The unique tryptophan residue of the vitamin D receptor is critical for ligand binding and transcriptional activation.

The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily of transcriptional regulators. Here we show that tryptophan 286 of the hVDR is critical for ligand binding and transactivation of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] target genes. Two mutants of the hVDR were produced, W286A and W286F, in which the tryptophan was replaced with an alanine or a phenylalanine, respectively. The W286A mutant did not bind 1,25(OH)2D3, interact with steroid receptor coactivator 1 (SRC-1) in vitro, or activate transcription. Moreover, the W286A receptor did not heterodimerize in a ligand-dependent manner with the human retinoid X receptor alpha (hRXRalpha). Although the W286F receptor heterodimerized with hRXRalpha, interacted with SRC-1, and bound 1,25(OH)2D3, its capacity to transactivate was attenuated severely. Thus, tryptophan 286 of hVDR plays an important role in specific 1,25(OH)2D3 ligand interaction and subsequently in hVDR/RXR interaction, SRC-1 binding, and ligand-dependent transactivation of 1,25(OH)2D3 target genes. These results identify the first amino acid that is absolutely required for ligand binding in the VDR and further define the structure-function relationship of 1,25(OH)2D3 interaction with its receptor.