title: In vitro farnesoid X receptor ligand sensor assay using surface plasmon resonance and based on ligand-induced coactivator association.
abstract: Ligand binding to nuclear receptors leads to a conformational change that increases the affinity of the receptors to coactivator proteins. We have developed a ligand sensor assay for farnesoid X receptor (FXR) in which the receptor-coactivator interaction can be directly monitored using surface plasmon resonance biosensor technology. A 25-mer peptide from coactivator SRC1 containing the LXXLL nuclear receptor interaction motif was immobilized on the surface of a BIAcore sensor chip. Injection of the FXR ligand binding domain (FXRLBD) with or without the most potent natural ligand, chenodeoxycholic acid (CDCA), over the surface of the chip resulted in a ligand- and LXXLL motif-dependent interaction. Kinetic analysis revealed that CDCA and its conjugates decreased the equilibrium dissociation constant (K(d)) by 8-11-fold, indicating an increased affinity. Using this technique, we found that a synthetic bile acid sulfonate, 3alpha,7alpha-dihydroxy-5beta-cholane-24-sulfonate, which was inactive in a FXR response element-driven luciferase assay using CV-1 cells, caused the most potent interaction, comparable to the reaction produced by CDCA. This method provides a rapid and reliable in vitro ligand assay for FXR. This kinetic analysis-featured technique may be applicable to mechanistic studies.
authors.affiliation: National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, 158-8501 Tokyo, Japan.
authors.forename: Tomofumi
authors.initials: T
authors.lastname: Fujino
authors.forename: Yoji
authors.initials: Y
authors.lastname: Sato
authors.forename: Mizuho
authors.initials: M
authors.lastname: Une
authors.forename: Toshie
authors.initials: T
authors.lastname: Kanayasu-Toyoda
authors.forename: Teruhide
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: Koichi
authors.initials: K
authors.lastname: Shudo
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Inoue
authors.forename: Tomoko
authors.initials: T
authors.lastname: Nishimaki-Mogami
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001647:Bile Acids and Salts / Q000737:chemistry / Q000494:pharmacology; D015374:Biosensing Techniques / Q000379:methods; D002460:Cell Line; D002635:Chenodeoxycholic Acid / Q000737:chemistry / Q000494:pharmacology; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D051548:Histone Acetyltransferases; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D056920:Nuclear Receptor Coactivator 1; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D020349:Surface Plasmon Resonance / Q000379:methods*; D014157:Transcription Factors / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D000099150:Receptor, Farnesoid X-Activated
chemical_list: D001647:Bile Acids and Salts; D002635:Chenodeoxycholic Acid; D004268:DNA-Binding Proteins; D051548:Histone Acetyltransferases; D008024:Ligands; D008156:Luciferases; D056920:Nuclear Receptor Coactivator 1; D010446:Peptide Fragments; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D000099150:Receptor, Farnesoid X-Activated; C538859:NCOA1 protein, human