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Alternative splicing within the ligand binding domain of the human constitutive androstane receptor
Molecular Genetics and Metabolism 80 (2003) 216–226 www.elsevier.com/locate/ymgme
Alternative splicing within the ligand binding domain of the human constitutive androstane receptorq Rajesh S. Savkur, Yifei Wu, Kelli S. Bramlett, Minmin Wang, Sufang Yao, Douglas Perkins, Michelle Totten, George Searfoss III, Timothy P. Ryan, Eric W. Su, and Thomas P. Burris* Gene Regulation Research, Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, IN 46285, USA Received 1 July 2003; accepted 14 August 2003
Abstract The human constitutive androstane receptor (hCAR; NR1I3) is a member of the nuclear receptor superfamily. The activity of hCAR is regulated by a variety of xenobiotics including clotrimazole and acetaminophen metabolites. hCAR, in turn, regulates a number of genes responsible for xenobiotic metabolism and transport including several cytochrome P450s (CYP 2B5, 2C9, and 3A4) and the multidrug resistance-associated protein 2 (MRP2, ABCC2). Thus, hCAR is believed to be a mediator of drug–drug interactions. We identified two novel hCAR splice variants: hCAR2 encodes a receptor in which alternative splice acceptor sites are utilized resulting in a 4 amino acid insert between exons 6 and 7, and a 5 amino acid insert between 7 and 8, and hCAR3 encodes a receptor with exon 7 completely deleted resulting in a 39 amino acid deletion. Both hCAR2 and hCAR3 mRNAs are expressed in a pattern similar to the initially described MB67 (hCAR1) with some key distinctions. Although the levels of expression vary depending on the tissue examined, hCAR2 and hCAR3 contribute 6–8% of total hCAR mRNA in liver. Analysis of the activity of these variants indicates that both hCAR2 and hCAR3 lose the ability to heterodimerize with RXR and lack transactivation activity in cotransfection experiments where either full-length receptor or GAL4 DNA-binding domain/CAR ligand binding domain chimeras were utilized. Although the role of hCAR2 and hCAR3 is currently unclear, these additional splice variants may provide for increased diversity in terms of responsiveness to xenobiotics. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Nuclear receptor; Xenobiotic; Steroid; Phenobarbital; Transcription; RXR; Clotrimazole
Introduction The superfamily of nuclear hormone receptors comprises a group of transcription factors that play significant roles in response to a number of biological regulators including steroids, retinoids, and thyroid hormones. Two of these nuclear receptors, the pregnane X receptor (PXR) [NR1I2] and the constitutive androstane receptor (CAR) [NR1I3] play a crucial role in response to xenobiotics [1,2]. This response is mediated q
The results of this manuscript were reported in abstract form at the 85th Annual Meeting of the Endocrine Society, June 19–22, 2003 (Abstract P3-266). * Corresponding author. Fax: 1-317-276-1414. E-mail address: [email protected] (T.P. Burris). 1096-7192/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2003.08.013
by these two nuclear hormone receptors either being directly or indirectly activated by xenobiotic chemicals leading to induction of expression of a series of genes involved in detoxification of these substances. The target genes for these xenobiotic nuclear receptors include a number of cytochrome P450 (CYP) monooxygenases as well as phase II xenobiotic-metabolizing enzymes and ATP binding cassette transporters associated with xenobiotic transport [3–5]. Activation of either PXR or CAR by pharmaceutical agents has also been shown to mediate drug–drug interactions resulting in potential difficulties in pharmacotherapy [6]. CAR and PXR are related members of the NR1I subfamily of nuclear hormone receptors [7] with 40% amino acid identity within their ligand binding domains (LBDs) [8]. Both human CAR (hCAR) and mouse CAR (mCAR) are
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most abundantly expressed in the liver, and are constitutively active in cell-based reporter assays in the absence of exogenously added ligands [9,10]. CAR has been implicated as the mediator of phenobarbital (PB) induction of the CYP2B genes based on the observation that CAR binds to the CYP2B PB-responsive enhancer, termed the PBRU or PB-responsive module [11]. Interestingly, at least a subset of the xenobiotics that activate CAR, including PB, appear to do so by causing translocation of the receptor from the cytoplasm to the nucleus without directly binding to CAR itself [12,13]. However, a number of xenobiotic activators of CAR do function through the more classical method of direct binding to the LBD [14]. It has also been demonstrated that CAR heterodimerizes with the retinoid X receptor (RXR) to bind to a wide variety of DNA response elements, and activates the transcription of target genes containing these elements in the absence of ligands [15]. It has been suggested that the LBD of CAR adopts a conformation where it binds to coactivators such as SRC-1 [16] or TIF2/GRIP1 [17] in the absence of ligands to exhibit its constitutive activity. However, this constitutive activity can be inhibited by inverse agonists such as androstanol, androstenol or clotrimazole as well as enhanced by agonists such as CITCO [16,18,19]. Alternative splicing generates segments of mRNA variability that can insert or delete amino acids, shift the reading frame, or introduce premature termination codons. Up to 59% of the human genes generate multiple mRNAs by alternative splicing [20], and 80% of alternative splicing results in changes in the encoded protein [21] revealing that it is likely to be the primary source of human proteomic diversity. A large fraction of alternative splicing events are regulated in a cell- or tissue-specific manner in which the splicing pathways are modulated according to cell-type, developmental stage, gender, or in response to external stimuli. Choi et al. [10], identified two isoforms of mCAR that arise as a result of alternative splicing of the single gene. The two splice variants mCAR1 and mCAR2 that are encoded from the single mCAR gene, contain identical DNA binding domains (DBD), but differ in the LBD. mCAR2 is truncated, lacking the Cterminal region of the conserved LBD that includes the AF-2 domain. This deletion results in both loss of transactivation activity (lack of AF2) and DNA binding activity due to deficits in the ability of mCAR2 to heterodimerize with RXR. Here we describe the identification and initial characterization of two novel splice variants of hCAR. These two novel isoforms (designated hCAR2 and hCAR3) differ from the previously identified MB67-hCAR (designated hCAR1) in their LBDs, but are identical in the DBD. These novel isoforms constitute 10% of the total hCAR mRNA, and display a differential tissue expres-
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sion profile relative to hCAR1. In contrast to the previously reported mCAR2, both hCAR2 and hCAR3 contain an intact AF-2. However, they are unable to bind DNA and transactivate target genes in cell-based reporter assays. Molecular modeling of hCAR2 and hCAR3 indicates that an alteration in the overall 3-dimensional shape and surface area of these splice variants is the most probable cause for the loss of their functional activity. Differences in the tissue expression, and function of the hCAR isoforms could reveal critical information for our understanding of xenobiotic metabolism as well as the basis for phenotypic variations in drug– drug interaction responses.
Materials and methods Cloning and identification of hCAR splice variants Human liver cDNA was subjected to PCR amplification and subcloned into pCR-II-Topo (Invitrogen, Carlsbad, CA). All clones were confirmed by sequencing. Quantification of hCAR splice variants Quantitative analysis of hCAR splice variants was performed using TaqMan QPCR (Applied Biosystems, Foster City, CA). For detection of hCAR1, forward and reverse primers spanned exon 6/exon 7 and exon 7/exon 8 junctions, respectively, with the TaqMan probe hybridizing to exon 7. To detect hCAR2, the forward and reverse primers were located at the exon 6/7 and exon 7/ 8 junctions, respectively, and contained the additional sequences specific for hCAR2. The TaqMan probe hybridized to exon 7. To detect hCAR3, the forward and reverse primers were located within exons 6 and 8, respectively. The TaqMan probe hybridized to exon 6/ exon 8 junction that is present only in hCAR3. Copy numbers for each of the splice variant transcripts were obtained by comparing sample counts to a 6-point standard curve of plasmid DNA with the specific primer set included in the same TaqMan plate. Electrophoretic mobility shift assays hCAR splice variants and hRXRa were expressed using coupled in vitro transcription and translation (Promega, Madison, WI) as recommended by the manufacturer. Gel-shift assays were performed using the [a-32 P]dCTP labeled DR-4 element from the CYP2B10 gene or the [a-32 P]dCTP labeled DR-5 element from the b-RARE promoter as previously described [15]. Super-shift assays were performed using the anti-hRXRa D-20 antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
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Plasmids Plasmid pG5-Luc containing five copies of the DNAbinding site of the minimal thymidine kinase (TK) promoter linked to a luciferase gene was purchased from Promega. The 5xDR-4–tk-Luc reporter, containing five copies of the DR-4 response element from the CYP2B10 gene [15], was generated by inserting the double-stranded oligonucleotide response element upstream of the minimal TK promoter linked to a luciferase gene (pTALuc, Clontech). Full-length expression vectors of hCAR splice variants and hRXRa were generated by PCR amplification of the respective cDNAs and subcloning into pcDNA3.1DVH vector (Invitrogen). Chimeras expressing the Gal4 DNA binding domain (DBD) fused to CAR LBDs were created utilizing the commercially available mammalian two-hybrid vector pM from Clontech. The pM-hCAR1LBD contains amino acids 77–348, pM-hCAR2LBD contains amino acids 77–357, and pM-hCAR3LBD contains amino acids 77–309. In each case the LBD fragments were generated by PCR from the full length cDNA and subcloned in frame downstream of the Gal 4 DBD into the pM vector. The expression vector for TIF2/GRIP1 has been previously described [22]. Cell culture and transient transfection assays CV1 cells were propagated in DulbeccoÕs modified EagleÕs/F12 (3:1) media without phenol red supplemented with 10% (v/v) dextran charcoal treated fetal bovine serum (Hyclone Laboratories, Logan, UT) and 2 mM glutamine at 37 °C under 5% CO2 . Prior to transfections, CV1 cells were plated in 96-well plates at a density of 12,000 cells/well. Transfection mixes contained 100 ng of reporter plasmids and 50 ng of receptor plasmids using FuGENE 6 (Roche Molecular Biochemicals). Forty-eight hour post-transfection, cells were harvested with Steady-Glo lysis buffer (Promega). Luciferase activity was measured in a Dynex luminometer. All experiments were done in triplicate, and unless otherwise indicated the data are displayed as the means SE of a single experiment representative of at least three independent experiments. Homology modeling of hCAR The 3-dimensional homology models for the LBD of hCAR1 and hCAR2 were constructed by using the automated comparative homology modeling program, Modeler [23], as implemented in InsightII2000 (Accelrys, San Diego, 2000). Unlike the conventional homology modeling scheme with time consuming stages of core region identification and loop region building, Modeler uses unique probability density functions (PDFs) to transfer the spatial restraints from the
template to the target proteins. Individual restraints are assembled into a single molecular PDF (MPDF) and optimized to give 3-dimensional coordinates for the target protein. The sequence alignment was generated by PSI-BLASTing of protein data bank (PDB) using the hCAR-LBD sequence as the query. The two best hits were PXR (1ilh) and VDR (1ie9) with the identity score of 49% and 37%, respectively. VDR was chosen as the template for the homology modeling because the sequence is fully aligned with the LBD of hCAR1 for 225 residues (as opposed to 192 aligned residues with PXR). Segments of overhanging sequence on hCARLBD were cut during the model building process. Two models were built with a high level of optimization for the conserved regions as well as a high level of optimization in the loop regions. None of the hydrogen atoms are built in the homology model. The model with the least )ln(PDF) value was selected as the final model and validated with Protein Health check in Quanta98 (Accelyrs, San Diego, 1999). This ensured that undesirable conformations were created on the backbones and sidechains in the ligand-binding pocket. The protein surface area was calculated using MOLCAD in Sybyl68 (Tripos, St. Louis, MO). The secondary structure prediction for the loop insertions on hCAR2 was generated by BIOPOLYMER module in Sybyl68 (Tripos, St. Louis, MO).
Results Isolation and characterization of the hCAR splice variants Previous studies identified two isoforms of the murine CAR (mCAR) that arise as a result of alternative splicing of the mCAR pre-mRNA [10]. In an attempt to obtain MB67 (hCAR), we obtained two full-length Genestorm cDNAs from Invitrogen, and interestingly both cDNAs appeared to be alternatively spliced variants of the initially described MB67 [9]. In order to obtain the initially described hCAR, we screened a human liver cDNA library by PCR amplification, and subcloned the products into pCR-II-Topo vector. Sequencing of several independent clones did not reveal any additional clones beyond these three characterized cDNAs. The three hCAR cDNAs, which we designated hCAR1 [9], hCAR2, and hCAR3 (Figs. 1A and B), are identical in the DBD and hinge regions, but differ in their LBDs. hCAR1, encoded by 9 exons is 348 amino acids in length, with a predicted molecular weight of 39.66 kDa. hCAR2 contains an additional 9 amino acids (with respect to hCAR1) in the LBD, and encodes a protein with a predicted molecular weight of 40.59 kDa, whereas hCAR3 lacks 39 amino acids in the LBD (relative to hCAR1), and has a predicted mass of 35.15 kDa. The amino acid sequence alignment of the
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Fig. 1. (A) Structure of human CAR gene. The 348 amino acid long wild-type human CAR (hCAR1) encoded by 9 exons comprised of a DNA binding domain (DBD), a hinge region, and a ligand binding domain (LBD). (B) Domain structural analysis of hCAR splice variants. hCAR2 contains an additional 9 amino acids in the LBD (depicted by white strips) whereas hCAR3 lacks 39 amino acids (relative to hCAR1) in the LBD. The hCAR splice variants are identical in the DBD and the hinge regions. (C) amino acid sequence alignment of the LBD of the hCAR splice variants. The 9 additional amino acids included in hCAR2 are highlighted. The bold dashes represent the 39 amino acids that are deleted in hCAR3. The numbers denote the positions of the amino acids in the hCAR proteins.
LBD of the three hCAR splice variants (Fig. 1C) depicts the positions of the additional 9 amino acids present in hCAR2 and the 39 amino acid deletion in hCAR3. To verify the authenticity of the hCAR splice variants, the exon–intron boundaries of exons 6 through 8 were analyzed (Fig. 2A). The nucleotide sequence alignment of the hCAR splice variants indicated that hCAR2 is derived from the use of the alternative splice donor/acceptor sites with insertions both upstream and downstream of exon 7. Interestingly, exon 7 is completely skipped to generate hCAR3. A comparison of the LBDs of the hCAR splice variants to the related members of the nuclear receptor superfamily (VDR and PXR) (Fig. 2B) indicated that of the additional 9 amino acids in the LBD of hCAR2, four (SPTV) are inserted amino-terminal to helix 7, and five (PAPYL) are carboxy-terminal to helix 8. The 39 amino acids that are deleted in the LBD of hCAR3 (relative to hCAR1) span helices 7 and 8. Expression profile analysis of hCAR splice variants To quantitatively differentiate between the hCAR splice variants, we used a TaqMan PCR based approach by designing probe sets that would selectively amplify each of the hCAR variants (see Materials and methods). Previous results have reported MB67 expression to be
predominant in the liver with low levels of expression in the heart and muscle followed by the kidneys and lung [9]. Consistent with these observations, the tissue expression profile revealed that hCAR1 is most abundantly expressed in the liver, followed by the ovary, lung, kidneys, and adrenal glands (Fig. 3A). In agreement with previous results [24], we also observed that the expression of hCAR1 dramatically increases in the adult liver compared to the fetal. The tissue expression profile of hCAR2 (Fig. 3B) also indicates that this splice variant is predominantly expressed in the kidney, lung, adrenal glands, and the liver. Interestingly, in contrast to hCAR1, the expression of hCAR2 is more abundant in the lung compared to the liver. The tissue expression profile of hCAR3 (Fig. 3C) reveals that this isoform is expressed at very low levels in most tissues, with the kidneys, small intestine, adrenal glands, and liver showing relatively increased expression. Since CAR mediates the hepatic effects of xenobiotics, the relative expression of the hCAR splice variants in the liver was determined using TaqMan QPCR analysis. As seen in Fig. 4, in all the three human liver samples tested, hCAR1 is the most abundantly expressed isoform. The other two splice variants, hCAR2 and hCAR3, comprise 6–8% of the total hCAR isoforms in the liver.
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Fig. 2. (A) Exon–intron boundaries for exons 6, 7, and 8 of hCAR. The exon–intron boundaries of hCAR1, hCAR2, and hCAR3 are depicted. The amino acid sequences for the respective receptors are indicated in bold. Alternative splice donor/acceptor sites (underlined nucleotide sequences) are utilized to derive hCAR2 with insertions both upstream and downstream of exon 7. Exon 7 is completely deleted in hCAR3. (B) Amino acid sequence comparison of the LBDs of the hCAR splice variants to related members of the nuclear receptor superfamily—VDR and PXR. Of the additional 9 amino acids in the LBD of hCAR2, 4 (SPTV) are inserted in helix 7, and 5 (PAPYL) are present in helix 8. The 39 amino acid deletion in the LBD of hCAR3 spans helices 7 and 8.
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Fig. 3. (A) Tissue expression profile of hCAR1. Origene Rapid Scan human tissue panel cDNAs were pre-normalized to equivalent levels of b-actin transcripts. Copy numbers were derived by comparing to a 6-point dilution standard curve of plasmid DNA containing hCAR1 included on the same TaqMan plate. hCAR1 is predominately expressed in the lung, ovary, adrenal glands, and liver. Expression of hCAR1 dramatically increases in adult liver compared to fetal. (B) Tissue expression profile of hCAR2. TaqMan QPCR analysis for hCAR2 was performed essentially as described for hCAR1. hCAR2 is highly expressed in the kidney, liver, adrenals and lung. Interestingly, in contrast to hCAR1, hCAR2 is abundantly expressed in the lung compared to the liver. (C) Tissue expression profile of hCAR3. TaqMan QPCR analysis for hCAR3 was performed as described above for hCAR1. hCAR3 is ubiquitously expressed in all tissues, with the kidneys, adrenals, small intestine, and liver showing relatively increased expression. Note the differences in the scales of the y-axis.
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Fig. 4. Relative expression of the hCAR splice variants in human liver. cDNA synthesized from 100 ng of total RNA isolated from three independent human liver samples were subjected to TaqMan QPCR. The copy numbers were obtained by comparing sample counts to a 6-point standard curve for each primer/probe set included in the same plate. In all three individual liver mRNAs, hCAR2, and hCAR3 comprise 6–8% of the total hCAR splice variants.
Transactivation and DNA binding of hCAR splice variants Previous studies have shown hCAR1 to exhibit a high constitutive activity in the absence of any exogenously added ligands [9]. To determine whether variations in
the LBD of hCAR, would affect this constitutive transactivation, the LBDs of the hCAR splice variants were fused to the yeast Gal4 DBD to create the chimeric fusion constructs, and tested in transient transfection assays. As seen in Fig. 5A, the Gal4–hCAR1-LBD is constitutively active in the absence of any added ligands. This apparently heterologous constitutive transactivation was observed in the presence of serum treated with charcoal to remove potential ligands. However, no significant transactivation was observed with hCAR2 and hCAR3. It has previously been shown that TIF2/GRIP1 is a potential coactivator of CAR, where it interacts with, and enhances the CAR-mediated transactivation [17]. To check whether GRIP1 is capable of transactivating the hCAR splice variants, an expression plasmid for GRIP1 was co-transfected with the Gal4–hCAR-LBD fusion plasmids and the reporter in CV1 cells. As seen in Fig. 5B, in agreement with previous results, GRIP1 dramatically enhances the transactivation of hCAR1. However, no increase in activation was observed with both hCAR2 and hCAR3. This suggests that variations in the LBD likely alter the structure of the LBD, and abolish the interaction of hCAR2 and hCAR3 with this coactivator. To determine whether the full-length hCAR splice variants are capable of transactivation, plasmids expressing full-length hCAR splice variants were co-transfected with a luciferase reporter plasmid in which 5 copies of the CYP2B10 (DR4) element were inserted upstream of the TK promoter. As seen in Fig. 5C, hCAR1 exhibited a twofold constitutive activity, consistent with the
Fig. 5. Transactivation by the hCAR splice variants. (A) CV-1 cells were co-transfected with the plasmids expressing the Gal4 fusion constructs with the LBDs of the hCAR splice variants or with the empty vector (vec) and pG5Luc reporter plasmid. Forty-eight hour post-transfection, cells were lysed and the luciferase activity was measured as described under Materials and methods. All experiments were done in triplicate, and the data are displayed as the means SE of a single experiment representative of three independent experiments. (B) CV-1 cells were co-transfected with the plasmids expressing the Gal4 fusion constructs with the LBDs of the hCAR splice variants and pG5Luc reporter in the presence or absence of GRIP1. Forty-eight hour post-transfection, cells were lysed and the luciferase activity was measured as described in section (A). All experiments were done in triplicate, and the data are displayed as the means SE of a single experiment representative of three independent experiments. (C). CV-1 cells were transfected with plasmids expressing the full-length hCAR splice variants or the empty vector (vec) and the 5xDR-4–tk-Luc reporter vector (see Materials and methods). Forty-eight hour post-transfection, cells were lysed and the luciferase activity was measured as described in section (A). For (C) data are reported as the combined means SE of three independent experiments.
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effect that has been reported with this element using mCAR [15]. However, hCAR2 and hCAR3 show a significant reduction in their ability to transactivate this element compared to hCAR1. To understand the basis for the reduced transactivation of hCAR2 and hCAR3 on the CYP2B10 (DR4) element, the ability of these proteins to bind the DR4 element was assessed using in vitro translated hCAR splice variants and assayed by gel-shift analysis. Consistent with previous observations, hCAR1 alone or hRXRa alone did not bind the DR4 element (Fig. 6A). However the hCAR1/hRXRa heterodimer bound to this element with a high affinity [15]. The mobility of this complex was further retarded by the addition of the anti-hRXRa antibody (D20), confirming the presence of hRXRa in the heterodimer complex. This hCAR1/ hRXRa heterodimer also bound with a high affinity to the DR5 response element of the bRARE [9] (Fig. 6B), and was also super-shifted by the addition of the antihRXRa antibody. In addition, longer exposure times also revealed that hCAR1 alone bound the DR5 element, although this interaction was much weaker than the hCAR1/RXR heterodimer (data not shown). However, neither hCAR2 nor hCAR3 bound the CYP2B10 (DR4) or the bRARE (DR5) elements, either alone or as a heterodimer with RXR. This suggested that despite the fact that the hCAR isoforms contain identical DBDs, variations in the LBD of hCAR2 and hCAR3 were adversely affecting the structure of these isoforms,
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and abolishing their ability to bind response elements and also reducing their ability to transactivate genes containing the elements. Model of LBD of hCAR splice variants Although the X-ray structure for hCAR1 is unavailable, X-ray structures have been solved for the closely related receptors PXR [25] and VDR [26]. VDR was chosen as a template for homology modeling for hCAR because a complete alignment of sequences was observed for the LBD of hCAR1 for 225 amino acid residues (as opposed to 192 for PXR). Sequence alignment between hVDR and hCAR1 revealed two gap insertions in the loop between the second and third b-strands. The overall RMSD for the 249 Ca atoms is . Visual inspection also indicated a major devi0.26 A ation between the second and third b-strands. Fig. 7 depicts the effect of the two insertions in hCAR2 (when compared to hCAR1). Both these insertions project onto the surface of the protein model. Neither insertion is able to form a secondary structure (a-helix or b-sheet) based on three prediction methods (Bayes Statistics [27], Information theory [28], and neural networks [29]). The 4 amino acids (SPTV) that are inserted amino-terminal to helix 7 generate Loop 1 (yellow arrow) between helix 6 and helix 7. This Loop 1 has the potential to form intramolecular interactions with the loop between helix 1 and helix 3 (marked in
Fig. 6. DNA binding by hCAR splice variants. hCAR splice variants and hRXRa proteins were expressed by in vitro translation, and used for electrophoretic mobility shift assays with a CYP2B10 (DR-4) probe (A) or with a b-RARE (DR-5) probe (B) as described under Materials and methods. Supershifts were performed using the anti-hRXRa (D20) antibody. Equivalent amounts of hCAR1, hCAR2, and hCAR3 were used in the binding reactions as determined by [35 S]methionine labeling.
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3-dimensional shape and surface area of hCAR2 and hCAR3 are consistent with our observation of loss of their activities.
Discussion
Fig. 7. Superimposition of hCAR1 (red) and hCAR2 (green) homology model. hCAR1 was modeled using hVDR as the template. Homologous residues are depicted in red. The 4 amino acid (SPTV) insertion creates Loop 1 (yellow arrow) between helix 6 and helix 7. This loop creates potential intramolecular interaction between Gln146 and Phe238 . This Loop 1 has the potential to form intramolecular interactions with the loop between helix 1 and helix 3 (marked in purple). The 5 amino acids (PAPYL) that are inserted carboxy-terminal to helix 8 generate Loop 2 (yellow arrow) between helix 8 and helix 9. This loop has the potential to interact with the DBD of hCAR2. These two extra loops 2 of additional surface area on hCAR2. create about 850 A
purple). The 5 amino acids (PAPYL) that are inserted carboxy-terminal to helix 8 generate Loop 2 (yellow arrow) between helix 8 and helix 9. This loop has the potential to interact with the DBD of hCAR2. These 2 of additional surtwo extra loops create about 850A face area on hCAR2 (the surface area of hCAR1 is 2 , and that of hCAR2 is 14040A 2 ). Molecular 13193A superimposition of hCAR2 and hPPARc/hRXRa heterodimer indicates that the two loop insertions are on the dimerization surface of the LBD. The complete loss of exon 7 spanning helices 7 and 8 in hCAR3 would likely lead to structural instability within the LBD. These drastic changes in the overall
Most human genes express more than one mRNA, either by alternative promoter usage or by alternative splicing. This permits the cell to generate functionally diverse protein isoforms that can be expressed according to different regulatory programs. Here, we identified two novel forms of hCAR, which are generated as a result of alternative splicing of the hCAR pre-mRNA. One of these, hCAR2, contains an additional 9 amino acids that are inserted both upstream and downstream of exon 7. Of these additional 9 amino acids that are present in the LBD of hCAR2, 4 are inserted amino-terminal to helix 7, and 5 are carboxy-terminal to helix 8. The other splice variant, hCAR3, lacks 39 amino acids as a result of removal of exon 7, with the deletion spanning helices 7 and 8. In addition to the splice variants that we observe, Auerbach et al. [30], indicated that two additional variants containing either the 4 amino acid insertion or the 5 amino acid insertion were expressed in liver as determine by RT-PCR. Our preliminary RT-PCR experiments did not detect these variants in the liver, which is consistent with our observation of only hCAR1, -2, and -3 being identified in our screen for novel variants of hCAR. Thus, based on our primer design the extensive TaqMan PCR analysis would not have detected these variants. Given that we detected distinct tissue expression patterns of hCAR1–3, it is possible that the two additional variants identified by Auerbach et al. [30] may also be significantly expressed in some tissues. With hCAR2 and hCAR3 contributing 6–8% of total hCAR mRNA in the liver, and sometimes more in other tissues (e.g., lung), the contribution of the additional variants may provide for an even greater contribution of the splice variants to total hCAR mRNA. In contrast to the previously reported isoform of mCAR (mCAR2) that lacked AF-2 [10], both hCAR2 and hCAR3 contain an intact AF-2 domain. This conserved AF-2 motif present in most nuclear receptors [31,32] has been directly associated with ligand-dependent transactivation in these receptors [33,34]. Since this AF-2 motif is involved not only in ligand-dependent, but also ligand-independent transactivation, we might expect the hCAR isoforms to exhibit similar constitutive activities. Surprisingly, our results indicate even though the two novel splice variants contain an intact AF-2 domain, they are unable to effectively transactivate. Furthermore, it is also interesting that although these variants have identical DBDs, both hCAR2 and hCAR3 are unable to heterodimerize with RXRa thus incapacitating their ability to bind to DNA. The lack of transactivation
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activity of hCAR2 and hCAR3, could potentially be attributed to lack of DNA binding activity of the fulllength receptor; however, we have also observed that the Gal4–LBD fusions of hCAR2 and hCAR3, which do not require RXR heterodimerization for DNA binding, are also unable to transactivate and are not coactivated by additional GRIP1. In the case of hCAR3, with its large deletion of helices 7 and 8 and predicted instability of structure, we were not surprised by these results. What was surprising is that the very short insertions between helices 6 and 7 and helices 8 and 9 in hCAR2 also result in the inability of the LBD to activate transcription. This is particularly intriguing since the coactivator binding surface does not appear to be directly affected. Indeed, our predicted models of the hCAR splice variants indicate that the 9 amino acid insertions in hCAR2 increase surface area, and generate two additional loops that can potentially form intramolecular interactions with the DBD. Furthermore, with the loop insertions localized to the dimerization surface of the LBD, we expect that the heterodimerization properties of CAR might be altered, which is consistent with our observation of the loss of RXR dimerization activity of hCAR2. hCAR3, on the other hand, completely lacks exon 7. This loss of 39 amino acids (relative to hCAR1) spanning helices 7 and 8, would likely undermine the structural integrity of the LBD even though we did not detect instability of the expressed protein (data not shown). The distribution of the hCAR splice variants reveals differences in their tissue expression patterns. hCAR1, the functionally active and abundant splice variant, are predominantly expressed in the liver, consistent with its role as a xenobiotic receptor. Of the two novel splice variants of hCAR, hCAR2 is predominantly expressed in the lung, whereas hCAR3 is expressed in most tissues at low levels. Tissue-specific differential splicing of the hCAR pre-mRNA implies the existence of one or more cis- and/or trans- acting factor(s) that regulate posttranscriptional modification of the pre-mRNA in different cell types [35]. The precise physiological significance of the hCAR splice variants is unknown, but the specificity in terms of the expression pattern suggests that there may be some degree of regulation. The fact that we cannot detect activity of these splice variants makes it very difficult to speculate on their role in xenobiotic metabolism, but it is possible that one mechanism to regulate hCAR responsiveness to xenobiotics may be to regulate the fraction of inactive hCAR2 and hCAR3 relative to total hCAR. In addition, given the well-characterized individual phenotypic responses to various xenobiotics in humans, it may be possible that there are variable levels of these splice variants mediating the distinctions in responsiveness. hCAR can also be activated through a translocation pathway independent of direct binding of the xenobiotic to the LBD and it is
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possible that the novel splice variant may be differentially active in this pathway. Although we would predict that delivery of hCAR2 or -3 through this pathway would unlikely yield a transcriptionally active receptor, the translocation pathway has been shown to be mediated by phosphorylation and the effect of post-translational modifications on these two variants has yet to be examined.
References [1] S.A. Kliewer, T.M. Willson, Regulation of xenobiotic and bile acid metabolism by the nuclear pregnane X receptor, J. Lipid Res. 43 (2002) 359–364. [2] S.A. Kliewer, B. Goodwin, T.M. Willson, The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism, Endocr. Rev. 23 (2002) 687–702. [3] H.R. Kast, B. Goodwin, P.T. Tarr, S.A. Jones, A.M. Anisfeld, C.M. Stoltz, P. Tontonoz, S. Kliewer, T.M. Willson, P.A. Edwards, Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor, J. Biol. Chem. 277 (2002) 2908–2915. [4] J. Sugatani, H. Kojima, A. Ueda, S. Kakizaki, K. Yoshinari, Q.H. Gong, I.S. Owens, M. Negishi, T. Sueyoshi, The phenobarbital response enhancer module in the human bilirubin UDP-glucuronosyltransferase UGT1A1 gene and regulation by the nuclear receptor CAR, Hepatology 33 (2001) 1232–1238. [5] J.M. Maglich, C.M. Stoltz, B. Goodwin, D. Hawkins-Brown, J.T. Moore, S.A. Kliewer, Nuclear pregnane X receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification, Mol. Pharmacol. 62 (2002) 638–646. [6] J.M. Lehmann, D.D. McKee, M.A. Watson, T.M. Willson, J.T. Moore, S.A. Kliewer, The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions, J. Clin. Invest. 102 (1998) 1016–1023. [7] A unified nomenclature system for the nuclear receptor superfamily. Cell 97 (1999) 161–163. [8] J.M. Maglich, A. Sluder, X. Guan, Y. Shi, D.D. McKee, K. Carrick, K. Kamdar, T.M. Willson, J.T. Moore, Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes, Genome Biol. 2 (2001) 1–7. [9] M. Baes, T. Gulick, H.S. Choi, M.G. Martinoli, D. Simha, D.D. Moore, A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements, Mol. Cell. Biol. 14 (1994) 1544–1551. [10] H.S. Choi, M. Chung, I. Tzameli, D. Simha, Y.K. Lee, W. Seol, D.D. Moore, Differential transactivation by two isoforms of the orphan nuclear hormone receptor CAR, J. Biol. Chem. 272 (1997) 23565–23571. [11] P. Honkakoski, I. Zelko, T. Sueyoshi, M. Negishi, The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene, Mol. Cell. Biol. 18 (1998) 5652–5658. [12] T. Kawamoto, T. Sueyoshi, I. Zelko, R. Moore, K. Washburn, M. Negishi, Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene, Mol. Cell. Biol. 19 (1999) 6318–6322. [13] I. Zelko, T. Sueyoshi, T. Kawamoto, R. Moore, M. Negishi, The peptide near the C terminus regulates receptor CAR nuclear translocation induced by xenochemicals in mouse liver, Mol. Cell. Biol. 21 (2001) 2838–2846.
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[14] L.B. Moore, J.M. Maglich, D.D. McKee, B. Wisely, T.M. Willson, S.A. Kliewer, M.H. Lambert, J.T. Moore, Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and benzoate X receptor (BXR) define three pharmacologically distinct classes of nuclear receptors, Mol. Endocrinol. 16 (2002) 977–986. [15] I. Tzameli, P. Pissios, E.G. Schuetz, D.D. Moore, The xenobiotic compound 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is an agonist ligand for the nuclear receptor CAR, Mol. Cell. Biol. 20 (2000) 2951–2958. [16] B.M. Forman, I. Tzameli, H.S. Choi, J. Chen, D. Simha, W. Seol, R.M. Evans, D.D. Moore, Androstane metabolites bind to and deactivate the nuclear receptor CAR-beta, Nature 395 (1998) 612– 615. [17] G. Min, J.K. Kemper, B. Kemper, Glucocorticoid receptorinteracting protein 1 mediates ligand-independent nuclear translocation and activation of constitutive androstane receptor in vivo, J. Biol. Chem. 277 (2002) 26356–26363. [18] J.M. Maglich, D.J. Parks, L.B. Moore, J.L. Collins, B. Goodwin, A.N. Billin, C.A. Stoltz, S.A. Kliewer, M.H. Lambert, T.M. Willson, J.T. Moore, Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes, J. Biol. Chem. 278 (2003) 17277–17283. [19] L.B. Moore, D.J. Parks, S.A. Jones, R.K. Bledsoe, T.G. Consler, J.B. Stimmel, B. Goodwin, C. Liddle, S.G. Blanchard, T.M. Willson, J.L. Collins, S.A. Kliewer, Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands, J. Biol. Chem. 275 (2000) 15122– 15127. [20] E.S. Lander, L.M. Linton, B. Birren, et al., Initial sequencing and analysis of the human genome, Nature 409 (2001) 860–921. [21] B. Modrek, C. Lee, A genomic view of alternative splicing, Nat. Genet. 30 (2002) 13–19. [22] Y.F. Wu, P. Delerive, W.W. Chin, T.P. Burris, Requirement of helix 1 and the AF-2 domain of the thyroid hormone receptor for coactivation by PGC-1, J. Biol. Chem. 277 (2002) 8898–8905. [23] A. Sali, T.L. Blundell, Comparative protein modelling by satisfaction of spatial restraints, J. Mol. Biol. 234 (1993) 779–815. [24] W. Huang, J. Zhang, S.S. Chua, M. Qatanani, Y. Han, R. Granata, D.D. Moore, Induction of bilirubin clearance by the
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Alternative splicing within the ligand binding domain of the human constitutive androstane receptorq Rajesh S. Savkur, Yifei Wu, Kelli S. Bramlett, Minmin Wang, Sufang Yao, Douglas Perkins, Michelle Totten, George Searfoss III, Timothy P. Ryan, Eric W. Su, and Thomas P. Burris* Gene Regulation Research, Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, IN 46285, USA Received 1 July 2003; accepted 14 August 2003
Abstract The human constitutive androstane receptor (hCAR; NR1I3) is a member of the nuclear receptor superfamily. The activity of hCAR is regulated by a variety of xenobiotics including clotrimazole and acetaminophen metabolites. hCAR, in turn, regulates a number of genes responsible for xenobiotic metabolism and transport including several cytochrome P450s (CYP 2B5, 2C9, and 3A4) and the multidrug resistance-associated protein 2 (MRP2, ABCC2). Thus, hCAR is believed to be a mediator of drug–drug interactions. We identified two novel hCAR splice variants: hCAR2 encodes a receptor in which alternative splice acceptor sites are utilized resulting in a 4 amino acid insert between exons 6 and 7, and a 5 amino acid insert between 7 and 8, and hCAR3 encodes a receptor with exon 7 completely deleted resulting in a 39 amino acid deletion. Both hCAR2 and hCAR3 mRNAs are expressed in a pattern similar to the initially described MB67 (hCAR1) with some key distinctions. Although the levels of expression vary depending on the tissue examined, hCAR2 and hCAR3 contribute 6–8% of total hCAR mRNA in liver. Analysis of the activity of these variants indicates that both hCAR2 and hCAR3 lose the ability to heterodimerize with RXR and lack transactivation activity in cotransfection experiments where either full-length receptor or GAL4 DNA-binding domain/CAR ligand binding domain chimeras were utilized. Although the role of hCAR2 and hCAR3 is currently unclear, these additional splice variants may provide for increased diversity in terms of responsiveness to xenobiotics. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Nuclear receptor; Xenobiotic; Steroid; Phenobarbital; Transcription; RXR; Clotrimazole
Introduction The superfamily of nuclear hormone receptors comprises a group of transcription factors that play significant roles in response to a number of biological regulators including steroids, retinoids, and thyroid hormones. Two of these nuclear receptors, the pregnane X receptor (PXR) [NR1I2] and the constitutive androstane receptor (CAR) [NR1I3] play a crucial role in response to xenobiotics [1,2]. This response is mediated q
The results of this manuscript were reported in abstract form at the 85th Annual Meeting of the Endocrine Society, June 19–22, 2003 (Abstract P3-266). * Corresponding author. Fax: 1-317-276-1414. E-mail address: [email protected] (T.P. Burris). 1096-7192/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2003.08.013
by these two nuclear hormone receptors either being directly or indirectly activated by xenobiotic chemicals leading to induction of expression of a series of genes involved in detoxification of these substances. The target genes for these xenobiotic nuclear receptors include a number of cytochrome P450 (CYP) monooxygenases as well as phase II xenobiotic-metabolizing enzymes and ATP binding cassette transporters associated with xenobiotic transport [3–5]. Activation of either PXR or CAR by pharmaceutical agents has also been shown to mediate drug–drug interactions resulting in potential difficulties in pharmacotherapy [6]. CAR and PXR are related members of the NR1I subfamily of nuclear hormone receptors [7] with 40% amino acid identity within their ligand binding domains (LBDs) [8]. Both human CAR (hCAR) and mouse CAR (mCAR) are
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most abundantly expressed in the liver, and are constitutively active in cell-based reporter assays in the absence of exogenously added ligands [9,10]. CAR has been implicated as the mediator of phenobarbital (PB) induction of the CYP2B genes based on the observation that CAR binds to the CYP2B PB-responsive enhancer, termed the PBRU or PB-responsive module [11]. Interestingly, at least a subset of the xenobiotics that activate CAR, including PB, appear to do so by causing translocation of the receptor from the cytoplasm to the nucleus without directly binding to CAR itself [12,13]. However, a number of xenobiotic activators of CAR do function through the more classical method of direct binding to the LBD [14]. It has also been demonstrated that CAR heterodimerizes with the retinoid X receptor (RXR) to bind to a wide variety of DNA response elements, and activates the transcription of target genes containing these elements in the absence of ligands [15]. It has been suggested that the LBD of CAR adopts a conformation where it binds to coactivators such as SRC-1 [16] or TIF2/GRIP1 [17] in the absence of ligands to exhibit its constitutive activity. However, this constitutive activity can be inhibited by inverse agonists such as androstanol, androstenol or clotrimazole as well as enhanced by agonists such as CITCO [16,18,19]. Alternative splicing generates segments of mRNA variability that can insert or delete amino acids, shift the reading frame, or introduce premature termination codons. Up to 59% of the human genes generate multiple mRNAs by alternative splicing [20], and 80% of alternative splicing results in changes in the encoded protein [21] revealing that it is likely to be the primary source of human proteomic diversity. A large fraction of alternative splicing events are regulated in a cell- or tissue-specific manner in which the splicing pathways are modulated according to cell-type, developmental stage, gender, or in response to external stimuli. Choi et al. [10], identified two isoforms of mCAR that arise as a result of alternative splicing of the single gene. The two splice variants mCAR1 and mCAR2 that are encoded from the single mCAR gene, contain identical DNA binding domains (DBD), but differ in the LBD. mCAR2 is truncated, lacking the Cterminal region of the conserved LBD that includes the AF-2 domain. This deletion results in both loss of transactivation activity (lack of AF2) and DNA binding activity due to deficits in the ability of mCAR2 to heterodimerize with RXR. Here we describe the identification and initial characterization of two novel splice variants of hCAR. These two novel isoforms (designated hCAR2 and hCAR3) differ from the previously identified MB67-hCAR (designated hCAR1) in their LBDs, but are identical in the DBD. These novel isoforms constitute 10% of the total hCAR mRNA, and display a differential tissue expres-
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sion profile relative to hCAR1. In contrast to the previously reported mCAR2, both hCAR2 and hCAR3 contain an intact AF-2. However, they are unable to bind DNA and transactivate target genes in cell-based reporter assays. Molecular modeling of hCAR2 and hCAR3 indicates that an alteration in the overall 3-dimensional shape and surface area of these splice variants is the most probable cause for the loss of their functional activity. Differences in the tissue expression, and function of the hCAR isoforms could reveal critical information for our understanding of xenobiotic metabolism as well as the basis for phenotypic variations in drug– drug interaction responses.
Materials and methods Cloning and identification of hCAR splice variants Human liver cDNA was subjected to PCR amplification and subcloned into pCR-II-Topo (Invitrogen, Carlsbad, CA). All clones were confirmed by sequencing. Quantification of hCAR splice variants Quantitative analysis of hCAR splice variants was performed using TaqMan QPCR (Applied Biosystems, Foster City, CA). For detection of hCAR1, forward and reverse primers spanned exon 6/exon 7 and exon 7/exon 8 junctions, respectively, with the TaqMan probe hybridizing to exon 7. To detect hCAR2, the forward and reverse primers were located at the exon 6/7 and exon 7/ 8 junctions, respectively, and contained the additional sequences specific for hCAR2. The TaqMan probe hybridized to exon 7. To detect hCAR3, the forward and reverse primers were located within exons 6 and 8, respectively. The TaqMan probe hybridized to exon 6/ exon 8 junction that is present only in hCAR3. Copy numbers for each of the splice variant transcripts were obtained by comparing sample counts to a 6-point standard curve of plasmid DNA with the specific primer set included in the same TaqMan plate. Electrophoretic mobility shift assays hCAR splice variants and hRXRa were expressed using coupled in vitro transcription and translation (Promega, Madison, WI) as recommended by the manufacturer. Gel-shift assays were performed using the [a-32 P]dCTP labeled DR-4 element from the CYP2B10 gene or the [a-32 P]dCTP labeled DR-5 element from the b-RARE promoter as previously described [15]. Super-shift assays were performed using the anti-hRXRa D-20 antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
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Plasmids Plasmid pG5-Luc containing five copies of the DNAbinding site of the minimal thymidine kinase (TK) promoter linked to a luciferase gene was purchased from Promega. The 5xDR-4–tk-Luc reporter, containing five copies of the DR-4 response element from the CYP2B10 gene [15], was generated by inserting the double-stranded oligonucleotide response element upstream of the minimal TK promoter linked to a luciferase gene (pTALuc, Clontech). Full-length expression vectors of hCAR splice variants and hRXRa were generated by PCR amplification of the respective cDNAs and subcloning into pcDNA3.1DVH vector (Invitrogen). Chimeras expressing the Gal4 DNA binding domain (DBD) fused to CAR LBDs were created utilizing the commercially available mammalian two-hybrid vector pM from Clontech. The pM-hCAR1LBD contains amino acids 77–348, pM-hCAR2LBD contains amino acids 77–357, and pM-hCAR3LBD contains amino acids 77–309. In each case the LBD fragments were generated by PCR from the full length cDNA and subcloned in frame downstream of the Gal 4 DBD into the pM vector. The expression vector for TIF2/GRIP1 has been previously described [22]. Cell culture and transient transfection assays CV1 cells were propagated in DulbeccoÕs modified EagleÕs/F12 (3:1) media without phenol red supplemented with 10% (v/v) dextran charcoal treated fetal bovine serum (Hyclone Laboratories, Logan, UT) and 2 mM glutamine at 37 °C under 5% CO2 . Prior to transfections, CV1 cells were plated in 96-well plates at a density of 12,000 cells/well. Transfection mixes contained 100 ng of reporter plasmids and 50 ng of receptor plasmids using FuGENE 6 (Roche Molecular Biochemicals). Forty-eight hour post-transfection, cells were harvested with Steady-Glo lysis buffer (Promega). Luciferase activity was measured in a Dynex luminometer. All experiments were done in triplicate, and unless otherwise indicated the data are displayed as the means SE of a single experiment representative of at least three independent experiments. Homology modeling of hCAR The 3-dimensional homology models for the LBD of hCAR1 and hCAR2 were constructed by using the automated comparative homology modeling program, Modeler [23], as implemented in InsightII2000 (Accelrys, San Diego, 2000). Unlike the conventional homology modeling scheme with time consuming stages of core region identification and loop region building, Modeler uses unique probability density functions (PDFs) to transfer the spatial restraints from the
template to the target proteins. Individual restraints are assembled into a single molecular PDF (MPDF) and optimized to give 3-dimensional coordinates for the target protein. The sequence alignment was generated by PSI-BLASTing of protein data bank (PDB) using the hCAR-LBD sequence as the query. The two best hits were PXR (1ilh) and VDR (1ie9) with the identity score of 49% and 37%, respectively. VDR was chosen as the template for the homology modeling because the sequence is fully aligned with the LBD of hCAR1 for 225 residues (as opposed to 192 aligned residues with PXR). Segments of overhanging sequence on hCARLBD were cut during the model building process. Two models were built with a high level of optimization for the conserved regions as well as a high level of optimization in the loop regions. None of the hydrogen atoms are built in the homology model. The model with the least )ln(PDF) value was selected as the final model and validated with Protein Health check in Quanta98 (Accelyrs, San Diego, 1999). This ensured that undesirable conformations were created on the backbones and sidechains in the ligand-binding pocket. The protein surface area was calculated using MOLCAD in Sybyl68 (Tripos, St. Louis, MO). The secondary structure prediction for the loop insertions on hCAR2 was generated by BIOPOLYMER module in Sybyl68 (Tripos, St. Louis, MO).
Results Isolation and characterization of the hCAR splice variants Previous studies identified two isoforms of the murine CAR (mCAR) that arise as a result of alternative splicing of the mCAR pre-mRNA [10]. In an attempt to obtain MB67 (hCAR), we obtained two full-length Genestorm cDNAs from Invitrogen, and interestingly both cDNAs appeared to be alternatively spliced variants of the initially described MB67 [9]. In order to obtain the initially described hCAR, we screened a human liver cDNA library by PCR amplification, and subcloned the products into pCR-II-Topo vector. Sequencing of several independent clones did not reveal any additional clones beyond these three characterized cDNAs. The three hCAR cDNAs, which we designated hCAR1 [9], hCAR2, and hCAR3 (Figs. 1A and B), are identical in the DBD and hinge regions, but differ in their LBDs. hCAR1, encoded by 9 exons is 348 amino acids in length, with a predicted molecular weight of 39.66 kDa. hCAR2 contains an additional 9 amino acids (with respect to hCAR1) in the LBD, and encodes a protein with a predicted molecular weight of 40.59 kDa, whereas hCAR3 lacks 39 amino acids in the LBD (relative to hCAR1), and has a predicted mass of 35.15 kDa. The amino acid sequence alignment of the
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Fig. 1. (A) Structure of human CAR gene. The 348 amino acid long wild-type human CAR (hCAR1) encoded by 9 exons comprised of a DNA binding domain (DBD), a hinge region, and a ligand binding domain (LBD). (B) Domain structural analysis of hCAR splice variants. hCAR2 contains an additional 9 amino acids in the LBD (depicted by white strips) whereas hCAR3 lacks 39 amino acids (relative to hCAR1) in the LBD. The hCAR splice variants are identical in the DBD and the hinge regions. (C) amino acid sequence alignment of the LBD of the hCAR splice variants. The 9 additional amino acids included in hCAR2 are highlighted. The bold dashes represent the 39 amino acids that are deleted in hCAR3. The numbers denote the positions of the amino acids in the hCAR proteins.
LBD of the three hCAR splice variants (Fig. 1C) depicts the positions of the additional 9 amino acids present in hCAR2 and the 39 amino acid deletion in hCAR3. To verify the authenticity of the hCAR splice variants, the exon–intron boundaries of exons 6 through 8 were analyzed (Fig. 2A). The nucleotide sequence alignment of the hCAR splice variants indicated that hCAR2 is derived from the use of the alternative splice donor/acceptor sites with insertions both upstream and downstream of exon 7. Interestingly, exon 7 is completely skipped to generate hCAR3. A comparison of the LBDs of the hCAR splice variants to the related members of the nuclear receptor superfamily (VDR and PXR) (Fig. 2B) indicated that of the additional 9 amino acids in the LBD of hCAR2, four (SPTV) are inserted amino-terminal to helix 7, and five (PAPYL) are carboxy-terminal to helix 8. The 39 amino acids that are deleted in the LBD of hCAR3 (relative to hCAR1) span helices 7 and 8. Expression profile analysis of hCAR splice variants To quantitatively differentiate between the hCAR splice variants, we used a TaqMan PCR based approach by designing probe sets that would selectively amplify each of the hCAR variants (see Materials and methods). Previous results have reported MB67 expression to be
predominant in the liver with low levels of expression in the heart and muscle followed by the kidneys and lung [9]. Consistent with these observations, the tissue expression profile revealed that hCAR1 is most abundantly expressed in the liver, followed by the ovary, lung, kidneys, and adrenal glands (Fig. 3A). In agreement with previous results [24], we also observed that the expression of hCAR1 dramatically increases in the adult liver compared to the fetal. The tissue expression profile of hCAR2 (Fig. 3B) also indicates that this splice variant is predominantly expressed in the kidney, lung, adrenal glands, and the liver. Interestingly, in contrast to hCAR1, the expression of hCAR2 is more abundant in the lung compared to the liver. The tissue expression profile of hCAR3 (Fig. 3C) reveals that this isoform is expressed at very low levels in most tissues, with the kidneys, small intestine, adrenal glands, and liver showing relatively increased expression. Since CAR mediates the hepatic effects of xenobiotics, the relative expression of the hCAR splice variants in the liver was determined using TaqMan QPCR analysis. As seen in Fig. 4, in all the three human liver samples tested, hCAR1 is the most abundantly expressed isoform. The other two splice variants, hCAR2 and hCAR3, comprise 6–8% of the total hCAR isoforms in the liver.
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Fig. 2. (A) Exon–intron boundaries for exons 6, 7, and 8 of hCAR. The exon–intron boundaries of hCAR1, hCAR2, and hCAR3 are depicted. The amino acid sequences for the respective receptors are indicated in bold. Alternative splice donor/acceptor sites (underlined nucleotide sequences) are utilized to derive hCAR2 with insertions both upstream and downstream of exon 7. Exon 7 is completely deleted in hCAR3. (B) Amino acid sequence comparison of the LBDs of the hCAR splice variants to related members of the nuclear receptor superfamily—VDR and PXR. Of the additional 9 amino acids in the LBD of hCAR2, 4 (SPTV) are inserted in helix 7, and 5 (PAPYL) are present in helix 8. The 39 amino acid deletion in the LBD of hCAR3 spans helices 7 and 8.
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Fig. 3. (A) Tissue expression profile of hCAR1. Origene Rapid Scan human tissue panel cDNAs were pre-normalized to equivalent levels of b-actin transcripts. Copy numbers were derived by comparing to a 6-point dilution standard curve of plasmid DNA containing hCAR1 included on the same TaqMan plate. hCAR1 is predominately expressed in the lung, ovary, adrenal glands, and liver. Expression of hCAR1 dramatically increases in adult liver compared to fetal. (B) Tissue expression profile of hCAR2. TaqMan QPCR analysis for hCAR2 was performed essentially as described for hCAR1. hCAR2 is highly expressed in the kidney, liver, adrenals and lung. Interestingly, in contrast to hCAR1, hCAR2 is abundantly expressed in the lung compared to the liver. (C) Tissue expression profile of hCAR3. TaqMan QPCR analysis for hCAR3 was performed as described above for hCAR1. hCAR3 is ubiquitously expressed in all tissues, with the kidneys, adrenals, small intestine, and liver showing relatively increased expression. Note the differences in the scales of the y-axis.
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Fig. 4. Relative expression of the hCAR splice variants in human liver. cDNA synthesized from 100 ng of total RNA isolated from three independent human liver samples were subjected to TaqMan QPCR. The copy numbers were obtained by comparing sample counts to a 6-point standard curve for each primer/probe set included in the same plate. In all three individual liver mRNAs, hCAR2, and hCAR3 comprise 6–8% of the total hCAR splice variants.
Transactivation and DNA binding of hCAR splice variants Previous studies have shown hCAR1 to exhibit a high constitutive activity in the absence of any exogenously added ligands [9]. To determine whether variations in
the LBD of hCAR, would affect this constitutive transactivation, the LBDs of the hCAR splice variants were fused to the yeast Gal4 DBD to create the chimeric fusion constructs, and tested in transient transfection assays. As seen in Fig. 5A, the Gal4–hCAR1-LBD is constitutively active in the absence of any added ligands. This apparently heterologous constitutive transactivation was observed in the presence of serum treated with charcoal to remove potential ligands. However, no significant transactivation was observed with hCAR2 and hCAR3. It has previously been shown that TIF2/GRIP1 is a potential coactivator of CAR, where it interacts with, and enhances the CAR-mediated transactivation [17]. To check whether GRIP1 is capable of transactivating the hCAR splice variants, an expression plasmid for GRIP1 was co-transfected with the Gal4–hCAR-LBD fusion plasmids and the reporter in CV1 cells. As seen in Fig. 5B, in agreement with previous results, GRIP1 dramatically enhances the transactivation of hCAR1. However, no increase in activation was observed with both hCAR2 and hCAR3. This suggests that variations in the LBD likely alter the structure of the LBD, and abolish the interaction of hCAR2 and hCAR3 with this coactivator. To determine whether the full-length hCAR splice variants are capable of transactivation, plasmids expressing full-length hCAR splice variants were co-transfected with a luciferase reporter plasmid in which 5 copies of the CYP2B10 (DR4) element were inserted upstream of the TK promoter. As seen in Fig. 5C, hCAR1 exhibited a twofold constitutive activity, consistent with the
Fig. 5. Transactivation by the hCAR splice variants. (A) CV-1 cells were co-transfected with the plasmids expressing the Gal4 fusion constructs with the LBDs of the hCAR splice variants or with the empty vector (vec) and pG5Luc reporter plasmid. Forty-eight hour post-transfection, cells were lysed and the luciferase activity was measured as described under Materials and methods. All experiments were done in triplicate, and the data are displayed as the means SE of a single experiment representative of three independent experiments. (B) CV-1 cells were co-transfected with the plasmids expressing the Gal4 fusion constructs with the LBDs of the hCAR splice variants and pG5Luc reporter in the presence or absence of GRIP1. Forty-eight hour post-transfection, cells were lysed and the luciferase activity was measured as described in section (A). All experiments were done in triplicate, and the data are displayed as the means SE of a single experiment representative of three independent experiments. (C). CV-1 cells were transfected with plasmids expressing the full-length hCAR splice variants or the empty vector (vec) and the 5xDR-4–tk-Luc reporter vector (see Materials and methods). Forty-eight hour post-transfection, cells were lysed and the luciferase activity was measured as described in section (A). For (C) data are reported as the combined means SE of three independent experiments.
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effect that has been reported with this element using mCAR [15]. However, hCAR2 and hCAR3 show a significant reduction in their ability to transactivate this element compared to hCAR1. To understand the basis for the reduced transactivation of hCAR2 and hCAR3 on the CYP2B10 (DR4) element, the ability of these proteins to bind the DR4 element was assessed using in vitro translated hCAR splice variants and assayed by gel-shift analysis. Consistent with previous observations, hCAR1 alone or hRXRa alone did not bind the DR4 element (Fig. 6A). However the hCAR1/hRXRa heterodimer bound to this element with a high affinity [15]. The mobility of this complex was further retarded by the addition of the anti-hRXRa antibody (D20), confirming the presence of hRXRa in the heterodimer complex. This hCAR1/ hRXRa heterodimer also bound with a high affinity to the DR5 response element of the bRARE [9] (Fig. 6B), and was also super-shifted by the addition of the antihRXRa antibody. In addition, longer exposure times also revealed that hCAR1 alone bound the DR5 element, although this interaction was much weaker than the hCAR1/RXR heterodimer (data not shown). However, neither hCAR2 nor hCAR3 bound the CYP2B10 (DR4) or the bRARE (DR5) elements, either alone or as a heterodimer with RXR. This suggested that despite the fact that the hCAR isoforms contain identical DBDs, variations in the LBD of hCAR2 and hCAR3 were adversely affecting the structure of these isoforms,
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and abolishing their ability to bind response elements and also reducing their ability to transactivate genes containing the elements. Model of LBD of hCAR splice variants Although the X-ray structure for hCAR1 is unavailable, X-ray structures have been solved for the closely related receptors PXR [25] and VDR [26]. VDR was chosen as a template for homology modeling for hCAR because a complete alignment of sequences was observed for the LBD of hCAR1 for 225 amino acid residues (as opposed to 192 for PXR). Sequence alignment between hVDR and hCAR1 revealed two gap insertions in the loop between the second and third b-strands. The overall RMSD for the 249 Ca atoms is . Visual inspection also indicated a major devi0.26 A ation between the second and third b-strands. Fig. 7 depicts the effect of the two insertions in hCAR2 (when compared to hCAR1). Both these insertions project onto the surface of the protein model. Neither insertion is able to form a secondary structure (a-helix or b-sheet) based on three prediction methods (Bayes Statistics [27], Information theory [28], and neural networks [29]). The 4 amino acids (SPTV) that are inserted amino-terminal to helix 7 generate Loop 1 (yellow arrow) between helix 6 and helix 7. This Loop 1 has the potential to form intramolecular interactions with the loop between helix 1 and helix 3 (marked in
Fig. 6. DNA binding by hCAR splice variants. hCAR splice variants and hRXRa proteins were expressed by in vitro translation, and used for electrophoretic mobility shift assays with a CYP2B10 (DR-4) probe (A) or with a b-RARE (DR-5) probe (B) as described under Materials and methods. Supershifts were performed using the anti-hRXRa (D20) antibody. Equivalent amounts of hCAR1, hCAR2, and hCAR3 were used in the binding reactions as determined by [35 S]methionine labeling.
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3-dimensional shape and surface area of hCAR2 and hCAR3 are consistent with our observation of loss of their activities.
Discussion
Fig. 7. Superimposition of hCAR1 (red) and hCAR2 (green) homology model. hCAR1 was modeled using hVDR as the template. Homologous residues are depicted in red. The 4 amino acid (SPTV) insertion creates Loop 1 (yellow arrow) between helix 6 and helix 7. This loop creates potential intramolecular interaction between Gln146 and Phe238 . This Loop 1 has the potential to form intramolecular interactions with the loop between helix 1 and helix 3 (marked in purple). The 5 amino acids (PAPYL) that are inserted carboxy-terminal to helix 8 generate Loop 2 (yellow arrow) between helix 8 and helix 9. This loop has the potential to interact with the DBD of hCAR2. These two extra loops 2 of additional surface area on hCAR2. create about 850 A
purple). The 5 amino acids (PAPYL) that are inserted carboxy-terminal to helix 8 generate Loop 2 (yellow arrow) between helix 8 and helix 9. This loop has the potential to interact with the DBD of hCAR2. These 2 of additional surtwo extra loops create about 850A face area on hCAR2 (the surface area of hCAR1 is 2 , and that of hCAR2 is 14040A 2 ). Molecular 13193A superimposition of hCAR2 and hPPARc/hRXRa heterodimer indicates that the two loop insertions are on the dimerization surface of the LBD. The complete loss of exon 7 spanning helices 7 and 8 in hCAR3 would likely lead to structural instability within the LBD. These drastic changes in the overall
Most human genes express more than one mRNA, either by alternative promoter usage or by alternative splicing. This permits the cell to generate functionally diverse protein isoforms that can be expressed according to different regulatory programs. Here, we identified two novel forms of hCAR, which are generated as a result of alternative splicing of the hCAR pre-mRNA. One of these, hCAR2, contains an additional 9 amino acids that are inserted both upstream and downstream of exon 7. Of these additional 9 amino acids that are present in the LBD of hCAR2, 4 are inserted amino-terminal to helix 7, and 5 are carboxy-terminal to helix 8. The other splice variant, hCAR3, lacks 39 amino acids as a result of removal of exon 7, with the deletion spanning helices 7 and 8. In addition to the splice variants that we observe, Auerbach et al. [30], indicated that two additional variants containing either the 4 amino acid insertion or the 5 amino acid insertion were expressed in liver as determine by RT-PCR. Our preliminary RT-PCR experiments did not detect these variants in the liver, which is consistent with our observation of only hCAR1, -2, and -3 being identified in our screen for novel variants of hCAR. Thus, based on our primer design the extensive TaqMan PCR analysis would not have detected these variants. Given that we detected distinct tissue expression patterns of hCAR1–3, it is possible that the two additional variants identified by Auerbach et al. [30] may also be significantly expressed in some tissues. With hCAR2 and hCAR3 contributing 6–8% of total hCAR mRNA in the liver, and sometimes more in other tissues (e.g., lung), the contribution of the additional variants may provide for an even greater contribution of the splice variants to total hCAR mRNA. In contrast to the previously reported isoform of mCAR (mCAR2) that lacked AF-2 [10], both hCAR2 and hCAR3 contain an intact AF-2 domain. This conserved AF-2 motif present in most nuclear receptors [31,32] has been directly associated with ligand-dependent transactivation in these receptors [33,34]. Since this AF-2 motif is involved not only in ligand-dependent, but also ligand-independent transactivation, we might expect the hCAR isoforms to exhibit similar constitutive activities. Surprisingly, our results indicate even though the two novel splice variants contain an intact AF-2 domain, they are unable to effectively transactivate. Furthermore, it is also interesting that although these variants have identical DBDs, both hCAR2 and hCAR3 are unable to heterodimerize with RXRa thus incapacitating their ability to bind to DNA. The lack of transactivation
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activity of hCAR2 and hCAR3, could potentially be attributed to lack of DNA binding activity of the fulllength receptor; however, we have also observed that the Gal4–LBD fusions of hCAR2 and hCAR3, which do not require RXR heterodimerization for DNA binding, are also unable to transactivate and are not coactivated by additional GRIP1. In the case of hCAR3, with its large deletion of helices 7 and 8 and predicted instability of structure, we were not surprised by these results. What was surprising is that the very short insertions between helices 6 and 7 and helices 8 and 9 in hCAR2 also result in the inability of the LBD to activate transcription. This is particularly intriguing since the coactivator binding surface does not appear to be directly affected. Indeed, our predicted models of the hCAR splice variants indicate that the 9 amino acid insertions in hCAR2 increase surface area, and generate two additional loops that can potentially form intramolecular interactions with the DBD. Furthermore, with the loop insertions localized to the dimerization surface of the LBD, we expect that the heterodimerization properties of CAR might be altered, which is consistent with our observation of the loss of RXR dimerization activity of hCAR2. hCAR3, on the other hand, completely lacks exon 7. This loss of 39 amino acids (relative to hCAR1) spanning helices 7 and 8, would likely undermine the structural integrity of the LBD even though we did not detect instability of the expressed protein (data not shown). The distribution of the hCAR splice variants reveals differences in their tissue expression patterns. hCAR1, the functionally active and abundant splice variant, are predominantly expressed in the liver, consistent with its role as a xenobiotic receptor. Of the two novel splice variants of hCAR, hCAR2 is predominantly expressed in the lung, whereas hCAR3 is expressed in most tissues at low levels. Tissue-specific differential splicing of the hCAR pre-mRNA implies the existence of one or more cis- and/or trans- acting factor(s) that regulate posttranscriptional modification of the pre-mRNA in different cell types [35]. The precise physiological significance of the hCAR splice variants is unknown, but the specificity in terms of the expression pattern suggests that there may be some degree of regulation. The fact that we cannot detect activity of these splice variants makes it very difficult to speculate on their role in xenobiotic metabolism, but it is possible that one mechanism to regulate hCAR responsiveness to xenobiotics may be to regulate the fraction of inactive hCAR2 and hCAR3 relative to total hCAR. In addition, given the well-characterized individual phenotypic responses to various xenobiotics in humans, it may be possible that there are variable levels of these splice variants mediating the distinctions in responsiveness. hCAR can also be activated through a translocation pathway independent of direct binding of the xenobiotic to the LBD and it is
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possible that the novel splice variant may be differentially active in this pathway. Although we would predict that delivery of hCAR2 or -3 through this pathway would unlikely yield a transcriptionally active receptor, the translocation pathway has been shown to be mediated by phosphorylation and the effect of post-translational modifications on these two variants has yet to be examined.
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