STRUCTURE/FUNCTION STUDIES OF THE VITAMIN D RECEPTOR

拟议的研究项目的主要目标是进一步获得 维生素D受体作用方式的信息和洞察力 (VDR)。这将通过开发一个模型系统来实现，该模型系统包括 A)将VDR基因克隆到载体中，这将允许 正常或突变受体在转基因细胞中的表达，b) 克隆钙结合蛋白(CABP)基因的5‘端，这是一个已知的基因 由VDR在转录水平上诱导，以及c)接收者 缺乏VDR的细胞系，如ROS 24/1细胞系。这 系统，其中很大一部分已经可用，将允许 对VDR的功能域进行广泛的测试，包括 用于激素结合、DNA结合和转录激活。 与VDR相互作用的CABP基因区域将是 首先通过测试CABP基因的部分缺失版本来描述， 然后通过更直接的方法，如DNase I足迹法。下一首, VDR中激素结合和VDR所必需的区域 已知的CABP基因上的结合位点(S) 根据早期的研究，将准确地确定部分删除 VDR基因，然后对特定的氨基酸进行定点突变 酸性密码子。最后，这种方法应该允许识别 VDR中负责转录激活的区域(S) CABP。VDR的功能域最终可以通过以下方式进行测试 将它们插入到异源蛋白质中，如糖皮质激素 受体，看看它们是否对其功能是必要的和充分的。 维生素D依赖患者成纤维细胞的可用性 II型软骨病将被开发为一种分析体内VDR- 缺陷型突变体。将会观察到，将人类VDR基因 将c DNA导入这些细胞可以恢复它们对维生素D的反应性 荷尔蒙，如24-羟基酶的诱导所示。如果恢复 ，以确认VDR是缺陷位置，则一个 将启动项目，以确定#年损害的确切性质 来自这些细胞的VDR。对政府的行动模式的刻画 VDR是类固醇和甲状腺激素受体家族的成员， 对基础生物学有重要意义，应该提高我们对 组织特异性基因调控。

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.