NATRIURETIC PEPTIDE RECEPTOR--STRUCTURE AND SIGNALING

心钠素(ANP)受体由单个 含有胞外心钠素结合域的多肽，单个 跨膜序列，以及包含一个 激酶同源结构域和鸟苷环化酶(GCase)结构域。 到目前为止，关于心钠素受体的研究大多是进行的。 通过缺失突变，主要集中在激酶的功能上- 调节受体活性的同源结构域。因此，我们的 对细胞外心钠素的结构和功能的认识 结合结构域基本上为零。这项提案的总体目标是 是为了了解ANP如何与结合域相互作用， 由这种相互作用产生的激活信号的性质是，以及 这种激活信号是如何传递到细胞内的 域。在对牛肾上腺ANP受体的研究中 膜，我们观察到受体的蛋白质降解过程 内源性膜结合蛋白水解酶。对这一现象的进一步研究 使我们在受体中识别出一种独特的“铰链”状结构 在ANP上经历明显构象变化的分子 结合，显然在受体信号转导中起着关键作用。 我们建议研究个别结构在铰链中所扮演的角色- 通过产生定点突变和评估在信号传递中的区域 它们对ANP结合、GCase激活以及ANP诱导的 铰链区的构象变化。以促进结构性的 经鉴定，我们已经产生了胞外ANP结合域 通过删除跨膜以可溶形式表达受体 序列和胞内结构域。我们还开发了一种新的 亲和标记程序，称为逐步亲和标记， 允许高度特定和近乎定量的化学标记 ANP结合部位。用这种方法和用微分技术 化学修饰，我们建议鉴定和确定几个 构成结合位点的受体序列的片段 结构。利用结构信息，我们进一步提出了 创建结合位点残基的定点突变以评估 它们在ANP结合和GCase激活中的作用。这些研究将 生成最终了解 受体-配体相互作用和受体激活的机制。

Atrial natriuretic peptide (ANP) receptor consists of a single polypeptide containing an extracellular ANP-binding domain, a single transmembrane sequence, and an intracellular region containing a kinase-homologous domain and a guanylate cyclase (GCase) domain. Studies of the ANP receptor reported to date have been performed mostly by deletion mutagenesis, focusing mainly on the function of the kinase- homologous domain that modulates receptor activity. consequently, our knowledge of the structure and function of the extracellular ANP- binding domain is essentially nil. The overall goal of this proposal is to understand how ANP interacts with the binding domain, what the nature of the activation signal generated by such interaction is, and how this activation signal is transmitted into the intracellular domain. In our studies of the ANP receptor in bovine adrenal membranes, we observed proteolytic processing of the receptor by endogenous membrane-bound proteases. Further studies of this phenomenon led us to identify a unique "hinge"-like structure in the receptor molecule that undergoes a distinct conformational change upon ANP binding and apparently plays a critical role in receptor signaling. We propose to examine the roles of individual structures in the hinge- region in signaling by producing site-directed mutations and evaluating their effects on ANP-binding, GCase-activation, and the ANP-induced conformational change in the hinge-region. To facilitate structural characterization, we have produced the extracellular ANP-binding domain of the receptor in a soluble form by deleting the transmembrane sequence and the intracellular domain. We have also developed a new affinity-labeling procedure, termed stepwise affinity-labeling, that allows highly specific and nearly quantitative chemical labeling of the ANP-binding site. By this method and by the technique of differential chemical modification, we propose to identify and determine several segments of the receptor sequence that constitute the binding-site structure. Utilizing the structural information, we further propose to create site-directed mutations of binding-site residues to evaluate their roles in ANP-binding and GCase-activation. These studies will generate information necessary for ultimate understanding of the mechanisms of the receptor-ligand interaction and receptor activation.