Alternative splicing within the ligand binding domain of the human constitutive androstane receptor

Savkur, Rajesh S.; Wu, Yingliang; Bramlett, Kelli; Wang, Minmin; Yao, Sufang; Perkins, Douglas; Totten, Michelle; Searfoss, George H.; Ryan, Timothy P.; Su, Eric W.; Burris, Thomas P.

doi:10.1016/j.ymgme.2003.08.013

Cited by 50 publications

(66 citation statements)

References 35 publications

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“…However, due to the use of heterologous promoters, these models do not provide bona fide levels of expression of PXR or CAR in all tissues of the mouse. Furthermore, splice variants of hPXR and hCAR have recently been described, some of which have been shown to have functional activity (19)(20)(21)(22)(23). None of these splice variants are expressed by mouse models in which pure cDNAs are used.…”

Section: Introductionmentioning

confidence: 99%

A novel panel of mouse models to evaluate the role of human pregnane X receptor and constitutive androstane receptor in drug response

Scheer¹,

Ross²,

Rode³

et al. 2008

J. Clin. Invest.

133

114

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The pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are closely related orphan nuclear hormone receptors that play a critical role as xenobiotic sensors in mammals. Both receptors regulate the expression of genes involved in the biotransformation of chemicals in a ligand-dependent manner. As the ligand specificity of PXR and CAR have diverged between species, the prediction of in vivo PXR and CAR interactions with a drug are difficult to extrapolate from animals to humans. We report the development of what we believe are novel PXR-and CAR-humanized mice, generated using a knockin strategy, and Pxr-and Car-KO mice as well as a panel of mice including all possible combinations of these genetic alterations. The expression of human CAR and PXR was in the predicted tissues at physiological levels, and splice variants of both human receptors were expressed. The panel of mice will allow the dissection of the crosstalk between PXR and CAR in the response to different drugs. To demonstrate the utility of this panel of mice, we used the mice to show that the in vivo induction of Cyp3a11 and Cyp2b10 by phenobarbital was only mediated by CAR, although this compound is described as a PXR and CAR activator in vitro. This panel of mouse models is a useful tool to evaluate the roles of CAR and PXR in drug bioavailability, toxicity, and efficacy in humans.

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Section: Introductionmentioning

confidence: 99%

A novel panel of mouse models to evaluate the role of human pregnane X receptor and constitutive androstane receptor in drug response

Scheer¹,

Ross²,

Rode³

et al. 2008

J. Clin. Invest.

133

114

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show abstract

“…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%

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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“…For example, some members of the cytochrome P450 family are involved in the metabolism of drugs and xenobiotics. Induction of some of these genes is mediated by NHRs (35) and distinct isoforms of these NHRs are generated through alternate splicing (36,37). Additionally, alternate splicing is used to generate either proapoptotic or antiapoptotic forms of the BCL-2 family of EC 50 is significantly different from EC 50 of samples in absence of modulator when compared using curve fit SEs on the log-EC 50 scale (P < 0.01).…”

Section: Discussionmentioning

confidence: 99%

Human Splicing Factor SPF45 (RBM17) Confers Broad Multidrug Resistance to Anticancer Drugs When Overexpressed— a Phenotype Partially Reversed By Selective Estrogen Receptor Modulators

et al. 2005

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The splicing factor SPF45 (RBM17) is frequently overexpressed in many solid tumors, and stable expression in HeLa cells confers resistance to doxorubicin and vincristine. In this study, we characterized stable transfectants of A2780 ovarian carcinoma cells. In a 3-day cytotoxicity assay, human SPF45 overexpression conferred 3-to 21-fold resistance to carboplatin, vinorelbine, doxorubicin, etoposide, mitoxantrone, and vincristine. In addition, resistance to gemcitabine and pemetrexed was observed at the highest drug concentrations tested. Knockdown of SPF45 in parental A2780 cells using a hammerhead ribozyme sensitized A2780 cells to etoposide by f5-fold relative to a catalytically inactive ribozyme control and untransfected cells, suggesting a role for SPF45 in intrinsic resistance to some drugs. A2780-SPF45 cells accumulated similar levels of doxorubicin as vector-transfected and parental A2780 cells, indicating that drug resistance is not due to differences in drug accumulation. Efforts to identify small molecules that could block SPF45-mediated drug resistance revealed that the selective estrogen receptor (ER) modulators tamoxifen and LY117018 (a raloxifene analogue) partially reversed SPF45-mediated drug resistance to mitoxantrone in A2780-SPF45 cells from 21-fold to 8-and 5-fold, respectively, but did not significantly affect the mitoxantrone sensitivity of vector control cells. Quantitative PCR showed that ERB but not ERA was expressed in A2780 transfectants. Coimmunoprecipitation experiments suggest that SPF45 and ERB physically interact in vivo. Thus, SPF45-mediated drug resistance in A2780 cells may result in part from effects of SPF45 on the transcription or alternate splicing of ERBregulated genes. (Cancer Res 2005; 65(15): 6593-600)

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Alternative splicing within the ligand binding domain of the human constitutive androstane receptor

Cited by 50 publications

References 35 publications

A novel panel of mouse models to evaluate the role of human pregnane X receptor and constitutive androstane receptor in drug response

A novel panel of mouse models to evaluate the role of human pregnane X receptor and constitutive androstane receptor in drug response

CAR2 Displays Unique Ligand Binding and RXRα Heterodimerization Characteristics

Human Splicing Factor SPF45 (RBM17) Confers Broad Multidrug Resistance to Anticancer Drugs When Overexpressed— a Phenotype Partially Reversed By Selective Estrogen Receptor Modulators

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