Differential regulation of heterodimerization by 1α,25-dihydroxyvitamin D3 and its 20-epi analog

Twenty-epi analogs of 1 alpha ,25-dihydroxyvitamin D-3 (1 alpha ,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 glulathione-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 RXR alpha and VDR was primarily deltanoid-dependent. The ED50 for induction of heterodimerization was 1-2 X 10(-9) M for 1 alpha ,25(OH)(2)D-3 and 0.5 x 10(-11) M for 20-epi 1 alpha ,25(OH)(2)D-3. Mutations in VDR's activation function 2 domain (AF-2) diminished the abilities of 1 alpha ,25(OH)(2)D-3 to induce a protease-resistant conformation and heterodimerization. These mutations changed neither the potency of 20-epi-1 alpha ,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 1 alpha ,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. (C) 2001 Elsevier Science Inc. All rights reserved.