The PDZ3 domain of the cellular scaffolding protein MAGI-1 interacts with the Coxsackievirus and adenovirus receptor (CAR)

Abstract

The Coxsackievirus and adenovirus receptor (CAR) is an essential cellular protein that is involved in cell-cell adhesion, protein trafficking, and viral infection. The major isoform of CAR is selectively sorted to the basolateral membrane of polarized epithelial cells where it co-localizes with the cellular scaffolding protein membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1). Previously, we demonstrated CAR interacts with MAGI-1 through a PDZ-domain dependent interaction. Here, we show that the PDZ3 domain of MAGI-1 is exclusively responsible for the high affinity interaction between the seven exon isoform of CAR and MAGI-1 using yeast-two-hybrid analysis and confirming this interaction biochemically and in cellular lysates by in vitro pull down assay and co-immunoprecipitation. The high affinity interaction between the PDZ3 domain and CAR C-terminus was measured by fluorescence resonance energy transfer. Further, we investigated the biological relevance of this high affinity interaction between CAR and the PDZ3 domain of MAGI-1 and found that it does not alter CAR-mediated adenovirus infection. By contrast, interruption of this high affinity interaction altered the localization of MAGI-1 indicating that CAR is able to traffic MAGI-1 to cell junctions. These data deepen the molecular understanding of the interaction between CAR and MAGI-1 and indicate that although CAR plays a role in trafficking PDZ-based scaffolding proteins to cellular junctions, association with a high affinity intracellular binding partner does not significantly alter adenovirus binding and entry via CAR.

Keywords: Adenovirus; Cell junction; Epithelia; PDZ domain.

Fig. 1. CAR has a high affinity interaction with MAGI-1 PDZ3

A) Yeast Y187 cells were transformed with pairs of pGBKT7-derived bait (CAR C-terminus) and pGADT7-derived prey vectors (PDZ domains), and plated on selective media containing X-α-gal. Blue colony indicates an interaction between CAR and PDZ3. B) In vitro pull down assay. Individual 35S-methionine-labeled MAGI-1 PDZ domains generated by in vitro translation were added to immunoprecipitated FLAG-tagged CAR from transfected COS-7 cells (CAR lysate). Western blot for immunoprecipitated CAR (FLAG-CAR), and autoradiography for PDZ domain binding (N, no PDZ domain). In vitro translation of all PDZ domains was confirmed by autoradiography. Emission spectra, linear plot of binding curve and average change in maximal fluorescence intensity at 570 nm (F0-F) of Cy3-PDZ domains and Cy5 CAR C-termini upon excitation of Cy3 at 550nm with increasing concentration of Cy5 CAR C-termini (0-500nM). C) Cy3-PDZ1 and Cy5-CAR (no interaction). D) Cy3-PDZ3 and Cy5-CAR. All emission spectrum were corrected for background signals. NA = no interaction. Representative data shown from 3 replicate experiments.

Fig. 2. CAR interacts with MAGI-1 PDZ3 in cells and does not alter adenovirus infection

COS-7 cells were co-transfected with each individual myc-tagged MAGI-1 PDZ domain and FLAG-tagged CAR. CAR was immunoprecipitated with A) mouse anti-CAR RmcB or B) anti-FLAG and probed with rabbit anti-FLAG (CAR, ~46 kDa) and rabbit anti-myc (MAGI-1 PDZ domains; 20-28kDa). Western blot of total lysates confirmed expression of all PDZ domains, CAR, and equal loading (actin). C) CAR-deficient CHO-K1 cells were co-transfected with CAR, MAGI-1 (dotted bars), PDZ3 (black bars), or PDZ1 (gray), and balanced with empty pcDNA3.1 plasmid (white bar, control), followed by AdV-β-Gal (MOI 100) transduction. D) CAR-deficient CHO-K1 cells were co-transfected with CAR, MAGI-1 (dotted bars), MAGI-1 PDZ3 mutant (GF844RE; black bars), or MAGI-1 PDZ1 mutant (GF673RE; gray), and balanced with empty pcDNA3.1 plasmid (white bar, control), followed by AdV-β-Gal (MOI 100) transduction. Representative data from 3 replicate experiments.

Fig. 3. MAGI-1 PDZ3 is required for CAR-mediated enrichment of MAGI-1 at cell junctions

COS-7 cells were transfected with A) MAGI-1, B) CAR and MAGI-1, C) CAR and MAGI-1 with the PDZ1 domain deleted, or D) CAR and MAGI-1 with the PDZ3 domain deleted, and immunostained with an anti-CAR Ab (1605p; red) and anti-HA Ab (directed against MAGI-1; green). Nuclei are stained with DAPI (blue). 60 x oil immersion confocal microscopy. Single X-Y optical sections are shown. Merge of optical sections shown in final panel. White arrow identifies a representative junction between two adjacent cells taken from 3 replicate experiments. White bar = 10 μm.