CAR2 Displays Unique Ligand Binding and RXR Heterodimerization Characteristics

Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary & Biomedical Sciences, The Pennsylvania State University, University Park, PA 16802, USA.
Drug Metabolism and Disposition (Impact Factor: 3.25). 04/2007; 35(3):428-39. DOI: 10.1124/dmd.106.012641
Source: PubMed


The constitutive androstane receptor (CAR; NR1I3) regulates the expression of genes involved in xenobiotic metabolism. Alternative splicing of the human CAR gene yields an array of mRNAs that encode structurally diverse proteins. One form of CAR, termed CAR2, contains an additional four amino acids (SPTV) that are predicted to reshape the ligand-binding pocket. The current studies show a marked, ligand-independent, CAR2-mediated transactivation of reporters containing optimal DR-3, DR-4, and DR-5 response elements, and reporters derived from the natural CYP2B6 and CYP3A4 gene promoters. Overexpression of the RXRalpha ligand binding domain was critical for achieving these effects. CAR2 interaction with SRC-1 was similarly dependent on the coexpression of RXRalpha. Mutagenesis of Ser233 (SPTV) to an alanine residue yielded a receptor possessing higher constitutive activity. Alternatively, mutating Ser233 to an aspartate residue drastically reduced the transactivation capacity of CAR2. The respective abilities of these mutagenized forms of CAR2 to transactivate a DR-4 x 3 reporter element correlated with their ability to interact with RxRalpha and to recruit SRC-1 in a ligand-regulated manner. Together, these results demonstrate a robust RXRalpha-dependent recruitment of coactivators and transactivation by CAR2. In addition, CAR2 displays novel dose responses to clotrimazole and androstanol compared with the reference form of the receptor while at the same time retaining the ability to bind CITCO. This result supports a hypothesis whereby the four-amino-acid insertion in CAR2 structurally modifies its ligand binding pocket, suggesting that CAR2 is regulated by a set of ligands distinct from those governing the activity of reference CAR.

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Available from: Scott S Auerbach, Oct 02, 2014
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    • "Expression vectors pCMV6- hPPAR␣, pCMV6-hPPAR␥ , and pCMV6-hCAR (CAR2) were purchased from Origene (Rockville, MD). The vectors, pTracer CMV2-hCAR1, pTracer CMV2-hCAR2, pcDNA 3.1-RXR␣, and pGL3-basic/TK CYP2B6-dervied XREM/PBREM were described previously (Auerbach et al., 2007). pTracer CMV2- CAR3 was also reported previously (Auerbach et al., 2005). "
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    ABSTRACT: Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of US adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα) and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 μg/mL) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 μg/mL. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187 and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 μg/mL). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 μg/mL) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2 and an antagonist for human PPARα.
    Full-text · Article · May 2014 · Toxicological Sciences
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    • "As presented in Figure 2, results of molecular modeling studies predict that the 5-aa insertion in CAR3 results in a receptor with virtually an identical LBP to that of wild-type CAR, suggesting that CAR3 may serve as unique surrogate for wildtype CAR in studies of ligand-specificity analyses. These modeling predictions for human CAR3 have been examined in functional assays reported previously (Auerbach et al., 2005; Faucette et al., 2007) and are further supported by the results presented in the current investigation. Interestingly, another recent report examined the functional activity of a CAR3-like receptor that possessed only the ''A'' residue of the naturally occurring CAR3 APYLT motif (Chen et al., 2010). "
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    ABSTRACT: The constitutive androstane receptor (CAR; NR1I3) is a member of the nuclear receptor superfamily and functions as an important xenochemical sensor and transcriptional modulator in mammalian cells. Upon chemical activation, CAR undergoes nuclear translocation and heterodimerization with the retinoid X receptor subsequent to its DNA target interaction. CAR is unusual among nuclear receptors in that it possesses a high level of constitutive activity in cell-based assays, obscuring the detection of ligand activators. However, a human splice variant of CAR, termed CAR3, exhibits negligible constitutive activity. In addition, CAR3 is activated by ligands with similar specificity as the reference form of the receptor. In this study, we hypothesized that similar CAR3 receptors could be constructed across various mammalian species' forms of CAR that would preserve species-specific ligand responses, thus enabling a more sensitive and differential screening assessment of CAR response among animal models. A battery of CAR3 receptors was produced in mouse, rat, and dog and comparatively evaluated with selected ligands together with human CAR1 and CAR3 in mammalian cell reporter assays. The results demonstrate that the 5-amino acid insertion that typifies human CAR3 also imparts ligand-activated receptor function in other species' CAR while maintaining signature responses in each species to select CAR ligands. These variant constructs permit in vitro evaluation of differential chemical effector responses across species and coupled with in vivo assays, the species-selective contributions of CAR in normal physiology and in disease processes such as hepatocarcinogenesis.
    Full-text · Article · Jul 2011 · Toxicological Sciences
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    • "As positive controls, CITCO for CAR variants (Maglich et al., 2003) or RFPM (Bertilsson et al., 1998) or TO (Mitro et al., 2007) for PXR were included. Because CAR1 is constitutively active, ANDRO (10lM), a mouse CAR (Forman et al., 1998) and human CAR1 (Auerbach et al., 2007) inverse agonist, was included to decrease its activity, which can be restored in the presence of an agonist. All chemical treatments were for 24 h and luciferase assays were performed as previously described (Dekeyser et al., 2009). "
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    ABSTRACT: Phthalates and other endocrine-disruptive chemicals are manufactured in large quantities for use as plasticizers and other commercial applications, resulting in ubiquitous human exposure and thus, concern regarding their toxicity. Innate defense against small molecule exposures is controlled in large part by the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). The human CAR gene undergoes multiple alternative splicing events resulting in the CAR2 and CAR3 variant receptors. Recent studies from our laboratory show that CAR2 is potently and specifically activated by di(2-ethylhexyl) phthalate (DEHP). We hypothesized that alternative splicing is a mechanism for increasing CAR's functional diversity, broadening the human receptors' repertoire of response to environmental xenobiotics. In these studies, we examine the interaction of alternatively spliced CARs and PXR with a range of suspected endocrine disruptors, including phthalates, bisphenol A (BPA), and 4-N-nonylphenol (NP). Transactivation and two-hybrid studies in COS-1 cells revealed differential selectivity of endocrine-disrupting chemicals for the variant CAR and PXR. Ex vivo studies showed DEHP and di-isononyl phthalate potently induced CYP2B6 and CYP3A4 expression in human hepatocytes. Mutation analysis of CAR2, in silico modeling, and ligand docking studies suggested that the SPTV amino acid insertion of CAR2 creates a unique ligand-binding pocket. Alternative gene splicing results in variant CAR receptors that selectively recognize phthalates and BPA. The interaction of phthalates with CAR and PXR suggests a xenobiotic response that is complex and biologically redundant.
    Full-text · Article · Mar 2011 · Toxicological Sciences
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