| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1994: ¥1,500,000 (Direct Cost: ¥1,500,000)
|
|---|
| Research Abstract |
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.
|
