Differential regulation of heterodimerization by 1alpha,25-dihydroxyvitamin D(3) and its 20-epi analog

Twenty-epi analogs of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) are 100-1000 times more potent transcriptionally than the natural hormone. To determine to what extent this enhanced activity is mediated through modulation of the dimerization process, we performed quantitative dimerization assays with in vitro translated vitamin D receptor (ivtVDR) and fusion proteins containing glutathione-S-transferase (GST) and either the ligand-binding domain of VDR (GST-VDR) or retinoid X receptor (RXR)alpha (GST-RXR). We found that VDR did not form homodimers in either the presence or absence of ligand, but heterodimerization of the ligand-binding domains of RXRalpha and VDR was primarily deltanoid-dependent. The ED(50) for induction of heterodimerization was 1-2 x 10(-)(9) M for 1alpha,25(OH)(2)D(3) and 0.5 x 10(-)(11) M for 20-epi 1alpha,25(OH)(2)D(3). Mutations in VDR's activation function 2 domain (AF-2) diminished the abilities of 1alpha,25(OH)(2)D(3) to induce a protease-resistant conformation and heterodimerization. These mutations changed neither the potency of 20-epi-1alpha,25(OH)(2)D(3) to induce protease-resistant conformation nor its potency to induce dimerization. Mutations in heptad 9/helix 10 abolished the ability of both 1alpha,25(OH)(2)D(3) and the 20-epi analog to induce dimerization, but not their potency to fold VDR into a protease-resistant conformation. We hypothesize that both the hormone and the analog stabilize receptor conformations that expose VDR's dimerization interface, and that interfaces exposed by these ligands are probably not significantly different. However, the mechanisms by which the two ligands expose the dimerization interface are different with respect to participation of the AF-2 domain.