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 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

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

In: Journal of Biological Chemistry, Vol. 272, No. 23, 06.06.1997, p. 14592-14599.

Research output: Contribution to journalArticle

@article{7a78383af00441c393694546d50894bf,
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 = "Peter Jurutka and Hsieh, {Jui Cheng} 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

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 -