mutagenesis resulting in a protein missing the last 4 amino acids (-ITVV). Similar to wild-type hCAREx8 (Figure 2B), COS-7 cells transfected with hCAREx8-PDZ showed robust junctional localiza- tion with some protein within intracellular vesicles (Figure 6A). Co-transfection of hCAREx8-PDZ with MAGI-1b-GFP resulted in co-expression of both proteins and did not alter the junctional localization of hCAREx8-PDZ (Figure 6B) or show diffuse localization of MAGI-1b-GFP (Figure 6C). These data indicate that the PDZ binding domain is required for the disappearance of hCAREx8. Thus, an interaction between hCAREx8 and MAGI-1b is required, raising the question of how this interaction differs from hCAREx7 and MAGI-1b. The upstream sequence plays a role in PDZ interactions We have previously shown a PDZ dependent interaction between hCAREx7 and PICK1-GFP [6]. Figure 3D demonstrates that there is no co-localization, and Figure 3E, shows there is no co- immunoprecipitation when hCAREx8 and PICK1-GFP are co- expressed. hCAREx7 and hCAREx8 differ by only the terminal 26 or 13 amino acids respectively. Since both hCAREx7 and hCAREx8 have type I PDZ binding domains we asked whether the PICK1 interaction was dependent on the terminal 4 amino acid sequence or the unique upstream sequences. To further define the role these sequences play in the PDZ domain-PDZ binding domain interaction, the hCAREx7 PDZ binding domain was swapped with the hCAREx8 PDZ binding domain (i.e. 22aa of hCAREx7 followed by ITVV, hCAREx7/8) or the hCAREx8 was swapped with hCAREx7 (i.e. 9aa of hCAREx8 followed by GSIV, hCAREx8/7). hCAREx7, hCAREx8, hCAREx7/8, or hCAREx8/7 were each co- transfected with PICK1-GFP. As previously shown, co-transfection of COS-7 cells with hCAREx7 and PICK1-GFP results in accumulation of hCAREx7 at the junctions of cells (Figure 7A) and hCAREx7 is able to pull PICK1-GFP (Figure 7B) from a perinuclear localization to the junctions of cells (Figure 7C). In contrast, as also demonstrated in Figure 3D, co-transfection of COS-7 cells with hCAREx8 and PICK1-GFP results in accumula- tion of hCAREx8 at the junctions of cells (Figure 7D). However, PICK1-GFP (Figure 7E) remains in a perinuclear localization (Figure 7F). Transfection of either hCAREx7/8 (Figure 7G) or hCAREx8/7 (Figure 7H) alone resulted in junctional localization. Interestingly, co-transfection of COS-7 cells with hCAREx7/8 and PICK1-GFP results in the co-localization of hCAREx7/8 (Figure 7I) and PICK1-GFP (Figure 7J) at the junctions of cells (Figure 7K). This suggests that the sequence ITVV is able to interact with PICK1-GFP but not in the context of the upstream hCAREx8 unique sequence. Finally, co-transfection of COS-7 cells with Novel CAR Exon 8 Interactions hCAREx8/7 and PICK1-GFP results in accumulation of hCAREx8/7 at the junctions of cells (Figure 8L), while PICK1-GFP (Figure 8M) remains in a perinuclear localization in a manner similar to wild type hCAREx8 (Figure 8N). These data indicate that the upstream sequence plays a significant role in the specificity of the PDZ domain-PDZ binding domain interaction and thus interactions must be defined experimentally. hCAREx8 PDZ binding domain and upstream nine amino acids define the MAGI-1 interaction The above data indicates that sequence immediately upstream of the PDZ binding domain may define the PDZ interaction. The difference between the respective interactions of hCAREx7 or hCAREx8 with MAGI-1b-GFP may also be due to interactions with the upstream 22 or 9 amino acids, the 4 terminal PDZ binding domain amino acids or a combination of both. Co- transfection of hCAREx7/8 with MAGI-1b-GFP resulted in the relocation and co-localization of MAGI-1b-GFP with hCAREx7/8 at the junctions of cells (Figure 8A-C). Interestingly, co- transfection of hCAREx8/7 with MAGI-1b-GFP revealed an intermediate phenotype (Figure 8D-F). Although there were depressed levels of hCAREx8/7 within the population of transfected cells (Figure 8D), some cells showed low levels of hCAREx8/7 expression at the junctions and low levels of co-localization with MAGI-1b-GFP at these junctions. Taken together with the results in Figure 7, these data suggest that although the PDZ binding domain is required for the interaction, both the exact sequence of the binding domain and upstream sequences may modulate the interactions of PDZ domain containing proteins. Discussion Adenoviruses are among the most studied viruses from the viral biology, cell biology and pathogenesis perspective. However, the failure of these viruses in applications for airway gene therapy and the identification of a basolaterally localized receptor, hCAR, made it clear that apical infection is inefficient. We and others found that once an epithelial cell is infected, progeny viruses can easily infect neighboring cells via the basolateral route, and moreover, disruption of cell adhesion by fiber allows virus escape into the lumen of the airway and back into the environment [4]. The question remained: is the initial infection random, requiring damage or does it use the adenovirus fiber-knob or another receptor? Human transcripts containing the predicted exon 8 have been described [2,28] but protein presence never verified. To better Figure 6. MAGI-1b-GFP-mediated loss of hCAREx8 requires the PDZ-binding domain (ITVV) of hCAREx8. COS-7 cells were transfected with hCAREx8-PDZ (A, red) or co-transfected with hCAREx8-PDZ (B, red) and MAGI-1b-GFP (C, green). Panel D shows the lack of co-localization of hCAREx8-PDZ (junctions) and MAGI-1b-GFP (cytoplasmic/diffuse). Confocal microscopy (60x oil immersion). doi:10.1371/journal.pone.0009909.g006 PLoS ONE | www.plosone.org 7 March 2010 | Volume 5 | Issue 3 | e9909