The splicing event to create the 8th exon form occurs within the 7th exon. Thus, these two isoforms contain identical extracellular and transmembrane domains, which predicts identical adenovirus binding and serotype preference. The majority of the cytoplasmic domain is identical except for the last 26 (CAREx7) or 13 (CAREx8) amino acids. Although comprised of distinct sequences, the last 4 amino acids of both isoforms encode class I PSD95/DlgA/ZO-1 (PDZ) binding domain sequences (-X-(S/T)-X-F, where X = any amino acid and F = any hydrophobic amino acid). Interacting PDZ- domain-containing proteins for human CAREx7 include MAGI-1b, PICK1, PSD-95, MUPP-1, LNX1, and ZO-1 [5–7,21]. Further- more, murine CAREx7 and CAREx8 interact with LNX2 and this interaction appears to be modulated by both the PDZ binding domain of each isoform as well as an upstream sequence common to both isoforms [22]. In contrast with the murine isoform, the protein for human CAREx8 has never been described. We used a computational approach to identify human exon 8 encoding CAR, and investigated the functional significance of this isoform in primary human airway epithelia. Results Human CAR exon 8 containing isoform Mouse CAR was originally cloned as an 8 exon protein. We hypothesized that human CAR may exist as an 8 exon protein as well (Figure 1A). We first determined whether the human genome contained homologous sequence for exon 8, by performing a BLAT search against the mouse sequence (www.genome.ucsc.edu). Similar sequences were identified for human, chimpanzee, dog and rat (Figure 1B). The predicted amino acid sequence for human and chimpanzee is identical. This sequence differs from mouse by one amino acid. Comparison of hCAR P343 to mCAR A343 using Conseq software identified this amino acid as an exposed or buried residue respectively with a non-structural role. Conservation could not be determined due to insufficient data. The score assigned by Conseq was validated using PolyPhen (http://www.bork.embl- heidelberg.de/polyPhen/), which assigns scores, derived from Novel CAR Exon 8 Interactions likelihood matrices, as ‘‘benign’’, ‘‘possibly damaging’’, ‘‘probably damaging’’, or ‘‘unknown’’. The change P343A was designated as ‘‘benign.’’ Although an intuitive difference between these two amino acids exists, based on these analyses, additional studies to determine whether protein function was affected were not pursued. Primers were designed for RT-PCR to evaluate the presence of hCAREx8 in several human cell lines including HeLa, 293, A549, Caco-2, and primary human airway epithelia. Transcripts for both hCAREx7 and hCAREx8 were detected in all cell lines and primary cells examined (data not shown). A panel of human RNA was subsequently screened for the full-length hCAREx7 (Figure 1D) or hCAREx8 (Figure 1E), and yielded full length as well as smaller bands for both transcripts. Semi-quantitative analysis of the bands for hCAREx7 suggested that heart.brain,lung.liver. In contrast, hCAREx8 transcripts showed a rank order of liver,heart. brain.lung. These data suggest that as in mice, human splice variant expression varies between organs. Localization and function of hCAREx8 is similar to hCAREx7 in non-polarized cells COS-7 cells were transfected with the cDNA for hCAREx7 (Figure 2A) or hCAREx8 (Figure 2B). Immuno-localization using the FLAG antibody showed a similar distribution for both with predominant junctional staining in addition to some perinuclear staining. CAR-negative CHO cells transiently expressing hCAREx7, hCAREx8, or GFP were infected with recombinant adenovirus containing the bacterial LacZ gene (Figure 2C). As previously described, CHO cells and CHO cells expressing GFP are refractory to adenovirus-mediated gene transfer [23]. In contrast, cells expressing hCAREx7 or hCAREx8 showed robust and similar levels of adenovirus-mediated gene transfer proving that both forms of CAR function as adenoviral receptors. Localization of hCAREx8 is distinct from hCAREx7 in polarized cells We hypothesized that hCAREx8 localization may differ from hCAREx7 in polarized cells. Primary well-differentiated human Figure 1. Human CAR is an 8 exon alternatively spliced protein. Panel A shows a schematic diagram of the published and predicted human and mouse exon arrangement. Panel B shows the alignment of the mouse CAR exon 8 with the predicted exon 8 from other species. Panel C and D show representative RT-PCR for human CAR Exon 8 or 7, respectively, in cells (HeLa) or tissues. doi:10.1371/journal.pone.0009909.g001 PLoS ONE | www.plosone.org 2 March 2010 | Volume 5 | Issue 3 | e9909