Functional domain of vitamin D receptor

1α，25-二羟基维生素D_3是维生素D_3最活跃的代谢物，是一种多功能制剂。1α，25-二羟基维生素D3的作用是通过其受体(VDR)以配体依赖的方式激活特定基因而实现的。为了研究VDR功能结构域的细节，我们建立了VDR各部分的高效表达和纯化系统以及全长VDR。首先，我们研究了DNA结合域。23-123位氨基酸足以使VDR与特定的DNA序列结合，称为维生素D负责元件。然而，VDR单独对DNA表现出一周的亲和力，而维甲酸X受体(RXR)明显增加了这种结合亲和力。其次，我们确定了配体结合域的位置。C端21个氨基酸的缺失完全降低了配体的结合活性。至于N-末端，第124个氨基酸之后的区域需要可检测到的配体结合活性。第三，我们绘制了参与同二聚体或异二聚体形成的VDR的二聚化界面。缺失分析表明，VDR至少有三个二聚化界面，其功能明显可区分。最后，我们利用结合位点选择的方法从大鼠基因组DNA中筛选出两个VDRE。其中一个为阴性VDRE，另一个为阳性VDRE。由于没有发现与数据库有显著相似性的序列，这两个基因都是新基因。对这些基因的进一步分析将提供有关维生素D的生物学功能的知识。

1alpha, 25-dihydroxyvitamin D_3, the most active metabolite of vitamin D_3, is a multifunctional agent. The actions of 1alpha, 25-dihydroxyvitamin D_3 are mediated through its receptor (VDR) that activates the specific genes in a ligand-dependent manner. In order to investigate the details of functional domains of VDR,we have developed the overexpression and purification system of various portions of VDR as well as a full-length VDR.At first, we investigated the DNA binding domain. The region of amino acids 23-123 is enough for binding of VDR to specific DNA sequence called as vitamin D responsible element. However, VDR alone showed a week affinity for DNA and retinoid X receptor (RXR) definitely increased the binding affinity. Second, we determined the location of ligand binding domain. The deletion of C-terminal 21 amino acids completely diminished the ligand binding activity. Regarding the N-terminus, the region following the 124th amino acid was required for a detectable level of ligand binding activity. Third, we have mapped the dimerization interfaces of VDR that is involved in homo- or heterodimer formation. The deletion analysis showed that the VDR has at least three dimerization interfaces whose functions are apparently distinguishable. Finally, we picked out two VDREs from rat genomic DNA using binding site selection method. One is identified as negative VDRE and another is positive VDRE.Both of them are novel genes because no sequence sharing significant similarity to databases was found. Further analyzes of these genes would provide knowledge about the biological function of vitamin D.