RECEPTOR-MEDIATED GENOMIC ACTION OF THE 1,25(OH)(2)D-3 HORMONE - EXPRESSION OF THE HUMAN VITAMIN-D-RECEPTOR IN ESCHERICHIA-COLI

The nuclear vitamin D receptor (VDR) binds the 1,25-dihydroxyvitamin D -3 (1,25(OH)(2)D-3) hormone with high affinity and elicits its actions to stimulate gene expression in target cells by binding to the vitami n D-responsive element (VDRE). VDREs in such positively controlled gen es as osteocalcin, osteopontin, beta(3) integrin and vitamin D-24-OHas e are direct hexanucleotide repeats with a spacer of three nucleotides . The present studies of VDR/VDRE interaction utilized full-length hum an vitamin D receptor (hVDR) that was overexpressed in E. coli, purifi ed to near homogeneity (>95%), and its authenticity confirmed by demon strating high affinity hormone binding and reactivity to monoclonal an tibody 9A7 gamma. The expressed hVDR displays strict dependence on the family of retinoid X receptors (RXRs) for binding to the vitamin D-re sponsive element (VDRE) in the rat osteocalcin gene. Similar overexpre ssion in E. coli of the DNA binding domain (Delta 134), containing onl y residues 4-133 of hVDR, generated a receptor species that possesses intrinsic DNA binding activity. Both full-length and Delta 134 hVDRs r etain similar DNA binding specificities when tested with several natur al hormone responsive elements, indicating that the N-terminal zinc fi nger region determines hVDR-DNA sequence selectivity. The C-terminal r egion of the molecule is required for hormone binding and confers the receptor with the property of very high affinity DNA binding, via hete rodimerization between hVDR and RXR. A natural ligand for the RXR co-r eceptor, 9-cis retinoic acid, suppresses both VDR-RXR binding to the V DRE and 1,25(OH)(2)D-3 stimulated transcription, indicating that 9-cis retinoic acid recruits RXR away from VDR to instead form RXR homodime rs.