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The critical steps in bile acid metabolism have remarkable differences between humans and mice. It is known that human cholesterol 7␣-hydroxylase, the enzyme catalyzing the rate-limiting step of bile acid synthesis, is more sensitive to bile acid suppression. In addition, hepatic bile acid export in humans is more dependent on the bile salt export pump (BSEP). To explore the molecular basis for these species differences, we analyzed the function of the ligand-binding domain (LBD) of human and murine farnesoid X receptor (FXR), a nuclear receptor for bile acids. We observed a strong interspecies difference in bile acid-mediated FXR function; in the coactivator association assay, chenodeoxycholate (CDCA) activated human FXR-LBD with 10-fold higher affinity and 3-fold higher maximum response than murine FXR-LBD. Consistently, in HepG2 cells human FXR-LBD increased reporter expression more robustly in the presence of CDCA. The basis for these differences was investigated by preparing chimeric receptors and by site-directed mutagenesis. are critically important for FXR function and that murine FXR can be "humanized" by substituting with the two corresponding residues of human FXR. Consistent with the difference in FXR-LBD transactivation, CDCA induced endogenous expression of human BSEP by 10 -12-fold and murine BSEP by 2-3-fold in primary hepatocytes. This study not only provides the identification of critical residues for FXR function but may also explain the species difference in bile acids/cholesterol metabolism.

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The dog GnRH receptor was cloned to facilitate the identification and characterization of selective nonpeptide GnRH antagonists. The dog receptor is 92% identical to the human GnRH receptor. Despite such high conservation, the quinolone-based nonpeptide GnRH antagonists were clearly differentiated by each receptor species. By contrast, peptide antagonist binding and functional activity were not differentiated by the two receptors. The basis of the differences was investigated by preparing chimeric receptors followed by site-directed mutagenesis. Remarkably, a single substitution of Phe313 to Leu313 in the dog receptor explained the major differences in binding affinities and functional activities. The single amino acid replacement of Phe313 of the human receptor with Leu313 resulted in a 160-fold decrease of binding affinity of the nonpeptide antagonist compound 1. Conversely, the replacement of Leu313 of the dog receptor with Phe313 resulted in a 360-fold increase of affinity for this compound. These results show that Phe313 of the GnRH receptor is critical for the binding of this structural class of GnRH antagonists and that the dog receptor can be "humanized" by substituting Leu for Phe. This study provides the first identification of a critical residue in the binding pocket occupied by nonpeptide GnRH antagonists and reinforces cautious extrapolation of ligand activity across highly conserved receptors.