Constitutive activity and ligand-dependent activation of the nuclear receptor CAR-insights from molecular dynamics simulations

The constitutive androstane receptor (CAR) possesses, unlike most other nuclear receptors, a pronounced basal activity in vitro whose structural basis is still not fully understood. Using comparative molecular dynamics simulations of CAR X-ray crystal structures, we evaluated the molecular basis for constitutive activity and ligand-dependent receptor activation. Our results suggest that a combination of van der Waals interactions and hydrogen bonds is required to maintain the activation helix in the active conformation also in absence of a ligand. Furthermore, we identified conformational rearrangements within the ligand-binding pocket upon agonist binding and an influence of CAR inducers pregnanedione and CITCO on the helical conformation of the activation helix. Based on the results a model for ligand-dependent CAR activation is suggested.     

Crystal structure of a heterodimeric complex of RAR and RXR ligand-binding domains

The crystal structure of a heterodimer between the ligand-binding domains (LBDs) of the human RARalpha bound to a selective antagonist and the constitutively active mouse RXRalphaF318A mutant shows that, pushed by a bulky extension of the ligand, RARalpha helix H12 adopts an antagonist position. The unexpected presence of a fatty acid in the ligand-binding pocket of RXRalpha(F318A is likely to account for its apparent "constitutivity." Specific conformational changes suggest the structural basis of pure and partial antagonism. The RAR-RXR heterodimer interface is similar to that observed in most nuclear receptor (NR) homodimers. A correlative analysis of 3D structures and sequences provides a novel view on dimerization among members of the nuclear receptor superfamily.     

Structural and Functional Similarity of Amphibian Constitutive Androstane Receptor with Mammalian Pregnane X Receptor

The nuclear receptors and xenosensors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2) induce the expression of xenobiotic metabolizing enzymes and transporters, which also affects various endobiotics. While human and mouse CAR feature a high basal activity and low induction upon ligand exposure, we recently identified two constitutive androstane receptors in Xenopus laevis (xlCARα and β) that possess PXR-like characteristics such as low basal activity and activation in response to structurally diverse compounds. Using a set of complementary computational and biochemical approaches we provide evidence for xlCARα being the structural and functional counterpart of mammalian PXR. A three-dimensional model of the xlCARα ligand-binding domain (LBD) reveals a human PXR-like L-shaped ligand binding pocket with a larger volume than the binding pockets in human and murine CAR. The shape and amino acid composition of the ligand-binding pocket of xlCAR suggests PXR-like binding of chemically diverse ligands which was confirmed by biochemical methods. Similarly to PXR, xlCARα possesses a flexible helix 11’. Modest increase in the recruitment of coactivator PGC-1α may contribute to the enhanced basal activity of three gain-of-function xlCARα mutants humanizing key LBD amino acid residues. xlCARα and PXR appear to constitute an example of convergent evolution.     

Functional and structural comparison of PXR and CAR

The nuclear receptors pregnane X receptor (PXR, NR1I2) and constitutive active receptor (CAR, NR1I3) have both been proposed to function as xenosensors, but the details of their respective physiological roles are still being elucidated. We have contrasted these two receptors in a variety of experiments including gene expression assays, cell-based ligand profiling assays, and crystallographic/structural modeling analyses. These data highlight key differences between PXR and CAR.     

Inflammation and drug metabolism: NF-kappB and the CAR and PXR xeno-receptors

Decreased drug metabolism, hyperbilirubinemia and intrahepatic cholestasis are frequently observed during inflammation. Additionally, it has long been appreciated that exposure to drug metabolism-inducing xenobiotics can impair immune function. The nuclear receptor CAR (constitutive androstane receptor or NR1I3) and PXR (pregnane X receptor, NR1I2) control phase I (cytochrome P450 2B and 3A), phase II (GSTA, UGT1A1), and transporter (MDR1, SLC21A6, MRP2) genes involved in drugs metabolism, bile acids and bilirubin clearance in response to xenobiotics. It is well known that inflammation, through the activation of NF-kappaB pathway, leads to a decrease of CAR, PXR and RXRalpha expression and the expression of their target genes. In addition, a new study reveals the mutual repression between PXR and NF-kappaB signaling pathways, providing a molecular mechanism linking xenobiotic metabolism and inflammation.     

Chromatin assembly enhances binding to the CYP2B1 phenobarbital-responsive unit (PBRU) of nuclear factor-1, which binds simultaneously with constitutive androstane receptor (CAR)/retinoid X receptor (RXR) and enhances CAR/RXR-mediated activation of the PBRU

Phenobarbital induction of CYP2B genes is mediated by a complex phenobarbital-responsive enhancer (PBRU), which contains a binding site for nuclear factor-1 (NF-1) flanked by two DR-4 nuclear receptor (NR) binding sites for a heterodimer of constitutive androstane receptor (CAR) and retinoid X receptor (RXR). To examine potential interactions between NF-1 and CAR/RXR, binding of purified recombinant proteins to DNA, or to chromatin assembled using Drosophila embryo extract, was examined. NF-1 and CAR/RXR bound simultaneously and independently to the overlapping NF-1 and NR-1 sites; binding of CAR/RXR to the NR-2 site was modestly increased by NF-1 binding; and CAR/RXR bound to a new site in the PBRU region, designated NR-3. Assembly of plasmid DNA into chromatin using Drosophila extract resulted in linearly phased nucleosomes in the PBRU region. The apparent binding affinity of NF-1 was increased by about 10-fold in assembled chromatin compared with DNA, whereas CAR/RXR binding was decreased. As observed for DNA, however, simultaneous, largely independent, binding to the NF-1 and NR sites was observed. CAR-mediated transactivation of the PBRU in cultured cells of hepatic origin was inhibited by mutations in the NF-1 site, and overexpression of NF-1 increased CAR transactivation in HepG2 cells. These studies demonstrate that NF-1 and CAR/RXR can both bind to the PBRU at the same time and that chromatin assembly increases NF-1 binding, which is consistent with previous in vivo footprinting studies in which the NF-1 site was occupied in untreated animals and the NF-1 and flanking NR sites were occupied after phenobarbital treatment. CAR-mediated trans-activation of the PBRU was increased by NF-1, analogous to NF-1 effects on phenobarbital induction in previous transient transfection studies and consistent with mediation of phenobarbital induction by CAR.     

Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and benzoate X receptor (BXR) define three pharmacologically distinct classes of nuclear receptors

The NR1I subfamily of nuclear receptors contains a phylogenetically diverse array of receptors related to the mammalian pregnane X receptor (PXR) (NR1I2) and constitutive androstane receptor (CAR) (NR1I3). We have carried out an extensive comparative analysis of this subgroup with representatives from fish, birds, amphibians, and mammals. Four novel receptors were isolated from fish, dog, pig, and monkey for this study and combined with a previously reported set of related receptors including human PXR, rabbit PXR, mouse PXR, chicken CXR, frog benzoate X receptors (BXRalpha, BXRbeta), and human and mouse CAR. A broad range of xenobiotics, steroids, and bile acids were tested for their ability to activate the ligand binding domain of each receptor. Three distinct groups of receptors were identified based on their pharmacological profiles: 1) the PXRs were activated by a broad range of xenobiotics and, along with the mammalian PXRs, included the chicken and fish receptors; 2) the CARs were less promiscuous, had high basal activities, and were generally repressed rather than activated by those compounds that modulated their activity; and 3) the BXRs were selectively activated by a subset of benzoate analogs and are likely to be specialized receptors for this chemical class of ligands. The PXRs are differentiated from the other NR1I receptors by a stretch of amino acids between helices 1 and 3, which we designate the H1-3 insert. This insert was present in the mammalian, chicken, and fish PXRs but absent in the CARs and BXRs. Modeling studies suggest that the H1-3 insert contributes to the promiscuity of the PXRs by facilitating the unwinding of helices-6 and -7, thereby expanding the ligand binding pocket.     

The Nuclear Orphan Receptor CAR-Retinoid X Receptor Heterodimer Activates the Phenobarbital-Responsive Enhancer Module of the CYP2B Gene

PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as a trans-acting factor for the phenobarbital-inducible Cyp2b10 gene.