STRUCTURE/FUNCTION STUDIES OF THE VITAMIN D RECEPTOR

维生素 D 受体的结构/功能研究

• 批准号: 3463503
• 负责人: G K WHITFIELD
• 金额: $ 9.92万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3463503
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA footprinting; aminoacid; brain mapping; calcium binding protein; chickens; complementary DNA; fibroblasts; gene deletion mutation; genetic enhancer element; genetic transcription; glucocorticoids; messenger RNA; model design /development; molecular cloning; mutant; proteolysis; receptor expression; reporter genes; site directed mutagenesis; steroid hormone; steroid hormone biosynthesis; steroid hormone receptor; transfection; transposon /insertion element; vitamin D; vitamin D resistant rickets

The main objective of the proposed research project is to gain further information and insight into the mode of action of the vitamin D receptor (VDR). This is to be approached by developing a model system consisting of a) the VDR cDNA cloned into a vector which will allow for the expression of normal or mutagenized receptor in transfected cells, b) the cloned 5' region of the calcium-binding protein (CaBP) gene, a gene known to be induced at the level of transcription by the VDR, and c) recipient cell lines which are VDR-deficient, such as the ROS 24/1 line. This system, a large portion of which is already available, will permit extensive testing of the functional domains of the VDR, including those for hormone-binding, DNA binding, and transcriptional activation. The regions of the CaBP gene which interact with the VDR will be delineated, first by testing partially deleted versions of the CaBP gene, and then by more direct methods, such as DNase I footprinting. Next, those regions of the VDR necessary for hormone binding and for VDR binding to the site(s) on the CaBP gene, already known in broad outline from earlier studies, will be precisely determined by partial deletion of the VDR cDNA, and then by site-directed mutagenesis of specific amino acid codons. Finally, this approach should permit the identification of the region(s) of the VDR responsible for transcriptional activation of CaBP. The functional domains of the VDR can ultimately be tested by inserting them into heterologous proteins, such as the glucocorticoid receptor, to see if they are necessary and sufficient for their function. The availability of fibroblasts from a patient with vitamin-D dependent rickets type II will be exploited as a means of analyzing an in vivo VDR- deficient mutant. It will be seen whether transfection of a human VDR cDNA into these cells can restore their responsiveness to the vitamin D hormone, as indicated by an induction of 24-hydroxylase. If restoration is achieved, confirming that the VDR is the site of defect, then a project will be initiated to determine the exact nature of the lesion in the VDR from these cells. A characterization of the mode of action of the VDR, a member of the family of steroid and thyroid hormone receptors, is significant to basic biology and should enhance our understanding of tissue-specific gene regulation.

拟议研究项目的主要目标是进一步获得 关于维生素 D 受体作用模式的信息和见解 （VDR）。这可以通过开发一个模型系统来解决，该系统包括 a) 将 VDR cDNA 克隆到载体中，该载体将允许 转染细胞中正常或诱变受体的表达，b) 钙结合蛋白 (CaBP) 基因的克隆 5' 区域，该基因已知 由 VDR 在转录水平上诱导，以及 c) 受体 VDR 缺陷的细胞系，例如 ROS 24/1 系。这 系统，其中很大一部分已经可用，将允许 对 VDR 的功能域进行广泛测试，包括 用于激素结合、DNA 结合和转录激活。 CaBP 基因中与 VDR 相互作用的区域是 首先通过测试 CaBP 基因的部分删除版本来描绘， 然后通过更直接的方法，例如 DNase I 足迹法。下一个， VDR 中激素结合和 VDR 所必需的区域 与 CaBP 基因上已知的位点结合 从早期的研究来看，将通过部分删除来精确确定 VDR cDNA，然后通过特定氨基的定点诱变 酸性密码子。最后，这种方法应该允许识别 VDR 的区域负责转录激活 钙结合蛋白。 VDR 的功能域最终可以通过以下方式进行测试： 将它们插入异源蛋白质，例如糖皮质激素 受体，看看它们对于其功能是否是必要和充分的。 维生素 D 依赖患者的成纤维细胞的可用性 II 型佝偻病将被用作分析体内 VDR- 缺陷突变体。将会看到是否转染人类VDR 将 cDNA 导入这些细胞可以恢复它们对维生素 D 的反应 激素，如 24-羟化酶的诱导所示。如果恢复 已实现，确认 VDR 是缺陷位置，然后 将启动项目以确定病变的确切性质 来自这些细胞的VDR。作用方式的表征 VDR 是类固醇和甲状腺激素受体家族的一员， 对基础生物学具有重要意义，应该增强我们对 组织特异性基因调控。