CALCITONIN RECEPTOR GENE EXPRESSION

我们最初的申请建议研究作用机制， 降钙素受体（CTR）基因的调节。 虽然最终 目标是利用这些信息来深入了解控制 破骨细胞的分化和功能，我们已经扩大了范围， 研究，包括调查的规则和机制， CTR在不直接参与矿物质的器官和组织中的作用 离子稳态 我们已经确定人类CTR基因编码 多种CTR同种型，其在功能上不同，并且在 以组织和细胞特异性的方式。 这些观测 提供了深入了解生物活动的多样性， 降钙素（CT）。 在竞争性的续约中，我们将继续专注于 注意破骨细胞CTR，但也将研究受体 在其他CT反应组织中。 本研究的目的如下：1.利用 逆转录聚合酶链反应（RT-PCR）和克隆 技术来完成测序和表征的 人CTR同种型的结构特性。 相应的 基因组结构将被确定，组织和细胞特异性 使用原位杂交建立的同种型的分布， 北方印迹分析和RNA酶保护。 2.完成 特定骨病变中细胞的表征，例如佩吉特病 骨和其他肉芽肿性疾病和肿瘤，其中 巨细胞的表型及其与破骨细胞的关系是 没有很好地建立。 这将有助于确定表型 生理性骨吸收细胞与 病理性重塑，并确定结构特征的 破骨细胞相关CTR。 3.因为CTR受体家族 表现出与其他成员不同的结构特征， G蛋白偶联受体超家族，结构/功能 这些受体的关系是特别感兴趣的。 我们 我还建议利用目前可用的不同CTR cDNA， 作为我们最近发现的其他CTR亚型，以定义 人CTR的独特信号传导特性和结合动力学 同种型。 4.在最后的具体目标，我们建议克隆和 表征人CTR基因并定义特异性和 可能独特的调控序列负责表达 破骨细胞和其他表达CTR的细胞和组织中的CTR。 含有推定启动子的CTR 5 '区的构建体 CTR基因的序列将与报告基因连接， 筛选使用转染诱导CTR表达的能力 在人单核细胞系或其它组成型 表达CTR（例如肾脏）。 这将定义结构基础 的CTR基因调控，并允许识别潜在的独特的 调节CTR的因素。

Our initial application proposed to study the mechanism of action and regulation of the calcitonin receptor (CTR) gene. Although the ultimate goal was to use this information to gain insights into the control of osteoclast differentiation and function, we have broadened the scope our studies to include investigation of the regulation and mechanism of action of the CTR in organs and tissues not directly involved in mineral ion homeostasis. We have established that the human CTR gene encodes multiple CTR isoforms that are functionally distinct and are expressed in a tissue- and cell-specific fashion. These observations have provided insights into the diversity of the biological activities of calcitonin (CT). In the competing renewal, we will continue to focus attention on the osteoclast CTR, but will also investigate the receptor in other CT-responsive tissues. The Aims are as follow: 1. Utilize the reverse transcriptase/polymerase chain reaction (RT-PCR) and cloning techniques to complete the sequencing and characterization of the structural properties of the human CTR isoforms. The corresponding genomic structure will be identified and the tissue and cell-specific distribution of the isoforms established using in situ hybridization, Northern blot analysis and RNase protection. 2. Complete the characterization of cells in specific bone lesions, e.g. Paget's disease of bone and other granulomatous diseases and tumors in which the phenotype of the giant cells and their relationship to osteoclasts is not well established. This will help to define the phenotypic relationship between bone resorbing cells in physiological and pathological remodeling and identify the structural features of the osteoclast-associated CTR . 3. Because the CTR family of receptors exhibit structural features that are different from other members of the G protein-coupled receptor superfamily, the structure/function relationships for these receptors are of particular interest. We propose to utilize the different CTR cDNAs presently available, as well as additional CTR isoforms that we have recently identified, to define the unique signaling properties and binding kinetics of the human CTR isoforms. 4. In the final Specific Aim, we propose to clone and characterize the human CTR gene and to define the specific and potentially unique regulatory sequences responsible for expression of the CTR in osteclasts and other CTR-expressing cells and tissues. Constructs of the CTR 5'-region containing the putative promoter sequences of the CTR gene will be linked to reporter gene(s) and screened for the capacity to induce CTR expression using transfection in human mononuclear cell lines or other cell types that constitutively express the CTR (e.g. kidney). This will define the structural basis of CTR gene regulation and permit identification of potentially unique factors that regulate the CTR.