airway epithelia [27,33]. In vitro, the C-terminus of hCAR is not required for adenovirus infection [27], however, considering the distinct localization and interactions between these isoforms, we cannot predict whether infection is identical in vivo. Furthermore, it is interesting to speculate that mutations altering the splicing or transcript abundance of the hCAREx8 isoform may be responsible for viral susceptibility. PDZ-based regulation has been described for other membrane proteins [34,35]. For example several PDZ-domain containing proteins are known to interact with the cystic fibrosis transmem- brane conductance regulator (CFTR) [36,37]. In contrast to hCAREx7, CFTR traffics to the apical membrane of airway epithelia where it behaves as a chloride channel. Interactions between the PDZ-binding domain of CFTR and the PDZ domain of Na+/H+ exchanger-3 regulatory factors 1 and 2 (NHERF1 and NHERF2) act to stabilize CFTR at the cell surface [38]. CFTR PDZ interactions with the CFTR-associated ligand (CAL) target CFTR for lysosomal degradation [39]. Cushing et al have recently shown that the delicate balance of interactions regulating cell surface maintenance and lysosomal degradation upon cycling is due to the relative affinity of the PDZ interactions [37]. The stronger interaction between CFTR and NHERF1/2 would be predicted to out compete the weaker interaction with CAL resulting in the relatively long CFTR half-life observed. This type of regulation differs from our data in at least two ways. One isoform (CAREx7) appears to be dominant in directing the localization of the PDZ-domain containing proteins while MAGI-1b is dominant in the interaction with hCAREx8 resulting in the loss of this protein. To our knowledge, all descriptions of PDZ-directed degradation have been during mature protein cycling. In contrast, we have observed immediate loss of hCAREx8 implying regulation during early protein synthesis or quality control stages. The question remains why differential compartmentalization of these hCAR isoforms would exist. The signals transduced by these isoforms, despite having the same extracellular ligands, may differ. Differential localization of these two isoforms could result in the cell being able to discern whether the signal is from the apical or basolateral compartment and mount the appropriate response. For example, both isoforms would recognize AMICA1/JAML present on the surface of neutrophils and dendritic cells. hCAREx7 may be a gatekeeper for neutrophil transmigration but hCAREx8 may either tether neutrophils to the apical surface or sense how many neutrophils are present. Alternatively, these two isoforms may play a role in dendritic cell surveillance and maintaining the seal around dendritic cell filopodia. These data also provide support for the importance of sequences upstream of the PDZ binding domain in dictating target PDZ domains and subsequent activity. Both the GSIV (Ex7) and ITVV (Ex8) sequences bind all three targets investigated, MAGI-1b, PICK1 and PSD95. However, the presence of the upstream 9 unique amino acids from hCAREx8 alters the resulting interaction such that PICK1 does not interact, PSD-95 does interact to localize at cellular junctions, and MAGI-1b results in degradation of the hCAREx8 protein. In contrast, the 22 amino acids from hCAREx7 allow a stable interaction between these three proteins and the ITVV PDZ-sequence from hCAREx8 resulting in co-localization at the junctions of cells. The exact mechanism requires further investigation and will lead to a greater understanding of this important class of protein interaction domains. In summary, human airway epithelia express several isoforms of hCAR. Importantly, human CAREx8 localizes to the apical surface where it may play a key role in the initiation of adenoviral, and Novel CAR Exon 8 Interactions potentially coxsackievirus, pulmonary infection. We propose a model for the regulation of this localization based upon isoform- specific PDZ binding domain interactions with MAGI-1b. Although PDZ based interactions are known to be key regulators of membrane microdomain structure, stability and signaling, we have shown that this interaction may also regulate protein expression. Furthermore, PDZ based interactions are influenced by several factors, including PDZ binding domain and upstream sequence context. Affinity of interaction between the PDZ domain and PDZ binding domain may be higher or lower depending on surrounding sequences resulting in the specificity of interaction. Surprisingly, either interacting partner can be dominant in dictating the result of the interaction (i.e. junctional trafficking versus degradation). Further elucidation of these mechanisms may provide a novel target for either down regulation of the adenovirus receptor to limit viral infection or alternatively up regulation for the purpose of adenoviral-based therapies. Materials and Methods Ethics statement This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board of the University of Iowa (IRB ID No. 9507432). Primary human airway epithelia were isolated from discarded and deidentified trachea and bronchi of donor lungs. This study used discarded lung tissue, thus the IRB deemed consent was not needed. Materials and constructs FLAG M2 antibody (Ab) was purchased from Sigma (F3165, St. Louis, MO), mouse anti-CD55 (DAF) was from BD Pharmingen (555691, San Jose, CA), mouse anti-Ezrin was from Santa Cruz (SC-58758, Santa Cruz, CA), mouse anti-acetylated a-tubulin was from Sigma (St. Louis, MO), Alexa-488 and -568 conjugated goat anti-mouse or anti-rabbit Abs, mouse and rabbit anti-GFP were from Molecular Probes (Eugene, OR). RmcB Ab (CRL-2379, ATCC, Manassas, VA) was produced by the University of Iowa Hybridoma Core. Rabbit anti-hCAR 1605 was produced in rabbits immunized with a GST fusion to the intracellular c- terminus (aa 261–365) as previously described [40]. Rabbit anti- hCAREx7 5490 and Rabbit anti-hCAREx8 5678 were produced in rabbits immunized with peptides of 13 c-terminal amino acids (CVMIPAQSKDGSIV and FKYPYKTDGITVVC respectively). COS-7 cells were from ATCC (Manassas, VA), and maintained under standard culture conditions (D-MEM with 10% FCS, penicillin and streptomycin). CHO-K1 cells were from BD Biosciences (Franklin Lakes, NJ) and maintained under standard culture conditions (D-MEM with 10% FCS, supplemented with tetracycline L-glutamine, penicillin and streptomycin). Ad serotype 5 containing the b-galactosidase (Ad-bGal), eGFP, RFP (peGFP- N1, pDSRed1, Clontech, Palo Alto, CA), or hCAR gene have previously been described [41,42]. The University of Iowa Gene Transfer Vector Core produced all viruses. Several cDNAs were kind gifts from the following investigators: hCAR was from Ronald Crystal; peGFP-MAGI-1b was from Irina Dobrosotskaya; PSD- 95-GFP was from David Bredt. The cDNA for PICK1-GFP has previously been described [43] and MAGI-1b-CMV-myc was subcloned and contained aa 642-1287. Web programs We performed a BLAT search with the mouse sequence (www. genome.ucsc.edu) to determine the human sequence for exon 8. Comparison of hCAR P343 to mCAR A343 using Conseq PLoS ONE | www.plosone.org 10 March 2010 | Volume 5 | Issue 3 | e9909