Previous reports have documented alternative splicing events in the mouse and human that likely impart structural and functional diversity within the NR1I subfamily (39
). PXR and VDR splice variants have been described that appear to possess altered receptor activities (40
). An alternatively spliced isoform of mCAR was identified in earlier studies (39
). More recently, data obtained from the use of ribonuclease protection assays suggested alternative splicing within the ligand binding domain of hCAR (41
). Combined, these observations lend support to the concept that alternative splicing may serve as an important mechanism for expanding the repertoire of xenobiotic receptors available to the mammalian cell.
In this investigation, we have identified and partially characterized activities associated with four novel isoforms of hCAR. These isoforms are encoded by alternatively spliced transcripts of the hCAR gene. One transcript incorporates 12 nucleotides of intron 6 through an alternative splice acceptor site, encoding an in-frame 4 amino acid insertion product, the hCAR-4aaINS isoform. Another transcript incorporates 15 nucleotides of intron 7 through an alternative splice acceptor site, encoding an in-frame 5 amino acid insertion variant, the hCAR-5aaINS isoform. A third transcript incorporates both splicing alterations simultaneously, encoding hCAR-dblINS. The fourth variant transcript, encoding hCAR-39aaΔ, results from a complete deletion of exon 7, also translationally in-frame. Each of the respective hCAR isoforms are predicted to possess altered structure, residing in the receptor’s E domain. The E domain, commonly referred to as the ligand-binding domain, performs integrated functions of dimerization, coregulator recruitment and transactivation (45
). In this report, these functions were evaluated for each of the variant hCAR receptors.
An important issue in defining receptor function involves demonstration of its stable expression, both in vivo
and in vitro
. We have presented evidence supporting the biological relevance of variant hCAR receptors. First, mRNA transcripts encoding each of the hCAR isoforms characterized herein were detected using RT–PCR analysis in 25 human liver tissues sampled to date. Secondly, each of the hCAR protein transcripts was efficiently and stably generated in multiple in vitro
expression systems, including transformed bacteria, reticulocyte lysates and transfected mammalian cells. Thirdly, each of the identified hCAR isoforms codes for in-frame translation variants and therefore would not be expected to be targeted by nonsense-mediated mRNA decay mechanisms that degrade premature termination products (46
). Fourthly, western blots derived from human liver extracts suggest the presence of multiple CAR protein isoforms.
Amplification of cDNA derived from liver samples indicated that variant isoforms of hCAR are likely to be present in all humans. However, the RT–PCR analysis presented here does not yield an accurate account of the abundance and ratios of these transcripts. These values may exhibit a large interindividual variability. It is already known that hCAR mRNA transcript levels vary significantly between liver samples (47
), and that activation of certain signaling cascades alters levels of CAR mRNA (41
). Consequently, one intriguing question is whether genetic variables play a role in expression of hCAR mRNA and/or the ratio of CAR transcripts. A precedent for signal transduction cascades that directly impinge on pre-mRNA splicing events has been presented (49
). Hence, a further intriguing question is whether treatments with dexamethasone or interleukin-6 affect the ratio of CAR transcripts and, in turn, what effect such exposures may have on responsiveness to inducing agents.
EMSA analysis revealed that of the hCAR variants assessed, the hCAR-4aaINS isoform was uniquely capable of interacting with both NR1 and NR2 elements, when heterodimerized with RXR. Based on our results from computer homology modeling, a potential explanation for the latter result is advanced. Insertion of the 5 amino acid sequence, APYLT, between helices 8 and 9 was predicted to stericly hinder the dimerization interface with RXR. Even more dramatically, removal of 39 amino acids, as encoded by the exon 7 deletion variant, would be likely to lead to the collapse of the dimerization interface.
In our studies, the DNA interaction of the hCAR-4aaINS–RXR was weak relative to the reference hCAR isoform complex. The hCAR modeling analysis predicts that the 4 amino acid insertion point for this variant receptor lies immediately adjacent to the dimerization interface and probably results in a C-terminal extension of helix 6, extending the helix in the direction of the dimerization interface. Thus the 4 amino acid insertion is also predicted to impact the receptor’s heterodimerization and DNA-binding functions, supporting the EMSA results presented here. Studies of hRXRβ3 that contains a 4 amino acid insertion in a structurally homologous position also reveal a compromised ability to form heterodimers with the retinoic acid receptor (35
). It is of interest to note that Mahajna et al
) also noted that the hRXRβ variant possessed an enhanced capacity to homodimerize, suggesting an alternative means by which the hCAR-4aaINS might interact with DNA (35
). In addition, it is possible that the rather weak interaction of the hCAR-4aaINS–RXR heterodimer with DNA in vitro
may be more robust in vivo.
For example, accessory factors may bind the PBREM sequence to form an enhanceosomal architecture, facilitating the interaction of the variant hCAR-4aaINS complex with its cognate response element. It is also possible that the hCAR-4aaINS isoform may be subjected to unique post-translational modifications in COS-1 cells, modifications that may modify its capacity to bind DNA. A number of nuclear receptors undergo post-translational modification by phosphorylation, consequently modifying receptor function (50
). Preliminary analysis of the of hCAR-4aaINS amino acid sequence using NetPhos 2.0 (51
) indicates that the inserted amino acids possess a high-scoring putative phosphorylation site (0.992) at the serine residue of the peptide GARVS
PTVG. Thus, alternative regulation strategies may impact hCAR receptor function and need to be evaluated further.
Studies conducted with the thyroid hormone receptor have suggested that structural alterations of the ligand-binding domain can alter the repertoire of DNA-binding elements involved in receptor interaction (52
). It therefore seems reasonable to speculate that variant isoforms of hCAR may bind to a different subset of response elements, distinct from previously defined hCAR–RXR response elements, as tested here. The biological implications of this suggestion may be important, as variant isoforms of hCAR may then be involved in the regulation of different subsets of human genes, possibly distinct from that of the reference form of hCAR. Efforts are underway in the laboratory to evaluate these possibilities.
Since all the hCAR isoforms assessed in the current studies were capable of interacting specifically with nuclear receptor coactivator SRC-1, two possibilities are suggested: (i) that the isoforms of hCAR may be capable of dominant-negative activity by acting to squelch coactivator recruitment; and/or (ii), as suggested above, that the variant hCAR isoforms may be capable of interacting with currently undefined DNA response elements, or might be recruited to promoters through other bridging factors, thereby modulating the transcriptional process through recruitment of coregulatory factors to target promoters. Results of transfection experiments presented in Figure B indicated that the former suggestion is not likely to be correct. In order to test the latter suggestion, experiments are currently underway examining the effects of artificial recruitment of hCAR isoforms to DNA using mammalian two-hybrid strategies that allow the evaluation of coactivator recruitment.
The variant isoforms of hCAR are predicted to encode structurally unique ligand-binding domains. Molecular modeling analysis reveals that the ligand-binding pocket structure would probably not be impacted in the hCAR-5aaINS insertion variant. However, the hCAR-4aaINS variant is predicted to impart a direct extension of helix 6, probably resulting in structural alteration of the ligand pocket. Receptors with this amino acid insertion would also be likely to possess an alteration in the pocket’s side chain composition, potentially resulting in altered ligand binding specificity of the isoform, an observation that would be agreement with studies of mouse RXRβ3 (34
). This prediction raises an important question: does this hCAR isoform respond to a set of ligands/pharmacophores that are distinct from the reference CAR isoform? One ligand evaluated in the current investigation, clotrimazole, exhibited inverse agonist activity with both the reference and the 4aaINS isoforms of hCAR. It remains curious that, unlike clotrimazole, PB and a number of PB-like inducing agents have been identified that are not ligands for the reference CAR isoforms (15
). This observation leads to the compelling question as to whether certain inducing compounds may interact alternatively with the ligand-binding pocket of hCAR-4aaINS, or other CAR isoforms. Assays are being developed to evaluate this intriguing question.
Alternative splicing of mRNA is one of the primary avenues for diversifying the proteome, and this process presents itself as a representation of nature’s efficiency, enabling an array of functions to be encoded in a single gene. The isoforms of hCAR presented in this study may be an example of this efficiency. Diversifying the proteome of the xenosensor system by mechanisms such as alternative splicing would probably facilitate an organism’s ability to adapt to its chemical environment. Further characterization of the variant isoforms of hCAR in animal models will be likely to lead to deciphering specific biological functions in the adaptive xenobiotic induction pathway.