PARATHYROID HORMONE RECEPTORS IN KIDNEY AND BONE

甲状旁腺激素（PTH）通过调节钙和磷酸盐稳态 对骨骼和肾脏的直接协调作用。 大多数，如果不是全部， 作用是由PTH受体介导的腺苷酸激活启动的 环化酶-环AMP级联反应。 尽管联合国发挥着主要和必要的作用， 对于PTH受体在激素中的作用，几乎一无所知 关于受体结合PTH能力的分子基础 （“识别”）或启动生物作用（“转导”）。 本提案是对这一问题的系统性处理办法。 具体目的是：1）溶解和部分纯化PTH 受体从骨和肾脏在功能上活跃的状态。 存在 在纯化过程中，将通过以下方式监测受体的浓度： 直接或重建后125 I-PTH（1-34）的结合 受体进入脂质体和缺乏PTH的人红细胞膜 受体。 溶解受体的功能活性保留 将通过其重建PTH敏感性腺苷酸的能力来证明 在cyc- S-49淋巴瘤细胞膜中缺乏环化酶， 刺激性鸟嘌呤核苷酸调节蛋白（Ns）。 纯化将 使用凝胶过滤和亲和层析的组合进行 色谱法，后者采用固定化PTH（1-34）以及 抗受体单克隆抗体。 2)为了确定功能域， 部分纯化的PTH受体。 在这些研究中，单克隆 特异性针对细胞膜的“识别”和“转导”区域的抗体， PTH受体将被开发并用作受体的分子探针 结构 3)使用单克隆抗体对抗“转换器”区域 的PTH受体，以确定是否其他腺苷酸环化酶偶联 激素受体具有同源转导结构域。 4)以确定 假性甲状旁腺功能减退症患者的Ns是否选择性 其重建高亲和力PTH受体的能力缺陷， 红细胞和S-49细胞膜中的PTH反应性腺苷酸环化酶。 这些实验的成功完成将描绘出 激素结合和信号传导所需的PTH受体的性质 以及PTH遗传缺陷的分子基础 受体-腺苷酸环化酶偶联存在于假性甲状旁腺功能减退症。 抗甲状旁腺素受体的单克隆抗体将有助于分离 PTH反应性骨和肾细胞的纯群体，并且，最终， 用于分离编码PTH受体的基因。

Parathyroid hormone (PTH) regulates calcium and phosphate homeostasis via direct, coordinated actions on bone and kidney. Most, if not all, of these effects are initiated by PTH receptor-mediated activation of the adenylate cyclase-cyclic AMP cascade. Despite the primary and necessary role of the PTH receptor in the hormone's effects, virtually nothing is known concerning the molecular basis of the receptor's ability to bind PTH ("recognition") or to initiate biologic action ("transduction"). The present proposal constitutes a systematic approach to this problem. The specific objectives are: 1) To solubilize and partially purify the PTH receptor from bone and kidney in a functionally active state. The presence of receptor during the course of purification will be monitored through binding of 125I-PTH(1-34) either directly or after reconstitution of the receptor into liposomes and human red cell membranes, which lack PTH receptors. Retention of functional activity of the solubilized receptor will be demonstrated by its ability to reconstitute PTH-sensitive adenylate cyclase in cyc- S-49 lymphoma cell membranes that are deficient in the stimulatory guanine nucleotide regulatory protein (Ns). Purification will be effected using a combination of gel filtration and affinity chromatography, the latter employing immobilized PTH(1-34) as well as anti-receptor monoclonal antibodies. 2) To identify functional domains of the partially purified PTH receptor. In these studies, monoclonal antibodies specific for the "recognition" and "transduction" regions of the PTH receptor will be developed and employed as molecular probes of receptor structure. 3) To use monoclonal antibodies against the "transducer" region of the PTH receptor to determine whether other adenylate cyclase-coupled hormone receptors have a homologous transduction domain. 4) To determine whether Ns from patients with pseudohypoparathyroidism is selectively defective in its ability to reconstitute high-affinity PTH receptors in erythrocytes, and PTH-responsive adenylate cyclase in S-49 cyc- membranes. Successful completion of these experiments would delineate the structural properties of PTH receptors required for hormone binding and signal generation, as well as the molecular basis for the genetic defect in PTH receptor-adenylate cyclase coupling present in pseudohypoparathyroidism. The anti-PTH receptor monoclonal antibodies will be useful for isolating pure populations of PTH-responsive bone and kidney cells, and, ultimately, for isolating genes encoding the PTH receptor.