Mechanisms of isoform specific localization and regulation of the Coxsackie virus and adenovirus receptor (CAR)

Abstract

<p>Adenoviruses are a common cause of respiratory illness including pneumonia, and are also prime candidate vectors for gene therapy. Adenoviruses share a receptor (CAR) with Coxsackie viruses, but the mechanism of their infection is not fully understood. This receptor exists in multiple isoforms in human airway epithelia. A seven-exon isoform (CAREx7) localizes primarily to the basolateral membrane, while a newly discovered eightexon form (CAR-Ex8) localizes to the apical and sub-apical compartments where it is more likely to mediate adenoviral infection. These two forms differ only in their C-terminus -26 amino acids unique to CAR-Ex7 are replaced by 13 in CAR-Ex8 -but they have been observed to interact differentially with another cellular protein, MAGI-lb. Whereas CAR-Ex7 colocalizes with MAGI at cell-cell junctions, CAREx8 is degraded upon co-expression with MAGI. We hypothesized that two tyrosines unique to CAR-Ex8 might be instrumental in this process and investigated this through site-directed mutagenesis, immunofluorescence microscopy, co-immunoprecipitation, Western blot, and adenoviral infection. We have discovered that, when either of these tyrosines is mutated individually, the differential interaction between CAR and MAGI persists, but when both are mutated together, the degradative interaction is ablated. We therefore conclude that the interaction between CAR-Ex8 and MAGI requires at least one of these tyrosines. Moreover, mutation of both tyrosines alters the activity of CAR-Ex8 as an adenovirus receptor. Further elucidation of the mechanism behind isoform specific localization of CAR may lead to a greater understanding of viral pathogenesis and new approaches to viral-mediated gene therapy.</p><p>This presentation occurred at the Wright State University Campus-Wide Celebration of Research, Scholarship and Creative Activities on April 16, 2010</p>