Characterization of the activation function-2 domain of the human 1,25-dihydroxyvitamin D3 receptor.
In this study, we determined the ligand-dependent activation function domain 2 (AF-2) of the human vitamin D receptor (hVDR) and characterized it using site-directed mutagenesis. A single mutation at glutamic acid-420 (E420Q) and an additional mutation at leucine-417 (L417A-E420Q) eliminated ligand-dependent transcriptional activation. In addition, lysine-264 was also demonstrated to be vital for ligand-induced transactivation. However, bacterial-overexpressed transcriptional factor IIB (TFIIB) was able to bind to both AF-2 and lysine-264 mutant hVDRs in vitro. The ligand-dependent transactivation via wild type hVDR was interfered with weakly only when a 10-fold molar excess of L417A-E420Q plasmid was co-transfected. This suppressive effect was diminished by introducing an additional mutation at a cysteine residue in the DNA binding domain. Thus, we conclude that the AF-2 domain of the hVDR located between amino acids 417 and 420, as well as lysine-264, are essential for ligand-dependent transactivation, and that TFIIB was not necessary for the function of these two regions of the hVDR. Our finding that AF-2 mutant hVDRs exhibit only very weak suppressive effect may indicate a difference in the molecular mechanism of the VDR-mediated transactivation from other nuclear receptors.
Department of Environmental Medicine, Research Institute, Osaka Medical Center for Maternal and Child Health, Izumi, Japan.
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S
Nakajima
M
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Yamagata
N
N
Sakai
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K
Ozono
D000818:Animals; D019556:COS Cells; D004247:DNA / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008239:Lysine / Q000502:physiology; D015675:Osteocalcin / Q000235:genetics; D012516:Osteosarcoma; D017354:Point Mutation; D051381:Rats; D018167:Receptors, Calcitriol / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D008024:Ligands; D018167:Receptors, Calcitriol; D011993:Recombinant Fusion Proteins; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D015675:Osteocalcin; D004247:DNA; D008239:Lysine