Response element and coactivator-mediated conformational change of the vitamin D-3 receptor permits sensitive interaction with agonists

The vitamin D receptor (VDR) is the nuclear receptor for 1,25-dihydroxyvita min D-3 [1 alpha,25(OH)(2)D-3] that acts as a ligand-dependent transcriptio n factor via combined contact with coactivator proteins (steroid receptor c oactivator-1, transcriptional intermediary factor 2, and receptor associate d coactivator 3) and specific DNA binding sites [vitamin D response element s (VDREs)]. Ligand-mediated conformational changes of the VDR contribute to the key mechanisms in this nuclear hormone signaling process. 1 alpha,25(O H)(2)D-3, MC1288 [20-epi-1 alpha,25(OH)(2)D-3], ZK161422 [20-methyl-1 alpha ,25(OH)(2)D-3], and Ro27-2310 (also called Gemini, having two side chains a t carbon 20) were used as model VDR agonists. The analysis of agonist-induc ed VDR conformations and coactivator interactions were found to be insuffic ient for extrapolating in vivo activities. In DNA-independent assays, such as classical limited protease digestions and glutathione S-transferase pull downs, Gemini seemed to be up to 10,000-fold and the other VDR agonists 10 - to 100-fold weaker than in functional in vivo assays. A more accurate des cription of the gene regulatory potential of VDR agonists was obtained with all tested VDR agonists by analyzing VDR conformations in the context of V DRE-bound VDR-retinoid X receptor heterodimers, in such assays as gel super shift, gel shift clipping, and limited protease digestion in the presence o f DNA and cofactor. Coactivators were found to shift the ligand sensitivity (by a factor of 4 for Gemini) and the ratio of VDR conformations in the pr esence of DNA toward the high-affinity ligand binding conformation (c1(LPD) ). In conclusion, the induction of response element- and coactivator-modula ted VDR conformations appears to be a key step for the gene regulatory func tion of a VDR agonist. The quantification of these effects would be of cent ral importance for the evaluation of the cell-specific efficacy of systemic ally applied 1 alpha,25(OH)(2)D-3 analogs.