Constitutive androstane receptor explained

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 NR1I3 gene.^[1] CAR is a member of the nuclear receptor superfamily and along with pregnane X receptor (PXR) functions as a sensor of and xenobiotic substances. In response, expression of proteins responsible for the metabolism and excretion of these substances is upregulated.^[2] Hence, CAR and PXR play a major role in the detoxification of foreign substances such as drugs.

Androstenol and several isomers of androstanol, androstanes, are endogenous antagonists of the CAR, and despite acting as antagonists, were the basis for the naming of this receptor.^[3] More recently, dehydroepiandrosterone (DHEA), also an androstane, has been found to be an endogenous agonist of the CAR.^[4]

Function

CAR is a member of the nuclear receptor superfamily, and is a key regulator of xenobiotic and 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.^[5] Ligands binding to CAR include bilirubin, a variety of foreign compounds, steroid hormones, and prescription drugs.^[6]

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.^[7] 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).^[8]

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.^[9] The consensus sequence of PBREM, containing direct repeat-4 motifs, was found to be conserved in mouse, rat and human 'Cyp2b' genes.^{[10] [11] [12]}

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.^[13] 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).^[14]

Indirect activation

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.^{[15] [16]} 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.^[17] 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.^[16]

Further reading

• Masuno M, Shimozawa N, Suzuki Y, Kondo N, Orii T, Tsukamoto T, Osumi T, Fujiki Y, Imaizumi K, Kuroki Y . Assignment of the human peroxisome assembly factor-1 gene (PXMP3) responsible for Zellweger syndrome to chromosome 8q21.1 by fluorescence in situ hybridization . Genomics . 20 . 1 . 141–2 . Mar 1994 . 8020947 . 10.1006/geno.1994.1144 .
• Choi HS, Seol W, Moore DD . 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 . Jan 1996 . 8603043 . 10.1016/0960-0760(95)00220-0 . 46464350 .
• Seol W, Choi HS, Moore DD . An orphan nuclear hormone receptor that lacks a DNA binding domain and heterodimerizes with other receptors . Science . 272 . 5266 . 1336–9 . May 1996 . 8650544 . 10.1126/science.272.5266.1336 . 1996Sci...272.1336S . 32853062 .
• Choi HS, Chung M, Tzameli I, Simha D, Lee YK, Seol W, Moore DD . Differential transactivation by two isoforms of the orphan nuclear hormone receptor CAR . The Journal of Biological Chemistry . 272 . 38 . 23565–71 . Sep 1997 . 9295294 . 10.1074/jbc.272.38.23565 . free .
• Seol W, Hanstein B, Brown M, Moore DD . Inhibition of estrogen receptor action by the orphan receptor SHP (short heterodimer partner) . Molecular Endocrinology . 12 . 10 . 1551–7 . Oct 1998 . 9773978 . 10.1210/mend.12.10.0184 . free .
• Forman BM, Tzameli I, Choi HS, Chen J, Simha D, Seol W, Evans RM, Moore DD . Androstane metabolites bind to and deactivate the nuclear receptor CAR-beta . Nature . 395 . 6702 . 612–5 . Oct 1998 . 9783588 . 10.1038/26996 . 1998Natur.395..612F . 4387623 .
• Gonzalez MM, Carlberg C . Cross-repression, a functional consequence of the physical interaction of non-liganded nuclear receptors and POU domain transcription factors . The Journal of Biological Chemistry . 277 . 21 . 18501–9 . May 2002 . 11891224 . 10.1074/jbc.M200205200 . free .
• Min G, Kim H, Bae Y, Petz L, Kemper JK . Inhibitory cross-talk between estrogen receptor (ER) and constitutively activated androstane receptor (CAR). CAR inhibits ER-mediated signaling pathway by squelching p160 coactivators . The Journal of Biological Chemistry . 277 . 37 . 34626–33 . Sep 2002 . 12114525 . 10.1074/jbc.M205239200 . free.
• Goodwin B, Hodgson E, D'Costa DJ, Robertson GR, Liddle C . Transcriptional regulation of the human CYP3A4 gene by the constitutive androstane receptor . Molecular Pharmacology . 62 . 2 . 359–65 . Aug 2002 . 12130689 . 10.1124/mol.62.2.359 .
• Ferguson SS, LeCluyse EL, Negishi M, Goldstein JA . Regulation of human CYP2C9 by the constitutive androstane receptor: discovery of a new distal binding site . Molecular Pharmacology . 62 . 3 . 737–46 . Sep 2002 . 12181452 . 10.1124/mol.62.3.737 . 25093374 .
• Zhang J, Huang W, Chua SS, Wei P, Moore DD . Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR . Science . 298 . 5592 . 422–4 . Oct 2002 . 12376703 . 10.1126/science.1073502 . 26269068 .
• Chang TK, Bandiera SM, Chen J . 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 . Jan 2003 . 12485946 . 10.1124/dmd.31.1.7 . 42133087 .
• Pascussi JM, Busson-Le Coniat M, Maurel P, Vilarem MJ . Transcriptional analysis of the orphan nuclear receptor constitutive androstane receptor (NR1I3) gene promoter: identification of a distal glucocorticoid response element . Molecular Endocrinology . 17 . 1 . 42–55 . Jan 2003 . 12511605 . 10.1210/me.2002-0244 . free .
• Shiraki T, Sakai N, Kanaya E, Jingami H . Activation of orphan nuclear constitutive androstane receptor requires subnuclear targeting by peroxisome proliferator-activated receptor gamma coactivator-1 alpha. A possible link between xenobiotic response and nutritional state . The Journal of Biological Chemistry . 278 . 13 . 11344–50 . Mar 2003 . 12551939 . 10.1074/jbc.M212859200 . free .
• Maglich JM, Parks DJ, Moore LB, Collins JL, Goodwin B, Billin AN, Stoltz CA, Kliewer SA, Lambert MH, Willson TM, Moore JT . Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes . The Journal of Biological Chemistry . 278 . 19 . 17277–83 . May 2003 . 12611900 . 10.1074/jbc.M300138200 . free .
• Xie W, Yeuh MF, Radominska-Pandya A, Saini SP, Negishi Y, Bottroff BS, Cabrera GY, Tukey RH, Evans RM . Control of steroid, heme, and carcinogen metabolism by nuclear pregnane X receptor and constitutive androstane receptor . Proceedings of the National Academy of Sciences of the United States of America . 100 . 7 . 4150–5 . Apr 2003 . 12644700 . 153063 . 10.1073/pnas.0438010100 . 2003PNAS..100.4150X . free .
• Huang W, Zhang J, Chua SS, Qatanani M, Han Y, Granata R, Moore DD . Induction of bilirubin clearance by the constitutive androstane receptor (CAR) . Proceedings of the National Academy of Sciences of the United States of America . 100 . 7 . 4156–61 . Apr 2003 . 12644704 . 153064 . 10.1073/pnas.0630614100 . 2003PNAS..100.4156H . free .
• Auerbach SS, Ramsden R, Stoner MA, Verlinde C, Hassett C, Omiecinski CJ . Alternatively spliced isoforms of the human constitutive androstane receptor . Nucleic Acids Research . 31 . 12 . 3194–207 . Jun 2003 . 12799447 . 162252 . 10.1093/nar/gkg419 .

Notes and References

1. Baes M, Gulick T, Choi HS, Martinoli MG, Simha D, Moore DD . A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements . Molecular and Cellular Biology . 14 . 3 . 1544–52 . Mar 1994 . 8114692 . 358513 . 10.1128/mcb.14.3.1544.
2. Wada T, Gao J, Xie W . PXR and CAR in energy metabolism . Trends in Endocrinology and Metabolism . 20 . 6 . 273–9 . Aug 2009 . 19595610 . 10.1016/j.tem.2009.03.003 . 25764831 .
3. Book: Nicholas A. Meanwell. Tactics in Contemporary Drug Design. 8 December 2014. Springer. 978-3-642-55041-6. 182–.
4. Kohalmy K, Tamási V, Kóbori L, Sárváry E, Pascussi JM, Porrogi P, Rozman D, Prough RA, Meyer UA, Monostory K . Dehydroepiandrosterone induces human CYP2B6 through the constitutive androstane receptor . Drug Metab. Dispos. . 35 . 9 . 1495–501 . 2007 . 17591676 . 2423426 . 10.1124/dmd.107.016303 .
5. Ueda A, Hamadeh HK, Webb HK, Yamamoto Y, Sueyoshi T, Afshari CA, Lehmann JM, Negishi M . Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital . Molecular Pharmacology . 61 . 1 . 1–6 . Jan 2002 . 11752199 . 10.1124/mol.61.1.1 . 20184152 .
6. Web site: Entrez Gene: NR1I3 nuclear receptor subfamily 1, group I, member 3.
7. Kodama S, Negishi M . Phenobarbital confers its diverse effects by activating the orphan nuclear receptor car . Drug Metabolism Reviews . 38 . 1–2 . 75–87 . 2006 . 16684649 . 10.1080/03602530600569851 . 43824300 .
8. Men S, Wang H . Phenobarbital in Nuclear Receptor Activation: An Update . Drug Metabolism and Disposition . 51 . 2 . 210–218 . February 2023 . 36351837 . 9900862 . 10.1124/dmd.122.000859 .
9. Kawamoto T, Sueyoshi T, Zelko I, Moore R, Washburn K, Negishi M . Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene . Molecular and Cellular Biology . 19 . 9 . 6318–22 . Sep 1999 . 10454578 . 84602 . 10.1128/mcb.19.9.6318.
10. Honkakoski P, Moore R, Washburn KA, Negishi M . Activation by diverse xenochemicals of the 51-base pair phenobarbital-responsive enhancer module in the CYP2B10 gene . Molecular Pharmacology . 53 . 4 . 597–601 . Apr 1998 . 9547348 . 10.1124/mol.53.4.597 . 38390000 .
11. Sueyoshi T, Kawamoto T, Zelko I, Honkakoski P, Negishi M . The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene . The Journal of Biological Chemistry . 274 . 10 . 6043–6 . Mar 1999 . 10037683 . 10.1074/jbc.274.10.6043 . free .
12. Mäkinen J, Frank C, Jyrkkärinne J, Gynther J, Carlberg C, Honkakoski P . Modulation of mouse and human phenobarbital-responsive enhancer module by nuclear receptors . Molecular Pharmacology . 62 . 2 . 366–78 . Aug 2002 . 12130690 . 10.1124/mol.62.2.366 .
13. Tzameli I, Pissios P, Schuetz EG, Moore DD . The xenobiotic compound 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is an agonist ligand for the nuclear receptor CAR . Molecular and Cellular Biology . 20 . 9 . 2951–8 . May 2000 . 10757780 . 85552 . 10.1128/MCB.20.9.2951-2958.2000 .
14. Maglich JM, Parks DJ, Moore LB, Collins JL, Goodwin B, Billin AN, Stoltz CA, Kliewer SA, Lambert MH, Willson TM, Moore JT . Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes . The Journal of Biological Chemistry . 278 . 19 . 17277–83 . May 2003 . 12611900 . 10.1074/jbc.M300138200 . free .
15. Yoshinari K, Kobayashi K, Moore R, Kawamoto T, Negishi M . 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 . Jul 2003 . 12885400 . 10.1016/s0014-5793(03)00720-8 . 2003FEBSL.548...17Y . 24859426 .
16. Mutoh S, Sobhany M, Moore R, Perera L, Pedersen L, Sueyoshi T, Negishi M . Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling . Science Signaling . 6 . 274 . ra31 . May 2013 . 23652203 . 10.1126/scisignal.2003705 . 5573139 .
17. Rencurel F, Stenhouse A, Hawley SA, Friedberg T, Hardie DG, Sutherland C, Wolf CR . AMP-activated protein kinase mediates phenobarbital induction of CYP2B gene expression in hepatocytes and a newly derived human hepatoma cell line . The Journal of Biological Chemistry . 280 . 6 . 4367–73 . Feb 2005 . 15572372 . 10.1074/jbc.M412711200 . free.