Vitamin D receptor zinc finger region binds to a direct repeat as a dimer and discriminates the spacing number between each half-site.
1 alpha,25-Dihydroxyvitamin D3, the most active metabolite of vitamin D3, is a multifunctional agent. The actions of 1 alpha,25-dihydroxyvitamin D3 are mediated through its receptor that activates the specific genes in a ligand-dependent manner. In order to investigate the details of DNA binding properties of vitamin D receptor, we have developed the overexpression and purification system of vitamin D receptor DNA binding domain. The purified peptide could specifically bind to the osteopontin-derived vitamin D responsible element (VDRE) but not to the osteocalcin and the calbindin D-9k-derived VDREs, as determined by bandshift analysis. The osteopontin VDRE contains a direct repeat of GGTTCA motif separated by 3 nucleotides, whereas the osteocalcin and calbindin D-9k VDREs have inadequate direct repeat. Further analyses using synthetic oligonucleotides revealed that vitamin D receptor DNA binding domain could discriminate the spacing number between the consensus steroid-responsible element motif and had different affinities to direct repeats that consisted of various related sequences. These studies give insight into ways in which vitamin D receptor mediates the signal of 1 alpha,25-dihydroxyvitamin D3.
Department of Environmental Biochemistry, Faculty of Pharmaceutical Sciences, Osaka University, Japan.
J
J
Nishikawa
M
M
Matsumoto
K
K
Sakoda
M
M
Kitaura
M
M
Imagawa
T
T
Nishihara
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D064026:Calbindins; D002117:Calcitriol / Q000494:pharmacology; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D046911:Macromolecular Substances; D008322:Mammals; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D015675:Osteocalcin / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D016335:Zinc Fingers / Q000502:physiology*
D064026:Calbindins; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D064030:S100 Calcium Binding Protein G; D015675:Osteocalcin; D002117:Calcitriol