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.     

Interaction of nectin with afadin is necessary for its clustering at cell-cell contact sites but not for its cis dimerization or trans interaction

We have recently found a novel functional unit of cell-cell adhesion at cadherin-based adherens junctions, consisting of at least nectin, a homophilic cell adhesion molecule, and afadin, an actin filament-binding protein, which connects nectin to the actin cytoskeleton. Here we studied a mechanism of cell-cell adhesion of the nectin-afadin system by use of a cadherin-deficient L cell line stably expressing the intact form of mouse nectin-2alpha, a truncated form of nectin-2alpha incapable of interacting with afadin (nectin-2alpha-DeltaC), or a point-mutated form of nectin-2alpha capable of interacting with afadin and a cadherin-expressing EL cell line, which transiently expressed the point-mutated form of nectin-2alpha. We found that the interaction of nectin-2alpha with afadin was necessary for their clustering at cell-cell contact sites. However, nectin-2alpha-DeltaC showed cis dimerization and trans interaction, both of which did not require the interaction of nectin-2alpha with afadin. We have previously shown in EL cells that the interaction of nectin-1 with afadin is necessary for its recruitment to adherens junctions. We found that the trans interaction of nectin-2alpha was furthermore necessary for this recruitment. On the basis of these observations, we propose a model for the mechanism of cell-cell adhesion of nectin and roles of afadin in this mechanism.     

Requirement of interaction of nectin-1alpha/HveC with afadin for efficient cell-cell spread of herpes simplex virus type 1

We recently found a novel cell-cell adhesion system at cadherin-based adherens junctions (AJs), consisting at least of nectin, a Ca(2+)-independent homophilic immunoglobulin-like adhesion molecule, and afadin, an actin filament-binding protein that connects nectin to the actin cytoskeleton. Nectin is associated with cadherin through afadin and alpha-catenin. The cadherin-catenin system increases the concentration of nectin at AJs in an afadin-dependent manner. Nectin constitutes a family consisting of three members: nectin-1, -2, and -3. Nectin-1 serves as an entry and cell-cell spread mediator of herpes simplex virus type 1 (HSV-1). We studied here a role of the interaction of nectin-1alpha with afadin in entry and/or cell-cell spread of HSV-1. By the use of cadherin-deficient L cells overexpressing the full length of nectin-1alpha capable of interacting with afadin and L cells overexpressing a truncated form of nectin-1alpha incapable of interacting with afadin, we found that the interaction of nectin-1alpha with afadin increased the efficiency of cell-cell spread, but not entry, of HSV-1. This interaction did not affect the binding to nectin-1alpha of glycoprotein D, a viral component mediating entry of HSV-1 into host cells. Furthermore, the cadherin-catenin system increased the efficiency of cell-cell spread of HSV-1, although it also increased the efficiency of entry of HSV-1. It is likely that efficient cell-cell spread of HSV-1 is caused by afadin-dependent concentrated localization of nectin-1alpha at cadherin-based AJs.     

Role of each immunoglobulin-like loop of nectin for its cell-cell adhesion activity

Nectins are Ca(2+)-independent immunoglobulin (Ig)-like cell-cell adhesion molecules that form cell-cell junctions, cooperatively with or independently of cadherins, in a variety of cells. Nectins comprise a family of four members, nectin-1, -2, -3, and -4. All nectins have one extracellular region with three Ig-like loops, one transmembrane segment, and one cytoplasmic tail. It has been shown mainly by use of cadherin-deficient L fibroblasts stably expressing each nectin that nectins first form homo-cis-dimers and then homo- or hetero-trans-dimers, causing cell-cell adhesion, and that the formation of the cis-dimers is necessary for the formation of the trans-dimers. However, kinetics of the formation of these dimers have not been examined biochemically by use of pure nectin proteins. We prepared here pure recombinant proteins of extracellular fragments of nectin-3 containing various combinations of Ig-like loops, all of which were fused to the Fc portion of IgG and formed homo-cis-dimers through the Fc portion, and of an extracellular fragment of nectin-1 containing three Ig-like loops which was fused to secreted alkaline phosphatase and formed homo-cis-dimers. We showed here by use of these proteins that the first Ig-like loop of nectin-3 was essential and sufficient for the formation of trans-dimers with nectin-1, but that the second Ig-like loop of nectin-3 was furthermore necessary for its cell-cell adhesion activity.     

Regulation of E-cadherin endocytosis by nectin through afadin, Rap1, and p120ctn

Adherens junctions (AJs) are a major cell-cell adhesion structure in epithelial cells that are formed by two major cell-cell adhesion molecules, E-cadherin and nectin. We have previously shown that nectin first forms cell-cell adhesion and then recruits non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites, which gradually trans-interact there, eventually forming AJs. We have examined here the effect of trans-interacting nectin on non-trans-interacting E-cadherin endocytosis. Trans-interacting nectin capable of associating with afadin, but not trans-interacting nectin mutant incapable of associating with afadin, inhibited non-trans-interacting E-cadherin endocytosis in intact cells. Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Studies on the mode of action of the nectin-afadin system using cell-free assay revealed that afadin associated with nectin bound Rap1 activated by trans-interacting nectin, interacted with p120ctn, and strengthened the binding of p120ctn to E-cadherin, eventually reducing non-trans-interacting E-cadherin endocytosis. Afadin, which did not bind Rap1, was inactive in this capacity. These results indicate that trans-interacting nectin inhibits non-trans-interacting E-cadherin endocytosis through afadin, Rap1, and p120ctn and thereby further accumulates non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites for the formation of AJs.     

Cellular localization of nectin-1 and glycoprotein D during herpes simplex virus infection

During viral entry, herpes simplex virus (HSV) glycoprotein D (gD) interacts with a specific cellular receptor such as nectin-1 (PRR1/HveC/CD111) or the herpesvirus entry mediator A (HVEM/HveA). Nectin-1 is involved in cell-to-cell adhesion. It is located at adherens junctions, where it bridges cells through homophilic or heterophilic interactions with other nectins. Binding of HSV gD prevents nectin-1-mediated cell aggregation. Since HSV gD affects the natural function of nectin-1, we further investigated the effects of gD expression on nectin-1 during HSV infection or in transfected cells. We also studied the importance of the interaction between nectin-1 and the cytoplasmic protein afadin for HSV entry and spread as well as the effects of infection on this interaction. In these investigations, we used a panel of cells expressing nectin-1 or nectin-1-green fluorescent protein fusions as the only mediators of HSV entry. During HSV infection, nectin-1 localization at adherens junction was dramatically altered in a manner dependent on gD expression. Nectin-1 and gD colocalized at cell contact areas between infected and noninfected cells and at the edges of plaques. This specific accumulation of gD at junctions was driven by expression of nectin-1 in trans on the surface of adjacent cells. Reciprocally, nectin-1 was maintained at junctions by the trans expression of gD in the absence of a cellular natural ligand. Our observations indicate that newly synthesized gD substitutes for nectin-1 of infected cells at junctions with noninfected cells. We propose that gD attracts and maintains the receptor at junctions where it can be used for virus spread.     

V-src kinase shifts the cadherin-based cell adhesion from the strong to the weak state and beta catenin is not required for the shift

The elevation of tyrosine phosphorylation level is thought to induce the dysfunction of cadherin through the tyrosine phosphorylation of beta catenin. We evaluated this assumption using two cell lines. First, using temperature-sensitive v-src-transfected MDCK cells, we analyzed the modulation of cadherin-based cell adhesion by tyrosine phosphorylation. Cell aggregation and dissociation assays at nonpermissive and permissive temperatures indicated that elevation of the tyrosine phosphorylation does not totally affect the cell adhesion ability of cadherin but shifts it from a strong to a weak state. The tyrosine phosphorylation levels of beta catenin, ZO-1, ERM (ezrin/radixin/moesin), but not alpha catenin, vinculin, and alpha- actinin, were elevated in the weak state. To evaluate the involvement of the tyrosine phosphorylation of beta catenin in this shift of cadherin-based cell adhesion, we introduced v-src kinase into L fibroblasts expressing the cadherin-alpha catenin fusion protein, in which beta catenin is not involved in cell adhesion. The introduction of v-src kinase in these cells shifted their adhesion from a strong to a weak state. These findings indicated that the tyrosine phosphorylation of beta catenin is not required for the strong-to-weak state shift of cadherin-based cell adhesion, but that the tyrosine phosphorylation of other junctional proteins, ERM, ZO-1 or unidentified proteins is involved.     

Recruitment of nectin-3 to cell-cell junctions through trans-heterophilic interaction with CD155, a vitronectin and poliovirus receptor that localizes to alpha(v)beta3 integrin-containing membrane microdomains

Nectins present a novel class of Ig superfamily adhesion molecules that, cooperatively with cadherins, establish and maintain cell-cell adherens junctions. CD155, the cognate receptor for poliovirus, undergoes cell-matrix contacts by binding to the extracellular matrix protein vitronectin. The significant homology of nectins with CD155 prompted us to investigate the possibility of their interaction. We determined that nectin-3 binds CD155 and its putative mouse homologue Tage4 in cell-based ligand binding assays. Coculture of nectin-3- and CD155-expressing HeLa cells led to CD155-dependent recruitment of nectin-3 to cell-cell contacts. In a heterologous coculture system with CD155 expressing mouse neuroblastoma cells, HeLa cell-expressed nectin-3 was recruited to contact sites with CD155 bearing neurites. CD155 and nectin-3 colocalized to epithelial cell-cell junctions in renal proximal tubules and in the amniotic membrane. Efficient interaction depended on CD155 dimerization, which appears to be aided by cell type-specific cofactors. We furthermore found CD155 to codistribute with alpha(v) integrin microdomains on the surface of transfected mouse fibroblasts and at amniotic epithelial cell junctions. Our findings demonstrate the possible trans-interaction between the bona fide cell-cell adherens type adhesion system (cadherin/nectin) and the cell-matrix adhesion system (integrin/CD155) by virtue of their nectin-3 and CD155 components, respectively.     

Arp2/3 activity is necessary for efficient formation of E-cadherin adhesive contacts

Classical cadherin adhesion molecules are fundamental determinants of cell-cell recognition that function in cooperation with the actin cytoskeleton. Productive cadherin-based cell recognition is characterized by a distinct morphological process of contact zone extension, where limited initial points of adhesion are progressively expanded into broad zones of contact. We recently demonstrated that E-cadherin ligation recruits the Arp2/3 actin nucleator complex to the plasma membrane in regions where cell contacts are undergoing protrusion and extension. This suggested that Arp2/3 might generate the protrusive forces necessary for cell surfaces to extend upon one another during contact assembly. We tested this hypothesis in mammalian cells by exogenously expressing the CA region of N-WASP. This fragment, which potently inhibits Arp2/3-mediated actin assembly in vitro, also effectively reduced actin assembly at cadherin adhesive contacts. Blocking Arp2/3 activity by this strategy profoundly reduced the ability of cells to extend cadherin adhesive contacts but did not affect cell adhesiveness. These findings demonstrate that Arp2/3 activity is necessary for cells to efficiently extend and assemble cadherin-based adhesive contacts.     

Direct binding of cell polarity protein PAR-3 to cell-cell adhesion molecule nectin at neuroepithelial cells of developing mouse

PAR-3 is a cell polarity protein that localizes at tight junctions (TJs) by direct binding to an immunoglobulin (Ig)-like cell-cell adhesion molecule JAM-1 in mammalian epithelial cells. Another Ig-like cell-cell adhesion molecule nectin plays a role in the localization of JAM-1 at TJs in epithelial cells. Nectin furthermore plays a role in the organization of adherens junctions (AJs) and TJs. Nectin comprises a family of four members, nectin-1, -2, -3, and -4. Nectins are associated with the actin cytoskeleton through afadin, of which the PDZ domain binds to nectins through their C-terminal four amino acids. We show here that PAR-3 binds to nectin-1 and -3 in neuroepithelial cells of the embryonic telencephalon, which are equipped with AJs, but not with typical TJs. Nectin-1, -2, -3, and afadin, but not JAM-1, were concentrated at AJs in neuroepithelial cells of the embryonic telencephalon at E13.5 and PAR-3 co-localized with nectins. PAR-3 was co-immunoprecipitated with nectin-1 and -3, but not with nectin-2 or JAM-1, from the mouse whole brain at E13.5. Recombinant PAR-3 stoichiometrically bound to recombinant nectin-1 and -3. The first one of the three PDZ domains of PAR-3 bound to the C-terminal four amino acids of nectin-1 and -3. The affinities of PAR-3 and afadin for nectin-1 and -3 were similar. Cadherin-deficient L cells expressing nectin-1 and -3 formed nectin-1- and -3-based cell-cell junctions, respectively, where PAR-3 as well as afadin was recruited. These results indicate that nectin-1 and -3 are involved in the localization of PAR-3 at AJs in the neuroepithelial cells of the embryonic telencephalon.     

Ectodomain shedding of nectin-1alpha by SF/HGF and TPA in MDCK cells

Nectin is a Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecule implicated in the organization of the junctional complex comprised of E-cadherin-based adherens junctions and claudin-based tight junctions in epithelial cells. Scatter factor (SF)/hepatocyte growth factor (HGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting phorbol ester, induce cell spreading, followed by cell-cell dissociation and cell scattering, in Madin-Darby canine kidney (MDCK) cells. We found here that SF/HGF and TPA induced proteolytic cleavage of nectin-1alpha in the ectodomain, resulting in generation of the 80-kDa extracellular fragment and the 33-kDa fragment composed of the transmembrane and cytoplasmic domains, in MDCK cells. This shedding of nectin-1alpha was inhibited by metalloprotease inhibitors. These results indicate that SF/HGF and TPA induce the ectodomain shedding of nectin-1alpha presumably by a metalloprotease, and have raised the possibility that this shedding is involved in the SF/HGF- and TPA-induced cell-cell dissociation.     

The signaling network of transforming growth factor beta1, protein kinase Cdelta, and integrin underlies the spreading and invasiveness of gastric carcinoma cells

Integrin-mediated cell adhesion and spreading enables cells to respond to extracellular stimuli for cellular functions. Using a gastric carcinoma cell line that is usually round in adhesion, we explored the mechanisms underlying the cell spreading process, separate from adhesion, and the biological consequences of the process. The cells exhibited spreading behavior through the collaboration of integrin-extracellular matrix interaction with a Smad-mediated transforming growth factor beta1 (TGFbeta1) pathway that is mediated by protein kinase Cdelta (PKCdelta). TGFbeta1 treatment of the cells replated on extracellular matrix caused the expression and phosphorylation of PKCdelta, which is required for expression and activation of integrins. Increased expression of integrins alpha2 and alpha3 correlated with the spreading, functioning in activation of focal adhesion molecules. Smad3, but not Smad2, overexpression enhanced the TGFbeta1 effects. Furthermore, TGFbeta1 treatment and PKCdelta activity were required for increased motility on fibronectin and invasion through matrigel, indicating their correlation with the spreading behavior. Altogether, this study clearly evidenced that the signaling network, involving the Smad-dependent TGFbeta pathway, PKCdelta expression and phosphorylation, and integrin expression and activation, regulates cell spreading, motility, and invasion of the SNU16mAd gastric carcinoma cell variant.     

The receptor tyrosine kinase ARK mediates cell aggregation by homophilic binding.

The ARK (AXL, UFO) receptor is a member of a new family of receptor tyrosine kinases whose extracellular domain contains a combination of fibronectin type III and immunoglobulin motifs similar to those found in many cell adhesion molecules. ARK mRNA is expressed at high levels in the mouse brain, prevalently in the hippocampus and cerebellum, and this pattern of expression resembles that of adhesion molecules that are capable of promoting cell aggregation through homophilic or heterophilic binding. We report here the ability of the murine ARK receptor to mediate homophilic binding. Expression of the ARK protein in Drosophila S2 cells induces formation of cell aggregates consisting of ARK-expressing cells, and aggregation leads to receptor activation, with an increase in receptor phosphorylation. Homophilic binding does not require ARK tyrosine kinase activity, since S2 cells expressing a receptor in which the intracellular domain was deleted were able to undergo aggregation as well as cells expressing the wild-type ARK receptor. Similar results were obtained with NIH 3T3 and CHO cells expressing high levels of ARK, although in this case ARK expression appeared to be accompanied by constitutive activation. The purified recombinant extracellular domain of ARK can induce homotypic aggregation of coated fluorescent beads (Covaspheres), and this protein can also function as a substrate for adhesion by S2 and NIH 3T3 cells expressing ARK. These results suggest that ARK represents a new cell adhesion molecule that through its homophilic interaction may regulate cellular functions during cell recognition.     

Nectin-like molecule-5/Tage4 enhances cell migration in an integrin-dependent, Nectin-3-independent manner

Cell migration plays roles in invasion of transformed cells and scattering of embryonic mesenchymal cells into surrounding tissues. We have found that Ig-like Necl-5/Tage4 is up-regulated in NIH3T3 cells transformed by an oncogenic Ras (V12Ras-NIH3T3 cells) and heterophilically trans-interacts with a Ca(2+)-independent Ig-like cell adhesion molecule nectin-3, eventually enhancing their intercellular motility. We show here that Necl-5 furthermore enhances cell migration in a nectin-3-independent manner. Studies using L fibroblasts expressing various mutants of Necl-5, NIH3T3 cells, and V12Ras-NIH3T3 cells have revealed that Necl-5 enhances serum- and platelet-derived growth factor-induced cell migration. The extracellular region of Necl-5 is necessary for directional cell migration, but not for random cell motility. The cytoplasmic region of Necl-5 is necessary for both directional and random cell movement. Necl-5 colocalizes with integrin alpha(V)beta(3) at leading edges of migrating cells. Analyses using an inhibitor or an activator of integrin alpha(V)beta(3) or a dominant negative mutant of Necl-5 have shown the functional association of Necl-5 with integrin alpha(V)beta(3) in cell motility. Cdc42 and Rac small G proteins are activated by the action of Necl-5 and required for the serum-induced, Necl-5-enhanced cell motility. These results indicate that Necl-5 regulates serum- and platelet-derived growth factor-induced cell migration in an integrin-dependent, nectin-3-independent manner, when cells do not contact other cells. We furthermore show here that enhanced motility and metastasis of V12Ras-NIH3T3 cells are at least partly the result of up-regulated Necl-5.     

Structure of herpes simplex virus glycoprotein D bound to the human receptor nectin-1

Binding of herpes simplex virus (HSV) glycoprotein D (gD) to a cell surface receptor is required to trigger membrane fusion during entry into host cells. Nectin-1 is a cell adhesion molecule and the main HSV receptor in neurons and epithelial cells. We report the structure of gD bound to nectin-1 determined by x-ray crystallography to 4.0 Å resolution. The structure reveals that the nectin-1 binding site on gD differs from the binding site of the HVEM receptor. A surface on the first Ig-domain of nectin-1, which mediates homophilic interactions of Ig-like cell adhesion molecules, buries an area composed by residues from both the gD N- and C-terminal extensions. Phenylalanine 129, at the tip of the loop connecting β-strands F and G of nectin-1, protrudes into a groove on gD, which is otherwise occupied by C-terminal residues in the unliganded gD and by N-terminal residues in the gD/HVEM complex. Notably, mutation of Phe129 to alanine prevents nectin-1 binding to gD and HSV entry. Together these data are consistent with previous studies showing that gD disrupts the normal nectin-1 homophilic interactions. Furthermore, the structure of the complex supports a model in which gD-receptor binding triggers HSV entry through receptor-mediated displacement of the gD C-terminal region.

Authors Summary

Herpes simplex virus (HSV) is a widespread human pathogen. Four viral glycoproteins (gD, gB, gH/gL) are required for HSV entry into host cells. gD binding to a cell surface receptor triggers conformational changes in the other viral glycoproteins leading to membrane fusion and viral entry. Two structurally unrelated cellular protein receptors, nectin-1 and HVEM, can mediate HSV entry upon binding to gD. The structure presented here reveals the molecular basis for the stable interaction between HSV-1 gD and the receptor nectin-1. Comparison with the previously determined structures of the gD/HVEM complex and unliganded gD shows that, despite the fact that the two receptors interact with different binding sites, they both cause a similar conformational change in gD. Therefore, our data point to a conserved mechanism for receptor mediated activation of the HSV entry process. In addition, the gD/Nectin-1 structure reveals that the gD-binding site overlaps with a surface involved in nectin-1 homo-dimerization. This observation explains how gD interferes with the cell adhesion function of nectin-1. Finally, the gD/Nectin-1 complex displays similarities with other viral ligands bound to immunoglobulin-like receptors suggesting a convergent mechanism for receptors selection and usage.     

Regulation of Cell–Cell Adhesion by Rac and Rho Small G Proteins in MDCK Cells

The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell–cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell–cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and β-catenin, adherens junctional proteins, at the cell–cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell–cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell– cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell–cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell–cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell– cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell– cell adhesion.     

Involvement of ZO-1 in Cadherin-based Cell Adhesion through Its Direct Binding to α Catenin and Actin Filaments

ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large (dlg)-like domain and a carboxyl-terminal half domain, is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. We introduced cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 were concentrated at cadherin-based cell–cell adhesion sites. In good agreement with these observations, N-ZO-1 was specifically coimmunoprecipitated from EL transfectants expressing N-ZO-1 (NZ-EL cells) with the E-cadherin/α, β catenin complex. In contrast, C-ZO-1 was localized along actin stress fibers. To examine the molecular basis of the behavior of these truncated ZO-1 molecules, N-ZO-1 and C-ZO-1 were produced in insect Sf9 cells by recombinant baculovirus infection, and their direct binding ability to the cadherin/catenin complex and the actin-based cytoskeleton, respectively, were examined in vitro. Recombinant N-ZO-1 bound directly to the glutathione-S-transferase fusion protein with α catenin, but not to that with β catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and α catenin was ~0.5 nM. On the other hand, recombinant C-ZO-1 was specifically cosedimented with actin filaments in vitro with a dissociation constant of ~10 nM. Finally, we compared the cadherin-based cell adhesion activity of NZ-EL cells with that of parent EL cells. Cell aggregation assay revealed no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, was significantly suppressed in NZ-EL cells. We conclude that in nonepithelial cells, ZO-1 works as a cross-linker between cadherin/catenin complex and the actin-based cytoskeleton through direct interaction with α catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a functional component in the cadherin-based cell adhesion system.     

Functional domains of alpha-catenin required for the strong state of cadherin-based cell adhesion

The interaction of cadherin-catenin complex with the actin-based cytoskeleton through alpha-catenin is indispensable for cadherin-based cell adhesion activity. We reported previously that E-cadherin-alpha-catenin fusion molecules showed cell adhesion and cytoskeleton binding activities when expressed in nonepithelial L cells. Here, we constructed deletion mutants of E-cadherin-alpha-catenin fusion molecules lacking various domains of alpha-catenin and introduced them into L cells. Detailed analysis identified three distinct functional domains of alpha-catenin: a vinculin/alpha-actinin-binding domain, a ZO-1-binding domain, and an adhesion-modulation domain. Furthermore, cell dissociation assay revealed that the fusion molecules containing the ZO-1-binding domain in addition to the adhesion-modulation domain conferred the strong state of cell adhesion activity on transfectants, although those lacking the ZO-1-binding domain conferred only the weak state. The disorganization of actin-based cytoskeleton by cytochalasin D treatment shifted the cadherin-based cell adhesion from the strong to the weak state. In the epithelial cells, where alpha-catenin was not precisely colocalized with ZO-1, the ZO-1-binding domain did not completely support the strong state of cell adhesion activity. Our studies showed that the interaction of alpha-catenin with the actin-based cytoskeleton through the ZO-1-binding domain is required for the strong state of E-cadherin-based cell adhesion activity.     

A zyxin-nectin interaction facilitates zyxin localization to cell-cell adhesions

Cell–cell junction remodeling is associated with dramatic actin reorganizations. Several actin regulatory systems have been implicated in actin remodeling events as cell–cell contacts are assembled and disassembled, including zyxin/LPP–VASP complexes. These complexes facilitate strong cell–cell adhesion by maintaining actin-membrane connections. It has been proposed that zyxin and LPP localize to cell–cell junctions via a well-defined interaction with alpha-actinin. This was recently confirmed for LPP, but zyxin localization at cell–cell contacts occurs independently of alpha-actinin binding. Here we seek to map the zyxin sequence responsible for localization to cell–cell contacts and identify the protein that docks zyxin at this cellular location. Previous results have shown that a zyxin fragment excluding the alpha-actin binding site and the LIM domains (amino acids 51–392) can independently localize to cell–cell contacts. Here, expression of smaller zyxin fragments show that zyxin localization requires amino acids 230–280. A yeast-two-hybrid screen, using the central region of zyxin as bait, resulted in the identification of the cell–cell adhesion receptor nectin-4 as a zyxin binding partner. Further demonstrating zyxin–nectin interactions, zyxin binds the intracellular domain of nectin-2 in vitro. Depletion of nectin-2 from L cells expressing E-cadherin results in a loss of zyxin localization to cell–cell contacts, demonstrating that the zyxin–nectin interaction plays a critical role in zyxin targeting to these sites.