The constitutive androstane receptor (CAR) also known as nuclear receptor subfamily 1, group I, member 3 is a protein that in humans is encoded by the NR1I3gene.[5] CAR is a member of the nuclear receptor superfamily and along with pregnane X receptor (PXR) functions as a sensor of endobiotic and xenobiotic substances. In response, expression of proteins responsible for the metabolism and excretion of these substances is upregulated.[6] Hence, CAR and PXR play a major role in the detoxification of foreign substances such as drugs.
CAR is a member of the nuclear receptor superfamily, and is a key regulator of xenobiotic and endobiotic metabolism. Unlike most nuclear receptors, this transcriptional regulator is constitutively active in the absence of ligand and is regulated by both agonists and inverse agonists. Ligand binding results in translocation of CAR from the cytosol into the nucleus, where the protein can bind to specific DNA sites, called response elements. Binding occurs both as a monomer and together with the retinoid X receptor (RXR) resulting in activation or repression of target gene transcription. CAR-regulated genes are involved in drug metabolism and bilirubin clearance. Examples for CAR-regulated genes are members of the CYP2B, CYP2C, and CYP3A subfamilies, sulfotransferases, and glutathione-S-transferases.[9] Ligands binding to CAR include bilirubin, a variety of foreign compounds, steroid hormones, and prescription drugs.[10]
Activation mechanism
Phosphorylated CAR forms a multiprotein complex with the heat shock protein 90 (hsp90) and the cytoplasmic CAR retention protein (CCRP) which keep CAR in the cytosol thereby inactivating it.[11] CAR can be activated in two ways: by direct binding of a ligand (e.g. TCPOBOP) or indirect regulation by phenobarbital (PB), a common seizure medication, facilitating the dephosphorylation of CAR through protein phosphatase 2 (PP2A) (Fig. 1).[12]
Both lead to the release of CAR from the multiprotein complex and its translocation into the nucleus. Here, CAR forms a heterodimer with retinoid X receptor (RXR) and interacts with the phenobarbital-responsive enhancer module (PBREM), a distal enhancer activating transcription of CAR target genes.[13]
The consensus sequence of PBREM, containing direct repeat-4 motifs, was found to be conserved in mouse, rat and human 'Cyp2b' genes.[14][15][16]
Direct activation
1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) is thought to bind directly to mouse CAR, thus inducing its translocation into the nucleus.[17] TCPOBOP does not bind to human CAR and hence has no effect on it. Human CAR can be activated by CITCO (6-(4-chlorophenyl)imidazo(2,1-b)(1,3)thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime).[18]
Indirect activation
Figure 1 - Activation mechanisms of CAR: Inactivated CAR is retained in the cytosol. Upon binding of TCPOBOP, CAR gets dephosphorylated by PP2A and translocates into the nucleus. Here, it forms a complex with RXR and binds to the PB-responsive enhancer module. Another possibility to activate CAR is the indirect activation through PB. PB binds competitively to EGFR, thus inducing the dephosphorylation of RACK-1. RACK-1 then stimulates PP2A to dephosphorylate CAR, which is then translocated into the nucleus.
Phenobarbital (PB), a widely used anticonvulsant, is used as a model ligand for indirect CAR activation. PB activates CAR, by inducing the dephosphorylation of CAR through PP2A. How PP2A is activated remains unclear, but several different mechanisms have been proposed.[19][20] The recruitment of PP2A has been shown to be mediated by the multiprotein complex. As PB is involved in the activation of AMP-activated protein kinase, it has been suggested that AMPK activates PP2A.[21]
Alternatively, PP2A might be activated through another pathway including the epidermal growth factor receptor (EGFR) and the receptor for activated C kinase 1 (RACK1). In the absence of PB, the epidermal growth factor (EGF) binds to EGFR, thereby activating the steroid receptor coactivator-1 (Src1), which in turn phosphorylates RACK1. Upon PB-exposure, PB binds competitively to EGFR and thus leads to inactivation of Src1. This results in a dephosphorylation of RACK1, which can subsequently stimulate PP2A to activate CAR.[20]
^Ueda A, Hamadeh HK, Webb HK, Yamamoto Y, Sueyoshi T, Afshari CA, Lehmann JM, Negishi M (Jan 2002). "Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital". Molecular Pharmacology. 61 (1): 1–6. doi:10.1124/mol.61.1.1. PMID11752199. S2CID20184152.
^Honkakoski P, Moore R, Washburn KA, Negishi M (Apr 1998). "Activation by diverse xenochemicals of the 51-base pair phenobarbital-responsive enhancer module in the CYP2B10 gene". Molecular Pharmacology. 53 (4): 597–601. doi:10.1124/mol.53.4.597. PMID9547348. S2CID38390000.
^Yoshinari K, Kobayashi K, Moore R, Kawamoto T, Negishi M (Jul 2003). "Identification of the nuclear receptor CAR:HSP90 complex in mouse liver and recruitment of protein phosphatase 2A in response to phenobarbital". FEBS Letters. 548 (1–3): 17–20. doi:10.1016/s0014-5793(03)00720-8. PMID12885400. S2CID24859426.
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Choi HS, Seol W, Moore DD (Jan 1996). "A component of the 26S proteasome binds on orphan member of the nuclear hormone receptor superfamily". The Journal of Steroid Biochemistry and Molecular Biology. 56 (1-6 Spec No): 23–30. doi:10.1016/0960-0760(95)00220-0. PMID8603043. S2CID46464350.
Goodwin B, Hodgson E, D'Costa DJ, Robertson GR, Liddle C (Aug 2002). "Transcriptional regulation of the human CYP3A4 gene by the constitutive androstane receptor". Molecular Pharmacology. 62 (2): 359–65. doi:10.1124/mol.62.2.359. PMID12130689.
Ferguson SS, LeCluyse EL, Negishi M, Goldstein JA (Sep 2002). "Regulation of human CYP2C9 by the constitutive androstane receptor: discovery of a new distal binding site". Molecular Pharmacology. 62 (3): 737–46. doi:10.1124/mol.62.3.737. PMID12181452. S2CID25093374.
Zhang J, Huang W, Chua SS, Wei P, Moore DD (Oct 2002). "Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR". Science. 298 (5592): 422–4. doi:10.1126/science.1073502. PMID12376703. S2CID26269068.
Chang TK, Bandiera SM, Chen J (Jan 2003). "Constitutive androstane receptor and pregnane X receptor gene expression in human liver: interindividual variability and correlation with CYP2B6 mRNA levels". Drug Metabolism and Disposition. 31 (1): 7–10. doi:10.1124/dmd.31.1.7. PMID12485946. S2CID42133087.
