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

Peter W. Jurutka, Jui-Cheng Hsieh, Lenore S. Remus, G Kerr Whitfield, Paul D. Thompson, Carol A. Haussler, Jorge C G Blanco, Keiko Ozato, Mark R Haussler

Research output: Contribution to journalArticle

74 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)14592-14599
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number23
DOIs
StatePublished - Jun 6 1997

Fingerprint

Transcription Factor TFIIB
Calcitriol Receptors
Furylfuramide
Transcriptional Activation
Chemical activation
Hormones
Amino Acids
Mutation
DNA
Ligands
Retinoid X Receptors
Mutagenesis
Calcitriol
Osteocalcin
Transcription
Glutathione Transferase
Vitamin D
Alanine
Sepharose
Machinery

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{cc6344c1aa434113979db284e6fda589,
title = "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",
abstract = "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.",
author = "Jurutka, {Peter W.} and Jui-Cheng Hsieh and Remus, {Lenore S.} and Whitfield, {G Kerr} and Thompson, {Paul D.} and Haussler, {Carol A.} and Blanco, {Jorge C G} and Keiko Ozato and Haussler, {Mark R}",
year = "1997",
month = "6",
day = "6",
doi = "10.1074/jbc.272.23.14592",
language = "English (US)",
volume = "272",
pages = "14592--14599",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "23",

}

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 W.

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

VL - 272

SP - 14592

EP - 14599

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 23

ER -