PMID- 1655763
OWN - NLM
STAT- MEDLINE
DCOM- 19911108
LR  - 20210210
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 266
IP  - 28
DP  - 1991 Oct 5
TI  - Baculovirus-mediated expression of the human vitamin D receptor. Functional 
      characterization, vitamin D response element interactions, and evidence for a 
      receptor auxiliary factor.
PG  - 18808-13
AB  - 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.
FAU - MacDonald, P N
AU  - MacDonald PN
AD  - Department of Biochemistry, College of Medicine, University of Arizona, Tucson 
      85724.
FAU - Haussler, C A
AU  - Haussler CA
FAU - Terpening, C M
AU  - Terpening CM
FAU - Galligan, M A
AU  - Galligan MA
FAU - Reeder, M C
AU  - Reeder MC
FAU - Whitfield, G K
AU  - Whitfield GK
FAU - Haussler, M R
AU  - Haussler MR
LA  - eng
GR  - 1-F32-GM13846-01/GM/NIGMS NIH HHS/United States
GR  - AR-15781/AR/NIAMS NIH HHS/United States
GR  - DK-33351/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (DNA-Binding Proteins)
RN  - 0 (Receptors, Calcitriol)
RN  - 0 (Receptors, Steroid)
RN  - 104982-03-8 (Osteocalcin)
RN  - 1406-16-2 (Vitamin D)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - Animals
MH  - Baculoviridae/*genetics
MH  - Base Sequence
MH  - Blotting, Western
MH  - Cloning, Molecular
MH  - DNA/metabolism
MH  - DNA-Binding Proteins/genetics/metabolism
MH  - Genetic Vectors
MH  - Humans
MH  - Methylation
MH  - Molecular Sequence Data
MH  - Osteocalcin/genetics/metabolism
MH  - Rats
MH  - Receptors, Calcitriol
MH  - Receptors, Steroid/*genetics/metabolism
MH  - Vitamin D/*metabolism
EDAT- 1991/10/05 00:00
MHDA- 1991/10/05 00:01
CRDT- 1991/10/05 00:00
PHST- 1991/10/05 00:00 [pubmed]
PHST- 1991/10/05 00:01 [medline]
PHST- 1991/10/05 00:00 [entrez]
AID - S0021-9258(18)55135-8 [pii]
PST - ppublish
SO  - J Biol Chem. 1991 Oct 5;266(28):18808-13.