Isoform-Specific Regulation and Localization of the Coxsackie and Adenovirus Receptor in Human Airway Epithelia Katherine J. D. A. Excoffon 1 , Nicholas D. Gansemer2, Matthew E. Mobily2, Philip H. Karp2, Kalpaj R. Parekh3, Joseph Zabner2* 1 Department of Biological Sciences, Wright State University, Dayton, Ohio, United States of America, 2 Department of Internal Medicine, University of Iowa, Iowa City, Iowa, United States of America, 3 Department of Cardiothoracic Surgery, University of Iowa, Iowa City, Iowa, United States of America Abstract Adenovirus is an important respiratory pathogen. Adenovirus fiber from most serotypes co-opts the Coxsackie-Adenovirus Receptor (CAR) to bind and enter cells. However, CAR is a cell adhesion molecule localized on the basolateral membrane of polarized epithelia. Separation from the lumen of the airways by tight junctions renders airway epithelia resistant to inhaled adenovirus infection. Although a role for CAR in viral spread and egress has been established, the mechanism of initial respiratory infection remains controversial. CAR exists in several protein isoforms including two transmembrane isoforms that differ only at the carboxy-terminus (CAREx7 and CAREx8). We found low-level expression of the CAREx8 isoform in well- differentiated human airway epithelia. Surprisingly, in contrast to CAREx7, CAREx8 localizes to the apical membrane of epithelia where it augments adenovirus infection. Interestingly, despite sharing a similar class of PDZ-binding domain with CAREx7, CAREx8 differentially interacts with PICK1, PSD-95, and MAGI-1b. MAGI-1b appears to stoichiometrically regulate the degradation of CAREx8 providing a potential mechanism for the apical localization of CAREx8 in airway epithelial. In summary, apical localization of CAREx8 may be responsible for initiation of respiratory adenoviral infections and this localization appears to be regulated by interactions with PDZ-domain containing proteins. Citation: Excoffon KJDA, Gansemer ND, Mobily ME, Karp PH, Parekh KR, et al. (2010) Isoform-Specific Regulation and Localization of the Coxsackie and Adenovirus Receptor in Human Airway Epithelia. PLoS ONE 5(3): e9909. doi:10.1371/journal.pone.0009909 Editor: Lisa F. P. Ng, Singapore Immunology Network, Singapore Received April 16, 2009; Accepted March 5, 2010; Published March 26, 2010 Copyright: 2010 Excoffon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a PPG grant from the NIH (HL51670-11). The Gene Transfer Vector Core is supported by the Roy J. Carver Charitable Trust, the NHLBI, CFF, and NIDDK. The In Vitro Cell Models Core is supported by the National Heart, Lung and Blood Institute and NIDDK [DK54759]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: joseph-zabner@uiowa.edu Introduction The Coxsackievirus and Adenovirus Receptor (CAR) plays a vital role in cell adhesion and viral infection [1–3]. The importance of CAR within epithelial junctions, where it behaves as an adhesion protein interacting with and potentially modulating the trafficking of key PDZ domain containing molecules, is becoming evident [4–7]. In contrast, how adenovirus initiates infection of the airway epithelium and whether CAR plays a role in initial adenoviral attachment and infection, when it is sequestered on the basolateral side of airway epithelia, remains unclear [8]. Alternative splicing plays an important role in eukaryotes. During pre-mRNA splicing, the spliceosome cleaves intron sequences, and joins exons together, forming an mRNA. Regulation of the spliceosome can result in alternative splicing of mRNA, determining which exons are present or absent in template mRNA. Alternative splicing not only regulates protein expression, but also allows multiple proteins to be expressed from the same gene resulting in significant proteomic diversity [9]. Alternatively spliced proteins may maintain similar activity, differing only in localization or interactions, or may vary widely in activity or regulation. It is estimated that alternative splicing occurs in 70–80% of human genes, but is more common in regulatory genes, and tissues with diverse cell types [10]. CAR is encoded by a highly conserved, alternatively spliced gene with five described transcripts. Three alternative transcripts encode CAR, lacking the transmembrane domain, yielding a soluble extracellular domain (splicing between exons 4/7, 3/7, 2/7) [11]. In experimental murine models, soluble CAR is able to inhibit viral infection but also results in toxicity [12–16]. Although the mechanism of toxicity is unknown, soluble CAR may be predicted to alter CAR- CAR interactions and thus epithelial cell adhesion [17]. Human CAR was first described by Bergelson et al as a 7 exon protein [1]. In contrast with other species, mouse CAR (mCAR) was initially cloned as a protein composed of 8 exons [18] and was named mCAR1. The 7 exon mouse form was subsequently identified and termed mCAR2. A detailed analysis of protein expression and localization in mice has revealed differential tissue- dependent expression and localization for the exon 7 and exon 8 isoforms [19,20]. This suggests that the interactions and potentially the functional importance of these two isoforms may be distinct. Furthermore, considering the emerging importance of signal transduction originating from microdomains within the cell membrane, these two isoforms would be predicted to differentially regulate cellular biology. PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9909