title: Quantitative analysis of the interaction of constitutive androstane receptor with chemicals and steroid receptor coactivator 1 using surface plasmon resonance biosensor systems: a case study of the Baikal seal (Pusa sibirica) and the mouse.
abstract: The constitutive androstane receptor (CAR) not only displays a high basal transcriptional activity but also acts as a ligand-dependent transcriptional factor. It is known that CAR exhibits different ligand profiles across species. However, the mechanisms underlying CAR activation by chemicals and the species-specific responses are not fully understood. The objectives of this study are to establish a high-throughput tool to screen CAR ligands and to clarify how CAR proteins from the Baikal seal (bsCAR) and the mouse (mCAR) interact with chemicals and steroid receptor coactivator 1 (SRC1). We developed the surface plasmon resonance (SPR) system to assess quantitatively the interaction of CAR with potential ligands and SRC1. The ligand-binding domain (LBD) of bsCAR and mCAR was synthesized in a wheat germ cell-free system. The purified CAR LBD was then immobilized on the sensor chip for the SPR assay, and the kinetics of direct interaction of CARs with ligand candidates was measured. Androstanol and androstenol, estrone, 17β-estradiol, TCPOBOP, and CITCO showed compound-specific but similar affinities for both CARs. The CAR-SRC1 interaction was ligand dependent but exhibited a different ligand profile between the seal and the mouse. The results of SRC1 interaction assay accounted for those of our previous in vitro CAR-mediated transactivation assay. In silico analyses also supported the results of CAR-SRC1 interaction; there is little structural difference in the ligand-binding pocket of bsCAR and mCAR, but there is a distinct discrimination in the helix 11 and 12 of these receptors, suggesting that the interaction of ligand-bound CAR and SRC1 is critical for determining species-specific and ligand-dependent transactivation over the basal activity. The SPR assays demonstrated a potential as a high-throughput screening tool of CAR ligands.
authors.affiliation: Center for Marine Environmental Studies, Ehime University, Matsuyama 790-8577, Japan.
authors.forename: Pham Thi
authors.initials: PT
authors.lastname: Dau
authors.forename: Hiroki
authors.initials: H
authors.lastname: Sakai
authors.forename: Masashi
authors.initials: M
authors.lastname: Hirano
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ishibashi
authors.forename: Yuki
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Kenji
authors.initials: K
authors.lastname: Kameda
authors.forename: Takahiro
authors.initials: T
authors.lastname: Fujino
authors.forename: Eun-Young
authors.initials: EY
authors.lastname: Kim
authors.forename: Hisato
authors.initials: H
authors.lastname: Iwata
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D000090702:Constitutive Androstane Receptor; D004785:Environmental Pollutants / Q000737:chemistry* / Q000378:metabolism; D057166:High-Throughput Screening Assays; D008024:Ligands; D051379:Mice; D056920:Nuclear Receptor Coactivator 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046048:Seals, Earless / Q000378:metabolism; D013045:Species Specificity; D020349:Surface Plasmon Resonance
chemical_list: D000090702:Constitutive Androstane Receptor; D004785:Environmental Pollutants; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D056920:Nuclear Receptor Coactivator 1