STRUCTURE/FUNCTION STUDIES OF THE VITAMIN D RECEPTOR

维生素 D 受体的结构/功能研究

• 批准号: 3463505
• 负责人: G K WHITFIELD
• 金额: $ 10.42万
• 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/3463505
• 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; oxygenases; 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 维生素D受体的作用方式的信息和见解 （VDR）。这是通过开发一个模型系统来实现的， a）克隆到载体中的VDR cDNA，所述载体将允许 正常或诱变受体在转染细胞中的表达，B） 克隆了钙结合蛋白（CaBP）基因的5'区，该基因是已知的 在转录水平上由VDR诱导，和c）受体 VDR缺陷的细胞系，如ROS 24/1系。这 系统，其中很大一部分已经可用，将允许 广泛测试VDR的功能域，包括 用于酶结合、DNA结合和转录激活。 与VDR相互作用的CaBP基因的区域将是 描绘，首先通过测试部分删除版本的CaBP基因， 然后通过更直接的方法，如DNA酶I足迹法。接下来， VDR的那些区域是激素结合和VDR 与CaBP基因上的位点结合，已经大致知道 从早期的研究中，将通过部分删除 VDR cDNA，然后通过特定氨基酸的定点突变 酸性密码子最后，这一方法应能确定 VDR中负责转录激活的区域 CaBP。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，如糖皮质激素， 受体，看看它们是否是必要的和足够的功能。 维生素D依赖性糖尿病患者成纤维细胞的可用性 将利用佝偻病II型作为分析体内VDR的手段， 缺陷突变体将看到转染人VDR是否 cDNA进入这些细胞可以恢复它们对维生素D的反应性， 激素，如24-羟化酶诱导所示。如果恢复 确认VDR是缺陷部位，则 将启动一个项目，以确定病变的确切性质， 这些细胞的VDR的作用方式的表征， VDR是类固醇和甲状腺激素受体家族的成员， 这对基础生物学意义重大，应该能增强我们对 组织特异性基因调控