title: Identification of functionally relevant lysine residues that modulate human farnesoid X receptor activation.
abstract: 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).
authors: Department of Pediatrics, University of Colorado Denver School of Medicine, Aurora, CO 80045, USA. An-Qiang.Sun@ucdenver.edu
authors: An-Qiang
authors: AQ
authors: Sun
authors: Yuhuan
authors: Y
authors: Luo
authors: Donald S
authors: DS
authors: Backos
authors: Shuhua
authors: S
authors: Xu
authors: Natarajan
authors: N
authors: Balasubramaniyan
authors: Philip
authors: P
authors: Reigan
authors: Frederick J
authors: FJ
authors: Suchy
mesh_terms: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D018938:Caco-2 Cells; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008239:Lysine / Q000235:genetics* / Q000378:metabolism*; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000187:drug effects / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism*; D047488:Retinoid X Receptors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics*; D000099150:Receptor, Farnesoid X-Activated
chemical_list: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; D001120:Arginine; D004268:DNA-Binding Proteins; D056572:Histone Deacetylase Inhibitors; D008024:Ligands; D008239:Lysine; D008780:Methyltransferases; D018160:Receptors, Cytoplasmic and Nuclear; D047488:Retinoid X Receptors; D000099150:Receptor, Farnesoid X-Activated; C093491:ABCB11 protein, human