Differential interaction of 1alpha,25-dihydroxyvitamin D3 analogues and their 20-epi homologues with the vitamin D receptor.
An important focus of structure-function studies of synthetic ligands for the vitamin D receptor (VDR) concerns the chiral center at carbon 20 of the steroid side chain; 20-epi analogues are 100-10, 000 times more potent transcriptionally than the natural hormone 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3). We have compared the binding properties of three pairs of analogues either with a natural (N) or 20-epi (E) orientation. In intact cells, 45-60% of VDR.N-analogue complexes, but only 5-20% of VDR.E-analogue complexes, dissociated over a 3-h interval. The two groups of ligands induced distinct changes in VDR conformation as revealed by protease clipping assays. Mapping of ligand-VDR binding activity by deletions indicated that amino acids 420-427 were important for high affinity of VDR.N-analogue complexes, but not for VDR.E-analogue complexes. Site-directed mutagenesis revealed that residues 421 and 422 were essential for 1alpha,25-(OH)2D3-induced conformational changes, high affinity of 1alpha,25-(OH)2D3 for VDR, and transcriptional activity, but not for binding of its 20-epi analogue. In contrast, deletion of residues 396-427 abolished binding of 1alpha,25-(OH)2D3, but binding of its 20-epi analogue was still detectable. The results suggest that the ligand-binding domain of VDR has multiple and different contact sites for the two families of side chain-modified ligands, resulting in VDR.ligand complexes with different half-lives and transcriptional activities.
Liu
Y Y
YY
Department of Medical Specialties, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Collins
E D
ED
Norman
A W
AW
Peleg
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry; D018167:Receptors, Calcitriol / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000737:chemistry; D047488:Retinoid X Receptors; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry; D014158:Transcription, Genetic; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014357:Trypsin; D002117:Calcitriol