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.     

A core function for p120-catenin in cadherin turnover

p120-catenin stabilizes epithelial cadherin (E-cadherin) in SW48 cells, but the mechanism has not been established. Here, we show that p120 acts at the cell surface to control cadherin turnover, thereby regulating cadherin levels. p120 knockdown by siRNA expression resulted in dose-dependent elimination of epithelial, placental, neuronal, and vascular endothelial cadherins, and complete loss of cell-cell adhesion. ARVCF and delta-catenin were functionally redundant, suggesting that proper cadherin-dependent adhesion requires the presence of at least one p120 family member. The data reveal a core function of p120 in cadherin complexes, and strongly predict a dose-dependent loss of E-cadherin in tumors that partially or completely down-regulate p120.     

Probing intercellular interactions between vascular endothelial cadherin pairs at single-molecule resolution and in living cells

Vascular endothelial (VE) cadherin is the surface glycoprotein cadherin specific to the endothelium that mediates cell-cell adhesion and plays a major role in the remodeling, gating, and maturation of vascular vessels. To investigate the contribution of individual VE-cadherins to endothelial cell-cell interactions and investigate whether different classical cadherins display different kinetics and micromechanical properties, we characterize the binding properties of VE-cadherin/VE-cadherin bonds at single-molecule resolution and in living human umbilical vein endothelial cells (HUVECs). Our single-molecule force spectroscopy measurements reveal that type II VE-cadherin molecules form bonds that are less prone to rupture and display a higher tensile strength than bonds formed by classical type I neuronal (N) cadherin and epithelial (E) cadherin. The equilibrium lifetime of the VE-cadherin/VE-cadherin bond is significantly longer than formed by N-cadherin/N-cadherin bonds and E-cadherin/E-cadherin bonds. These results indicate that VE-cadherins form bonds that have kinetics and mechanical properties that are significantly different from those formed by classical type I cadherins, properties that are particularly well adapted to the barrier and adhesive functions of VE-cadherin in endothelial cell-cell junctions.     

Cadherins mediate both the association between PS1 and beta-catenin and the effects of PS1 on beta-catenin stability

Presenilin1 (PS1), a protein involved in cellular development, forms functional complexes with beta-catenin, a regulator of Wnt signaling and cell-cell adhesion. In addition, both proteins have been shown to play important roles in disease including cancer and Alzheimer disease. Although PS1 and beta-catenin are found in the same complexes, it is not clear whether they bind directly to each other or a third complex component, like cadherin, may mediate their interactions. Here we show that PS1 and beta-catenin form no detectable complexes in cells that express no cadherin. In contrast, these complexes are readily found in E-cadherin containing cells. Furthermore, binding of both PS1 and beta-catenin to E-cadherin is necessary for the formation of PS1/beta-catenin complexes. Importantly, our data show that binding of PS1 to cadherin mediates the effects of PS1 on the phosphorylation, ubiquitination, and destabilization of beta-catenin. Thus, cadherins mediate both the association of PS1 and beta-catenin and the effects of PS1 on the cellular levels of beta-catenin.     

Nonchordate classic cadherins have a structurally and functionally unique domain that is absent from chordate classic cadherins

Classic cadherins, which are adhesion molecules in cell-cell adherens junctions, have a large contribution to the construction of the animal body. Their molecular structures show clear differences between chordate and nonchordate metazoans. Although nonchordate classic cadherins have cadherin superfamily-specific extracellular repeats (CRs) and a highly conserved cytoplasmic domain (CP), these cadherins have a unique extracellular domain that is absent from vertebrate and ascidian classic cadherins. We called this the primitive classic cadherin domain (PCCD). To understand the roles of the PCCD, we constructed and characterized a series of mutant forms of the Drosophila classic cadherin DE-cadherin. Biochemical analyses indicated that the last two CRs and PCCD form a special structure with proteolytic cleavage. Mutations in the PCCD did not eliminate the cell-cell-binding function of DE-cadherin in cultured cells, but prevented the cadherin from efficiently translocating to the plasma membrane in epithelial cells of the developing embryo. In addition, genetic rescue assays suggested that although CP-mediated control plays a central role in tracheal fusion, the role of the PCCD in efficient recruitment of DE-cadherin to apical areas of the plasma membranes is also important for dynamic epithelial morphogenesis. We propose that there is a fundamental difference in the mode of classic cadherin-mediated cell-cell adhesion between chordate and nonchordate metazoans.     

Disruption of epithelial cell-cell adhesion by exogenous expression of a mutated nonfunctional N-cadherin.

Cadherins, a family of transmembrane cell-cell adhesion receptors, require interactions with the cytoskeleton for normal function. To assess the mechanisms of these interactions, we studied the effect of exogenous expression of a mutant N-cadherin, cN390 delta; on epithelial cell-cell adhesion. The intracellular domain of cN390 delta was intact but its extracellular domain was largely deleted so that this molecule was not functional for cell adhesion. cDNA of cN390 delta was attached to the metallothionein promoter, and introduced into the keratinocyte line PAM212 expressing endogenous E- and P-cadherin. When the expression of cN390 delta was induced by Zn2+, cadherin-dependent adhesion of the transfected cells was inhibited, resulting in the dispersion of cell colonies, although their contacts were maintained under high cell density conditions. In these cultures, cN390 delta was expressed not only on the free surfaces of the cells but also at cell-cell junctions. The endogenous cadherins were concentrated at cell-cell junctions under normal conditions. As a result of cN390 delta expression, however, the endogenous cadherins localizing at the cell-cell junctions were largely diminished, suggesting that these molecules were replaced by the mutant molecules at these sites. As a control, we transfected the same cell line with cDNA of a truncated form of N-cadherin cadherin whose intracellular C terminus had been deleted leaving the extracellular domain intact. This molecule had no effect on cell-cell adhesion, nor did it localize to cell-cell contact sites. We also found that the association of the endogenous cadherins with alpha- and beta-catenins and plakoglobin was not affected by the expression of cN390 delta, which also formed a complex with these molecules, suggesting that no competition occurred between the endogenous and exogenous cadherins for these cytoplasmic proteins. These and other additional results suggest that the nonfunctional cadherins whose intracellular domain is intact occupy the sites where the endogenous cadherins should localize, through interactions with the cytoskeleton, and inhibit the cadherin adhesion system.     

The cadherins: cell-cell adhesion molecules controlling animal morphogenesis

Cadherins are a family of glycoproteins involved in the Ca2+-dependent cell-cell adhesion mechanism which is detected in most kinds of tissues. Inhibition of the cadherin activity with antibodies induces dissociation of cell layers, indicating a fundamental importance of these molecules in maintaining the multicellular structure. Cadherins are divided into subclasses, including E-, N- and P-cadherins. While all subclasses are similar in molecular weight, Ca2+- and protease-sensitivity, each subclass is characterized by a unique tissue distribution pattern and immunological specificity. Analysis of amino acid sequences deduced from cDNA encoding these molecules showed that they are integral membrane proteins of 723-748 amino acids long and share common sequences; similarity in the sequences between subclasses is in a range of 50-60% when compared within a single animal species. L cells, with very little endogenous cadherin activity, transfected with the cadherin cDNA acquired high cadherin-mediated aggregating activity. Their colony morphology was altered by the ectopic expression of cadherins from the dispersed type to the compact type, providing direct evidence for a key role of cadherins in cell-cell adhesion. It has been suggested that cadherins bind cells by their homophilic interactions at the extracellular domain and are associated with actin bundles at the cytoplasmic domain. It appears that each cadherin subclass has binding specificity and this molecular family is involved in selective cell-cell adhesion. In development, the expression of each cadherin subclass is spatiotemporally regulated and associated with a variety of morphogenetic events; e.g. the termination or initiation of expression of a cadherin subclass in a given cell collective is correlated with its segregation from or connection with other cell collectives. Antibodies to cadherins were shown to perturb the morphogenesis of some embryonic organs in vitro. These observations suggest that cadherins play a crucial role in construction of tissues and the whole animal body.     

Once upon a time there was beta-catenin in cadherin-mediated signalling

beta-Catenin was initially characterized as a protein interacting with the cadherin cytoplasmic tail and regulating cell-cell contacts and actin cytoskeleton interactions. Moreover, the gene coding for the Drosophila orthologue of beta-catenin, armadillo, was independently identified downstream of wingless in the segment-polarity signalling pathway. In fact, beta-catenin/Armadillo turned out to be key mediators of the Wnt/Wingless pathways in vertebrates and invertebrates. beta-Catenin participates in both adhesion and signalling functions in a mutually exclusive manner; bound to cadherins at the plasma membrane or 'unbound' in cytosolic or nuclear complexes. This model had placed beta-catenin at the crossroads between cadherin and Wnt signalling, leading to the dogma of inhibition of beta-catenin signalling by cadherins.     

Cadherin-Mediated Adhesion Is Required for Normal Growth Regulation of Human Gingival Epithelial Cells

The cadherins are a family of cell membrane proteins that mediate calcium-dependent cell-cell adhesion. E-cadherin is required for the formation, differentiation, polarization and stratification of epithelia; P-cadherin is also expressed on many epithelia. We report here the first study of cadherin expression in immortalized human gingival epithelial cells (IHGK) and examine the role of cadherins in growth regulation of these cells. We found that the IHGK cells are similar to normal gingival epithelial cells in their cadherin expression and density-dependent inhibition of growth.

The IHGK cells proliferate more rapidly at low calcium concentration (0.15 mM) than at physiological concentrations of calcium (1.8 mM) and magnesium (0.65 mM; Ca/Mg medium) suggesting that calcium is required for density-dependent regulation of proliferation. To evaluate the possibility that cadherin function is required for contact inhibition in these cells, we grew them in Ca/Mg medium in the presence of adhesion-blocking anti-cadherin monoclonal antibodies. At anti-E-cadherin concentrations sufficient to disrupt cell-cell adhesion, the proliferation of the IHGK cells was similar to that observed in medium containing 0.2 mM EDTA. Anti-P-cadherin had a much weaker effect on cell proliferation than anti-E-cadherin, and cells grown in medium containing both antibodies grew at intermediate rates. The increased proliferation of the IHGK cells in either low calcium medium or Ca/Mg medium containing adhesion-blocking anti-cadherin antibodies suggests that cadherin-medi-ated adhesion is required for density-dependent regulation of growth of these cells.     

Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts

Cadherin cell adhesion molecules are major determinants of tissue patterning which function in cooperation with the actin cytoskeleton. In the context of stable adhesion, cadherin/catenin complexes are often envisaged to passively scaffold onto cortical actin filaments. However, cadherins also form dynamic adhesive contacts during wound healing and morphogenesis. Here actin polymerization has been proposed to drive cell surfaces together, although F-actin reorganization also occurs as cell contacts mature. The interaction between cadherins and actin is therefore likely to depend on the functional state of adhesion. We sought to analyze the relationship between cadherin homophilic binding and cytoskeletal activity during early cadherin adhesive contacts. Dissecting the specific effect of cadherin ligation alone on actin regulation is difficult in native cell-cell contacts, due to the range of juxtacrine signals that can arise when two cell surfaces adhere. We therefore activated homophilic ligation using a specific functional recombinant protein. We report the first evidence that E-cadherin associates with the Arp2/3 complex actin nucleator and demonstrate that cadherin binding can exert an active, instructive influence on cells to mark sites for actin assembly at the cell surface.     

Interaction of alpha-actinin with the cadherin/catenin cell-cell adhesion complex via alpha-catenin

Cadherins are Ca(2+)-dependent, cell surface glycoproteins involved in cell-cell adhesion. Extracellularly, transmembrane cadherins such as E- , P-, and N-cadherin self-associate, while intracellularly they interact indirectly with the actin-based cytoskeleton. Several intracellular proteins termed catenins, including alpha-catenin, beta- catenin, and plakoglobin, are tightly associated with these cadherins and serve to link them to the cytoskeleton. Here, we present evidence that in fibroblasts alpha-actinin, but not vinculin, colocalizes extensively with the N-cadherin/catenin complex. This is in contrast to epithelial cells where both cytoskeletal proteins colocalize extensively with E-cadherin and catenins. We further show that alpha- actinin, but not vinculin, coimmunoprecipitates specifically with alpha- and beta-catenin from N- and E-cadherin-expressing cells, but only if alpha-catenin is present. Moreover, we show that alpha-actinin coimmunoprecipitates with the N-cadherin/catenin complex in an actin- independent manner. We therefore propose that cadherin/catenin complexes are linked to the actin cytoskeleton via a direct association between alpha-actinin and alpha-catenin.     

Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules

A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E- cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types.     

Tyrosine phosphorylation and cadherin/catenin function

Cadherin-mediated cell-cell adhesion is perturbed in protein tyrosine kinase (PTK)-transformed cells. While cadherins themselves appear to be poor PTK substrates, their cytoplasmic binding partners, the Arm catenins, are excellent PTK substrates and therefore good candidates for mediating PTK-induced changes in cadherin behavior. These proteins, p120^ctn, β-catenin and plakoglobin, bind to the cytoplasmic region of classical cadherins and function to modulate adhesion and/or bridge cadherins to the actin cytoskeleton. In addition, as demonstrated recently for β-catenin, these proteins also have crucial signaling roles that may or may not be related to their effects on cell-cell adhesion. Tyrosine phosphorylation of cadherin complexes is well documented and widely believed to modulate cell adhesiveness. The data to date, however, is largely correlative and the mechanism of action remains unresolved. In this review, we discuss the current literature and suggest models whereby tyrosine phosphorylation of Arm catenins contribute to regulation or perturbation of cadherin function.     

Expression of N-cadherin by human squamous carcinoma cells induces a scattered fibroblastic phenotype with disrupted cell-cell adhesion

E-cadherin is a transmembrane glycoprotein that mediates calcium- dependent, homotypic cell-cell adhesion and plays an important role in maintaining the normal phenotype of epithelial cells. Disruption of E- cadherin activity in epithelial cells correlates with formation of metastatic tumors. Decreased adhesive function may be implemented in a number of ways including: (a) decreased expression of E-cadherin; (b) mutations in the gene encoding E-cadherin; or (c) mutations in the genes that encode the catenins, proteins that link the cadherins to the cytoskeleton and are essential for cadherin mediated cell-cell adhesion. In this study, we explored the possibility that inappropriate expression of a nonepithelial cadherin by an epithelial cell might also result in disruption of cell-cell adhesion. We showed that a squamous cell carcinoma-derived cell line expressed N-cadherin and displayed a scattered fibroblastic phenotype along with decreased expression of E- and P-cadherin. Transfection of this cell line with antisense N- cadherin resulted in reversion to a normal-appearing squamous epithelial cell with increased E- and P-cadherin expression. In addition, transfection of a normal-appearing squamous epithelial cell line with N-cadherin resulted in downregulation of both E- and P- cadherin and a scattered fibroblastic phenotype. In all cases, the levels of expression of N-cadherin and E-cadherin were inversely related to one another. In addition, we showed that some squamous cell carcinomas expressed N-cadherin in situ and those tumors expressing N- cadherin were invasive. These studies led us to propose a novel mechanism for tumorigenesis in squamous epithelial cells; i.e., inadvertent expression of a nonepithelial cadherin.     

Diversity of the cadherin family: evidence for eight new cadherins in nervous tissue.

To examine the diversity of the cadherin family, we isolated cDNAs from brain and retina cDNA preparations with the aid of polymerase chain reaction. The products obtained included cDNAs for two of three known cadherins as well as eight distinct cDNAs, of which deduced amino acid sequences show significant similarity with the known cadherin sequences. Larger cDNA clones were isolated from human cDNA libraries for six of the eight new molecules. The deduced amino acid sequences show that the overall structure of these molecules is very similar to that of the known cadherins, indicating that these molecules are new members of the cadherin family. We have tentatively designated these cadherins as cadherin-4 through -11. The new molecules, with the exception of cadherin-4, exhibit features that distinguish them as a group from previously cloned cadherins; they may belong to a new subfamily of cadherins. Northern blot analysis showed that most of these cadherins are expressed mainly in brain, although some are expressed in other tissues as well. These findings show that the cadherin family of adhesion molecules is much larger than previously thought, and suggest that the new cadherins may play an important role in cell-cell interactions within the central nervous system.     

Cadherin-mediated cell-cell adhesion: sticking together as a family

The cadherins comprise a family of single-pass transmembrane proteins critical for cell-cell adhesion in vertebrates and invertebrates. The recently determined structure of the whole ectodomain from C-cadherin suggests that the adhesion of cadherins presented by juxtaposed cells is mediated by a strand-swapped dimer in which core hydrophobic elements are exchanged between the partner molecules. Sequence analysis suggests that several cadherin subfamilies share this adhesive mechanism. Recent work has shed new light on the molecular basis of cadherin adhesion, although understanding the specificity of these interactions remains a major challenge.     

Homoassociation of VE-cadherin follows a mechanism common to "classical" cadherins

Vascular endothelial cadherin (VE-cadherin/cadherin5) is specifically expressed in adherens junctions of endothelial cells and exerts important functions in cell-cell adhesion as well as signal transduction. To analyze the mechanism of VE-cadherin homoassociation, the ectodomains CAD1-5 were connected by linker sequences to the N terminus of the coiled-coil domain of cartilage matrix protein (CMP). The chimera VECADCMP were expressed in mammalian cells. The trimeric coiled-coil domain leads to high intrinsic domain concentrations and multivalency promoting self-association. Ca(2+)-dependent homophilic association of VECADCMP was detected in solid phase assays and cross-linking experiments. A striking analogy to homoassociation of type I ("classical") cadherins like E, N or P-cadherin was observed when interactions in VECADCMP and between these trimeric proteins were analyzed by electron microscopy. Ca(2+)-dependent ring-like and double ring-like arrangements suggest interactions between domains 1 and 2 of the ectodomains, which may be correlated with lateral and adhesive contacts in the adhesion process. Association to complexes composed of two VECADCMP molecules was also demonstrated by chemical cross-linking. No indication for an antiparallel association of VECAD ectodomains to hexameric complexes as proposed by Legrand et al. was found. Instead the data suggest that homoassociation of VE-cadherin follows the conserved mechanism of type I cadherins.     

Regulation of Rho family GTPases by cell-cell and cell-matrix adhesion

Integrins and cadherins are transmembrane adhesion receptors that are necessary for cells to interact with the extracellular matrix or adjacent cells, respectively. Integrins and cadherins initiate signaling pathways that modulate the activity of Rho family GTPases. The Rho proteins Cdc42, Rac1, and RhoA regulate the actin cytoskeleton. Cdc42 and Rac1 are primarily involved in the formation of protrusive structures, while RhoA generates myosin-based contractility. Here we examine the differential regulation of RhoA, Cdc42, and Rac1 by integrin and cadherin signaling. Integrin and cadherin signaling leads to a decrease in RhoA activity and activation of Cdc42 and Rac1. When the normal RhoA suppression is antagonized or RhoA signaling is increased, cells exhibited impaired spreading on the matrix protein fibronectin and decreased cell-cell adhesion. Spreading on fibronectin and the formation of cell-cell adhesions is decreased in cells expressing dominant negative forms of Cdc42 or Rac1. These data demonstrate that integrins and cadherins regulate Rho proteins in a comparable manner and lead us to speculate that these changes in Rho protein activity participate in a feedback mechanism that promotes further cell-matrix or cell-cell interaction, respectively.     

Transmembrane control of cadherin-mediated cell-cell adhesion

The cadherin family of cell-cell adhesion molecules plays a central role in organization of cells into multicellular structures. An important feature of the action of cadherins is that they form a complex with cytoskeletal proteins, and the formation of this complex is crucial for their adhesive function. Cadherin-mediated cell adhesion is thus controlled through the interaction with cytoplasmic proteins, and, for such control, phosphorylation of these proteins and also cadherins themselves might be involved. This regulatory mechanism of cell adhesion is perhaps fundamental to a variety of morphogenetic processes.