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.