Functional effects of coxsackievirus and adenovirus receptor glycosylation on homophilic adhesion and adenoviral infection

The coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and homophilic adhesion protein. The extracellular portion of CAR consists of two immunoglobulin (Ig)-like domains, each with a consensus sequence for N-glycosylation. We used chemical, genetic, and biochemical studies to show that both sites are glycosylated and contribute to the function of CAR. Although the glycosylation of CAR does not alter cell surface levels or junctional localization, it affects both adhesion and adenovirus infection in unique ways. CAR-mediated adhesion appears to require at least one site of glycosylation since cells expressing CAR without glycosylation do not cluster with each other. In contrast, glycosylation of the Ig-like domain proximal to the membrane is key to the cooperative behavior of adenovirus binding and infection. Contrary to the hypothesis that cooperativity improves viral infection, our data show that although glycosylation of the D2 domain is required for adenovirus cooperative binding, it has a negative consequence upon infection. This is the first report dissecting the adhesion and receptor activities of CAR, revealing that factors other than the binding interface play a significant role in the function of CAR. These data have important implications for both cancers with altered glycosylation states and cancer treatments using oncolytic adenovirus.     

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

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.     

Lipid raft localization of GABA A receptor and Na+, K+-ATPase in discrete microdomain clusters in rat cerebellar granule cells

The microdomain localization of the GABA(A) receptor in rat cerebellar granule cells was studied by subcellular fractionation and fluorescence- and immunogold electron microscopy. The receptor resided in lipid rafts, prepared at 37 degrees C by extraction with the nonionic detergent Brij 98, but the raft fraction, defined by the marker ganglioside GM(1) in the floating fractions following density gradient centrifugation, was heterogeneous in density and protein composition. Thus, another major raft-associated membrane protein, the Na(+), K(+)-ATPase, was found in discrete rafts of lower density, reflecting clustering of the two proteins in separate membrane microdomains. Both proteins were observed in patchy "hot spots" at the cell surface as well as in isolated lipid rafts. Their insolubility in Brij 98 was only marginally affected by methyl-beta-cyclodextrin. In contrast, both the GABA(A) receptor and Na(+), K(+)-ATPase were largely soluble in ice cold Triton X-100. This indicates that Brij 98 extraction defines an unusual type of cholesterol-independent lipid rafts that harbour membrane proteins also associated with underlying scaffolding/cytoskeletal proteins such as gephyrin (GABA(A) receptor) and ankyrin G (Na(+), K(+)-ATPase). By providing an ordered membrane microenvironment, lipid rafts may contribute to the clustering of the GABA(A) receptor and the Na(+), K(+)-ATPase at distinct functional locations on the cell surface.     

The coxsackie- and adenovirus receptor (CAR) is an in vivo marker for epithelial tight junctions, with a potential role in regulating permeability and tissue homeostasis

The coxsackie- and adenovirus receptor (CAR) is a transmembrane protein belonging to the immunoglobulin superfamily. The function of CAR as a virus receptor has been extensively analyzed, while its physiological role and expression pattern in adult tissues have remained less clear. CAR associates with epithelial tight junctions in vitro and mediates cell-cell adhesion. Using a set of affinity-purified antibodies, we show that CAR is predominantly expressed in epithelial cells lining the body cavities in adult mice, where it specifically co-localizes with the tight junction components ZO-1 and occludin. Notably, CAR could not be detected in endothelial cells of the vasculature, including brain capillaries. CAR expression correlated positively with the maturity of tight junctions and inversely with permeability. With a few exceptions, the two known CAR isoforms were co-expressed in most epithelial cells analyzed. A CAR mutant lacking the intracellular tail over-expressed in transgenic mice was diffusely localized over the plasma membrane, showing the importance of this domain for correct subcellular localization in vivo. We conclude that CAR is localized to epithelial tight junctions in vivo where it may play a role in the regulation of epithelial permeability and tissue homeostasis.     

Fatty acid modification of the coxsackievirus and adenovirus receptor

Membrane-proximal cysteines 259 and 260 in the cytoplasmic tail of the coxsackievirus and adenovirus receptor (CAR) are known to be essential for the tumor suppression activity of CAR. We demonstrate that these residues provide an S-acylation motif for modification of CAR with the fatty acid palmitate. Substitution of alanine for cysteines 259 and 260 results in the additional localization of CAR in perinuclear compartments with no effect on the efficiency of adenovirus infection. The results indicate that palmitylation is important for stable plasma membrane expression and biological activity of CAR but is not critical for adenovirus receptor performance.     

Direct visualization of Ras proteins in spatially distinct cell surface microdomains

Localization of signaling complexes to specific microdomains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains, including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.     

Effects of integrin-mediated cell adhesion on plasma membrane lipid raft components and signaling

Anchorage dependence of cell growth, which is mediated by multiple integrin-regulated signaling pathways, is a key defense against cancer metastasis. Detachment of cells from the extracellular matrix triggers caveolin-1-dependent internalization of lipid raft components, which mediates suppression of Rho GTPases, Erk, and phosphatidylinositol 3-kinase in suspended cells. Elevation of cyclic adenosine monophosphate (cAMP) following cell detachment is also implicated in termination of growth signaling in suspended cells. Studies of integrins and lipid rafts, however, examined mainly ganglioside GM1 and glycosylphosphatidylinositol-linked proteins as lipid raft markers. In this study, we examine a wider range of lipid raft components. Whereas many raft components internalized with GM1 following cell detachment, flotillin2, connexin43, and Gα(s) remained in the plasma membrane. Loss of cell adhesion caused movement of many components from the lipid raft to the nonraft fractions on sucrose gradients, although flotillin2, connexin43, and H-Ras were resistant. Gα(s) lost its raft association, concomitant with cAMP production. Modification of the lipid tail of Gα(s) to increase its association with ordered domains blocked the detachment-induced increase in cAMP. These data define the effects of that integrin-mediated adhesion on the localization and behavior of a variety of lipid raft components and reveal the mechanism of the previously described elevation of cAMP after cell detachment.     

Sorting motifs in the intracellular domain of the low density lipoprotein receptor interact with a novel domain of sorting nexin-17

The low density lipoprotein (LDL) receptor plays a major role in maintaining human plasma cholesterol levels and mutations in the gene cause familial hypercholesterolemia. The LDL receptor (LDLR) pathway has been well characterized, but little is known of proteins involved in its complex intracellular sorting and trafficking. Sorting nexin 17 (SNX17) has recently been implicated in LDLR intracellular trafficking. We show here that endogenous SNX17 is highly expressed in several cell types and is localized partially in early endosomes. We found that the PX domain of SNX17 is required for its endosomal localization but does not interact directly with the LDL receptor. A novel domain containing a FERM-like domain of SNX17 is needed for its interaction with the LDL receptor. Mutations in the NPXY motif of the LDL-receptor cytoplasmic tail that disrupt internalization also disrupt its interaction with SNX17, whereas mutations elsewhere had little effect. When transiently overexpressed in Chinese hamster ovary cells, SNX17 localized to large vesicular structures and disrupted normal trafficking of the LDL receptor in a PX domain-dependent manner. These results suggest that SNX17 plays a role in the cellular trafficking of the LDL receptor through interaction with the NPVY motif in its cytoplasmic domain and interaction of the PX domain with subcellular membrane compartments.     

Identification of phospholipid scramblase 1 as a novel interacting molecule with beta -secretase (beta -site amyloid precursor protein (APP) cleaving enzyme (BACE))

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) is an integral membrane aspartic proteinase responsible for beta-site processing of APP, and its cytoplasmic region composed of 24 amino acid residues has been shown to be involved in the endosomal localization of BACE. With the yeast two-hybrid screening, we found that the cytoplasmic domain of phospholipid scramblase 1 (PLSCR1), a type II integral membrane protein, interacts with the cytoplasmic region of BACE. In cultured cells, BACE and PLSCR1 were colocalized in the Golgi area and in endosomal compartments, whereas they were co-redistributed in late endosome-derived multivesicular bodies when treated with U18666A, suggesting that both proteins share a common trafficking pathway in cells. Co-immunoprecipitation analysis showed that both proteins form a protein complex at an endogenous expression level in the human neuroblastoma SH-SY5Ycells, and the dileucine residue of the BACE tail is also revealed to be essential for the physical interaction with PLSCR1 in vitro and in vivo. Moreover, both BACE and PLSCR1 were localized in a low buoyant lipid microdomain in SH-SY5Y cells. The dileucine-defective BACE mutant was also fractionated into the lipid microdomain, but much less stably than wild-type BACE. Taken together, our current study suggests the functional involvement of PLSCR1 in the intracellular distribution of BACE and/or recruitment of BACE into the detergent-insoluble lipid raft.     

Molecular characterization of the detergent-insoluble cholesterol-rich membrane microdomain (raft) of the central nervous system

Many fundamental neurological issues such as neuronal polarity, the formation and remodeling of synapses, synaptic transmission, and the pathogenesis of the neuronal cell death are closely related to the membrane dynamics. The elucidation of functional roles of a detergent-insoluble cholesterol-rich domain (raft) could therefore provide good clues to the molecular understanding of these important phenomena, for the participation of the raft in the fundamental cell functions, such as signal transduction and selective transport of lipids and proteins, has been elucidated in nonneural cells. Interestingly, the brain is rich in raft and the brain-derived raft differs in its lipid and protein components from other tissue-derived rafts. Since many excellent reviews are written on the membrane lipid dynamics of this microdomain, signal transduction, and neuronal glycolipids, we review on the characterization of the raft proteins recovered in the detergent-insoluble low-density fraction from rat brain. Special focus is addressed on the biochemical characterization of a neuronal enriched protein, NAP-22, for the lipid organizing activity of this protein has become increasingly clear.     

Molecular characterization of the detergent-insoluble cholesterol-rich membrane microdomain (raft) of the central nervous system

Many fundamental neurological issues such as neuronal polarity, the formation and remodeling of synapses, synaptic transmission, and the pathogenesis of the neuronal cell death are closely related to the membrane dynamics. The elucidation of functional roles of a detergent-insoluble cholesterol-rich domain (raft) could therefore provide good clues to the molecular understanding of these important phenomena, for the participation of the raft in the fundamental cell functions, such as signal transduction and selective transport of lipids and proteins, has been elucidated in nonneural cells. Interestingly, the brain is rich in raft and the brain-derived raft differs in its lipid and protein components from other tissue-derived rafts. Since many excellent reviews are written on the membrane lipid dynamics of this microdomain, signal transduction, and neuronal glycolipids, we review on the characterization of the raft proteins recovered in the detergent-insoluble low-density fraction from rat brain. Special focus is addressed on the biochemical characterization of a neuronal enriched protein, NAP-22, for the lipid organizing activity of this protein has become increasingly clear.     

The receptor-bound N-terminal ectodomain of the amyloid precursor protein is associated with membrane rafts

The soluble N-terminal ectodomain of amyloid precursor protein (sAPP), resulting from alpha-secretase-mediated proteolytic processing, has been shown to function as a growth factor for epithelial cells, including keratinocytes and thyrocytes. Extracellularly applied sAPP binds to a cell surface receptor and exhibits a patchy binding pattern reminiscent of that observed for raft proteins. Here we show that (i) the receptor-bound sAPP resides in a detergent-insoluble membrane microdomain which cofractionates in density gradients with cholesterol-rich membrane rafts and caveolae; (ii) the sAPP-binding microdomains are different from caveolae; and (iii) sAPP is capable of binding to isolated rafts and inducing tyrosine phosphorylation of some raft proteins. These observations suggest that a novel type of membrane raft is involved in sAPP signaling.     

The cell surface protein coxsackie- and adenovirus receptor (CAR) directly associates with the Ligand-of-Numb Protein-X2 (LNX2)

The coxsackievirus and adenovirus receptor (CAR) is a cell surface protein that is proposed to be involved in cell-cell adhesion. Based on a yeast two-hybrid screen, co-immunoprecipitation and binding experiments, the intracellular tail of CAR was found to interact both in vivo and in vitro with the Ligand-of-Numb Protein-X2 (LNX2). The interacting domains between the two proteins were identified by truncation analyses and affinity chromatography. CAR and LNX2 protein expression in embryonic mouse tissues was analyzed by immunohistochemistry. The results suggest that CAR is a partner in a protein complex organized at specific subcellular sites by LNX2.     

The PDZ1 and PDZ3 Domains of MAGI-1 Regulate the Eight-Exon Isoform of the Coxsackievirus and Adenovirus Receptor

Epithelial integrity is essential for homeostasis and poses a formidable barrier to pathogen entry. Major factors for viral entry into epithelial cells are the localization and abundance of the primary receptor. The coxsackievirus and adenovirus receptor (CAR) is a primary receptor for these two pathogenic groups of viruses. In polarized epithelia, a low-abundance, alternatively spliced eight-exon isoform of CAR, CAR(Ex8), is localized apically where it can support viral infection from the air-exposed surface. Using biochemical, cell biology, genetic, and spectroscopic approaches, we show that the levels of apical CAR(Ex8) are negatively regulated by the PDZ domain-containing protein MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1) and that two MAGI-1 PDZ domains, PDZ1 and PDZ3, regulate CAR(Ex8) levels in opposing ways. Similar to full-length MAGI-1, expression of the isolated PDZ3 domain significantly reduces cell surface CAR(Ex8) abundance and adenovirus infection. In contrast, the PDZ1 domain is able to rescue CAR(Ex8) and adenovirus infection from MAGI-1-mediated suppression. These data suggest a novel cell-based strategy to either suppress viral infection or augment adenovirus-based gene therapy.     

A novel approach to examining compositional heterogeneity of detergent-resistant lipid rafts

Lipid rafts play an important role in cell signalling, cell adhesion and other cellular functions. Compositional heterogeneity of lipid rafts provides one mechanism of how lipid rafts provide the spatial and temporal regulation of cell signalling and cell adhesion. The constitutive presence of some signalling receptors/molecules and accumulation of others in the lipid raft allows them to interact with each other and thereby facilitate relay of signals from the plasma membrane to the cell interior. Devising a method that can analyze these lipid microdomains for the presence of signalling receptors/molecules on an individual raft basis is required to address the issue of lipid raft heterogeneity. SDS-PAGE analysis, currently used for analyses of detergent-resistant lipid rafts, does not address this question. We have designed a cell-free assay that captures detergent-resistant lipid rafts with an antibody against a raft-resident molecule and detects the presence of another lipid raft molecule. Our results suggest that detergent-resistant lipid rafts, also known as detergent-resistant membranes, are heterogeneous populations on an immortalized mouse T-cell plasma membrane with respect to antigen receptor/signalling complex and other signalling/adhesion proteins. This cell-free assay provides a simple and quick way to examine the simultaneous presence of two proteins in the lipid rafts and has the potential to estimate trafficking of molecules in and out of the lipid microdomains during cell signalling on a single detergent-resistant lipid raft basis.     

Dimeric structure of the coxsackievirus and adenovirus receptor D1 domain at 1.7 A resolution

BACKGROUND: The coxsackievirus and adenovirus receptor (CAR) comprises two extracellular immunoglobulin domains, a transmembrane helix and a C-terminal intracellular domain. The amino-terminal immunoglobulin domain (D1) of CAR is necessary and sufficient for adenovirus binding, whereas the site of coxsackievirus attachment has not yet been localized. The normal cellular role of CAR is currently unknown, although CAR was recently proposed to function as a homophilic cell adhesion molecule. RESULTS: The human CAR D1 domain was bacterially expressed and crystallized. The structure was solved by molecular replacement using the structure of CAR D1 bound to the adenovirus type 12 fiber head and refined to 1.7 A resolution, including individual anisotropic temperature factors. The two CAR D1 structures are virtually identical, apart from the BC, C"D, and FG loops that are involved both in fiber head binding and homodimerization in the crystal. Analytical equilibrium ultracentrifugation shows that a dimer also exists in solution, with a dissociation constant of 16 microM. CONCLUSIONS: The CAR D1 domain forms homodimers in the crystal using the same GFCC'C" surface that interacts with the adenovirus fiber head. The homodimer is very similar to the CD2 D1-CD58 D1 heterodimer. CAR D1 also forms dimers in solution with a dissociation constant typical of other cell adhesion complexes. These results are consistent with reports that CAR may function physiologically as a homophilic cell adhesion molecule in the developing mouse brain. Adenovirus may thus have recruited an existing and conserved interaction surface of CAR to use for its own cell attachment.     

Disruption of the Coxsackievirus and Adenovirus Receptor-Homodimeric Interaction Triggers Lipid Microdomain- and Dynamin-dependent Endocytosis and Lysosomal Targeting

The coxsackievirus and adenovirus receptor (CAR) serves as a docking factor for some adenovirus (AdV) types and group B coxsackieviruses. Its role in AdV internalization is unclear as studies suggest that its intracellular domain is dispensable for some AdV infection. We previously showed that in motor neurons, AdV induced CAR internalization and co-transport in axons, suggesting that CAR was linked to endocytic and long-range transport machineries. Here, we characterized the mechanisms of CAR endocytosis in neurons and neuronal cells. We found that CAR internalization was lipid microdomain-, actin-, and dynamin-dependent, and subsequently followed by CAR degradation in lysosomes. Moreover, ligands that disrupted the homodimeric CAR interactions in its D1 domains triggered an internalization cascade involving sequences in its intracellular tail.     

Multiple regions within the coxsackievirus and adenovirus receptor cytoplasmic domain are required for basolateral sorting

The coxsackievirus and adenovirus receptor (CAR) mediates attachment and infection by coxsackie B viruses and many adenoviruses. In human airway epithelia, as well as in transfected Madin-Darby canine kidney cells, CAR is expressed exclusively on the basolateral surface. Variants of CAR that lack the cytoplasmic domain or are attached to the cell membrane by a glycosylphosphatidylinositol anchor are expressed on both the apical and basolateral surfaces. We have examined the localization of CAR variants with progressive truncations of the cytoplasmic domain, as well as with mutations that ablate a potential PDZ (PSD95/dlg/ZO-1) interaction motif and a putative tyrosine-based sorting signal. In addition, we have examined the targeting of two murine CAR isoforms, with different C-terminal sequences. The results suggest that multiple regions within the CAR cytoplasmic domain contain information that is necessary for basolateral targeting.     

Apical Localization of the Coxsackie-Adenovirus Receptor by Glycosyl-Phosphatidylinositol Modification Is Sufficient for Adenovirus-Mediated Gene Transfer through the Apical Surface of Human Airway Epithelia

In well-differentiated human airway epithelia, the coxsackie B and adenovirus type 2 and 5 receptor (CAR) resides primarily on the basolateral membrane. This location may explain the observation that gene transfer is inefficient when adenovirus vectors are applied to the apical surface. To further test this hypothesis and to investigate requirements and barriers to apical gene transfer to differentiated human airway epithelia, we expressed CAR in which the transmembrane and cytoplasmic tail were replaced by a glycosyl-phosphatidylinositol (GPI) anchor (GPI-CAR). As controls, we expressed wild-type CAR and CAR lacking the cytoplasmic domain (Tailless-CAR). All three constructs enhanced gene transfer with similar efficiencies in fibroblasts. In airway epithelia, GPI-CAR localized specifically to the apical membrane, where it bound adenovirus and enhanced gene transfer to levels obtained when vector was applied to the basolateral membrane. Moreover, GPI-CAR facilitated gene transfer of the cystic fibrosis transmembrane conductance regulator to cystic fibrosis airway epithelia, correcting the Cl(-) transport defect. In contrast, when we expressed wild-type CAR it localized to the basolateral membrane and failed to increase apical gene transfer. Only a small amount of Tailless-CAR resided in the apical membrane, and the effects on apical virus binding and gene transfer were minimal. These data indicate that binding of adenovirus to an apical membrane receptor is sufficient to mediate effective gene transfer to human airway epithelia and that the cytoplasmic domain of CAR is not required for this process. The results suggest that targeting apical receptors in differentiated airway epithelia may be sufficient for gene transfer in the genetic disease cystic fibrosis.     

Isoform-Specific Regulation and Localization of the Coxsackie and Adenovirus Receptor in Human Airway Epithelia

Adenovirus is an important respiratory pathogen. Adenovirus fiber from most serotypes co-opts the Coxsackie-Adenovirus Receptor (CAR) to bind and enter cells. However, CAR is a cell adhesion molecule localized on the basolateral membrane of polarized epithelia. Separation from the lumen of the airways by tight junctions renders airway epithelia resistant to inhaled adenovirus infection. Although a role for CAR in viral spread and egress has been established, the mechanism of initial respiratory infection remains controversial. CAR exists in several protein isoforms including two transmembrane isoforms that differ only at the carboxy-terminus (CAR^Ex7 and CAR^Ex8). We found low-level expression of the CAR^Ex8 isoform in well-differentiated human airway epithelia. Surprisingly, in contrast to CAR^Ex7, CAR^Ex8 localizes to the apical membrane of epithelia where it augments adenovirus infection. Interestingly, despite sharing a similar class of PDZ-binding domain with CAR^Ex7, CAR^Ex8 differentially interacts with PICK1, PSD-95, and MAGI-1b. MAGI-1b appears to stoichiometrically regulate the degradation of CAR^Ex8 providing a potential mechanism for the apical localization of CAR^Ex8 in airway epithelial. In summary, apical localization of CAR^Ex8 may be responsible for initiation of respiratory adenoviral infections and this localization appears to be regulated by interactions with PDZ-domain containing proteins.