The Role of the Extracellular Domain in the Biology of the Coxsackievirus and Adenovirus Receptor

Abstract

The Coxsackievirus B and Adenovirus Receptor (CAR) plays a dual role as a homotypic junctional adhesion protein and as a viral receptor. CAR is a transmembrane protein and a member of the Immunoglobulin (Ig) superfamily with two extracellular Ig-like domains. The most distal Ig-like domain (D1) mediates the homophilic interaction and is also responsible for the high-affinity binding of the adenovirus (Ad) fiber protein. Currently, no activity has been ascribed to the proximal Ig-like domain (D2). To further understand the function of the extracellular domain in the biological activities of CAR, we created extracellular deletion mutants and evaluated cellular localization, adhesion, and viral infection. Deletion of any segment of the extracellular domain results in loss of adhesion and mislocalization as explained by a model, termed "diffusion trapping," that suggests adhesion is the driving force in junctional localization. Loss of junctional localization and adhesion was particularly apparent in polarized human airway epithelia, where mutant CAR expression was basolateral but not limited to the lateral junctions between cells. Surprisingly, the D2 domain was required for adenovirus fiber-knob binding and infection. In summary, the entire extracellular domain of CAR is of vital importance to the biology of this highly conserved and important protein.

Functional Effects of Coxsackievirus and Adenovirus Receptor Glycosylation on Homophilic Adhesion and Adenoviral Infection

“…We have previously shown that the D2 domain of CAR is important for Ad infection and that deletion of this domain reduces Ad binding and infection significantly (11). Additionally, we hypothesized that this domain played a role only in spacing the D1 domain appropriately from the membrane to facilitate an efficient interaction with the Ad fiber knob.…”

“…CHO-K1 cells (BD Biosciences, Franklin Lakes, NJ) were maintained under standard culture conditions (DMEM and FCS supplemented with tetracycline L-glutamine, penicillin, and streptomycin). Ad serotype 5 containing the ␤-galactosidase (Ad-␤-Gal), green fluorescent protein (peGFP-N1; Clontech, Mountain View, CA), or CAR gene has previously been described (10,11). All recombinant Ads were generated by the University of Iowa Gene Transfer Vector Core.…”

“…Transfected CHO-K1 cells were infected with Ad-␤-Gal (MOI as indicated in figures) for 1 h at 37°C and lysed after 24 h. ␤-Gal expression per milligram of protein was determined, as previously described (10). Binding of Ad was determined by 3 H-labeled Ad as previously described (11). Briefly, cells were transfected as above, washed twice with either ice-cold or room temperature (RT) PBS, incubated with 3 H-labeled Ad at various MOIs for 1 h on ice or at RT.…”

The Coxsackievirus and Adenovirus Receptor: Glycosylation and the Extracellular D2 Domain Are Not Required for Coxsackievirus B3 Infection

“…The chimeric hCAR-D1/IgSF11-D2 domain was inserted into phCAR as described above. The plasmids phCAR N106Q , phCAR N201Q , and phCAR N106/201Q containing the hCAR glycosylation mutants hCAR N106Q , hCAR N201Q , and hCAR N106/201Q , respectively, were generated by site-directed mutagenesis of phCAR following the protocol described previously (31).…”

Cystic Fibrosis Transmembrane Conductance Regulator in Sarcoplasmic Reticulum of Airway Smooth Muscle. Implications for Airway Contractility

“…Although stimulated emission depletion microscopy showed low levels of plasma membraneassociated CFTR-GFP, most CFTR-GFP had a high degree of colocalization with ER-Tracker (Invitrogen, Carlsbad, CA), which has previously been used to label the SR compartment (Figures 2A and 2B) (36,37). As a control, we overexpressed the coxsackievirus and adenovirus receptor (GFP-labeled) (38). In ASM cells, coxsackievirus and adenovirus receptor-GFP predominantly localized to the plasma membrane and we found minimal colocalization with ER-Tracker dye ( Figures 2C and 2D).…”