title: Serine 350 of human pregnane X receptor is crucial for its heterodimerization with retinoid X receptor alpha and transactivation of target genes in vitro and in vivo.
abstract: The human pregnane X receptor (hPXR), a member of the nuclear receptor superfamily, senses xenobiotics and controls the transcription of genes encoding drug-metabolizing enzymes and transporters. The regulation of hPXR's transcriptional activation of its target genes is important for xenobiotic detoxification and endobiotic metabolism, and hPXR dysregulation can cause various adverse drug effects. Studies have implicated the putative phosphorylation site serine 350 (Ser(350)) in regulating hPXR transcriptional activity, but the mechanism of regulation remains elusive. Here we investigated the transactivation of hPXR target genes in vitro and in vivo by hPXR with a phosphomimetic mutation at Ser(350) (hPXR(S350D)). The S350D phosphomimetic mutation reduced the endogenous expression of cytochrome P450 3A4 (an hPXR target gene) in HepG2 and LS180 cells. Biochemical assays and structural modeling revealed that Ser(350) of hPXR is crucial for formation of the hPXR-retinoid X receptor alpha (RXRα) heterodimer. The S350D mutation abrogated heterodimerization in a ligand-independent manner, impairing hPXR-mediated transactivation. Further, in a novel humanized transgenic mouse model expressing the hPXR(S350D) transgene, we demonstrated that the S350D mutation alone is sufficient to impair hPXR transcriptional activity in mouse liver. This transgenic mouse model provides a unique tool to investigate the regulation and function of hPXR, including its non-genomic function, in vivo. Our finding that phosphorylation regulates hPXR activity has implications for development of novel hPXR antagonists and for safety evaluation during drug development.
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Yue-Ming
authors.initials: YM
authors.lastname: Wang
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Sergio C
authors.initials: SC
authors.lastname: Chai
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Wenwei
authors.initials: W
authors.lastname: Lin
authors.affiliation: Center for Pharmacogenetics, Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261, USA; Department of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Chai
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Ayesha
authors.initials: A
authors.lastname: Elias
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Su Sien
authors.initials: SS
authors.lastname: Ong
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Satyanarayana R
authors.initials: SR
authors.lastname: Pondugula
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA; Integrated Biomedical Sciences Program, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
authors.forename: Jordan A
authors.initials: JA
authors.lastname: Beard
authors.affiliation: Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Erin G
authors.initials: EG
authors.lastname: Schuetz
authors.affiliation: Department of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Su
authors.initials: S
authors.lastname: Zeng
authors.affiliation: Center for Pharmacogenetics, Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Xie
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA; Integrated Biomedical Sciences Program, University of Tennessee Health Science Center, Memphis, TN 38163, USA. Electronic address: taosheng.chen@stjude.org.
authors.forename: Taosheng
authors.initials: T
authors.lastname: Chen
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008822:Mice, Transgenic; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D000077297:Pregnane X Receptor; D055503:Protein Multimerization; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D047490:Retinoid X Receptor alpha / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics*; D015533:Transcriptional Activation
chemical_list: D008565:Membrane Proteins; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid; D047490:Retinoid X Receptor alpha; D012694:Serine; C496508:Cyp3a11 protein, mouse; D051544:Cytochrome P-450 CYP3A
keywords: Gene regulation; Nuclear receptor; Receptor regulation; Transcription regulation; Xenobiotic