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 sup erfamily of transcriptional regulators, Here we show that tryptophan 286 of the hVDR Is critical for ligand binding and transactivation of 1,25-dihydr oxyvitamin D-3 [1,25(OH)(2)D-3] target genes. Two mutants of the hVDR were produced, W286A and W286F, in which the tryptophan was replaced with an ala nine or a phenylalanine, respectively. The W286A mutant did not bind 1,25(O H)2D3, interact with steroid receptor coactivator 1 (SRC-1) in vitro, or ac tivate transcription. Moreover, the W286A receptor did not heterodimerize i n a ligand-dependent manner with the human retinoid X receptor alpha (hRXR alpha). Although the W286F receptor heterodimerized with hRXR alpha, intera cted with SRC-1, and bound 1,25(OH)(2)D-3 its capacity to transactivate was attenuated severely. Thus, tryptophan 286 of hVDR plays an important role in specific 1,25(OH)(2)D-3 ligand interaction and subsequently in hVDR/RXR interaction, SRC-1 binding, and ligand-dependent transactivation of 1,25(OH )(2)D-3 target genes, These results identify the first amino acid that is a bsolutely required for ligand binding in the VDR and further define the str ucture-function relationship of 1,25(OH)(2)D-3 interaction with its recepto r.