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.
Solomon
C
C
Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada.
Macoritto
M
M
Gao
X L
XL
White
J H
JH
Kremer
R
R
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D019281:Dimerization; D051548:Histone Acetyltransferases; D006801:Humans; D008024:Ligands; D009154:Mutation / Q000235:genetics; D056920:Nuclear Receptor Coactivator 1; D011485:Protein Binding; D018167:Receptors, Calcitriol / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000378:metabolism
D008024:Ligands; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014807:Vitamin D; C097949:1,25-dihydroxyvitamin D; D014364:Tryptophan; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1