FIG. 5.

Identification of ligands for mFXRβ. (A) Lanosterol selectively induces SRC1 binding to mFXRβ at physiological concentrations. GST-mFXRβ LBD and biotinylated mSRC1 peptide were mixed, and the effect of lanosterol and the FXR agonist GW4064 on the interaction was determined by HTRF assay. Cofactor interaction is induced by lanosterol with an EC₅₀ of 1 μM, whereas GW4064 only acts as a weak agonist. Signals are plotted as means of triplicates, and error bars indicate SDs. (B) GST-hFXR LBD was mixed with biotinylated, His-tagged hTif2, and the effects of lanosterol and GW4064 on the respective interaction were assayed. The FXR agonist GW4064 induces cofactor interaction with an EC₅₀ of 70 nM, whereas lanosterol does not show any effect. (C) A diverse set of compounds induces cofactor interaction with mFXRβ but not FXR. Shown are EC₅₀s and the relative efficacy of SRC1 recruitment (the efficacy of lanosterol was set to 100%) calculated from dose-response experiments for a selected group of compounds found to be active for FXRβ. (D) Cholates and cholic acids do not induce significant SRC1 binding to mFXRβ, whereas lanosterol does induce SRC1 recruitment. GST-mFXRβ LBD and biotinylated mSRC1 peptide were mixed. The effects of various compounds at a concentration of 40 μM on the interaction was determined by HTRF assay. Ratios of signals versus DMSO controls are plotted as means of triplicates, and SDs are indicated. CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; UDCA, ursodeoxycholic acid; CA, cholic acid.