| dc.description.abstract |
The Coxsackievirus and adenovirus receptor (CAR) has two membrane encoding isoforms. The seven exon isoform of CAR (CAR^Ex7) is localized at the basolateral surface of polarized epithelia where it behaves as a homophilic adhesion protein and is inaccessible for adenoviral infection. The eight exon isoform (CAR^Ex8) can be found on the apical surface where it may mediate apical viral infection. The two isoforms differ only in the last 26 (CAR^Ex7) or 13 (CAR^Ex8) amino acids of the cytoplasmic domain. Although both isoforms contain the same AP-1b signal, which is involved in basolateral sorting, CAR^Ex8 is not localized on the basolateral surface which suggests that the CAR^Ex8-specific 13 amino acid sequence may be dominant over AP-1b-mediated basolateral trafficking. We hypothesized that novel proteins interact specifically with CAREx8. The C-terminus of each isoform of CAR was cloned into the pHH2 plasmid, which included glutathione S-transferase (GST) and 6x-His tags, and transformed into competent Rosetta E. Coli cells. GST-CAR^Ex7, GST-CAR^Ex8 or GST alone were synthesized and purified on GST-sepharose columns. Lysates from Calu-3 cells were pre- cleared and incubated with the GST-fusion proteins or GST (as control) and complexes were isolated with GST-sepharose beads. Proteins were separated by SDS-PAGE and stained with Coommasie blue. GST-CAR^Ex7 and GST-CAR^Ex8 bands were compared and a band, unique to CAR^Ex8, resulting from the Calu-3 cell lysate was sent to MS Bioworks for protein identification by mass spectrophotometry. 94 different proteins were detected in the band. The most prominent types of proteins identified were: actin associated (14/94), chaperone associated (14/94), ubiquitin associated (5/94), clathrin associated (5/94) and DNA/RNA/protein catalytic enzymes (15/94). In summary, CAREx8 interacts with a large number of proteins that will be investigated on a candidate basis. |
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