Regulation of ligand-induced heterodimerization and coactivator interaction by the activation function-2 domain of the vitamin D receptor.

Twenty-epi analogs of 1 alpha ,25-dihydroxyvitamin D-3 (1,25D(3)) are 100-1000 times more potent transcriptionally than the natural hormone. To determine whether this enhanced activity is mediated through modulation of the dimerization process or through interaction with coactivators, we performed quantitative protein-protein interaction assays with in vitro translated vitamin D receptor (ivtVDR) and fusion proteins containing glutathione-S-transferase (GST) and either the ligand-binding domain of retinoid X receptor (RXR alpha), or the nuclear receptor-interacting domain of the steroid receptor coactivator 1 (SRC-1), or the glucocorticoid receptor-interacting protein 1 (GRIP-1). We found that heterodimerization of the ligand-binding domains of RXR alpha and VDR was primarily deltanoid dependent as was the interaction of VDR with the SRC-1 or with GRIP-1. The ED50 for induction of heterodimerization was 2 nM for 1,25D(3) and 0.05 nM for 20-epi-1,25D(3). However, the ED50 for induction of VDR interaction with SRC-1 was similar for both 1,25D(3) and the 20-epi analog (ED50 = 0.7-1.0 nM) as was the ED50 for ligand-mediated interaction of VDR with GRIP-1 (ED50 = 0.1-0.3 nM). Mutations in heptad 9 diminished both 1,25D(3) and the 20-epi analog-mediated dimerization, without changing binding of these ligands to VDR. Mutations in VDR's activation function 2 (AF-2) domain/helix 12 residues diminished the ability of 1,25D(3) to induce heterodimerization and interaction with SRC-1. These mutations did not change the ability of 20-epi-1,25D(3) to induce dimerization but did diminish its ability to induce interaction with SRC-1. We hypothesize that both the hormone and the analog stabilize receptor conformations that expose VDR's functional interfaces. The mechanisms by which the two ligands expose these functional interfaces differ with respect to participation of the AF-2 domain.