Receptor mediated genomic action of the 1,25(OH)2D3 hormone: expression of the human vitamin D receptor in E. coli.
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, beta 3 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 9A7 gamma. 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 (delta 134), containing only residues 4-133 of hVDR, generated a receptor species that possesses intrinsic DNA binding activity. Both full-length and delta 134 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.
Hsieh
J C
JC
Department of Biochemistry, College of Medicine, University of Arizona, Tucson 85724, USA.
Nakajima
S
S
Galligan
M A
MA
Jurutka
P W
PW
Haussler
C A
CA
Whitfield
G K
GK
Haussler
M R
MR
D001483:Base Sequence; D002117:Calcitriol / Q000502:physiology*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D018167:Receptors, Calcitriol; D011994:Recombinant Proteins; D002117:Calcitriol