The calcitropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] also regulates cellular growth and differentiation, and so it has been considered as a treatment for malignancy and psoriasis. Several analogs of 1,25(OH)2D3 are up to 10,000 times more potent that 1,25(OH)2D3 as growth-inhibitory and differentiating agents, but their enhanced activity is not correlated with greater affinity to the nuclear vitamin D receptor (VDR) or with enhanced calcium mobilization. Instead, these activities are correlated with enhanced transcriptional activation of VDR. The objective of this study is to dissect the mechanisms that lead to maximal activation of the VDR by these analogs so that more effective, clinically applicable compounds can be developed. The hypothesis is that the mode of ligand interaction with the VDR can be changed by chemical and stereochemical modifications of 1,25(OH)2D3; these structural changes in the ligand also modulate the levels and the spectrum of VDR-mediated transcriptional activities by changing dimerization and DNA-binding preferences of VDR/ligand complexes. To test the hypothesis the 1,25(OH)2D3-binding site will be mapped by site-directed mutagenesis and its contact points with the VDR will be confirmed by covalent labeling of the ligand-binding site with a 1,25(OH)2D3 derivative that can be photoactivated. The binding activity of 1,25(OH)2D3 to wild-type and mutated VDR will be compared with that of the analogs, so that the effect of chemical and stereochemical modifications in the ligand on its binding requirements can be determined. To determine whether or not analogs can modify VDR interaction with nuclear receptors their effect on dimerization preferences of VDR will be examined in vitro. Bacterially-expressed VDR or RXR will be used to capture ligand-activated VDR complexes, and the latter will be quantified. The effect of 1,25(OH)2D3 and analogs on dimerization interfaces will be examined by mutation analysis of the ligand-dependent dimerization domain. To study the effect of differential ligand interaction on the type of response element used for VDR action, cellular or synthetic VDR-ligand complexes will be incubated with random oligonucleotides. The specific DNA sequences that bind will be extracted from the receptor/DNA complexes, amplified by the polymerase chain reaction, sequenced and tested for transcriptional activity by DNA-transfer methods.
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