Baculovirus-mediated expression of the human vitamin D receptor. Functional characterization, vitamin D response element interactions, and evidence for a receptor auxiliary factor.
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 immunologic 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.
MacDonald
P N
PN
Department of Biochemistry, College of Medicine, University of Arizona, Tucson 85724.
Haussler
C A
CA
Terpening
C M
CM
Galligan
M A
MA
Reeder
M C
MC
Whitfield
G K
GK
Haussler
M R
MR
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000235:genetics* / Q000378:metabolism; D014807:Vitamin D / Q000378:metabolism*
D004268:DNA-Binding Proteins; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D015675:Osteocalcin; D014807:Vitamin D; D004247:DNA