level with apical membrane markers decay accelerating factor (DAF, Figure 2L) and ezrin (Figure 2M) [24–26]. We hypothesized that endogenous hCAREx8 may be responsible for the inefficient, albeit detectable, level of adenovirus infection from the apical surface of airway epithelia. To determine if augmenting expression would augment adenovirus infection, dissociated airway epithelia were transduced with hCAREx7, hCAREx8 or GFP and seeded on semi-permeable filters. Cultures were allowed to polarize and form an epithelium over 1 week. When the resistance of all cultures was above 300 mVNcm22, cells were infected from the apical surface with adenovirus containing the LacZ gene (Figure 2N). Human airway epithelia expressing GFP showed baseline low level adenovirus-mediated gene transfer. Epithelia expressing hCAREx8 showed approximately 5-fold greater gene transfer than epithelia expressing GFP (Figure 2N) or mock transduced (Figure S3) and close to a 100% increase in gene transfer compared to epithelia expressing hCAREx7 (Figure 2N) This increase in infection is similar to previously published results for glycophosphatidylinosi- tol-linked hCAR which is missing the transmembrane and cytoplasmic domains and localizes explicitly to the apical surface of polarized airway epithelia [27]. This suggests that hCAREx8 explains the low-level baseline apical adenovirus infection and that there may be a maximal amount of infection possible through apically localized receptor. These data also raise the question why hCAREx8 does not localize to the basolateral surface. hCAREx8 interacts with PSD-95 but not PICK1 via the PDZ binding domain PDZ-interactions may modulate localization as well as function. It is known that both the sequence of the binding domain and the upstream sequences affect PDZ domain interactions. The mechanism by which the upstream sequences affect the specificity of interaction remains unclear. Although hCAREx7 and hCAREx8 Novel CAR Exon 8 Interactions have type 1 PDZ binding domains (X(S/T)XW) at the C-terminus (hCAREx7 –GSIV; hCAREx8 –ITVV), these and the upstream sequences are distinct. Thus we hypothesized that PDZ-interac- tions may be responsible for altered localization in human airway epithelia. We have previously shown that hCAREx7 interacts with PICK1 and PSD-95 via a PDZ binding domain specific interaction such that co-localization by immunocytochemistry reveals hCAREx7 is able to pull these proteins out of the cytoplasm and co-localize at the junctions between cells. We further hypothesized that hCAREx8 may interact with some hCAREx7 partners. COS-7 cells were co-transfected with hCAREx8 and PSD-95-GFP and immunocytochemistry was performed to determine localization. hCAREx8 localized to the junctions as observed in Figure 2B. In the absence of hCAREx8, PSD-95-GFP localizes diffusely within the cytoplasm [6]. In the presence of hCAREx8, the localization of PSD-95-GFP was altered to co- localize with hCAREx8 at the junctions of cells (Figure 3A). Localization of a PDZ mutant form of hCAREx8 (hCAREx8-PDZ) was similar to full length hCAREx8 (Figure 3B) and was unable to alter the diffuse localization of PSD-95-GFP upon co-transfection. Whereas an interaction with full length hCAREx8 was evident by co-immunoprecipitation (Figure 3C), no interaction was observed by coimmunoprecipitation with hCAREx8-PDZ, indicating that this interaction requires the ITVV PDZ binding domain sequence. Next, hCAREx8 and PICK1-GFP were co-transfected into COS-7 cells. Immunocytochemistry for hCAREx8 revealed the lack of interaction between PICK1-GFP and CAREx8 at the junctions where the majority of hCAREx8 was localized (Figure 3D). In some cells there was co-localization in the perinuclear region. To determine if there was a physical interaction, each protein was immunoprecipitated and evaluated by Western blot (Figure 3E). No interaction was detected. Taken together, these data suggest that either there is no interaction or the interaction is too weak to Figure 3. hCAREx8 co-localizes and interacts with PSD-95 but not PICK1. Panel A shows co-localization (yellow) of hCAREx8 (red) and PSD-95- GFP (green). In contrast, in panel B, hCAREx8-PDZ does not co-localize at the junctions of cells. hCAREx8-PDZ localizes to the junctions between cells whereas PSD-95-GFP fluorescence remains diffuse. Panel C shows immunoprecipitation of PSD-95-GFP with the hCAR specific extracellular domain monoclonal antibody RmcB, GFP antibody, but not a control antibody (MopC). Panels D and E shows the lack of co-localization and immunoprecipitation between hCAREx8 (junctional) and PICK1-GFP (perinuclear). Confocal microscopy (60x oil immersion). doi:10.1371/journal.pone.0009909.g003 PLoS ONE | www.plosone.org 4 March 2010 | Volume 5 | Issue 3 | e9909