Localization of MAGI-1 Domains in Non-polarized Cells

Abstract

<p>MAGI-l is an essential member of the membrane-associated guanylate kinase family of scaffolding proteins responsible for organizing proteins at cell-to-cell junctions. MAGI-l has an inverted domain structure including an inactive guanylate kinase domain, two WW domains, and six PDZ domains. It is alternatively spliced near the c-terminus resulting in three distinct isoforms (MAGI-la, b or c). Each isoform has distinct tissue distribution with MAGI-lb being present at the basolateral junctions of epithelial tissues. We hypothesized that the cellular localization of MAGI-lb was due to a specific domain within MAGI-l. Primers were designed for each individual MAGI-l domain (PDZO aa 1-110; GuK aa 106-300; WW aa 296-440; PDZl aa 441-620; PDZ2 aa 621-814; PDZ3 aa 815-960; PDZ4 aa 961-1100; PDZ5 aa 1101-1232; c-terminus aa 1232-1471). Each domain was PCR amplified and cloned into the pcDNA 3.1/V5/GW/D-TOPO (Invitrogen) plasmid. Inserted MAGI-l domains were verified by PCR, restriction digestion, and DNA sequencing. Appropriate plasm ids were transfected into COS7 cells with Xfect transfection reagent (Clontech) and the cells incubated at 3rc for 48 hours followed by Western blotting analysis, immunocytochemistry and confocal microscopy to confirm expression and identify the localization of each isolated domain, respectively. Bands corresponding to the expected molecular weight (~20kDa and 30kDa) were present after blotting with VS epitope antibodies. Interestingly, all domains were localized in the cytoplasm except for the cterminus, which was found in the nucleus. These data suggest that either an intact protein or interacting proteins may be responsible for junctional localization of MAGI-l. The ability to express the individual domains of the protein MAGI-l is an important first step in studying how other proteins, such as the Coxsakievirus and adenovirus receptor, interact with MAGI.</p><p>This presentation occurred at the Wright State University Campus-Wide Celebration of Research, Scholarship and Creative Activities on April 8, 2011</p>