PSD-95-GFP, PICK1-GFP, or GFP (Figure 4G-I). hCAREx8 was detectable by Western blot, presumably due to the relatively few cells transfected with hCAREx8 but not MAGI-1b-GFP. No interaction between hCAREx8 and MAGI-1b-GFP was observed by co-immunoprecipitation even when cells were treated with proteosomal inhibitors (data not shown). These data suggest that a transient interaction between MAGI-1b and hCAREx8 results in the disappearance of hCAREx8. Quantitation of the stoichiometric hCAREx8 interaction with MAGI-1b-GFP Since some MAGI-1b-GFP expressing cells also expressed some, albeit mislocalized, hCAREx8, we hypothesized that there would be a stoichiometric relationship between these proteins. COS-7 cells were electroporated with a dose response of hCAREx8 (0-30 mg plasmid DNA) in the presence of constant (15 mg plasmid) MAGI-1b-GFP or GFP. hCAREx8 expression was evaluated by immunocytochemistry (red fluorescence) using fluorescence microscopy and Western blot. Fluorescence intensity was calculated as average pixel count per field of view (n = 6, Figure 5A). Increasing the amount of hCAREx8 plasmid, in the presence of constant GFP plasmid, resulted in a relatively linear increase in hCAR fluorescence (Figure 5A). In contrast, when hCAREx8 was co-transfected with a constant amount of MAGI-1b-GFP, the dose response curve was shifted significantly to the right, indicating that hCAREx8 expression was suppressed in the presence of MAGI-1b-GFP relative to co-expression with GFP. Western blot using the hCAREx8 specific antibody was quantitated by chemiluminescence imaging, relative to b-actin protein expression (Figure 5B), and revealed a similar relationship. Although a rapid increase in hCAREx8 protein in the presence of GFP was observed, the sensitivity of detection was apparent by the plateau of the curve. The dose response curve for hCAREx8, when co- transfected with MAGI-1b-GFP, was again shifted significantly to the right. The observed difference in hCAREx8 protein was not due to transfection or transcription since quantitative PCR for plasmid and RT-PCR for hCAREx8 RNA were similar (data not shown). Moreover, no effect was observed on GFP or MAGI-1b-GFP fluorescence upon increasing hCAREx8 (data not shown). MAGI-1b-GFP interaction with CAREx8 decreases adenovirus infection To determine a physiological response to the loss of hCAREx8 in the presence of MAGI-1b-GFP, CHO-K1 cells were transfected with hCAREx8 or MAGI-1b-GFP, or co-transfected with hCAREx8, at a constant dose, with MAGI-1b-GFP at increasing doses. All transfections were balanced with GFP plasmid to maintain equal amounts of DNA. Transfected cells were transduced with Ad-b-gal 48h later. b-galactosidase expression was determined 24 hr post-transduction (Figure 5C). Similar to Figure 2C, transfection of CHO-K1 cells with hCAREx8 renders them susceptible to adenovirus infection while transfection with MAGI-1b-GFP does not. Co-transfection of hCAREx8 with a dose response of MAGI-1b-GFP resulted in a dose-related reduction of susceptibility to Ad-bgal-mediated gene expression, indicating that there was a reduction of cell surface hCAREx8 available as a receptor. Taken together with the previous data, we concluded that in contrast to hCAREx7, co-expression of hCAREx8 with MAGI-1b-GFP results in the disappearance of hCAREx8 and may explain the absence of hCAREx8 in the adherens junctions of airway epithelia. Novel CAR Exon 8 Interactions Figure 5. Co-expression of hCAREx8 with MAGI-1b-GFP results in less immunofluorescence, protein, and adenovirus-mediated gene transfer. COS-7 cells were transfected with 0 to 30 mg of hCAREx8 +/2 15 mg MAGI-1b-GFP and evaluated for hCAREx8 specific immuno- fluorescence (A) or CAREx8 specific protein by Western blot (B). In the presence of MAGI-1b-GFP, the hCAREx8 expression curve is shifted to the right suggesting a loss of hCAREx8 protein. Panel C shows CHO cells transfected with varying amounts of hCAREx8 and MAGI-1b-GFP, and evaluated for Ad-b-galactosidase gene transfer. Co-expression of MAGI- 1b-GFP resulted in a decrease of adenovirus-mediated gene transfer in a dose response relationship. *p,0.03. doi:10.1371/journal.pone.0009909.g005 The hCAREx8-MAGI-1b-GFP interaction requires the hCAREx8 PDZ binding domain The interaction between the hCAREx7 isoform with MAGI-1b- GFP requires the CAREx7 PDZ binding domain (-GSIV). To determine the requirement of the hCAREx8 PDZ binding domain, a stop codon was added to the cDNA of hCAREx8 by site-directed PLoS ONE | www.plosone.org 6 March 2010 | Volume 5 | Issue 3 | e9909