F3/contactin,a neuronal cell adhesion molecule implicated in axogenesis and myelination

A general feature of the cell adhesion molecules belonging to the immunoglobulin family (Ig-CAMs) is to display a modular structure that provides a framework for multiple binding sites for other recognition molecules. Among this family, F3/contactin is a glycan phosphatidyl-inositol (GPI)-anchored molecule expressed by neurons that displays the distinctiveness to exert heterophilic but no homophilic binding activities. The Ig domains of F3/contactin were shown to interact with the L1 family of Ig-CAMs, including L1, NrCAM, and neurofascin. Binding between F3/contactin and NrCAM is known to modulate axonal elongation of the cerebellar granule cells and to control sensory axon guidance. F3/contactin mediates neuron-glial contacts through its association with extracellular matrix components (tenascin-R, tenascin-C) and RPTPbeta/phosphacan, influencing axonal growth and fasciculation. Another major role of F3/contactin is to organize axonal subdomains at the node of Ranvier of myelinated fibers in interplay with other Ig-CAMs, through its binding with caspr/paranodin at paranodes and the voltage-gated sodium channels in the nodal region. The F3/contactin deficient mice display a severe ataxia correlated with defects in axonal and dendritic projections in the cerebellum. These mice also display defects in nerve influx conduction due to the disruption of the axo-glial contacts at paranodes. Finally, the recent identification of a Drosophila homologue of F3/contactin indicated that this family of GPI-anchored CAMs plays a conserved function in axonal insulation.     

Association between the first two immunoglobulin-like domains of the neural cell adhesion molecule N-CAM.

The extracellular domain of N-CAM contains five immunoglobulin-like (Ig) and two fibronectin type III-like domains and facilitates cell-cell binding through multiple, weak interdomain interactions. NMR spectroscopy indicated that the two N-terminal Ig-like domains from chicken N-CAM (Ig I and Ig II) interact with millimolar affinity. Physico-chemical studies show that this interaction is significantly amplified when the domains are covalently linked, consistent with an antiparallel domain arrangement. The binding of the two individual domains and the dimerization of the concatenated protein were essentially independent of salt, up to a concentration of 200 mM. The residues in Ig I involved in the interaction map to the BED strands of the beta sandwich, and delineate a largely hydrophobic patch.     

Interaction of the immunoglobulin-like cell adhesion molecule hepaCAM with caveolin-1

Subsequent to our identification of the novel immunoglobulin-like cell adhesion molecule hepaCAM, we demonstrated that hepaCAM is capable of modulating cell growth and cell–extracellular matrix interactions. In this study, we examined the localization of hepaCAM in lipid rafts/caveolae as well as the interaction of hepaCAM with the caveolar structural protein caveolin-1 (Cav-1). Our results revealed that a portion of hepaCAM resided in detergent-resistant membranes and co-partitioned with Cav-1 to low buoyant density fractions characteristic of lipid rafts/caveolae. In addition, co-localization and coimmunoprecipitation assays confirmed the association of hepaCAM with Cav-1. Deletion analysis of hepaCAM showed that the extracellular first immunoglobulin domain of hepaCAM was required for binding Cav-1. Furthermore, when co-expressed, Cav-1 induced the expression of hepaCAM as well as distributed hepaCAM to intracellular Cav-1-positive caveolar structures. Taken together, our findings indicate that hepaCAM is partially localized in the lipid rafts/caveolae and interacts with Cav-1 through its first immunoglobulin domain.     

The neural cell adhesion molecule binds to fibroblast growth factor receptor 2

The neural cell adhesion molecule (NCAM) can bind to and activate fibroblast growth factor receptor 1 (FGFR1). However, there are four major FGFR isoforms (FGFR1-FGFR4), and it is not known whether NCAM also interacts directly with the other three FGFR isoforms. In this study, we show by surface plasmon resonance analysis that NCAM can bind to FGFR2 with an affinity similar to that for the NCAM-FGFR1 interaction. However, the kinetic parameters for the NCAM-FGFR2 binding are different from those of the NCAM-FGFR1 binding. Both receptors were shown to cycle relatively fast between the NCAM bound and unbound states, although FGFR2 cycling was clearly faster (13 times) than the FGFR1 cycling. Moreover, ATP was more effective in inhibiting the binding of NCAM to FGFR1 than to FGFR2, indicating that the binding sites in NCAM for the two receptors are similar, but not identical.     

The long isoform of the cell adhesion molecule C-CAM binds to actin

C-CAM is a member of the carcinoembryonic antigen family (CEA) of the rat, which mediates cell adhesion in vitro and binds to signal transduction molecules. In many tissues C-CAM is expressed in the apical domain of the plasma membrane in close contact with intracellular cortical microfilaments, e.g., in the microvilli of the brush borders of enterocytes. Regarding this subcellular localisation, we have investigated the C-CAM interaction with the cytoskeleton. The association of C-CAM with detergent-insoluble structures increased when the small intestinal mucosa was extracted under conditions known to preserve the cytoskeleton of the brush borders. We found a co-immunoprecipitation of actin with C-CAM of the small intestine mucosa which increased in the presence of the chemical cross-linker DSP, allowing the demonstration of complexes between C-CAM and actin of different molecular masses. A recombinant fusion protein of the cytoplasmic domain of the long isoform of C-CAM bound specifically to purified actin in a co-sedimentation assay. These results suggest an intrinsic actin-binding activity of C-CAM.     

The glycosylphosphatidyl inositol-anchored adhesion molecule F3/contactin is required for surface transport of paranodin/contactin-associated protein (caspr)

Paranodin/contactin-associated protein (caspr) is a transmembrane glycoprotein of the neurexin superfamily that is highly enriched in the paranodal regions of myelinated axons. We have investigated the role of its association with F3/contactin, a glycosylphosphatidyl inositol (GPI)-anchored neuronal adhesion molecule of the Ig superfamily. Paranodin was not expressed at the cell surface when transfected alone in CHO or neuroblastoma cells. Cotransfection with F3 resulted in plasma membrane delivery of paranodin, as analyzed by confocal microscopy and cell surface biotinylation. The region that mediates association with paranodin was mapped to the Ig domains of F3 by coimmunoprecipitation experiments. The association of paranodin with F3 allowed its recruitment to Triton X-100-insoluble microdomains. The GPI anchor of F3 was necessary, but not sufficient for surface expression of paranodin. F3-Ig, a form of F3 deleted of the fibronectin type III (FNIII) repeats, although GPI-linked and expressed at the cell surface, was not recovered in the microdomain fraction and was unable to promote cell surface targeting of paranodin. Thus, a cooperative effect between the GPI anchor, the FNIII repeats, and the Ig regions of F3 is required for recruitment of paranodin into lipid rafts and its sorting to the plasma membrane.     

The paranodal complex of F3/contactin and caspr/paranodin traffics to the cell surface via a non-conventional pathway

During myelination, membrane-specialized domains are generated by complex interactions between axon and glial cells. The cell adhesion molecules caspr/paranodin and F3/contactin play a crucial role in the generation of functional septate-like junctions at paranodes. We have previously demonstrated that association with the glycosylphosphatidylinositol-linked F3/contactin is required for the recruitment of caspr/paranodin into the lipid rafts and its targeting to the cell surface. When transfected alone in neuroblastoma N2a cells, caspr/paranodin is retained in the endoplasmic reticulum (ER). Using chimerical constructs, we show that the cytoplasmic region does not contain any ER retention signal, whereas the ectodomain plays a crucial role in caspr/paranodin trafficking. A series of truncations encompassing the extracellular region of caspr/paranodin was unable to abolish ER retention. We show that N-glycosylation and quality control by the lectin-chaperone calnexin are required for the cell surface delivery of caspr/paranodin. Cell surface transport of F3/contactin and caspr/paranodin is insensitive to brefeldin A and the two glycoproteins are endoglycosidase H-sensitive when associated in complex, recruited into the lipid rafts, and expressed on the cell surface. Our results indicate a Golgi-independent pathway for the paranodal cell adhesion complex that may be implicated in the segregation of axonal subdomains.     

Neural cell recognition molecule F11: homology with fibronectin type III and immunoglobulin type C domains

We report here the complete cDNA sequence of F11 130 kd polypeptide, a chick neural cell surface-associated glycoprotein implicated in neurite fasciculation and elongation. The predicted protein sequence of 1010 amino acids includes an amino-terminal signal peptide and a carboxy-terminal hydrophobic stretch, which is compatible with the consensus motif for covalent attachment of glycosyl-phosphatidylinositol. Accordingly, F11 lacks an intracellular domain, which is consistent with evidence obtained from protease protection experiments on isolated microsomes. In addition, the molecule comprises six domains related to the immunoglobulin domain type C and four resembling fibronectin repeat type III. Both types of repeats resemble those present in neural cell adhesion molecules L1 and N-CAM. The possible identity of F11 with the chick neural glycoprotein contactin is discussed.     

Neural cell adhesion molecule modulates mesenchymal stromal cell migration via activation of MAPK/ERK signaling

Mesenchymal Stromal Cells (MSCs) represent promising tools for cellular therapy owing to their multipotentiality and ability to localize to injured, inflamed sites and tumor. Various approaches to manipulate expression of MSC surface markers, including adhesion molecules and chemokine receptors, have been explored to enhance homing of MSCs. Recently, Neural Cell Adhesion Molecule (NCAM) has been found to be expressed on MSCs yet its function remains largely elusive. Herein, we show that bone marrow-derived MSCs from NCAM deficient mice exhibit defective migratory ability and significantly impaired adipogenic and osteogenic differentiation potential. We further explore the mechanism governing NCAM mediated migration of MSCs by showing the interplay between NCAM and Fibroblast Growth Factor Receptor (FGFR) induces activation of MAPK/ERK signaling, thereby the migration of MSCs. In addition, re-expression of NCAM180, but not NCAM140, could restore the defective MAPK/ERK signaling thereby the migration of NCAM deficient MSCs. Finally, we demonstrate that NCAM180 expression level could be manipulated by pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-α treatment. Overall, our data reveal the vital function of NCAM in MSCs migration and differentiation thus raising the possibility of manipulating NCAM expression to enhance homing and therapeutic potential of MSCs in cellular therapy.     

Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein

We have isolated a novel actin filament-binding protein, named afadin, localized at cadherin-based cell-cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament-binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor-related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell-cell AJs in various tissues and cell lines. In E-cadherin-expressing EL cells, PRR was recruited to cadherin-based cell-cell AJs through interaction with afadin. PRR showed Ca2+-independent cell-cell adhesion activity. These results indicate that PRR is a cell-cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell-cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word "necto" meaning "to connect").     

Protein F: an adhesin of Streptococcus pyogenes binds fibronectin via two distinct domains

The binding of Streptococcus pyogenes to fibronectin (FN) enables the adherence of this pathogen to target epithelial cells, which is the first necessary step for initiation of infection. Binding is mediated by a bacterial surface protein termed protein F. Here we provide the complete structure of protein F and identify two domains responsible for binding to fibronectin. The first domain is located towards the C-terminal end of the molecule and is composed of five repeats of 37 amino acids that are completely repeated four times and a fifth time partially. The second domain is adjacent to the first domain and is located on the /V-terminal side of it. It is composed of a single stretch of 43 amino acids. Protein F expressed in Escherichia coli completely blocked the binding of fibronectin to S. pyogenes. However, mutant proteins that contained only one or the other of the two domains were only capable of partial blockage of binding. Complete blockage of binding of fibronectin could be achieved when a protein extract containing the N-terminal domain was mixed in a binding reaction with a protein extract containing the C-terminal domain. Similarly, a purified recombinant protein containing the two domains only, blocked the binding completely. In contrast, a purified recombinant protein containing just the C-terminal domain, blocked the binding partially. A clone exclusively expressing the C-terminal domain, completely blocked the binding of the 30 kDa N-terminal fragment of fibronectin to S. pyogenes, whereas a clone expressing the N-terminal domain failed to block the binding of this FN fragment. Thus, the two FN-binding domains of protein F are necessary for maximal bacterial binding and act in concert to enhance the binding to fibronectin. The possibility that the two domains bind to two different regions on the fibronectin molecule is discussed.     

Endothelial adhesion molecule ESAM binds directly to the multidomain adaptor MAGI-1 and recruits it to cell contacts

Endothelial cell-selective adhesion molecule (ESAM) is an immunoglobulin-like transmembrane protein associated with endothelial tight junctions (TJ). Based on a yeast two-hybrid screen, we have identified the membrane-associated guanylate kinase protein MAGI-1 as an intracellular binding partner of ESAM. MAGI-1 is a multidomain adaptor protein, which binds to transmembrane, cytoskeletal, and signaling molecules, and has been localized to tight junctions in epithelial cells. MAGI-1 associates with the very C-terminal sequence of ESAM most likely through a PDZ domain-mediated interaction. The direct interaction between ESAM and MAGI-1 was confirmed by pull-down experiments. The two proteins formed stable complexes in transfected Chinese hamster ovary (CHO) cells, which could be immunoisolated. We found MAGI-1 to be associated with cell-cell contacts in human umbilical vein endothelial cells (HUVECs) and in mouse endothelium, where it colocalizes with ESAM. In CHO cells, recruitment of MAGI-1 to cell contacts required the presence of ESAM. Hence, ESAM may be involved in anchoring MAGI-1 at endothelial tight junctions.     

Transfected F3/F11 neuronal cell surface protein mediates intercellular adhesion and promotes neurite outgrowth

The mouse neuronal F3 glycoprotein and its chicken homolog F11 belong to a subclass of proteins of the immunoglobulin superfamily with preferential localization on axons and neurites. We have transfected F3 cDNA into CHO cells. Biochemical analysis establishes that the cDNA we have cloned codes for a 130 kd phosphatidylinositol-anchored polypeptide. F3-expressing transfectants exhibited enhanced self-adhesive properties, aggregating with faster kinetics and forming larger aggregates than F3-negative control cells. When used as a culture substrate for sensory neurons, F3-transfected cells showed a markedly enhanced ability to promote neurite outgrowth compared with nontransfected cells. The results support the idea that F3/F11 and other closely similar proteins function as cell adhesion molecules that play a role in axonal growth and guidance.     

Coxsackievirus A21 binds to decay-accelerating factor but requires intercellular adhesion molecule 1 for cell entry.

It is becoming increasingly apparent that many viruses employ multiple receptor molecules in their cell entry mechanisms. The human enterovirus coxsackievirus A21 (CAV21) has been reported to bind to the N-terminal domain of intercellular adhesion molecule 1 (ICAM-1) and undergo limited replication in ICAM-1-expressing murine L cells. In this study, we show that in addition to binding to ICAM-1, CAV21 binds to the first short consensus repeat (SCR) of decay-accelerating factor (DAF). Dual antibody blockade using both anti-ICAM-1 (domain 1) and anti-DAF (SCR1) monoclonal antibodies (MAbs) is required to completely abolish binding and replication of high-titered CAV21. However, the binding of CAV21 to DAF, unlike that to ICAM-1, does not initiate a productive cell infection. The capacity of an anti-DAF (SCR3) MAb to block CAV21 infection but not binding, coupled with immunoprecipitation data from chemical cross-linking studies, indicates that DAF and ICAM-1 are closely associated on the cell surface. It is therefore suggested that DAF may function as a low-affinity attachment receptor either enhancing viral presentation or providing a viral sequestration site for subsequent high-affinity binding to ICAM-1.     

Solubility and posttranslational regulation of GP130/F11--a neuronal GPI-linked cell adhesion molecule enriched in the neuronal membrane skeleton.

GP130 (renamed contactin) has previously been identified by its detergent insolubility and retention with the actin-containing "membrane skeleton" isolated from chicken neurons and brain. The contactin sequence predicted a transmembrane and cytoplasmic domain for the molecule. Recently, F11 was shown to have an identical sequence except for the C terminus, and it was predicted to be linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) group. Here we describe that GP130 can be released both from brain membranes and the detergent-insoluble membrane skeleton by a phosphoinositol-specific phospholipase C (PI-PLC) indicating that F11 and GP130/contactin are probably identical and that surprisingly the lipid anchor is partly or totally responsible for its non-ionic detergent insolubility. The "membrane skeleton" is a rich source of GPI-linked glycoproteins as judged by 1) most glycoproteins can be released by a PI-PLC and 2) most [3H]ethanolamine-labeled glycoproteins are present in, or enriched in the membrane skeleton. Thus, detergent insolubility appears to be a characteristic of GPI-anchored glycoproteins. No evidence has been obtained that GP130/F11 is released or secreted in vivo or in culture. In addition, GP130/F11 has an unusually long half-life in culture of greater than 3 days. The structure of the neuronal membrane skeleton and the potential function of GPI-anchored glycoproteins is discussed.     

Structure of the tandem fibronectin type 3 domains of neural cell adhesion molecule

Activation of the fibroblast growth factor receptor (FGFR) by neural cell adhesion molecule (NCAM) is essential for NCAM-mediated neurite outgrowth. Previous peptide studies have identified two regions in the fibronectin type 3 (FN3)-like domains of NCAM as being important for these activities. Here we report the crystal structure of the NCAM FN3 domain tandem, which reveals an acutely bent domain arrangement. Mutation of a non-conserved surface residue (M610R) led to a second crystal form showing a substantially different conformation. Thus, the FN3 domain linker is highly flexible, suggesting that it corresponds to the hinge seen in electron micrographs of NCAM. The two putative FGFR1-binding segments, one in each NCAM FN3 domain, are situated close to the domain interface. They form a contiguous patch in the more severely bent conformation but become separated upon straightening of the FN3 tandem, suggesting that conformational changes within NCAM may modulate FGFR1 activation. Surface plasmon resonance experiments demonstrated only a very weak interaction between the NCAM FN3 tandem and soluble FGFR1 proteins expressed in mammalian cells (dissociation constant > 100 μM). Thus, the NCAM-FGFR1 interaction at the cell surface is likely to depend upon avidity effects due to receptor clustering.     

Antibodies to the mammalian adhesion molecule J1/tenascin and its carbohydrate epitope L2/HNK-1 label the receptor lymph cavities of insect sensilla

Data from light- and electronimmunocytochemistry gave evidence that the antibodies to the mammalian adhesion molecule J1/tenascin and its carbohydrate structure L2/HNK-1 react with immunoreactive structures present in the inner and outer receptor lymph cavities of antennal sensilla of the honey bee. Immunoreactivity was additionally present in the cytoplasm of the enveloping cells surrounding the receptor lymph cavities. Cell contacts between enveloping cells and between dendrites and enveloping cells were never observed to be antigen positive.