TY - JOUR
T1 - Mutations in the 1,25-dihydroxyvitamin D3 receptor identifying C- terminal amino acids required for transcriptional activation that are functionally dissociated from hormone binding, heterodimeric DNA binding, and interaction with basal transcription factor IIB, in vitro
AU - Jurutka, Peter
AU - Hsieh, Jui Cheng
AU - Remus, Lenore S.
AU - Whitfield, G. Kerr
AU - Thompson, Paul D.
AU - Haussler, Carol A.
AU - Blanco, Jorge C G
AU - Ozato, Keiko
AU - Haussler, Mark R.
PY - 1997/6/6
Y1 - 1997/6/6
N2 - To investigate a potential ligand-dependent transcriptional activation domain (AF-2) in the C-terminal region of the human vitamin D receptor (hVDR), two conserved residues, Leu-417 and Glu-420, were replaced with alanines by site-directed mutagenesis (L417A and E420A). Transcriptional activation in response to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was virtually eliminated when either point mutant was transfected into several mammalian cell lines. Furthermore, both mutants exhibited a dominant negative phenotype when expressed in COS-7 cells. Scatchard analysis at 4 °C and a ligand-dependent DNA binding assay at 25 °C revealed essentially normal 1,25-(OH)2D3 binding for the mutant hVDRs, which were also equivalent to native receptor in associating with the rat osteocalcin vitamin D responsive element as a presumed heterodimer with retinoid X receptor. Glutathione S- transferase-human transcription factor IIB (TFIIB) fusion protein linked to Sepharose equally coprecipitated the wild-type hVDR and the AF-2 mutants. These data implicate amino acids Leu-417 and Glu-420, residing in a putative α-helical region at the extreme C terminus of hVDR, as critical in the mechanism of 1,25-(OH)2D3-stimulated transcription, likely mediating an interaction with a coactivator(s) or a component of the basal transcriptional machinery distinct from TFIIB.
AB - To investigate a potential ligand-dependent transcriptional activation domain (AF-2) in the C-terminal region of the human vitamin D receptor (hVDR), two conserved residues, Leu-417 and Glu-420, were replaced with alanines by site-directed mutagenesis (L417A and E420A). Transcriptional activation in response to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was virtually eliminated when either point mutant was transfected into several mammalian cell lines. Furthermore, both mutants exhibited a dominant negative phenotype when expressed in COS-7 cells. Scatchard analysis at 4 °C and a ligand-dependent DNA binding assay at 25 °C revealed essentially normal 1,25-(OH)2D3 binding for the mutant hVDRs, which were also equivalent to native receptor in associating with the rat osteocalcin vitamin D responsive element as a presumed heterodimer with retinoid X receptor. Glutathione S- transferase-human transcription factor IIB (TFIIB) fusion protein linked to Sepharose equally coprecipitated the wild-type hVDR and the AF-2 mutants. These data implicate amino acids Leu-417 and Glu-420, residing in a putative α-helical region at the extreme C terminus of hVDR, as critical in the mechanism of 1,25-(OH)2D3-stimulated transcription, likely mediating an interaction with a coactivator(s) or a component of the basal transcriptional machinery distinct from TFIIB.
UR - http://www.scopus.com/inward/record.url?scp=0030980888&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030980888&partnerID=8YFLogxK
U2 - 10.1074/jbc.272.23.14592
DO - 10.1074/jbc.272.23.14592
M3 - Article
C2 - 9169418
AN - SCOPUS:0030980888
SN - 0021-9258
VL - 272
SP - 14592
EP - 14599
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 23
ER -