RT Journal Article
SR Electronic
T1 Identification of Functionally Relevant Lysine Residues That Modulate Human Farnesoid X Receptor Activation
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1078
OP 1086
DO 10.1124/mol.113.084772
VO 83
IS 5
A1 An-Qiang Sun
A1 Yuhuan Luo
A1 Donald S. Backos
A1 Shuhua Xu
A1 Natarajan Balasubramaniyan
A1 Philip Reigan
A1 Frederick J. Suchy
YR 2013
UL http://molpharm.aspetjournals.org/content/83/5/1078.abstract
AB Base amino acid lysine residues play an important role in regulation of nuclear receptors [e.g., farnesyl X receptor (FXR)], leading to enhanced or suppressed biologic activity. To understand the molecular mechanisms and the subsequent effects in modulating FXR functions in diverse biologic processes, we individually replaced eight highly conserved lysine residues of human FXR (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 [organic solute transporter α/β and bile salt export pump (BSEP)] promoter reporter activity in a ligand-dependent fashion. None of the four mutants affected the nuclear localization of FXR. Protein interaction studies show that K210R slightly but significantly decreased FXR/retinoid X receptor (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 had 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 of hFXR, K122, K210, K339, and K460, have been identified that play a critical role in FXR target gene regulation and molecular interaction (protein-protein and protein-DNA).