Isoform-specific expression of the Coxsackie and adenovirus receptor (CAR) in neuromuscular junction and cardiac intercalated discs
© Shaw et al; licensee BioMed Central Ltd. 2004
Received: 03 August 2004
Accepted: 08 November 2004
Published: 08 November 2004
The Coxsackie and adenovirus receptor (CAR) has a restricted expression pattern in the adult. In skeletal muscle, although CAR is expressed in immature fibers, its transcript levels are barely detectable in mature muscle. This is in contrast to the robust expression observed in the heart. However, both heart and skeletal muscle are susceptible to infection with the Coxsackie B virus which utilizes primarily CAR for cellular internalization. The specific point of viral entry in skeletal and heart muscle remains unknown.
Using antibodies directed against the extracellular and the cytoplasmic domains of CAR, we show CAR in normal human and mouse skeletal muscle to be a novel component of the neuromuscular junction. In cardiac muscle, CAR immunoreactivity is observed at the level of intercalated discs. We demonstrate a single isoform of CAR to be expressed exclusively at the human neuromuscular junction whereas both predominant CAR isoforms are expressed at the intercalated discs of non-diseased human heart.
The localization of CAR to these important junctional complexes suggests that CAR may play both a structural and a regulatory role in skeletal and cardiac muscle, and that these complexes may serve as a point of entry for Coxsackie B virus.
The Coxsackie and adenovirus receptor (CAR) [1, 2], a transmembrane protein of the immunoglobulin super-family, serves as a receptor for adenovirus (Ad) subgroups A, C, D, E and F  as well as Coxsackie B viruses (CVB) . CAR is a highly conserved protein with two predominant isoforms, produced through differential splicing, and having cytoplasmic domains of either 107 residues (ending in SIV) or 94 residues (ending in TVV) [2, 5]. The extracellular domain mediates homophilic cell adhesion [6–8] and ectopically-expressed CAR localizes to homotypic intercellular contacts . The expression of CAR is regulated developmentally [6, 9–12] as well as in a tissue-specific manner [2, 5]. To date, most studies on CAR expression in the adult have resorted to analysis of transcript levels. These have revealed that the pattern of tissue-specific expression differs between humans and mice. In humans, a predominant transcript of ~6 – 6.5 kb is observed in heart, testis, prostate and pancreas while much less expression is detected in liver, brain, colon and small intestine. In the mouse on the other hand, the most abundant expression is in liver, kidney, lung and heart.
Results and Discussion
Both of the cytoplasmic variants contain a PDZ recognition motif at their distal end, implicating CAR as a putative member of multiprotein complexes. This is indeed the case in polarized epithelial cells in which CAR is expressed at the tight junction where it associates with the tight junction scaffolding protein ZO-1  and contributes to maintenance of transepithelial resistance. The localization of CAR to cardiac intercalated discs is in agreement with CAR having a structural or regulatory role as a transmembrane member of junctional complexes. The intercalated discs are composed of at least three structurally distinct cellular junctions – desmosomes, the adherens junctions, and gap junctions . The SIV and TVV isoforms may localize to separate components of the intercalated disc, or given that CAR has been implicated as a transmembrane component in tight junctions , both isoforms may be localized to the adherens junction, a structure analogous to tight junctions.
It is interesting that a correlation can be drawn between disease incidence and expression levels of the CAR receptor. Cases of viral myocarditis in the human population outnumber those of myositis. This may be attributable to the difference of endogenous CAR expression between the two tissues. Considering that induction of CAR accompanies myocarditis  and its dramatic upregulation has recently been demonstrated in patients suffering from multiple diseases of the heart  including dilated cardiomyopathy, a pathological phenotype linked to persistent acute myocarditis, upregulation of the receptor may render the heart even more susceptible to further viral infection. Conversely, the low level of endogenous CAR expression in skeletal muscle may safeguard against the wide-spread viral infection seen in myocarditis and may be responsible for the less severe clinical features of myositis.
CAR is a novel member of the neuromuscular junction. In cardiac muscle, both CAR isoforms are found at the intercalated discs. The localization of CAR to these important junctional complexes suggests that CAR may play both a structural and a regulatory role in skeletal and cardiac muscle, and that these complexes may serve as a point of entry for Coxsackie B virus.
Figure 1 depicts the sequences used as immunogen to generate the various antibodies. There was no overlap (i.e. common epitopes) between any of the antibodies. The rabbit polyclonal antibodies used in this study were described previously [21, 24]. Briefly, the N-terminal polyclonal antibody (ab 2240) was prepared against a His-tagged fusion protein which encoded amino acid residues 22–208 of the extracellular domain of mouse CAR . This antiserum cross-reacts with human CAR on Western blots and in indirect immunofluorescence. The two C-terminal polyclonal antibodies  were generated using peptides encompassing the last 13 amino acids of the two predominant human CAR isoforms [2, 5]. Antiserum RP194 was raised against the sequence FKYAYKTDGITVV while RP291 was raised against the sequence VMIPAQSKDGSIV. Both these antisera cross-react with the mouse CAR homologs (the peptides are conserved 100% between the two species ). All antisera were affinity purified prior to use.
To raise the chicken anti-CAR antibody (ChCT), purified His-tagged fusion protein encoding the C-terminal portion of CAR that is common to both isoforms (amino acids 259–339) was emulsified in an equal volume of TiterMax Gold adjuvant (CytRx Corp., Norcross, GA) and injected intramuscularly into chickens. One month post-injection, IgY antibodies to CAR were obtained from the eggs of injected chickens and subjected to affinity purification.
Immunolabelling was performed using standard techniques. Briefly, frozen sections (5 μm) of normal human skeletal and cardiac muscle biopsies, murine skeletal and cardiac muscle were fixed in 2% paraformaldehyde (pH 6.8) for 1–2 minutes, followed by overnight incubation at 4°C with the primary antibodies (a 1:30 dilution was used for ab 2240, and 1:200 dilution for the abs RP291 and RP194, in blocking solution made of 3% bovine serum albumin and 0.05% Tween-20 in phosphate-buffered saline). Incubation with a mouse anti-rabbit biotin-conjugated secondary ab (1:120; Jackson Immunoresearch Laboratories, West Grove, PA) was followed by Cy-3-conjugated streptavidin (1:1000; Jackson Immunoresearch Laboratories). Controls consisted of sections treated in the absence of primary antibody. Neuromuscular junctions were revealed with Alexa-488-conjugated-α-bungarotoxin [α BTX] (1:40) (Molecular Probes, Eugene, OR). Slides were viewed on a Leica microscope-based imaging system using OpenLab imaging software (Quorum Technologies, St Catharines, ON).
Western blot analysis and immunoprecipitation
Cardiac muscle tissue was homogenized in extraction buffer [1% Triton X-100; 0.1 mM EDTA; 0.1 mM EGTA; 50 mM Tris-HCl; pH 8.0; with protease inhibitors (Roche)] at 4°C. After a 30 second sonication, samples were centrifuged at 3000 × g for 30 seconds at 4°C. Protein samples (10 μg) were anayzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% (w/v) polyacrylamide gels, followed by electrotransfer to nitrocellulose. The blots were blocked in 10% BLOTTO (skim milk powder) in Tris buffered saline – Tween 20 (TBS-T) for 45 minutes at room temperature. Anti-CAR antibody was added in 10% BLOTTO at a dilution of 1:2500. Following incubation with peroxidase-labeled goat-anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories), the signal was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Baie d'Urfe, QC).
Immunoprecipitation was carried out on cardiac muscle homogenates that had been pre-cleared with Protein-A Agarose slurry (Sigma), followed by overnight incubation at 4°C with RP291 or RP194. The samples were further incubated with Protein-A Agarose for 2 hours, washed twice with extraction buffer and then eluted with 2 X Laemmli SDS sample buffer with 5% mercaptoethanol. Following SDS-PAGE and electrotransfer, nitrocellulose membranes were probed with a primary polyclonal chicken anti-CAR (ChCT) in blocking solution (diluted 1:500) overnight at 4°C. Signal was revealed following incubation with a peroxidase-conjugated donkey anti-chicken IgY (Jackson Immunoresearch Laboratories) at a dilution of 1:2500 for 40 minutes, and enhanced chemiluminescence.
This work was supported by a grant from the Canadian Institutes of Health Research and the Muscular Dystrophy Association (USA). J. N. is a National Research Scholar of the FRSQ and a Killam Scholar.
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