The Coxsackievirus–Adenovirus Receptor Reveals Complex Homophilic and Heterophilic Interactions on Neural Cells

Abstract

The coxsackievirus-adenovirus receptor (CAR) is a member of the Ig superfamily strongly expressed in the developing nervous system. Our histological investigations during development reveal an initial uniform distribution of CAR on all neural cells with a concentration on membranes that face the margins of the nervous system (e.g., the basal laminae and the ventricular side). At more advanced stages, CAR becomes downregulated and restricted to specific regions including areas rich in axonal and dendritic surfaces.To study the function of CAR on neural cells, we used the fiber knob of the adenovirus, extracellular CAR domains, blocking antibodies to CAR, as well as CAR-deficient neural cells. Blocking antibodies were found to inhibit neurite extension in retina organ and retinal explant cultures, whereas the application of the recombinant fiber knob of the adenovirus subtype Ad2 or extracellular CAR domains promoted neurite extension and adhesion to extracellular matrices.We observed a promiscuous interaction of CAR with extracellular matrix glycoproteins, which was deduced from analytical ultracentrifugation experiments, affinity chromatography, and adhesion assays. The membrane proximal Ig domain of CAR, termed D2, was found to bind to a fibronectin fragment, including the heparin-binding domain 2, which promotes neurite extension of wild type, but not of CAR-deficient neural cells. In contrast to heterophilic interactions, homophilic association of CAR involves both Ig domains, as was revealed by ultracentrifugation, chemical cross-linking, and adhesion studies. The results of these functional and binding studies are correlated to a U-shaped homodimer of the complete extracellular domains of CAR detected by x-ray crystallography.

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

“…Although we cannot exclude the possibility that some shared D2 sequences participate in virus binding, the sequence identity among these domains is quite low (31% for CAR and IgSF11), and most of the identical residues are remote from D1 in the available crystal structure (data not shown) (10). We believe it more likely that replacement of CAR-D2 with D2 domains of CD55 or CD80 affected the structure or orientation of CAR-D1.…”

“…Whereas D1 shows a typical V-type fold structure, D2 has a C2-type immunoglobulin fold (5,6). Several studies indicate a primary role for the D1 domain in CAR interactions with CVB3 (7) and adenovirus (8), as well as in CAR/CAR homophilic interactions (9)(10)(11). Although the isolated D1 domain, produced in Escherichia coli, binds adenovirus efficiently (8), the same D1 domain was found to bind poorly to CVB3 (7), suggesting a possible supporting role for the D2 domain during CAR/ CVB3 interaction.…”

Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells

“…The extracellular N-terminal domain of CAR is known to exist as a homo-dimer in solution and various biochemical evidence suggest this may contribute to cis and trans interactions between receptors, i.e. within the same cell and across cell-cell junctions [51][52][53][54]. However, the receptor state in intact cells and the potential role of self-association in controlling cell-cell adhesion and adenovirus docking is currently unknown [54,55].…”

“…within the same cell and across cell-cell junctions [51][52][53][54]. However, the receptor state in intact cells and the potential role of self-association in controlling cell-cell adhesion and adenovirus docking is currently unknown [54,55]. We have therefore applied combined FRAP, FLIM, tr-FAIM microscopy to investigate the dynamics and dimerisation of CAR in living cells.…”

Kinetic and Structural Analysis of Coxsackievirus B3 Receptor Interactions and Formation of the A-Particle

“…CAR is comprised of an extracellular domain folded into subdomains, D1 and D2, a single membrane-spanning sequence, and a C-terminal intracellular domain. Crystal structures for both human CAR D1 (PDB ID 1F5W and 1KAC) and murine CAR D1D2 (PDB ID 3MJ7 and 3JZ7) have been reported (29)(30)(31)(32).…”