The impact of functional vitamin D(3) receptor conformations on DNA-dependent vitamin D(3) signaling.

The vitamin D(3) receptor (VDR) is the nuclear receptor for 1alpha, 25-dihydroxyvitamin D(3) (VD) that acts primarily as a heterodimer with the retinoid X receptor (RXR) on different types of VD response elements, i.e., DNA-bound VDR-RXR heterodimers are the molecular switches in nuclear VD signaling pathways. In this study, DNA-dependent limited protease digestion assays and gel shift clipping assays were used for the analysis of VDR conformations and showed the same high ligand sensitivity for VD response element-bound VDR-RXR heterodimers (EC(50) of 0.1 nM for VD). In contrast, DNA-independent limited protease digestion assays clearly demonstrated a reduced ligand sensitivity for monomeric VDR in solution. Interestingly, the relative amount of reduction was found to be specific for each VDR agonist. Moreover, complex formation of the VDR on DNA resulted in a shift from the receptor's low-affinity ligand binding conformation (c3(LPD)) to its high affinity conformation (c1(LPD)). Finally, the characterization of the conformations of N- and C-terminally truncated VDR proteins defined the high-affinity ligand binding domain of the VDR as being positioned between amino acids 128 and 427. Taken together, the analysis of VDR conformations in solution in comparison to those of DNA-complexed VDR-RXR heterodimers allows a differentiation to be drawn between DNA-dependent and DNA-independent VD signaling pathways that can in turn be used for the identification of pathway selective VDR agonists.