In vitro farnesoid X receptor ligand sensor assay using surface plasmon resonance and based on ligand-induced coactivator association

Tomofumi Fujino, Yoji Sato, Mizuho Une, Toshie Kanayasu-Toyoda, Teruhide Yamaguchi, Koichi Shudo, Kazuhide Inoue, Tomoko Nishimaki-Mogami

Research output: Contribution to journalArticle

24 Citations (Scopus)

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 (Kd) by 8-11-fold, indicating an increased affinity. Using this technique, we found that a synthetic bile acid sulfonate, 3α,7α-dihydroxy-5β- 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.

Original languageEnglish
Pages (from-to)247-252
Number of pages6
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume87
Issue number4-5
DOIs
Publication statusPublished - Dec 2003
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Endocrinology
  • Clinical Biochemistry
  • Cell Biology

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