Nancy Bigley (Committee Member), Katherine Excoffon (Advisor), Michael Leffak (Committee Chair), Mill Miller (Committee Member), Robert Putnam (Committee Member)
Doctor of Philosophy (PhD)
A major factor in virus entry into cells is localization and abundance of the primary receptor. The Coxsackievirus and adenovirus receptor (CAR) is the primary receptor for group B coxsackievirus and many serotypes of adenovirus. In most epithelia, a seven exon isoform of CAR (CAREx7) is exclusively localized at the basolateral surface where it behaves as a homophilic adhesion protein and is inaccessible for viral infection. However, in well-differentiated human airway epithelia, we recently discovered an alternatively spliced, low abundance isoform of CAR (CAREx8) that is apically localized where it may initiate apical viral infection. The two isoforms differ only in the last 26 (CAREx7) or 13 (CAREx8) amino acids of the cytoplasmic domain, which suggests that some intracellular interactions may differ. One such differential interaction involves MAGI-1, an essential PDZ-domain containing protein known to be involved in cell polarization and cancer. In non-polarized COS7 cells, the CAREx8 protein level is regulated by MAGI-1b. CAR-MAGI-1 interactions were investigated by MAGI-1 siRNA knockdown, in vitro translation, immunocytochemistry, co-immunoprecipitation-Western blot analysis, fluorescence resonance energy transfer, direct binding assays, and adenovirus infection. Data showed that both CAR isoforms were expressed in several cell lines with CAREx7 RNA consistently more highly expressed than CAREx8 and MAGI-1 siRNA knockdown improved adenovirus infection in polarized cells. Both isoforms strongly interacted with PDZ3. CAREx8 also interacted with PDZ1. Whereas co-expression of PDZ1 with CAREx8 did not affect adenovirus infection, it could compete with full length MAGI-1 to protect CAREx8 from loss. Co-expression of CAREx8 with PDZ3 significantly reduced CAREx8 cell surface expression and adenovirus infection but did not reduce total CAREx8 protein levels suggesting that this may be the MAGI-1 PDZ domain responsible for holding CAREx8 within the cell but it is likely that other MAGI-1 interacting proteins/domains are required for loss of CAREx8. This dissertation provides the molecular basis for MAGI-1-mediated regulation of CAREx8 which will allow further investigation into the mechanisms of CAREx8 cell surface expression and hence viral infection of polarized epithelia.
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