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

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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.