PT - JOURNAL ARTICLE AU - An-Qiang Sun AU - Yuhuan Luo AU - Donald S Backos AU - Shuhua Xu AU - Natarajan Balasubramaniyan AU - Philip Reigan AU - Frederick J Suchy TI - Identification of Functional Relevant Lysine Residues that Modulate Human Farnesoid X Receptor (Hfxr) Activation AID - 10.1124/mol.113.084772 DP - 2013 Jan 01 TA - Molecular Pharmacology PG - mol.113.084772 4099 - http://molpharm.aspetjournals.org/content/early/2013/03/07/mol.113.084772.short 4100 - http://molpharm.aspetjournals.org/content/early/2013/03/07/mol.113.084772.full AB - Base amino acid lysine residues play an important role in regulation of nuclear receptors (e.g. FXR) leading to enhanced or suppressed biological activity. To understand the molecular mechanisms and the subsequent effects in modulating FXR functions in diverse biological processes, we individually replaced eight highly conserved lysine residues of hFXR with arginine. The effects of each mutated FXR on target gene activation, subcellular localization, protein-protein association, and protein-DNA interaction were investigated. Results demonstrated that K122R, K210R, K339R, and K460R mutants of hFXR, significantly impaired target gene (OSTalpha/beta and BSEP) promoter reporter activity in a ligand-dependent fashion. All of the four mutants did not affect the nuclear localization of FXR. Protein interaction studies show that K210R slightly but significantly decreased FXR/RXR binding affinity, but enhanced the interaction of FXR with lysine methyltransferase Set7/9 by ~21%. K460R decreased the FXR interaction with Set7/9 by ~45%, but has no significant effects on interaction with RXR. Electrophoretic mobility shift assays demonstrated that hFXR-K210R and -K339R reduced the protein-DNA (IR1 element at hBSEP promoter) binding affinity by ~80% and ~90%, respectively. Computational-based protein modeling studies were consistent with these results and provided further insights into the potential underlying mechanisms responsible for these results. In conclusion, four highly conserved lysine residues, K122, K210, K339, and K460 of hFXR have been identified that play a critical role in FXR target gene regulation and molecular interaction (protein-protein and protein-DNA).