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First published online 10 August 2004
doi: 10.1242/jcs.01300

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A role for the PDZ-binding domain of the coxsackie B virus and adenovirus receptor (CAR) in cell adhesion and growth

¹ Department of Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, USA
² Howard Hughes Medical Institute, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, USA

View larger version (16K): [in a new window]   Fig. 1. Correlation between CAR mRNA transcript levels and transepithelial resistance in human airway epithelia from eight different organ donors. (A) Relationship between CAR transcript levels as determined by array analysis and transepithelial resistance measured by Ussing chamber studies in well-differentiated primary human airway epithelia cultures grown at the air-liquid interface (R2=0.8037, n=8). (B) No correlation was evident between ß-Actin transcript levels and transepithelial resistance (R2=9x10–5, n=8).

View larger version (9K): [in a new window]   Fig. 2. Transepithelial resistance in human airway epithelia expressing CAR, CARGSIV, or GFP. Primary human airway epithelia were infected with adenoviruses expressing CAR, CARGSIV, or eGFP and transepithelial resistance was measured 3 days after infection (n=12). CARGSIV failed to increase the transepithelial resistance to the same degree as wild-type CAR. Mean resistances differ significantly from each other (*P<0.002). Cultures infected with Ad-eGFP did not differ significantly from baseline transepithelial resistance.

View larger version (14K): [in a new window]   Fig. 3. Characteristics of L-cells stably transfected with pcDNA3.1-CARGSIV, CAR, or –eGFP. (A) Elevation of reporter gene expression in CAR and CARGSIV stable cell lines in comparison to GFP 2 days after infection with an adenovirus containing the ß-galactosidase gene (*P<0.001). (B) Growth comparison between these cell lines by protein concentration (*P<0.001). (C) Relative increase in cell count at day 5 versus day 3 (*P<0.00001). CAR significantly reduced cell growth in comparison to CARGSIV and eGFP stable cells.

View larger version (56K): [in a new window]   Fig. 4. CAR does not interact with LIN-7b, GRIP1, or PIST. Panel A shows the immunofluorescent staining of COS-7 cells expressing CAR, LIN-7b, GRIP1-myc, or PIST-HA alone. CAR primarily localizes to the junctions between cells while LIN-7b, GRIP1, and PIST show a diffuse staining. Panel B shows no alteration in staining and no significant colocalization between LIN-7b (green), GRIP1-myc (green), or PIST-HA (green) when co-expressed with CAR (red). Panel C shows western blots of COS-7 cells co-transfected with CAR and LIN7b, GRIP1, or PIST. Each protein is detected after IP with LIN-7, myc, or HA Ab respectively but do not co-IP with CAR using the CAR-specific RmcB Ab or a nonspecific control Ab.

View larger version (87K): [in a new window]   Fig. 5. CAR does not interact with SAP97. Panel A shows the immunofluorescent staining of COS-7 cells expressing CAR, CARGSIV, or SAP97-GFP alone. CAR, CARGSIV, and SAP97-GFP primarily localize to the junctions between cells. Panel B shows that there is no alteration in the localization of SAP97-GFP (green), CAR (red), or CARGSIV (red) when co-expressed. This similar compartmentalization resulted in significant colocalization (yellow). Panel C shows a western blot/IP of COS-7 cells co-expressing CAR and SAP97-GFP. SAP97-GFP is detected after IP with GFP Ab but not with the CAR specific Ab RmcB or control Ab.

View larger version (82K): [in a new window]   Fig. 6. The CAR interaction with MAGI-1b, PICK1 and PSD-95 depends on the CAR PDZ-binding motif GSIV. (A) shows the immunofluorescent staining of COS-7 cells expressing CAR, CARGSIV, MAGI-1b-GFP, PICK1-GFP, or PSD-95-GFP alone. CAR and CARGSIV primarily localize to the junctions between cells while MAGI-1b-GFP, PICK1-GFP, and PSD-95-GFP show mainly diffuse staining. (B) shows significant colocalization (yellow) of MAGI-1b-GFP, PICK1-GFP or PSD-95-GFP with CAR (red in merged images) upon co-transfection. By contrast, there was no alteration in staining and no significant colocalization between these proteins when co-expressed with CARGSIV (red in merged images).

View larger version (37K): [in a new window]   Fig. 7. CAR co-IP with MAGI-1b, PICK1, and PSD-95. Panel A shows western blots of COS-7 cells co-transfected with CAR and GFP-tagged MAGI-1b, PICK1, or PSD-95. IP was performed using a GFP Ab, to confirm the presence of the PDZ-domain-containing protein, the CAR specific monoclonal Ab RmcB and an unrelated control Ab (IgG or myc). Bands with the appropriate molecular weight were present for each of the PDZ-domain-containing proteins after IP with both GFP and RmcB but not the control Ab, indicating co-IP with CAR. Panel B shows western blots of COS-7 cells co-transfected with MAGI-1b-GFP and CAR or CARGSIV. IP for the FLAG epitope on CAR or CARGSIV resulted in a co-IP between MAGI-1b-GFP and CAR but not CARGSIV.

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