ARTICLES High-Throughput System for Analyzing Ligand-Induced Cofactor Recruitment by Vitamin D Receptor Midori A. Arai, †,⊥ Ken-ichi Takeyama, ‡ Saya Ito, ‡ Shigeaki Kato, ‡ Tai C. Chen, § and Atsushi Kittaka* ,† Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan, Institute of Molecular and Cellular Bioscience, The University of Tokyo, Yayoi, Tokyo 113-0032, Japan, and Boston University School of Medicine, Boston, Massachusetts 02118. Received April 30, 2006; Revised Manuscript Received February 4, 2007 A high-throughput screening system for analyzing small molecule-induced coactivator (CoA) recruitment by the vitamin D receptor (VDR) has been developed. The vitamin D-induced protein-protein interactions between VDR and fluorophore (Cy3 or Cy5)-labeled TIF2 or SRC-1 were successfully detected by using a new HCHO fixing method of the protein complex on microplates. The results obtained from this screening of our synthetic vitamin D analogues suggest that the CoA-recruiting activities play an important role in determining the biological activity of various vitamin D analogues and explain the discrepancies between the VDR binding affinity and their biological activity. INTRODUCTION The superfamily of nuclear receptors (NRs) activates tran- scription by recruiting coactivators in a ligand-dependent manner to assemble regulatory complexes on target genes (1, 2). The structure of a NR comprises four regions (A/B, C, D, and E/F) including a highly conserved DNA-binding domain and a C-terminal ligand-binding domain (LBD). This C-terminal E/F region contains the activation function-2 (AF-2) motif that mediates ligand-dependent transcriptional activation. The steroid hormones, thyroid hormone, retinoic acids, and 1R,25-dihy- droxyvitamin D 3 [1R,25(OH) 2 D 3 ] all bind to their respective NRs, which include the androgen receptor, estrogen receptors, retinoic acid receptors, retinoid X receptors, and the vitamin D receptor (VDR). 1R,25(OH) 2 D 3 (Figure 1A, 1), the active form of vitamin D, binds specifically to VDR and plays a vital role in calcium homeostasis (3, 4), osteoclastogenesis (5, 6), kera- tinocyte differentiation (7, 8), phosphate homeostasis (9), immune response, cellular proliferation, differentiation, and apoptosis (10, 11). Crystal structures of the VDR-LBD, alone and in complex with its ligands (12), with coactivator peptides (13), or as heterodimers, have been reported (14). The LBD is generally a three-layered R-helical sandwich of R-helices (H1-H12). When the ligand binds to LBD, H12 moves to seal the ligand-binding cavity, creating the AF-2 motif and a hydrophobic cleft (Figure 1B). This hydrophobic region, together with other surface residues, accommodates the receptor interaction domain (RID) of coactivators (15, 16), such as transcriptional factor 2 (TIF2) (17), steroid receptor coactivating factor 1 (SRC-1) (18), TRAP220 (19), and AIB-1 (20) (Figure 1C). The RID includes short helical regions called “NR boxes”, which contain the consensus sequence LXXLL (where X is any amino acid). The resulting conformational changes in the VDR lead to the removal of corepressors and the recruitment of various coactivators followed by histone acetylation and target gene promoter activation (21). The active form of vitamin D or its analogues have been used clinically for treating psoriasis (22), osteoporosis (23, 24), and secondary hyperparathyroidism (25). Furthermore, 1R,25- (OH) 2 D 3 or its analogues have been shown to inhibit the proliferation of breast, colon, and prostate cancer cells in cultures and tumor cell growth in animal models and stimulate HL-60 cell differentiation. However, the mechanism of interaction between ligand-inducible NRs and cofactor proteins (coactivator, coregulator, and corepressor) remains unknown except in a few cases (26, 27). Such ligand-induced cofactor recruitment has been investigated in cells using a two-hybrid assay and transactivation activity under the expression of a specific coactivator. For direct detection of the complex, a gel shift assay using radio-labeled vitamin D response element and a pull-down assay of radiolabeled coactivator have been used. Other methods using flow cytometry (28) and fluorescence resonance energy transfer (29, 30) have also been reported. A high-throughput system would be extremely useful for examining the correlation between molecular mechanisms and the phenotypes induced by small ligands. Despite the growing success in forward and reverse chemical genetics studies, including protein microarrays, small-molecule microarrays, and cell-based high-throughput assays (31, 32), it remains a key challenge to develop such a system for analyzing ligand-induced interactions between NRs and cofactors. Nishikawa and co- workers reported excellent pioneering work on a high-throughput detection system that used alkaline phosphatase-fused TIF2 on the microplate (33). Here we describe a rapid in vitro high- throughput system to examine ligand-induced coactivator (CoA) recruitment using fluorophore-labeled cofactors and a formal- dehyde (HCHO)-based fixing method (Figure 2). Moreover, using this system, we are able to explain some of the discrep- * Corresponding author. akittaka@pharm.teikyo-u.ac.jp. † Teikyo University. ‡ The University of Tokyo. § Boston University School of Medicine. ⊥ Current address: Chiba University, Inage, Chiba, 263-8522, Japan. 614 Bioconjugate Chem. 2007, 18, 614-620 10.1021/bc0601121 CCC: $37.00 © 2007 American Chemical Society Published on Web 04/05/2007