Receptor mediated genomic action of the 1,25(OH)2D3 hormone: Expression of the human vitamin D receptor in E. coli

Jui-Cheng Hsieh, Shigeo Nakajima, Michael A. Galligan, Peter W. Jurutka, Carol A. Haussler, G Kerr Whitfield, Mark R Haussler

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

The nuclear vitamin D receptor (VDR) binds the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone with high affinity and elicits its actions to stimulate gene expression in target cells by binding to the vitamin D-responsive element (VDRE). VDREs in such positively controlled genes as osteocalcin, osteopontin, β3 integrin and vitamin D-24-OHase are direct hexanucleotide repeats with a spacer of three nucleotides. The present studies of VDR VDRE interaction utilized full-length human vitamin D receptor (hVDR) that was overexpressed in E. coli, purified to near homogeneity (> 95%), and its authenticity confirmed by demonstrating high affinity hormone binding and reactivity to monoclonal antibody 9A7γ. The expressed hVDR displays strict dependence on the family of retinoid X receptors (RXRs) for binding to the vitamin D-responsive element (VDRE) in the rat osteocalcin gene. Similar overexpression in E. coli of the DNA binding domain (Δ134), containing only residues 4-133 of hVDR, generated a receptor species that possesses intrinsic DNA binding activity. Both full-length and Δ134 hVDRs retain similar DNA binding specificities when tested with several natural hormone responsive elements, indicating that the N-terminal zinc finger region determines hVDR-DNA sequence selectivity. The C-terminal region of the molecule is required for hormone binding and confers the receptor with the property of very high affinity DNA binding, via heterodimerization between hVDR and RXR. A natural ligand for the RXR co-receptor, 9-cis retinoic acid, suppresses both VDR-RXR binding to the VDRE and 1,25(OH)2D3 stimulated transcription, indicating that 9-cis retinoic acid recruits RXR away from VDR to instead form RXR homodimers.

Original languageEnglish (US)
Pages (from-to)583-594
Number of pages12
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume53
Issue number1-6
DOIs
StatePublished - 1995

Fingerprint

Calcitriol Receptors
Retinoid X Receptors
Escherichia coli
Hormones
Vitamin D
DNA
Osteocalcin
Genes
Osteopontin
Calcitriol
Cholecalciferol
Nucleic Acid Repetitive Sequences
DNA sequences
Zinc Fingers
Transcription
Gene expression
Integrins
Zinc
Rats
Nucleotides

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Clinical Biochemistry
  • Endocrinology
  • Molecular Biology
  • Molecular Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

Receptor mediated genomic action of the 1,25(OH)2D3 hormone : Expression of the human vitamin D receptor in E. coli. / Hsieh, Jui-Cheng; Nakajima, Shigeo; Galligan, Michael A.; Jurutka, Peter W.; Haussler, Carol A.; Whitfield, G Kerr; Haussler, Mark R.

In: Journal of Steroid Biochemistry and Molecular Biology, Vol. 53, No. 1-6, 1995, p. 583-594.

Research output: Contribution to journalArticle

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abstract = "The nuclear vitamin D receptor (VDR) binds the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone with high affinity and elicits its actions to stimulate gene expression in target cells by binding to the vitamin D-responsive element (VDRE). VDREs in such positively controlled genes as osteocalcin, osteopontin, β3 integrin and vitamin D-24-OHase are direct hexanucleotide repeats with a spacer of three nucleotides. The present studies of VDR VDRE interaction utilized full-length human vitamin D receptor (hVDR) that was overexpressed in E. coli, purified to near homogeneity (> 95{\%}), and its authenticity confirmed by demonstrating high affinity hormone binding and reactivity to monoclonal antibody 9A7γ. The expressed hVDR displays strict dependence on the family of retinoid X receptors (RXRs) for binding to the vitamin D-responsive element (VDRE) in the rat osteocalcin gene. Similar overexpression in E. coli of the DNA binding domain (Δ134), containing only residues 4-133 of hVDR, generated a receptor species that possesses intrinsic DNA binding activity. Both full-length and Δ134 hVDRs retain similar DNA binding specificities when tested with several natural hormone responsive elements, indicating that the N-terminal zinc finger region determines hVDR-DNA sequence selectivity. The C-terminal region of the molecule is required for hormone binding and confers the receptor with the property of very high affinity DNA binding, via heterodimerization between hVDR and RXR. A natural ligand for the RXR co-receptor, 9-cis retinoic acid, suppresses both VDR-RXR binding to the VDRE and 1,25(OH)2D3 stimulated transcription, indicating that 9-cis retinoic acid recruits RXR away from VDR to instead form RXR homodimers.",
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