The C-terminus of the Coxsackievirus and adenovirus receptor directly interacts with the PDZ1 and PDZ3 domains of MAGI-1 with high affinity

Abstract

In most epithelia, a seven exon isoform of the coxsackievirus and adenovirus receptor (CAREx7) is localized at the basolateral surface where it behaves as a homophilic adhesion protein and is inaccessible for viral infection. However, we have recently discovered an alternatively spliced, low abundance isoform of CAR (CAREx8) that is present at the apical surface of well- differentiated human airway epithelia. The two isoforms differ only in the last 26 (CAREx7) or 13 (CAREx8) amino acids of the cytoplasmic domain. MAGI-1 is an essential PDZ domain containing protein known to be involved in cell polarization and cancer. We have previously demonstrated the interaction of both CAR isoforms with the third PDZ domain of MAGI-1 (PDZ3), and additionally, CAREx8 with PDZ1. We hypothesized that CAREx8 binds PDZ1 and PDZ3 directly with high affinity. To test this, the CAR C-termini and individual PDZ1, PDZ3, and PDZ2 (as control) domains were cloned into a prokaryotic expression plasmid and transformed into competent E. coli cells. Each of the proteins were synthesized, purified, and labeled with Cy3 or Cy5 fluorophores. Direct fluorescent binding assay and fluorescence resonance energy transfer (FRET) were used to detect the affinities between Cy3 labeled PDZ domains and Cy5 labeled CAR C- terminus proteins. Ligand binding curves indicated a high affinity binding for PDZ1-CAREx8, PDZ3-CAREx8 and PDZ3-CAREx7 with Kd values less than 10nM. In transfected CHO-K1 cells, PDZ3 was able to decrease the cell surface expression of CAREx8 and adenovirus infection, without causing a decrease in total CAREx8 protein levels. In contrast, PDZ1 was able to rescue CAREx8 from MAGI-1 mediated degradation and thus allow adenovirus infection. These data suggest that the PDZ1 and PDZ3 domains represent potential druggable small molecules that are able to regulate the levels of CAREx8 and hence adenovirus infection.