Alternatively spliced isoforms of the human constitutive androstane receptor

Auerbach, Scott S.; Ramsden, R. T.; Stoner, Michael W.; Verlinde, Christophe L. M. J.; Hassett, Christopher; Omiecinski, Curtis J.

doi:10.1093/nar/gkg419

Cited by 114 publications

(168 citation statements)

References 52 publications

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“…We and others have recently described a number of mRNA splice variants of the human CAR gene that potentially represent a large expansion of the CAR proteome (Auerbach et al, 2003;Savkur et al, 2003;Arnold et al, 2004;Jinno et al, 2004;Lamba et al, 2004). One of the variant forms results from the use of an alternative splice acceptor site in intron 6, leading to the insertion of 12 additional nucleotides.…”

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confidence: 99%

“…The resultant mRNA has been reported to make up 6 to 10% of the total CAR transcript in human liver (Jinno et al, 2004). This transcript encodes a protein containing an additional four amino acids (SPTV) that are predicted to extend helix 6 of the ligand binding domain and potentially affect the structure of the ligand binding pocket (Auerbach et al, 2003;Savkur et al, 2003). We term this form of the receptor CAR2 [CAR1 being the reference form of the receptor (Baes et al, 1994)].…”

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confidence: 99%

“…We term this form of the receptor CAR2 [CAR1 being the reference form of the receptor (Baes et al, 1994)]. CAR2 retains a limited ability to transactivate CAR-responsive reporters (Auerbach et al, 2003;Arnold et al, 2004;Jinno et al, 2004), a result that correlates with a reduced affinity for RXR␣ and in turn a compromised ability to interact with DNA (Auerbach et al, 2003;Arnold et al, 2004). Ligand studies of CAR2 demonstrated that clotrimazole deactivated the receptor, whereas CITCO produced a weak, albeit significant activation of CAR2 (Jinno et al, 2004)-a result that is contrary to mammalian two-hybrid studies published separately (Arnold et al, 2004).…”

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confidence: 99%

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CAR2 Displays Unique Ligand Binding and RXRα Heterodimerization Characteristics

Auerbach¹,

DeKeyser²,

Stoner³

et al. 2006

Drug Metab Dispos

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ABSTRACT: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 RXR␣ ligand binding domain was critical for achieving these effects. CAR2 interaction with SRC-1 was similarly dependent on the coexpression of RXR␣. 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 ؋ 3 reporter element correlated with their ability to interact with RxR␣ and to recruit SRC-1 in a ligand-regulated manner. Together, these results demonstrate a robust RXR␣-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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CAR2 Displays Unique Ligand Binding and RXRα Heterodimerization Characteristics

Auerbach¹,

DeKeyser²,

Stoner³

et al. 2006

Drug Metab Dispos

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“…In order to investigate whether select CAR splice variants activate the hCTSE promoter, the largest promoter construct was used to examine potential hCTSE transcriptional modulation by the prominent hCAR variants, CAR2 and CAR3 [22]. In these studies, expression plasmids for CAR1, CAR2, CAR3, and RXRα were used to transiently transfect COS-1 cells and assessed for their respective transactivation potential with the …”

Section: Activation Of Hctse By Car Splice Variantsmentioning

confidence: 99%

“…The human CAR gene produces several splice variants that exhibit unique functional characteristics and are expressed in liver tissues simultaneously with the reference form of CAR, CAR1 [22][23][24][25]. In order to investigate whether select CAR splice variants activate the hCTSE promoter, the largest promoter construct was used to examine potential hCTSE transcriptional modulation by the prominent hCAR variants, CAR2 and CAR3 [22].…”

Section: Activation Of Hctse By Car Splice Variantsmentioning

confidence: 99%

Regulation of the human cathepsin E gene by the constitutive androstane receptor

Page

Strom

Omiecinski

2007

Archives of Biochemistry and Biophysics

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Cathepsin E (CTSE) is an aspartic protease that has been linked to antigen processing and innate immunity. Elevated levels of CTSE expression have also been associated with several forms of cancer, including carcinomas exhibiting highly invasive character. In this study, we performed DNA microarray experiments, together with quantitative reverse transcriptase PCR analyses and enzymatic activity determinations to identify human CTSE as a novel target gene for regulation by the constitutive androstane receptor (CAR), a nuclear receptor activated by the liver tumor promoting agent, phenobarbital. In particular, two motifs within the 5′-flanking region of the human CTSE gene were identified as direct sites of interaction with CAR/RXRα heterodimers, a direct repeat-3 site at position −766 and a direct repeat-4 site at position −1407. Thus, these studies demonstrate CAR-mediated regulation of CTSE within primary hepatocyte cultures from several individual donors and suggest that elevated CTSE activity may play a functional role in the etiology of hepatocarcinogenesis. KeywordsCathepsin E; Constitutive androstane receptor; Hepatocytes; Primary hepatocytes; Liver; Human; Hepatocytes Cathepsin E (CTSE) 1 is an intracellular aspartic protease that hydrolyzes various biologically active peptides [1]. Unlike related proteases, CTSE is localized outside of the lysosomal system and likely functions in extralysosomal proteolysis [2]. Although its exact biological functions remain elusive, several physiological roles of CTSE have been reported, including roles in immune defense and antigen processing [3][4][5]. In this respect, CTSEdeficient mice exhibit markedly increased susceptibility to both gram positive and gram negative bacterial infection, likely due to impaired regulation of bacterial elimination [5]. Given the apparent association of enhanced CTSE expression with hepatocellular carcinoma, we hypothesize that liver tumor promoting agents may function to enhance CTSE expression levels in liver and that increased proteolytic activity may underlie certain mechanistic aspects of the tumor promotion process. In the studies reported here, we identify CTSE expression in human hepatoctyes. In particular, microarray expression profiling analyses indicated that exposures to the classical liver tumor promoter, phenobarbital (PB), resulted in increased CTSE transcript levels within primary hepatocyte cultures from several individual donors, a result that was confirmed by quantitative RT-PCR experiments. Further, we demonstrate that the constitutive androstane receptor (CAR) acts as a transcriptional regulator of the human CTSE gene and that these events lead to PB-inducible increases in functional hepatic CTSE enzymatic activity. Materials and methods ChemicalsPhenobarbital sodium salt was obtained from UWMC Drug Services (Seattle, WA). Clotrimazole, meclizine and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). CITCO was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Cell cultureCO...

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Genetic Variants of Xenobiotic Receptors and their Implications in Drug Metabolism and Pharmacogenetics

Lamba

Schuetz

2008

Nuclear Receptors in Drug Metabolism

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Alternatively spliced isoforms of the human constitutive androstane receptor

Cited by 114 publications

References 52 publications

CAR2 Displays Unique Ligand Binding and RXRα Heterodimerization Characteristics

CAR2 Displays Unique Ligand Binding and RXRα Heterodimerization Characteristics

Regulation of the human cathepsin E gene by the constitutive androstane receptor

Genetic Variants of Xenobiotic Receptors and their Implications in Drug Metabolism and Pharmacogenetics

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