Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

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Fujino, T. et al.

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

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.[1]

References

In vitro farnesoid X receptor ligand sensor assay using surface plasmon resonance and based on ligand-induced coactivator association. Fujino, T., Sato, Y., Une, M., Kanayasu-Toyoda, T., Yamaguchi, T., Shudo, K., Inoue, K., Nishimaki-Mogami, T. J. Steroid Biochem. Mol. Biol. (2003) [Pubmed]

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