Synthesis of the CAR c-terminus and MAGI-1 PDZ Domains Proteins

Abstract

<p>The Coxsackievirus and adenovirus receptor (CAR) is an epithelial junctional transmembrane protein that is involved in cell adhesion and growth. CAR is also important for viral binding to cells and hence its abundance and localization is important for adenovirus infection. It is known that a seven exon isoform of CAR (CAREx7) localizes to the basolateral surface in polarized cells and provides an innate barrier to viral infection. However, we have recently discovered that an alternatively spliced, low-abundance, isoform of CAR (CAREx8) localizes to the apical membrane of polarized primary human airway epithelia where it can mediate initiation of adenovirus infection from the apical surface. Both isoforms differ only at the c-terminus. Our lab has shown that both isoforms of CAR interact with MAGIlb, a membrane-associated guanylate kinase scaffolding protein, in a PDZ dependent manner. Whereas CAREx7 pulls MAGI-lb to the junctions, MAGI-1b causes loss of CAREx8. We hypothesize that each CAR isoform interacts with different MAGI-1 PDl-binding domains (PDlO-PDl5). N-terminally His-GST-tagged CAREx7-c-terminus (aa 261-365), CAREx8-cterminus (aa 261-352), and individual MAGI-1 PDl domains (aa 20-110, 465-555, 630-730, 840-930, 990-1080, 1140-1230) were prepared by cloning PCR fragments for CAR and MAGI-1 into the vector pHH2. Clones were verified by PCR, restriction digestion and DNA sequencing. We then transformed appropriate plasm ids into Rosetta2 competent cells (EM D). Protein synthesis was induced with IPTG followed by six samples taken hourly. Proteins were extracted with PBS and run on SDS-PAGE. Coomassie blue staining/destaining demonstrated protein bands at the expected size of between 30kDa and 46kDa for all clones after 1 hour of induction. Isolated GST-fusion proteins will be used to study CAR-MAGI-1 interaction using in vitro FRET analysis.</p><p>This presentation occurred at the Wright State University Campus-Wide Celebration of Research, Scholarship and Creative Activities on April 8, 2011</p>