CALCITONIN RECEPTOR GENE EXPRESSION

我们最初的应用是研究作用机制和 降钙素受体(CTR)基因调控。虽然终极的 目标是利用这些信息来深入了解对 破骨细胞的分化和功能，我们扩大了我们的范围 研究将包括对生物多样性的调节和机制的研究 CTR在未直接与矿物质有关的器官和组织中的作用 离子动态平衡。我们已经确定人类CTR基因编码 多种功能不同的CTR亚型，并表达 以特定于组织和细胞的方式。这些观察结果已经 提供了对生物活动的多样性的见解 降钙素(CT)。在竞争的更新中，我们将继续专注于 关注破骨细胞CTR，但也将研究受体 在其他对CT敏感的组织中。目标如下：1.利用 逆转录聚合酶链式反应(RT-PCR)及其克隆 完成序列测定和表征的技术 人CTR亚型的结构特性。相应的 将确定基因组结构，并针对组织和细胞 用原位杂交确定的异构体的分布， Northern杂交分析和核糖核酸酶保护。2.填写以下表格 特定骨骼病变中的细胞特征，例如Paget病 骨骼和其他肉芽肿性疾病和肿瘤的 巨细胞的表型及其与破骨细胞的关系为 还不是很成熟。这将有助于定义表型 生理性骨吸收细胞与骨吸收细胞的关系 病理重塑并确定关节的结构特征 破骨细胞相关CTR。3.因为CTR受体家族 展示不同于其他成员的结构特征 G蛋白偶联受体超家族的结构/功能 这些受体的关系尤其令人感兴趣。我们 还建议利用现有的不同CTR cDNA 作为我们最近发现的其他CTR亚型，以定义 人CTR独特的信号特性和结合动力学 异构体。4.在最终的具体目标中，我们建议克隆和 鉴定人CTR基因并确定其特异性和 潜在独特的调控序列负责表达 成骨细胞和其他表达CTR的细胞和组织中的CTR。 含可能启动子的CTR5‘-区的构建 Ctr基因序列将与报告基因连锁(S)和 利用转染法筛选诱导CTR表达的能力 在人单核细胞系或其他类型的细胞中 表示CTR(例如肾脏)。这将定义结构基础 CTR基因调控并允许识别潜在的独一无二 规范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.