Novel CAR Exon 8 Interactions Figure 7. The result of the MAGI-1b-GFP interaction with hCAR requires both the PDZ binding domain and the upstream isoform specific amino acids. The PDZ binding domain of hCAREx7 and hCAREx8 were swapped by PCR as shown in panel A. Both constructs contained identical upstream sequences. The localization was determined in transfected COS-7 cells either alone, hCAREx7/8 (B), hCAREx8/7 (C) or upon co- transfection with MAGI-1b-GFP (D-I). Panels D and E show junctional expression of hCAREx7/8 and MAGI-1b-GFP, respectively, and co-localization in panel F. Panel G shows minor expression of hCAREx8/7 in the presence of robust MAGI-1b-GFP expression in panel H. Some co-localization is observed in panel I. Confocal microscopy (60x oil immersion). doi:10.1371/journal.pone.0009909.g007 understand the function of hCAR we sought to investigate the presence, localization and interactions of human CAREx8 and their relevance in the human airway epithelium. Proteins exhibit regulation at multiple levels. Here we show that not only is the transmembrane form of human CAR regulated by splicing, but also the exon 8 specific isoform is subsequently regulated at the protein level by the PDZ domain containing protein MAGI-1b. Furthermore, hCAREx8 localizes to the apical compartment of human airway epithelia where it could serve as the receptor initiating adenovirus infection. RNA splicing is an important event that significantly increases proteomic diversity and may impart cell specificity. Despite the importance of hCAR in viral infection, cell adhesion, and development, the genetic and splicing regulation of the gene for hCAR, CXADR, have not been well studied. It is known that expression of hCAR is upregulated by histone deacetylase (HDAC) inhibitors, a finding with significant implications for oncolytic adenoviral cancer therapy [29–31]. Whether HDAC inhibitors affect the splicing of hCAR is also currently unknown. Several transcripts have been described for hCAR. However, the importance of each splice variant is unclear. Secreted splice variants that interact with the extracellular domain of the transmembrane form of hCAR could clearly alter homophilic transmembrane hCAR interactions and hence modulate junction- al remodeling. These variants could also play a physiological role modulating interactions between hCAR and its ligand, AMICA1/ JAML, found on transmigrating lymphocytes and dendritic cells [32]. Considering the importance of junctional organization, inter- actions, and signaling, alternative transmembrane forms could play equally important roles in junctional remodeling and responses to ligation with soluble isoforms or ligands on other cell types. Two transmembrane isoforms are known for CAR. Although several interactions have been discovered for both the mouse and human CAREx7 isoform, only one interaction has been described for the alternative, less prevalent mouse CAREx8 isoform. Both mouse CAREx7 and CAREx8 interact with Ligand- of-Numb Protein-X2 (LNX2), an intracellular scaffolding protein that may play a role in Notch signaling [22]. Interestingly, both isoforms interact with LNX2 through two different regions within the intracellular domain of mCAR; each unique C-terminal PDZ binding domain and a region in common just upstream of the splice junction. Our previous work revealed that interaction with hCAREx7 results in junctional localization of MAGI-1b [6]. In contrast, this work shows that the interaction with hCAREx8 is unique to other MAGI-1b interactions since it results in the loss of hCAREx8. Epithelia express MAGI-1b where it localizes to the basolateral junctions. hCAREx7 also localizes to the basolateral junctional adhesion complex. The data presented herein is consistent with PLoS ONE | www.plosone.org 8 March 2010 | Volume 5 | Issue 3 | e9909