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Regulation of Ligand-Induced Heterodimerization and Coactivator Interaction by the Activation Function-2 Domain of the Vitamin D Receptor

Department of Medical Specialties The University of Texas M. D. Anderson Cancer Center Houston Texas, 77030

Twenty-epi analogs of 1,25-dihydroxyvitamin D₃ (1,25D₃) 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), 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 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 1,25D₃ and 0.05 nM for 20-epi-1,25D₃. However, the ED₅₀ for induction of VDR interaction with SRC-1 was similar for both 1,25D₃ 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 1,25D₃ 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 1,25D₃ to induce heterodimerization and interaction with SRC-1. These mutations did not change the ability of 20-epi-1,25D₃ 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.

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