that ANP binding to its receptor is chloride concentration dependent. This finding suggest a possible chloride mediated feedback control of the ANP receptor playing a role in salt-fluid homeostasis. To understand the mechanisms of signaling and its control by chloride, we propose 1) to elucidate the mechanism of receptor-ANP interaction and the ANP-induced conformational changes responsible for signal transduction by a combination of site-directed mutagenesis and crystallographic determination of the structures of the BCD bound with ANP and with the inactiveanalog AP-I, 2) to elucidate the mechanism of chloride-control of ANP binding by characterizing the chloride-binding site by mutagenesis, analyzingchloride effects on the BCD conformation and dimerization by biophysical analyses, and determining the crystal structure of the BCD lacking chloride, and 3) to express and purify the ICD and to characterize its activities and molecular behavior to understand the mechanism of ICD action in ANP receptor signaling. These studies will provide us a more accurate understanding of the mechanisms of ANP-receptor interaction and signaling, chloride-control of the ANP receptor, and the mechanism of GCase activation in response to ANP binding signal. Such knowledge will give us a better understanding of the physiological actions of ANP that, in turn, will promote development of more reliable diagnostic methods and more effective therapies for the cardiovascular diseases. PERFORMANCE SITE ========================================Section End===========================================