Previous studies of CAR variants demonstrated that not all of the detected protein products were likely attributed to alternatively spliced mRNA species (23
). For examples, when COS-1 cells were transfected with plasmids expressing various CAR mRNA splice variants, lower molecular weight bands were consistently observed in SDS-PAGE gels. Even upon expressing a CAR splice variant possessing a partial deletion of exon 7, a proportionally lower molecular weight band was detected. From these observations, we hypothesized that isoforms of CAR may arise from the use of internal translation start sites. In the present study, we identify new protein translational variants of human CAR that differ from those reported previously. Specifically, we demonstrate that downstream in-frame start codons produce N-terminally truncated CAR variants (ΔNCAR) from wild-type as well as alternatively spliced mRNA transcripts. We determined that CAR1, CAR76 and CAR80 proteins all functionally activate luciferase reporter activity when driven by a DR-1-type PPRE promoter element.
Specifically, since strong Kozak sequences (36
) and translational start sites were predicted from the use of methionines at positions 1, 76, 80, 125, 128, 168 and 265, we generated plasmid constructs with optimized Kozak sequences to enable overexpression of CAR proteins from each putative internal start site, and expressed these constructs in transiently transfected COS-1 cells, in rabbit reticulocyte lysates and in wheat germ in vitro
translation systems. In each case, protein bands were observed that migrated with the expected molecular weights of the truncated CAR76 and CAR80 proteins, and also co-migrated with the more rapidly migrating bands detectable upon co-expression of the full-length protein encoded by the CAR1 template. A methionine to lysine mutation at codon 1 (CAR M1K) abolished expression of full-length CAR, but yielded prominent bands resulting from CAR76 and CAR80 initiation. Similarly, when the codons for amino acids 76 and 80 were mutated from methionine to lysine in CAR1, the CAR76 and CAR80 bands were abolished, with proteins predicted to initiate at amino acids 125 and 128 becoming readily detectable. Dramatic sequential mutations of upstream methionines always led to increases of protein expression from downstream internal translation start sites, supporting a model of ‘cap-dependent’ translation of CAR mRNA, whereas internal ribosome entry site (IRES)-dependent levels of protein translation of ΔNCAR would be expected to proceed independently of mutations of upstream initiator codons (38
Others have reported that full-length mouse CAR can activate a DR-2-type PPRE, but failed to show any significant transactivation on a DR-1 (13
). Rather, mouse CAR repressed ligand-dependent PPAR α/RXRα activity on a DR-1 (13
). A more recent study suggested that full-length CAR binds to the promoters of the CYP7A1 and PEPCK genes, decreasing their expression by competing with the transcription factor HNF4 for binding to DR-1 elements (39
). Perhaps the specific sequence context of a DR-1 element may influence whether CAR exerts a positive or negative effect on transcription activity. The reported discrepancies may also have resulted from differences in the cell lines used or from the inclusion/exclusion of RXRα co-transfection. It is interesting that another laboratory reported that alternative splicing of human CAR may result in an mRNA variant that lacks the usual protein translational start site in exon 2, instead enabling translation from an AUG start codon in exon 1 (24
). In this latter case, the use of an alternative exon 1 translation initiation codon predicts the inclusion of unique N-terminal amino acids that are in-frame with amino acids encoded by exon 3. These investigators also predicted CAR translation from codons 125 and 128, rather than codons 76 and 80 that we identify here as the actual start sites used to generate ΔNCARs possessing unique transcriptional activities (24
In our experiments, full-length CAR and the ΔNCARs, CAR76 and CAR80, exhibited constitutive activity on a special DR-1 PPRE, and the receptor activities were downregulated by treatment with the CAR inverse agonist 5α-androstan-3α-ol (androstanol). Co-treatment with androstanol and the human CAR-specific agonist, CITCO, completely restored CAR and ΔNCAR activities to constitutive levels. Transient co-expression of RXRα variants and CAR or ΔNCARs revealed that receptor heterodimerization was the most important criterion for gene promoter activation. In this respect, the RXRα AF-1 domain was not required for transactivation by the ΔNCARs, nor was RXRα's AF-2 domain. When point-mutated RXRα expression constructs were co-transfected with the ΔNCARs, the RXRα heterodimer-defective variant, Y397A, completely ablated the ability of the ΔNCARs to activate a PPRE response element, yet the Y397A RXRα could still homodimerize and function in reporter transactivation. Conversely, a mutation of RXRα that abolishes RXRα homodimerization, L430F, still allowed for efficient heterodimer formation with CAR and the ΔNCARs, reflected by subsequent reporter gene activation, but effectively inhibited RXRα homodimer transactivation in the presence of chemical agonist.
Therefore, our results demonstrate that the lack of a CAR-DNA-binding domain in a CAR-RXRα receptor heterodimer does not inactivate the dimer's transactivation function on a DR-1 or in a PPRE promoter context. In fact, examples of classical nuclear receptors have been reported previously that retain transactivation ability on gene promoters without direct DNA-binding interactions, including ER (40
), the retinoic acid receptor (RAR) (41
) and the glucocorticoid receptor (42
). In the latter case, transgenic mice possessing DNA-binding defective GRs were still viable and the modified receptors retained functional ability to cross-talk with other transcription factors, thereby participating in processes such as transrepression. For CAR, and perhaps for other nuclear receptors as well, we predict that truncated forms of the receptor may exhibit full or partial activation function, or even repression, and that the spectrum of these activities may manifest on an altered repertoire of response elements. Specifically, we propose a new model of CAR DBD-independent transactivation, whereby only the RXRα component of the heterodimer binds directly to a DR-1-containing PPRE, tethered to CAR or ΔNCARs (CAR76 or CAR80), with functional contributions from the CAR AF-2 domain providing the basis for co-activator recruitment and subsequent gene transactivation function. This type of heterodimer–DNA interaction is in striking contrast to that reported previously for the APYLT-CAR splice variant, a variant that possesses a 5 aa insertion in the receptor's dimerization interface (14
). For the latter receptor, the RXRα DBD, rather than the APYLT-CAR DBD, is expendable for transcriptional activation, implying a model invoking direct contact of only the CAR portion of the dimer with DNA, not the RXRα component (14
). Taken together, along with the recently identified phosphorylation modifications that impact the ability of CAR to translocate (43
), it is becoming clear that the post-transcriptional regulation of CAR expression is an important means of generating a diversity of receptor modalities that likely function to enhance its complex role as a receptor integrator of xenobiotic and endobiotic sensing in mammalian cells.