Alternative splicing affects the function and tissue-specific expression of the human constitutive androstane receptor

Abstract

BACKGROUND: The constitutive androstane receptor (CAR) plays a key role in the control of drug metabolism and transport by mediating the phenobarbital-type induction of many phase I and II drug metabolizing enzymes and drug transporters. RESULTS: We identified transcripts generated by four different alternative splicing events in the human CAR gene. Two of the corresponding ligand binding domain isoforms demonstrated novel functional properties: First, CAR(SV3), which is encoded by a transcript containing an lengthened exon 7, differentially transactivated target gene promoters. Second, CAR(SV2), which results from the use of an alternative 3' splice site lengthening exon 8, showed ligand-dependent instead of constitutive interaction with coactivators. Furthermore, alternatively spliced transcripts demonstrated a tissue-specific expression pattern. In most tissues, only transcripts generated by alternative splicing within exon 9 were expressed. The encoded variant demonstrated a loss-of-function phenotype. Correct splicing of exon 8 to exon 9 is restricted to only a few tissues, among them liver and small intestine for which CAR function has been demonstrated, and is associated with the induction of CAR expression during differentiation of intestinal cells. CONCLUSION: Due to their specific activities, CAR variant proteins SV2 and SV3 may modulate the activity of reference CAR(SV1). Furthermore, we propose that transcriptional activation and regulation of splicing of exon 9 may be coupled to ensure appropriate tissue- and differentiation state-specific expression of transcripts encoding functional CAR protein. Altogether, alternative splicing seems to be of utmost importance for the regulation of CAR expression and function.

Figure 1

Genomic organization and structure of alternatively spliced transcripts of human CAR (A) Genomic organization of the gene (top) and structure of transcripts (bottom). Exons are depicted as open boxes. Introns are shown by horizontal lines. Extension of exons 7 and 8 by use of alternative intronic 3' splice sites is depicted by black boxes. The 5' part of exon 9 which is deleted by use of an alternative 3'splice site within the exon is shown by a gray box with dotted line. Start and stop codons are indicated. TGA* indicates the stop codon of transcripts with the deletion in exon 9. Arrowheads represent the position of the indicated primers (see Table 1). Alternatively spliced transcripts are referred to as SV1 to SV12. (B) Detailed description of the alternative splicing in exon 9. An alternative 3'splice site resides within exon 9. Use of this site results in the out-of-frame deletion of the first 76 bp of the exon. 5' and 3' splice sites are shown in bold lower case letters. Exon sequences represented in the transcripts with the deletion are in upper case letters. Encoded amino acids are shown below. The asterisk denotes the new stop codon.

Figure 2

Structure and intracellular distribution of CAR isoforms (A) Schematic representation of the indicated CAR isoforms. The insertion of 4 or/and 5 additional amino acids in CAR isoforms SV2, SV3 and SV6 is depicted by black boxes. SV4 lacks the 39 amino acids encoded by exon 7. The last 42 amino acids are replaced by 7 new ones in SV5 (depicted by a light gray box). The numbers denote positions of amino acids in reference CAR(SV1). (B) Western Blot analysis of cytoplasmic and nuclear proteins of COS1 cells transiently transfected with the expression plasmids of the indicated CAR isoforms. Protein amounts were adjusted according to transfection efficiency as estimated by the activity of β-galactosidase encoded by the co-transfected expression plasmid pCMVβ. Blots were probed with a CAR-specific antibody.

Figure 3

Impaired heterodimerization and DNA binding activities of CAR isoforms (A) Mammalian two hybrid assays were performed in COS1 cells transfected with combinations of an expression plasmid encoding a GAL4-DBD/RXRα–LBD fusion protein and expression plasmids encoding VP16-AD/CAR-LBD fusion proteins of CAR isoforms, as indicated. The columns show the mean activation factors (± S.D.) of the co-transfected GAL4-dependent reporter through interaction of the GAL4-DBD/RXRα–LBD fusion protein with VP16-AD/CAR-LBD fusion proteins of the CAR isoforms SV1 to SV6. The activity of the GAL4-DBD/RXRα–LBD fusion in the presence of empty expression vector pVP16-AD was designated as 1. (B) Gel electrophoretic analysis of [³⁵S]-methionine labeled in vitro synthesized CAR protein isoforms, as indicated. Equal amounts of DNA of the respective expression plasmids were transcribed and translated in vitro and proteins were analyzed, as described in Experimental Procedures. (C) Electrophoretic mobility shift assays were performed using in vitro translated proteins bound to a radiolabeled doublestranded oligonucleotide corresponding to the DR3 motif of the XREM of CYP3A4. Binding reactions contained (+) or lacked (-) the indicated proteins. Complexes of CAR/RXRα heterodimers with the oligonucleotide are marked by an arrow.

Figure 4

CAR(SV3) differentially transactivates promoter reporter genes COS1 cells were co-transfected with enhancer/promoter reporter gene plasmids and expression plasmids encoding CAR isoforms, as indicated. The columns show the mean activation factors (± S.D.) of the respective reporter genes by the indicated CAR isoforms. The activity of each reporter in the presence of empty expression vector pcDNA3 was designated as 1. Statistically significant differences are indicated by asterisks (***, p < 0.001).

Figure 5

Constitutive and ligand-dependent coactivator interactions of CAR isoforms Mammalian two hybrid assays were performed in COS1 cells transiently transfected with combinations of expression plasmids encoding VP16-AD/CAR-LBD fusion proteins and GAL4-DBD/coactivator-RID fusion proteins, as indicated, together with the reporter gene plasmid pGL3-G5. The columns show the mean activation factors (± S.D.) of the GAL4-dependent reporter through interaction of GAL4-DBD/coactivator-RID fusion proteins (SRC-1, TIF-2, ACTR, DRIP205) with VP16-AD/CAR-LBD fusion proteins of the CAR isoforms SV1 to SV6. Open and filled columns indicate treatment with vehicle Me₂SO and CITCO (1 μM), respectively. The activity of each GAL4-DBD/coactivator-RID fusion in the presence of the empty expression vector pVP16-AD (VP16) treated with Me₂SO was designated as 1.

Figure 6

Tissue-specific splicing of exons 8 and 9. Qualitative RT-PCR analysis of the expression of transcripts with alternatively spliced exon 8 (A) and exon 9 (B) in the indicated human tissues. Small intestine and colon mucosa samples were derived from one individual each. In contrast, the samples of the other tissues represent pools (Human Total RNA Master Panel II, Clontech). The assays were performed as described in Experimental Procedures, with random hexamer primed cDNA of total RNA and primer pairs F1/R1 (A) or F2/R2 (B) (see Table 1). SV1, SV2, SV5 denote control reactions performed with DNA of the corresponding CAR isoform expression plasmids.

Figure 7

CAR expression and alternative splicing of exon 9 during enterocytic differentiation of Caco-2 TC7 cells. (A) Northern Blot analysis with polyadenylated RNA of the indicated cell lines. Caco-2 TC7 cells were analyzed from subconfluent (sub), confluent (confl) and 15 days post-confluent (15d pc) cultures. The blot was sequentially hybridized with probes for the genes indicated. The arrow marks the major CAR mRNA species of 1.4 to 1.7 kb. (B) Analysis of the expression of transcripts with alternatively spliced exon 9 by qualitative RT-PCR with random hexamer primed cDNA of polyadenylated RNA of Caco-2 TC7 cells cultured until confluence (confl) and for 15 days post-confluent (15d pc). PCR was performed with primers F2/R2 (Table 1). SV1 and SV5 denote control reactions performed with DNA of the corresponding CAR isoform expression plasmids. The lane on the left shows a 50 bp ladder size marker. By mixing DNA of SV1 and SV5 CAR isoform expression plasmids in different molar ratios, we confirmed that both fragments were amplified with equal efficiency (data not shown).