The contact site A glycoprotein mediates cell-cell adhesion by homophilic binding in Dictyostelium discoideum

Dictyostelium discoideum expresses a developmentally regulated cell surface glycoprotein of Mr 80,000 (gp80), which has been implicated in the formation of the EDTA-resistant contact sites A at the cell aggregation stage. To determine whether gp80 participates directly in cell binding and, if so, its mode of action, we conjugated purified gp80 to Covaspheres (Covalent Technology Corp., Ann Arbor, MI) and investigated their ability to bind to cells. The binding of gp80- Covaspheres was dependent on the developmental stage of the cells, with maximal interaction at the late aggregation stage. Scanning electron microscopic studies revealed the clustering of gp80-Covaspheres at the polar ends of these cells, similar to the pattern of gp80 distribution on the cell surface as reported earlier (Choi, A. H. C., and Siu, C.- H., 1987, J. Cell Biol., 104:1375-1387). Precoating cells with an adhesion-blocking anti-gp80 monoclonal antibody inhibited the binding of gp80-Covaspheres, suggesting that Covasphere-associated gp80 might undergo homophilic interaction with gp80 on the cell surface. Quantitative binding of 125I-labeled gp80 to intact cells gave an estimate of 1.5 X 10(5) binding sites per cell at the aggregation stage. Binding of soluble gp80 to cells was blocked by precoating cells with the anti-gp80 monoclonal antibody. The ability of gp80 to undergo homophilic interaction was further tested in a filter-binding assay, which showed that 125I-labeled gp80 was able to interact with gp80 bound on nitrocellulose in a dosage-dependent manner. In addition, reassociation of cells was significantly inhibited in the presence of soluble gp80, suggesting that gp80 has a single cell-binding site. These results are consistent with the notion that gp80 mediates cell- cell binding at the aggregation stage of development via homophilic interaction.     

Structural requirements for interaction of sodium channel beta 1 subunits with ankyrin

Sodium channel beta subunits modulate channel kinetic properties and cell surface expression levels and function as cell adhesion molecules. beta 1 and beta 2 participate in homophilic cell adhesion resulting in ankyrin recruitment to cell contact sites. We hypothesized that a tyrosine residue in the cytoplasmic domain of beta 1 may be important for ankyrin recruitment and tested our hypothesis using beta 1 mutants replacing Tyr(181) with alanine (beta 1Y181A), phenylalanine (beta 1Y181F), or glutamate (beta 1Y181E), or a truncated construct deleting all residues beyond Tyr(181) (beta 1L182(STOP)). Ankyrin recruitment was observed in beta 1L182(STOP), showing that residues Ile(166)-Tyr(181) contain the major ankyrin recruiting activity of beta 1. Ankyrin recruitment was abolished in beta 1Y181E, suggesting that tyrosine phosphorylation of beta 1 may inhibit beta 1-ankyrin interactions. Ankyrin(G) and beta 1 associate in rat brain membranes and in transfected cells expressing beta 1 and ankyrin(G) in the absence of sodium channel alpha subunits. beta 1 subunits are recognized by anti-phosphotyrosine antibodies following treatment of these cell lines with fibroblast growth factor. beta 1 and ankryin(G) association is not detectable in cells following treatment with fibroblast growth factor. Ankyrin(G) and beta 1Y181E do not associate even in the absence of fibroblast growth factor treatment. beta 1 subunit-mediated cell adhesion and ankyrin recruitment may contribute to sodium channel placement at nodes of Ranvier. The phosphorylation state of beta 1Y181 may be a critical regulatory step in these developmental processes.     

Nectin-3, a new member of immunoglobulin-like cell adhesion molecules that shows homophilic and heterophilic cell-cell adhesion activities

We have isolated a novel cell-cell adhesion system localized at cadherin-based adherens junctions (AJs). This system consists of at least nectin, a Ca(2+)-independent immunoglobulin-like adhesion molecule, and afadin, an actin filament-binding protein, that connects nectin to the actin cytoskeleton. Nectin constitutes a family consisting of two members, nectin-1 and -2. We have isolated here a third member of the nectin family and named it nectin-3. Nectin-3 has three splicing variants, nectin-3alpha (biggest), -3beta (middle), and -3gamma (smallest). Like nectin-1 and -2, nectin-3alpha consists of three extracellular immunoglobulin-like domains, a transmembrane segment, and a cytoplasmic region with the C-terminal consensus motif for binding to the PDZ domain. Nectin-3alpha formed a cis-homo-dimer and showed Ca(2+)-independent trans-homo-interaction to cause homophilic cell-cell adhesion. Nectin-3alpha furthermore showed trans-hetero-interaction with nectin-1 or -2 but did not form a cis-hetero-dimer with nectin-1 or -2. Nectin-1 did not show trans-hetero-interaction with nectin-2. The affinity of trans-hetero-interaction of nectin-3alpha with nectin-1 or -2 was higher than that of trans-homo-interaction of nectin-1, -2, or -3alpha. Nectin-2 and -3 were ubiquitously expressed, whereas nectin-1 was abundantly expressed in brain. Nectin-3alpha was colocalized with nectin-2 at cadherin-based AJs and interacted with afadin. These results indicate that the nectin family consists of at least three members, nectin-1, -2, and -3, all of which show homophilic and heterophilic cell-cell adhesion activities and are localized at cadherin-based AJs.     

Heterophilic interactions of sodium channel beta1 subunits with axonal and glial cell adhesion molecules

Voltage-gated sodium channels localize at high density in axon initial segments and nodes of Ranvier in myelinated axons. Sodium channels consist of a pore-forming alpha subunit and at least one beta subunit. beta1 is a member of the immunoglobulin superfamily of cell adhesion molecules and interacts homophilically and heterophilically with contactin and Nf186. In this study, we characterized beta1 interactions with contactin and Nf186 in greater detail and investigated interactions of beta1 with NrCAM, Nf155, and sodium channel beta2 and beta3 subunits. Using Fc fusion proteins and immunocytochemical techniques, we show that beta1 interacts with the fibronectin-like domains of contactin. beta1 also interacts with NrCAM, Nf155, sodium channel beta2, and Nf186 but not with sodium channel beta3. The interaction of the extracellular domains of beta1 and beta2 requires the region 169TEEEGKTDGEGNA181 located in the intracellular domain of beta2. Interaction of beta1 with Nf186 results in increased Nav).2 cell surface density over alpha alone, similar to that shown previously for contactin and beta2. We propose that beta1 is the critical communication link between sodium channels, nodal cell adhesion molecules, and ankyrinG.     

Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1-EC5). Studies on cadherin specificity have implicated the NH(2)-terminal EC1 domain in the homophilic binding interaction, but the roles of the other extracellular cadherin (EC) domains have not been evaluated. We have undertaken a systematic analysis of the binding properties of the entire cadherin extracellular domain and the contributions of the other EC domains to homophilic binding. Lateral (cis) dimerization of the extracellular domain is thought to be required for adhesive function. Sedimentation analysis of the soluble extracellular segment of C-cadherin revealed that it exists in a monomer-dimer equilibrium with an affinity constant of approximately 64 microm. No higher order oligomers were detected, indicating that homophilic binding between cis-dimers is of significantly lower affinity. The homophilic binding properties of a series of deletion constructs, lacking successive or individual EC domains fused at the COOH terminus to an Fc domain, were analyzed using a bead aggregation assay and a cell attachment-based adhesion assay. A protein with only the first two NH(2)-terminal EC domains (CEC1-2Fc) exhibited very low activity compared with the entire extracellular domain (CEC1-5Fc), demonstrating that EC1 alone is not sufficient for effective homophilic binding. CEC1-3Fc exhibited high activity, but not as much as CEC1-4Fc or CEC1-5Fc. EC3 is not required for homophilic binding, however, since CEC1-2-4Fc and CEC1-2-4-5Fc exhibited high activity in both assays. These and experiments using additional EC combinations show that many, if not all, the EC domains contribute to the formation of the cadherin homophilic bond, and specific one-to-one interaction between particular EC domains may not be required. These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions (Sivasankar, S., W. Brieher, N. Lavrik, B. Gumbiner, and D. Leckband. 1999. PROC: Natl. Acad. Sci. USA. 96:11820-11824; Sivasankar, S., B. Gumbiner, and D. Leckband. 2001. Biophys J. 80:1758-68). We propose new models for how the cadherin extracellular repeats may contribute to adhesive specificity and function.     

Clusters of neural cell adhesion molecule at sites of cell-cell contact

I have examined the distribution of neural cell adhesion molecule (N-CAM) in cultured C2 myogenic cells and other cell lines to determine if N-CAM accumulates at sites of cell-cell contact. C2 cells growing in log phase display large clusters of neural cell adhesion molecule where they contact each other. These clusters are remarkably stable, do not form at cell-substrate contacts, and appear not to be enriched in a number of other cytoskeletal, membrane, or extracellular proteins. Thus, N-CAM clusters form preferentially in response to cell-cell contact and are specifically enriched in N-CAM. As C2 cultures mature and differentiate, clusters persist at contacts between aligning myoblasts and between myotubes, consistent with a role in myogenesis. N-CAM is also enriched at cell-cell contacts in cultures of PC12, NRK, and CHO cells. These cells have significant amounts of N-CAM as detected on immunoblots. Clusters are not seen in L929 cells, which do not have detectable amounts of N-CAM. Coculture of these cells with C2 cells results in the clustering of N-CAM at heterologous contacts between C2 cells and NRK, CHO, or PC12 cells, but not between C2 cells and L929 cells. These results suggest that N-CAM specifically accumulates where N-CAM-bearing cells contact one another. Clustering of N-CAM may be an important step in strengthening intercellular adhesion.     

Sodium channel beta1 and beta3 subunits associate with neurofascin through their extracellular immunoglobulin-like domain

Sequence homology predicts that the extracellular domain of the sodium channel beta1 subunit forms an immunoglobulin (Ig) fold and functions as a cell adhesion molecule. We show here that beta1 subunits associate with neurofascin, a neuronal cell adhesion molecule that plays a key role in the assembly of nodes of Ranvier. The first Ig-like domain and second fibronectin type III-like domain of neurofascin mediate the interaction with the extracellular Ig-like domain of beta1, confirming the proposed function of this domain as a cell adhesion molecule. beta1 subunits localize to nodes of Ranvier with neurofascin in sciatic nerve axons, and beta1 and neurofascin are associated as early as postnatal day 5, during the period that nodes of Ranvier are forming. This association of beta1 subunit extracellular domains with neurofascin in developing axons may facilitate recruitment and concentration of sodium channel complexes at nodes of Ranvier.     

Sodium channel beta1 subunits promote neurite outgrowth in cerebellar granule neurons

Many immunoglobulin superfamily members are integral in development through regulation of processes such as growth cone guidance, cell migration, and neurite outgrowth. We demonstrate that homophilic interactions between voltage-gated sodium channel beta1 subunits promote neurite extension in cerebellar granule neurons. Neurons isolated from wild-type or beta1(-/-) mice were plated on top of parental, mock-, or beta1-transfected fibroblasts. Wild-type neurons consistently showed increased neurite length when grown on beta1-transfected monolayers, whereas beta1(-/-) neurons showed no increase compared with control conditions. beta1-mediated neurite extension was mimicked using a soluble beta1 extracellular domain and was blocked by antibodies directed against the beta1 extracellular domain. Immunohistochemical analysis suggests that the beta1 and beta4 subunits, but not beta2 and beta3, are expressed in cerebellar Bergmann glia as well as granule neurons. These results suggest a novel role for beta1 during neuronal development and are the first demonstration of a functional role for sodium channel beta subunit-mediated cell adhesive interactions.     

Sodium channel beta1 subunit-mediated modulation of Nav1.2 currents and cell surface density is dependent on interactions with contactin and ankyrin

Voltage-gated sodium channels are composed of a pore-forming alpha subunit and at least one auxiliary beta subunit. Both beta1 and beta2 are cell adhesion molecules that interact homophilically, resulting in ankyrin recruitment. In contrast, beta1, but not beta2, interacts heterophilically with contactin, resulting in increased levels of cell surface sodium channels. We took advantage of these results to investigate the molecular basis of beta1-mediated enhancement of sodium channel cell surface density, including elucidating structure-function relationships for beta1 association with contactin, ankyrin, and Nav1.2. beta1/beta2 subunit chimeras were used to assign putative sites of contactin interaction to two regions of the beta1 Ig loop. Recent studies have shown that glutathione S-transferase fusion proteins containing portions of Nav1.2 intracellular domains interact directly with ankyrinG. We show that native Nav1.2 associates with ankyrinG in cells in the absence of beta subunits and that this interaction is enhanced in the presence of beta1 but not beta1Y181E, a mutant that does not interact with ankyrinG. beta1Y181E does not modulate Nav1.2 channel function despite efficient association with Nav1.2 and contactin. beta1Y181E increases Nav1.2 cell surface expression, but not as efficiently as wild type beta1. beta1/beta2 chimeras exchanging various regions of the beta1 Ig loop were all ineffective in increasing Nav1.2 cell surface density. Our results demonstrate that full-length beta1 is required for channel modulation and enhancement of sodium channel cell surface expression.     

Activation of beta1 integrins induces cell-cell adhesion

Integrins are highly regulated receptors that can function in both cell-substrate and cell-cell adhesion. We have found that the activating anti-beta1 mAb, 12G10, can specifically and rapidly induce both cell-substrate and cell-cell adhesion of HT-1080 human fibrosarcoma and other cell types. Binding of mAb 12G10 induced clustering of cell-surface integrins, and the preferential localization of beta1 integrins expressing the 12G10 epitope at cell-cell adhesion sites. Fab fragments of mAb 12G10 induced HT-1080 cell-cell adhesion as effectively as did intact antibodies, suggesting that integrin clustering was not due to direct antibody crosslinking. Latrunculin B, an inhibitor of F-actin polymerization, inhibited cell-cell adhesion but not the clustering of integrins. Results from a novel, two-color cell-cell adhesion assay suggested that nonactivated cells can bind to activated cells and that integrin activation-induced HT-1080 cell-cell adhesion minimally requires the interaction of activated alpha2beta1 with nonactivated alpha3beta1. These findings suggest that HT-1080 cell-cell adhesion induced by integrin activation require a signaling process involving integrin clustering and the subsequent organization of the cytoskeleton. Integrin activation could therefore play a key role in cell-cell adhesion.     

Distinct ligand binding sites in integrin alpha3beta1 regulate matrix adhesion and cell-cell contact

The integrin alpha3beta1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with alpha3beta1 via a surface loop within the alpha3 beta-propeller (residues 242-246) but outside the laminin binding region, suggesting that uPAR-integrin interactions could signal differently from matrix engagement. To explore this, alpha3-/- epithelial cells were reconstituted with wild-type (wt) alpha3 or alpha3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt alpha3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and gamma-catenin. Src kinase inhibition or expression of Src 1-251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that alpha3beta1 regulates both cell-cell contact and matrix adhesion, but through distinct protein interaction sites within its beta-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition.     

Drosophila fasciclin I is a novel homophilic adhesion molecule that along with fasciclin III can mediate cell sorting

Fasciclin I is a membrane-associated glycoprotein that is regionally expressed on a subset of fasciculating axons during neuronal development in insects; it is expressed on apposing cell surfaces, suggesting a role in specific cell adhesion. In this paper we show that Drosophila fasciclin I is a novel homophilic cell adhesion molecule. When the nonadhesive Drosophila S2 cells are transfected with the fasciclin I cDNA, they form aggregates that are blocked by antisera against fasciclin I. When cells expressing fasciclin I are mixed with cells expressing fasciclin III, another Drosophila homophilic adhesion molecule, the mixture sorts into aggregates homogeneous for either fasciclin I- or fasciclin III-expressing cells. The ability of these two novel adhesion molecules to mediate cell sorting in vitro suggests that they might play a similar role during neuronal development.     

Rapid molecular and morphological responses of prostate cell lines to cell-cell contact

Cell-cell adhesion in 2-D PZ-HPV-7 prostate epithelial and DU-145 prostate cancer cell aggregates (monolayers), synchronously and rapidly (within 30 s) formed in suspension in an ultrasound trap has been examined over 60 min. The intracellular distributions of the cadherin/catenin complex components for both cell lines were time-dependent and were clearly identifiable as early as 150 s following cell-cell contact in the trap, while equilibrium positions were reached within 60 min following cell-cell contact. The accumulation of E-cadherin at the cell-cell interface was greater for PZ-HPV-7 than for DU-145 cells over 60 min in the trap, with the apparent formation of adherens junctions over that time scale in PZ-HPV-7 but not in DU-145 cells. The amounts of F-actin, alpha-, beta-, and gamma-catenins recruited to the cell-cell interface of PZ-HPV-7 cells were on average 2.4 times higher than those of DU-145 cells. The ability of different cell types to spread along neighboring cells was 1.5-fold greater for the PZ-HPV-7 than for the DU-145 cells. These results, discussed also in the context of earlier studies of cell adhesion in an ultrasound trap, characterize a reduced adhesiveness of DU-145 cells compared to PZ-HPV-7 cells.     

Lateral clustering of cadherin-4 without homophilic interaction: possible involvement in the concentration process at cell-cell adhesion sites as well as in the cell adhesion activity

It is thought that the concentration of classic cadherins at cell-cell adhesion sites is essential for generating strong cell-cell adhesion activity, but the mechanism is not well understood. To clarify the structural basis of the concentration process and the cell adhesion activity, we constructed various mutants of cadherin-4 and examined the adhesion properties of the transfectants. A deletion mutant lacking the entire cytoplasmic domain had weak, but significant Ca(2+)-dependent cell adhesion activity. Interestingly, the deletion mutant showed intrinsic cluster formation in the absence of cell-cell adhesion, possible lateral cluster formation. The cytoplasmic domain-deleted cadherin-4 containing the mutation of Trp-2 to Ala, which is known to inhibit the strand dimer formation required for the cell-cell adhesion, retained the possible activity of lateral cluster formation, supporting this notion. These results suggest that the extracellular domain has intrinsic activity of lateral cluster formation. Indeed, deletion of a cadherin repeat in the extracellular domain significantly reduced or abolished the lateral cluster formation as well as the concentration of cadherin-4 at cell-cell contact sites and cell adhesion activity. When transfectants of the cytoplasmic domain-deleted cadherin-4 made cell-cell contact and formed intimate cell-cell adhesion, the lateral clusters of cadherin-4 initially gathered at cell-cell contact sites, and a smooth linear concentration was gradually formed along the cell-cell adhesion interface. The results suggest that the lateral cluster formation is involved in the concentration process of cadherin-4 at cell-cell adhesion sites, hence in the strong cell adhesion activity of cadherin-4 as well.     

Characterization of the kinetics of neural cell adhesion molecule homophilic binding

A solid-phase assay has been developed for the investigation of the kinetics of neural cell adhesion molecule (NCAM) binding. Using this assay we can show that NCAM binds to itself in a time-dependent and saturable manner. Binding constants (KB values) of 6.9 x 10(-8) M and 1.23 x 10(-6) M, respectively, were obtained for adult and newborn rat NCAM homophilic binding. Binding is specifically inhibited by Fab' fragments of polyclonal anti-NCAM antibodies but is unaffected by heparin or chondroitin sulphate. This indicates that the NCAM homophilic binding site is separate from and independent of the heparin-binding site and that a developmental modification, probably polysialation, gives rise to marked differences in the adhesive properties of NCAM.     

Amphiphilic cationic peptides mediate cell adhesion to plastic surfaces

Four amphiphilic peptides, each with net charges of +2 or more at neutrality and molecular weights under 4 kilodaltons, were found to mediate the adhesion of normal rat kidney fibroblasts to polystyrene surfaces. Two of these peptides, a model for calcitonin (peptide 1, MCT) and melittin (peptide 2, MEL), form amphiphilic alpha-helical structures at aqueous/nonpolar interfaces. The other two, a luteinizing hormone-releasing hormone model (peptide 3, LHM) and a platelet factor model (peptide 4, MPF) form beta-strand structures in amphiphilic environments. Although it contains only 10 residues, LHM mediated adhesion to surfaces coated with solutions containing as little as 10 pmoles/ml of peptide. All four of these peptides were capable of forming monolayers at air-buffer interfaces with collapse pressures greater than 20 dynes/cm. None of these four peptides contains the tetrapeptide sequence Arg-Gly-Asp-Ser, which has been associated with fibronectin-mediated cell adhesion. Ten polypeptides that also lacked the sequence Arg-Gly-Asp-Ser but were nonamphiphilic and/or had net charges less than +2 at neutrality were all incapable of mediating cell adhesion (Pierschbacher and Ruoslahti, 1984). The morphologies of NRK cells spread on polystyrene coated with peptide LHM resemble the morphologies on fibronectin-coated surfaces, whereas cells spread on surfaces coated with MCT or MEL exhibit strikingly different morphologies. The adhesiveness of MCT, MEL, LHM, and MPF implies that many amphiphilic cationic peptides could prove useful as well defined adhesive substrata for cell culture and for studies of the mechanism of cell adhesion.     

Differential effects of beta 1 and beta 2 subunits on BK channel activity

High conductance, calcium- and voltage-activated potassium (BK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (beta) subunits. beta1 and beta2 subunits increase apparent channel calcium sensitivity. The beta1 subunit also decreases the voltage sensitivity of the channel and the beta2 subunit produces an N-type inactivation of BK currents. We further characterized the effects of the beta1 and beta2 subunits on the calcium and voltage sensitivity of the channel, analyzing the data in the context of an allosteric model for BK channel activation by calcium and voltage (Horrigan and Aldrich, 2002). In this study, we used a beta2 subunit without its N-type inactivation domain (beta2IR). The results indicate that the beta2IR subunit, like the beta1 subunit, has a small effect on the calcium binding affinity of the channel. Unlike the beta1 subunit, the beta2IR subunit also has no effect on the voltage sensitivity of the channel. The limiting voltage dependence for steady-state channel activation, unrelated to voltage sensor movements, is unaffected by any of the studied beta subunits. The same is observed for the limiting voltage dependence of the deactivation time constant. Thus, the beta1 subunit must affect the voltage sensitivity by altering the function of the voltage sensors of the channel. Both beta subunits reduce the intrinsic equilibrium constant for channel opening (L0). In the allosteric activation model, the reduction of the voltage dependence for the activation of the voltage sensors accounts for most of the macroscopic steady-state effects of the beta1 subunit, including the increase of the apparent calcium sensitivity of the BK channel. All allosteric coupling factors need to be increased in order to explain the observed effects when the alpha subunit is coexpressed with the beta2IR subunit.     

Structure and function of voltage-dependent ion channel regulatory beta subunits

Voltage-dependent K(+), Ca(2+), and Na(+) channels play vital roles in basic physiological processes, including management of the action potential, signal transduction, and secretion. They share the common function of passively transporting ions across cell membranes; thus, it would not be surprising if they should exhibit similarities of both structure and mechanism. Indeed, the principal pore-forming (alpha) subunits of each show either exact or approximate 4-fold symmetry and share a similar transmembrane topology, and all are gated by changes in membrane potential. Furthermore, these channels all possess an auxiliary polypeptide, designated the beta subunit, which plays an important role in their regulation. Despite considerable functional semblences and abilities to interact with structurally similar alpha subunits, however, there is considerable structural diversity among the beta subunits. In this review, we discuss the similarities and differences in the structures and functions of the beta subunits of the voltage-dependent K(+), Ca(2+), and Na(+) channels.