Studies on response element of vitamin D target genes and discrimination of vitamin D actions

本文采用二维丙氨酸扫描突变分析法（2D-ASMA）研究了维生素D受体（VDR）及其配体之间的相互作用。我们对VDR配体结合口袋的所有氨基酸残基进行了丙氨酸的一点突变，并通过荧光素酶测定方法使用12个配体评估了它们的转录活性。结果呈现在二维表格上，每个突变体垂直排列，化合物水平排列。从该表中，我们得到以下结果：1）具有维生素D结构的配体显示出彼此相似的反式激活模式。2)具有维生素D结构的配体具有用于反式激活的10个必需残基（Y143、D144、L233、I271、R274、W286、Y295、H397、Y 401、F422）。3)各组结构相似的配体均表现出特征性的活性模式。4)对于活性弱的配体，必需残基的数量增加。5)具有比野生型更强活性的突变体增加维生素D超激动剂。6)没有维生素D结构的LCAs显示出与维生素D配体不同的活性模式。基于这些结果，我们将探讨维生素D作用的识别机制。

We investigated the interactions between the vitamin D receptor (VDR) and its ligands by a new method which is termed two-dimensional alanine scanning mutational analysis (2D-ASMA). We made one-point mutants to alanine of all amino acid residues lining the ligand binding pocket of the VDR and evaluated their transcriptional activity using 12 ligands by luciferase assay method. The results were presented on two-dimensional table with each mutant arranged at the vertical and compounds at the horizontal. From this table we obtained the followings: 1) Ligands with vitamin D structure showed similar transactivation pattern each other. 2) Ligands with vitamin D structure have ten essential residues (Y143, D144, L233, 1271, R274, W286, Y295, H397, Y401, F422) for transactivation. 3) Each group of ligands with similar structure showed characteristic activity pattern. 4) Number of essential residues increases for ligands with weak activity. 5) Mutants having stronger activity than the wild type increase in vitamin D super-agonists. 6) LCAs without vitamin D structure show different activity pattern from vitamin D ligands. Based on these results we will investigate discrimination mechanism of vitamin D actions.