Baculovirus-mediated expression of the human vitamin D receptor: Functional characterization, vitamin D response element interactions, and evidence for a receptor auxiliary factor

Paul N. MacDonald, Carol A. Haussler, Christopher M. Terpening, Michael A. Galligan, Mina C. Reeder, G Kerr Whitfield, Mark R Haussler

Research output: Contribution to journalArticle

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Abstract

A baculovirus expression vector system (BEVS) was used to overproduce the full-length human vitamin D receptor (hVDR) in Spodoptera frugiperda ovarian cells. hVDR was expressed to a level of 0.5% of the total soluble protein in this system. Western analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunologie properties equivalent to those of hVDR expressed in mammalian cells. The BEVS-derived receptor displayed specificity and high affinity (apparent Kd = 0.7 nM) for the 1,25(OH)2D3 ligand. Recombinant hVDR generated a specific protein-DNA complex with a duplex oligomer containing a vitamin D-responsive element (VDRE) in gel mobility shift assays. The intensity of the VDR-VDRE complex was not affected by 1,25(OH)2D3. However, the complex exhibited increased mobility in the presence of hormone, possibly the result of a 1,25(OH)2D3-dependent conformational change. A nuclear extract obtained from CV-1 cells markedly enhanced the intensity of this VDR·VDRE complex and produced an additional distinct VDR-dependent complex, thus implicating a role for nuclear auxiliary factors in multiple high affinity VDR·VDRE interactions. Finally, methylation interference studies defined the guanine residues contacted when the putative VDR-auxiliary factor complex associates with the rat osteocalcin VDRE; specifically, all of the GC base pairs in the sequence GGGTGAATGAGGACA. Therefore, these results show that the BEV system elicits high level expression of hVDR with critical functional characteristics being preserved.

Original languageEnglish (US)
Pages (from-to)18808-18813
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number28
StatePublished - Oct 5 1991

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Vitamin D Response Element
Calcitriol Receptors
Baculoviridae
Vitamin D
Spodoptera
Proteins
Methylation
Osteocalcin
Guanine
Electrophoretic Mobility Shift Assay
Oligomers
Base Pairing
Rats
Assays
Gels
Cells
Hormones
Ligands
DNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Baculovirus-mediated expression of the human vitamin D receptor : Functional characterization, vitamin D response element interactions, and evidence for a receptor auxiliary factor. / MacDonald, Paul N.; Haussler, Carol A.; Terpening, Christopher M.; Galligan, Michael A.; Reeder, Mina C.; Whitfield, G Kerr; Haussler, Mark R.

In: Journal of Biological Chemistry, Vol. 266, No. 28, 05.10.1991, p. 18808-18813.

Research output: Contribution to journalArticle

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abstract = "A baculovirus expression vector system (BEVS) was used to overproduce the full-length human vitamin D receptor (hVDR) in Spodoptera frugiperda ovarian cells. hVDR was expressed to a level of 0.5{\%} of the total soluble protein in this system. Western analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunologie properties equivalent to those of hVDR expressed in mammalian cells. The BEVS-derived receptor displayed specificity and high affinity (apparent Kd = 0.7 nM) for the 1,25(OH)2D3 ligand. Recombinant hVDR generated a specific protein-DNA complex with a duplex oligomer containing a vitamin D-responsive element (VDRE) in gel mobility shift assays. The intensity of the VDR-VDRE complex was not affected by 1,25(OH)2D3. However, the complex exhibited increased mobility in the presence of hormone, possibly the result of a 1,25(OH)2D3-dependent conformational change. A nuclear extract obtained from CV-1 cells markedly enhanced the intensity of this VDR·VDRE complex and produced an additional distinct VDR-dependent complex, thus implicating a role for nuclear auxiliary factors in multiple high affinity VDR·VDRE interactions. Finally, methylation interference studies defined the guanine residues contacted when the putative VDR-auxiliary factor complex associates with the rat osteocalcin VDRE; specifically, all of the GC base pairs in the sequence GGGTGAATGAGGACA. Therefore, these results show that the BEV system elicits high level expression of hVDR with critical functional characteristics being preserved.",
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