Adhesive and lateral E-cadherin dimers are mediated by the same interface

E-cadherin is a transmembrane protein that mediates Ca(2+)-dependent cell-cell adhesion. To study cadherin-cadherin interactions that may underlie the adhesive process, a recombinant E-cadherin lacking free sulfhydryl groups and its mutants with novel cysteines were expressed in epithelial A-431 cells. These cysteine mutants, designed according to various structural models of cadherin dimers, were constructed to reveal cadherin dimerization by the bifunctional sulfhydryl-specific cross-linker BM[PE0]3. Cross-linking experiments with the mutants containing a cysteine at strand B of their EC1 domains did show cadherin dimerization. By their properties these dimers correspond to those which have been characterized by co-immunoprecipitation assay. Under standard culture conditions the adhesive dimer is a dominant form. Calcium depletion dissociates adhesive dimers and promotes the formation of lateral dimers. Our data show that both dimers are mediated by the amino-terminal cadherin domain. Furthermore, the interfaces involved in both adhesive and lateral dimerization appear to be the same. The coexistence of the structurally identical adhesive and lateral dimers suggests some flexibility of the extracellular cadherin region.     

Both the dimerization and immunochemical properties of E-cadherin EC1 domain depend on Trp(156) residue

Using site-directed mutagenesis, we show in this paper that the adhesive interface detected in cadherin crystals is unlikely to mediate adhesive interaction between myc- and flag-tagged E-cadherin molecules in human A-431 cells. We also found that a critical residue within this interface, His(233), is part of the epitope for mAb SHE78-7. This epitope was accessible to the antibody in the adhesive E-cadherin dimers, which is consistent with uninvolvement of the site containing His(233) in cell-cell adhesion. However, both the adhesive dimerization and the integrity of the SHE78-7 epitope depended on the same intramolecular interaction between Trp(156) and its hydrophobic pocket. Our data suggest that this interaction may have an important regulatory function in controlling the surface topology of the NH(2)-terminal domain of E-cadherin.     

Endocytosis of cadherin from intracellular junctions is the driving force for cadherin adhesive dimer disassembly

The adhesion receptor E-cadherin maintains cell-cell junctions by continuously forming short-lived adhesive dimers. Here mixed culture cross-linking and coimmunoprecipitation assays were used to determine the dynamics of adhesive dimer assembly. We showed that the amount of these dimers increased dramatically minutes after the inhibition of endocytosis by ATP depletion or by hypertonic sucrose. This increase was accompanied by the efficient recruitment of E-cadherin into adherens junctions. After 10 min, when the adhesive dimer amount had reached a plateau, the assembly of new dimers stalled completely. These cells, in a striking difference from the control, became unable to disintegrate both their intercellular contacts and adhesive dimers in response to calcium depletion. The same effects, but after a slightly longer time course, were obtained using acidic media, another potent approach inhibiting endocytosis. These data suggest that endocytosis is the main pathway for the dissociation of E-cadherin adhesive dimers. Its inhibition blocks the replenishment of the monomeric cadherin pool, thereby inhibiting new dimer formation. This suggestion has been corroborated by immunoelectron microscopy, which revealed cadherin-enriched coated pit-like structures in close association with adherens junctions.     

Adhesive But Not Lateral E-cadherin Complexes Require Calcium and Catenins for Their Formation

We examined intercadherin interactions in epithelial A-431 cells producing endogenous E-cadherin and recombinant forms of E-cadherin tagged either by myc or by flag epitopes. Three distinct E-cadherin complexes were found. The first is a conventional E-cadherin–catenin complex consisting of one E-cadherin molecule linked either to β-catenin/α-catenin or to plakoglobin/α-catenin dimers. The second is a lateral E-cadherin complex incorporating two E-cadherin– catenin conventional complexes combined in parallel fashion via dimerization of the NH₂-terminal extracellular domain of E-cadherin. The third complex is likely to contain two E-cadherin–catenin conventional complexes derived from two opposing cells and arranged in an antiparallel fashion. Formation of the antiparallel but not lateral complex strictly depends on extracellular calcium and E-cadherin binding to catenins. Double amino acid substitution Trp156Ala/Val157Gly within the extracellular NH₂-terminal E-cadherin domain completely abolished both lateral and antiparallel inter–E-cadherin association. These data support an idea that the antiparallel complex has the adhesion function. Furthermore, they allow us to suggest that antiparallel complexes derive from lateral dimers and this complex process requires catenins and calcium ions.     

Stable and unstable cadherin dimers: mechanisms of formation and roles in cell adhesion

Numerous attempts to elucidate the strength of cadherin dimerization that mediates intercellular adhesion have produced controversial and inconclusive results. To clarify this issue, we compared E-cadherin dimerization on the surface of living cells with how the same process unfolds on agarose beads. In both cases, dimerization was monitored by the same site-specific cross-linking assay, greatly simplifying data interpretation. We showed that on the agarose surface under physiological conditions, E-cadherin produced a weak dimer that immediately dissociated after the depletion of calcium ions. However, either at pH 5 or in the presence of cadmium ions, E-cadherin produced a strong dimer that was unable to dissociate upon calcium depletion. Both types of dimers were W156-dependent. Remarkably, only the strong dimer was found on the surface of living cells. We also showed that the intracellular cadherin region, the clustering of which through catenins had been proposed as stabilizer of weak intercadherin interactions, was not needed, in fact, for cadherin junction assembly. Taken together, our data present convincing evidence that cadherin adhesion is based on high-affinity cadherin-cadherin interactions.     

Conformational epitopes at cadherin calcium-binding sites and p120-catenin phosphorylation regulate cell adhesion

We investigated changes in cadherin structure at the cell surface that regulate its adhesive activity. Colo 205 cells are nonadhesive cells with a full but inactive complement of E-cadherin-catenin complexes at the cell surface, but they can be triggered to adhere and form monolayers. We were able to distinguish the inactive and active states of E-cadherin at the cell surface by using a special set of monoclonal antibodies (mAbs). Another set of mAbs binds E-cadherin and strongly activates adhesion. In other epithelial cell types these activating mAbs inhibit growth factor-induced down-regulation of adhesion and epithelial morphogenesis, indicating that these phenomena are also controlled by E-cadherin activity at the cell surface. Both types of mAbs recognize conformational epitopes at different interfaces between extracellular cadherin repeat domains (ECs), especially near calcium-binding sites. Activation also induces p120-catenin dephosphorylation, as well as changes in the cadherin cytoplasmic domain. Moreover, phospho-site mutations indicate that dephosphorylation of specific Ser/Thr residues in the N-terminal domain of p120-catenin mediate adhesion activation. Thus physiological regulation of the adhesive state of E-cadherin involves physical and/or conformational changes in the EC interface regions of the ectodomain at the cell surface that are mediated by catenin-associated changes across the membrane.     

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.     

Defining interactions and distributions of cadherin and catenin complexes in polarized epithelial cells

The cadherin/catenin complex plays important roles in cell adhesion, signal transduction, as well as the initiation and maintenance of structural and functional organization of cells and tissues. In the preceding study, we showed that the assembly of the cadherin/catenin complex is temporally regulated, and that novel combinations of catenin and cadherin complexes are formed in both Triton X-100-soluble and - insoluble fractions; we proposed a model in which pools of catenins are important in regulating assembly of E-cadherin/catenin and catenin complexes. Here, we sought to determine the spatial distributions of E- cadherin, alpha-catenin, beta-catenin, and plakoglobin, and whether different complexes of these proteins accumulate at steady state in polarized Madin-Darby canine kidney cells. Protein distributions were visualized by wide field, optical sectioning, and double immunofluorescence microscopy, followed by reconstruction of three- dimensional images. In cells that were extracted with Triton X-100 and then fixed (Triton X-100-insoluble fraction), more E-cadherin was concentrated at the apical junction relative to other areas of the lateral membrane. alpha-Catenin and beta-catenin colocalize with E- cadherin at the apical junctional complex. There is some overlap in the distribution of these proteins in the lateral membrane, but there are also areas where the distributions are distinct. Plakoglobin is excluded from the apical junctional complex, and its distribution in the lateral membrane is different from that of E-cadherin. Cells were also fixed and then permeabilized to reveal the total cellular pool of each protein (Triton X-100-soluble and -insoluble fractions). This analysis showed lateral membrane localization of alpha-catenin, beta- catenin, and plakoglobin, and it also revealed that they are distributed throughout the cell. Chemical cross-linking of proteins and analysis with specific antibodies confirmed the presence at steady state of E-cadherin/catenin complexes containing either beta-catenin or plakoglobin, and catenin complexes devoid of E-cadherin. Complexes containing E-cadherin/beta-catenin and E-cadherin/alpha-catenin are present in both the Triton X-100-soluble and -insoluble fractions, but E-cadherin/plakoglobin complexes are not detected in the Triton X-100- insoluble fraction. Taken together, these results show that different complexes of cadherin and catenins accumulate in fully polarized epithelial cells, and that they distribute to different sites. We suggest that cadherin/catenin and catenin complexes at different sites have specialized roles in establishing and maintaining the structural and functional organization of polarized epithelial cells.     

Intestinal LI-cadherin acts as a Ca2+-dependent adhesion switch

Cadherins are Ca(2+)-dependent transmembrane glycoproteins that mediate cell-cell adhesion and are important for the structural integrity of epithelia. LI-cadherin and the classical E-cadherin are the predominant two cadherins in the intestinal epithelium. LI-cadherin consists of seven extracellular cadherin repeats and a short cytoplasmic part that does not interact with catenins. In contrast, E-cadherin is composed of five cadherin repeats and a large cytoplasmic domain that is linked via catenins to the actin cytoskeleton. Whereas E-cadherin is concentrated in adherens junctions, LI-cadherin is evenly distributed along the lateral contact area of intestinal epithelial cells. To investigate if the particular structural properties of LI-cadherin result in a divergent homotypic adhesion mechanism, we analyzed the binding parameters of LI-cadherin on the single molecule and the cellular level using atomic force microscopy, affinity chromatography and laser tweezer experiments. Homotypic trans-interaction of LI-cadherin exhibits low affinity binding with a short lifetime of only 1.4 s. Interestingly, LI-cadherin binding responds to small changes in extracellular Ca(2+) below the physiological plasma concentration with a high degree of cooperativity. Thus, LI-cadherin might serve as a Ca(2+)-regulated switch for the adhesive system on basolateral membranes of the intestinal epithelium.     

Recognition of E-cadherin by integrin alpha(E)beta(7): requirement for cadherin dimerization and implications for cadherin and integrin function.

We have investigated the importance of dimerization of E-cadherin in the heterophilic adhesive interaction between E-cadherin and integrin alpha(E)beta(7). Dimerization of cadherin molecules in parallel alignment is known to be essential for homophilic adhesion and has been attributed to Ca(2+)-dependent interactions in the domain 1-2 junction or to cross-intercalation of Trp2 from one molecule to the other. We have disrupted either or both of these proposed mechanisms by point mutations in E-cadherin-Fc and have tested the modified proteins for alpha(E)beta(7)-mediated cell adhesion. Prevention of Trp2 intercalation had no adverse effect on integrin-mediated adhesion, whereas disruption of Ca(2+) binding permitted adhesion but with reduced efficiency. Both modifications in combination abolished recognition by alpha(E)beta(7). In EGTA, alpha(E)beta(7) adhered to wild type E-cadherin but not to the Trp2 deletion mutant. Independent evidence that the mutations prevented either or both mechanisms for dimerization is presented. The data show that dimerization is required for recognition by alpha(E)beta(7) and that it can take place by either of two mechanisms. Implications for the roles of the alpha(E) and beta(7) integrin subunits in ligand binding and for Trp2 and Ca(2+) in the assembly of cadherin complexes are discussed.     

Profiling of E-cadherin, beta-catenin and Ca(2+) in embryo-uterine interactions at implantation

Establishment of early pregnancy is promoted by a complex network of signalling molecules that mediate cell-to-cell and cell-to-extracellular matrix communications between the receptive endometrium and the invasive trophectoderm. In this study, we have attempted to evaluate the expression profiles of cadherin and catenin during embryo implantation in the mouse. Western blotting studies along with immunocytochemical analysis revealed that E-cadherin is expressed rather ubiquitously in the uterine epithelial cells, distinct enrichment is observed on the apical membrane in the endometrium of peri-implantation uterus specifically at the implantation sites and not at the inter-implanation sites. beta-Catenin also is upregulated and is specifically restricted to apical membrane of epithelial cells of implantation sites. Progesterone induced expression of E-cadherin and 17beta-estradiol regulated the expression of catenin in implantation-delayed uteri. Interestingly, estradiol imparted negative modulation on cadherin expression when co-administered with progesterone. On the contrary, trophoblast exhibits a striking down regulation of cadherin, catenin and Ca(2+) at peri implanting stage. These observations suggest that the trophoblasts exhibited an invasive phenotype while the endometrial epithelium displayed an adhesive phenotype during the window of implantation. Thus, embryo implantation presents an instance where two interacting surfaces showed mutually complementing interaction phenotypes.     

Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling

The epithelial cadherin (E-cadherin)-catenin complex binds to cytoskeletal components and regulatory and signaling molecules to form a mature adherens junction (AJ). This dynamic structure physically connects neighboring epithelial cells, couples intercellular adhesive contacts to the cytoskeleton, and helps define each cell's apical-basal axis. Together these activities coordinate the form, polarity, and function of all cells in an epithelium. Several molecules regulate AJ formation and integrity, including Rho family GTPases and Par polarity proteins. However, only recently, with the development of live-cell imaging, has the extent to which E-cadherin is actively turned over at junctions begun to be appreciated. This turnover contributes to junction formation and to the maintenance of epithelial integrity during tissue homeostasis and remodeling.     

The crystal structure of human E-cadherin domains 1 and 2, and comparison with other cadherins in the context of adhesion mechanism

Cell adhesion mediated by type I cadherins involves homophilic "trans" interactions that are thought to be brought about by a strand exchange mechanism involving the N-terminal extracellular domain. Here, we present the high-resolution crystal structure of the N-terminal two domains of human E-cadherin. Comparison of this structure with other type I cadherin structures reveals features that are likely to be critical to facilitate dimerization by strand exchange as well as dimer flexibility. We integrate this structural knowledge to provide a model for type I cadherin adhesive interactions. Intra-molecular docking of the conserved N-terminal "adhesion arm" into the acceptor pocket in monomeric E-cadherin appears largely identical to inter-molecular docking of the adhesion arm in adhesive trans dimers. A strained conformation of the adhesion arm in the monomer, however, may create an equilibrium between "open" and "closed" forms that primes the cadherin for formation of adhesive interactions, which are then stabilized by additional dimer-specific contacts. By contrast, in type II cadherins, strain in the adhesion arm appears absent and a much larger surface area is involved in trans adhesion, which may compensate the activation energy required to peel off the intra-molecularly docked arm. It seems that evolution has selected slightly different adhesion mechanisms for type I and type II cadherins.     

Low-expression of E-cadherin in leukaemia cells causes loss of homophilic adhesion and promotes cell growth

E-cadherin (epithelial cadherin) belongs to the calcium-dependent adhesion molecule superfamily and is implicated in the interactions of haematopoietic progenitors and bone marrow stromal cells. Adhesion capacity to bone marrow stroma was impaired for leukaemia cells, suggesting that a breakdown of adhesive mechanisms governed by an adhesion molecule may exist in leukaemic microenvironment. We previously found that E-cadherin was low expressed in primary acute leukaemia cells compared with normal bone marrow mononuclear cells. In this study, we investigate the functional importance of low E-cadherin expression in leukaemia cell behaviours and investigate its effects in the abnormal interaction of leukaemic cells with stromal cells. After expression of E-cadherin was restored by a demethylating agent in leukaemia cells, E-cadherin-specific adhesion was enhanced. Additionally, siRNA (small interfering RNA)-mediated silencing of E-cadherin in Raji cells resulted in a reduction of cell homophilic adhesion and enhancement of cell proliferation and colony formation. These results suggest that low expression of E-cadherin contributes to the vigorous growth and transforming ability of leukaemic cells.     

Cadherin exits the junction by switching its adhesive bond

The plasticity of cell-cell adhesive structures is crucial to all normal and pathological morphogenetic processes. The molecular principles of this plasticity remain unknown. Here we study the roles of two dimerization interfaces, the so-called strand-swap and X dimer interfaces of E-cadherin, in the dynamic remodeling of adherens junctions using photoactivation, calcium switch, and coimmunoprecipitation assays. We show that the targeted inactivation of the X dimer interface blocks the turnover of catenin-uncoupled cadherin mutants in the junctions of A-431 cells. In contrast, the junctions formed by strand-swap dimer interface mutants exhibit high instability. Collectively, our data demonstrate that the strand-swap interaction is a principal cadherin adhesive bond that keeps cells in firm contact. However, to leave the adherens junction, cadherin reconfigures its adhesive bond from the strand swap to the X dimer type. Such a structural transition, controlled by intercellular traction forces or by lateral cadherin alignment, may be the key event regulating adherens junction dynamics.     

CAS/CSE 1 stimulates E-cadhrin-dependent cell polarity in HT-29 human colon epithelial cells

The establishment and maintenance of epithelial polarity are crucial for tissue organization and function in mammals. Epithelial cadherin (E-cadherin) is expressed in epithelial cell membrane and is important for cell-cell adhesion, intercellular junctions formation, as well as epithelial cell polarization. We report herein that CAS (CAS/CSE 1), the human cellular apoptosis susceptibility protein, interacts with E-cadherin and stimulates polarization of HT-29 human colon epithelial cells. CAS binds with E-cadherin but not with beta-catenin in the immunoprecipitation assays. Interaction of CAS with E-cadherin enhances the formation of E-cadherin/beta-catenin cell-cell adhesive complex. Electron microscopic study demonstrated that CAS overexpression in cells stimulates intercellular junction complex formation. The disorganization of cellular cytoskeleton by cytochalasin D, colchicine, or acrylamide treatment disrupts CAS-stimulated HT-29 cell polarization. CAS-mediated HT-29 cell polarity is also inhibited by antisense E-cadherin DNA expression. Our results indicate that CAS cooperates with E-cadherin and plays a role in the establishment of epithelial cell polarity.     

A novel cadherin cell adhesion molecule: its expression patterns associated with implantation and organogenesis of mouse embryos

The Ca2+-dependent cell adhesion molecules, termed cadherins, were previously divided into two subclasses, E- and N-types, with different adhesive specificity. In this study, we identified a novel class of cadherin, termed P-cadherin, using a visceral endoderm cell line PSA5- E. This cadherin was a 118,000-D glycoprotein and distinct from E- and N-cadherins in immunological specificity and molecular mass. In accord with these findings, cells with P-cadherin did not cross-adhere with cells with E-cadherin. P-Cadherin first appeared in developing mouse embryos in the extraembryonic ectoderm and the visceral endoderm at the egg cylinder stage and later was expressed in various tissues. The placenta and the uterine decidua most abundantly expressed this cadherin. The expression of P-cadherin was transient in many tissues, and its permanent expression was limited to certain tissues such as the epidermis, the mesothelium, and the corneal endothelium. When the tissue distribution of P-cadherin was compared with that of E-cadherin, we found that: each cadherin displayed a unique spatio-temporal pattern of expression; P-cadherin was co-expressed with E-cadherin in local regions of various tissues; and onset or termination of expression of P- cadherin was closely associated with connection or segregation of cell layers, as found with other cadherins. These results suggested that differential expression of multiple classes of cadherins play a role in implantation and morphogenesis of embryos by providing cells with heterogenous adhesive specificity.     

Misregulated E-cadherin expression associated with an aggressive brain tumor phenotype

Background

Cadherins are essential components of the adherens junction complexes that mediate cell-cell adhesion and regulate cell motility. During tissue morphogenesis, changes in cadherin expression (known as cadherin switching) are a common mechanism for altering cell fate. Cadherin switching is also common during epithelial tumor progression, where it is thought to promote tumor invasion and metastasis. E-cadherin is the predominant cadherin expressed in epithelial tissues, but its expression is very limited in normal brain.

Methodology/Principal Findings

We identified E-cadherin expression in a retrospective series of glioblastomas exhibiting epithelial or pseudoepithelial differentiation. Unlike in epithelial tissues, E-cadherin expression in gliomas correlated with an unfavorable clinical outcome. Western blotting of two panels of human GBM cell lines propagated either as xenografts in nude mice or grown under conventional cell culture conditions confirmed that E-cadherin expression is rare. However, a small number of xenograft lines did express E-cadherin, its expression correlating with increased invasiveness when the cells were implanted orthotopically in mouse brain. In the conventionally cultured SF767 glioma cell line, E-cadherin expression was localized throughout the plasma membrane rather than being restricted to areas of cell-cell contact. ShRNA knockdown of E-cadherin in these cells resulted in decreased proliferation and migration in vitro.

Conclusions/Significance

Our data shows an unexpected correlation between the abnormal expression of E-cadherin in a subset of GBM tumor cells and the growth and migration of this aggressive brain tumor subtype.     

N-cadherin-mediated cell motility requires cis dimers

Cadherins are expressed on the cell surface as a dimer in the membrane of one cell (cis dimer) that interacts with a cis dimer on an adjacent cell to form an adhesive trans dimer. It is well established that both cis and trans dimers must form for the cadherin to be an effective adhesion protein. In addition to their adhesive activity cadherins also play an important role in modulating cell behavior by regulating cell motility and signal transduction. Whether or not cis or trans dimers are necessary for the nonadhesive functions of cadherins has not been addressed. Here we show that N-cadherin cis dimers are necessary to induce cell motility in epithelial cells and that N-cadherin's ability to modulate the steady state levels of activated small GTPases requires both cis and trans dimers.     

E-cadherin homophilic ligation directly signals through Rac and phosphatidylinositol 3-kinase to regulate adhesive contacts.

Classical cadherins mediate cell recognition and cohesion in many tissues of the body. It is increasingly apparent that dynamic cadherin contacts play key roles during morphogenesis and that a range of cell signals are activated as cells form contacts with one another. It has been difficult, however, to determine whether these signals represent direct downstream consequences of cadherin ligation or are juxtacrine signals that are activated when cadherin adhesion brings cell surfaces together but are not direct downstream targets of cadherin signaling. In this study, we used a functional cadherin ligand (hE/Fc) to directly test whether E-cadherin ligation regulates phosphatidylinositol 3-kinase (PI 3-kinase) and Rac signaling. We report that homophilic cadherin ligation recruits Rac to nascent adhesive contacts and specifically stimulates Rac signaling. Adhesion to hE/Fc also recruits PI 3-kinase to the cadherin complex, leading to the production of phosphatidylinositol 3,4,5-trisphosphate in nascent cadherin contacts. Rac activation involved an early phase, which was PI 3-kinase-independent, and a later amplification phase, which was inhibited by wortmannin. PI 3-kinase and Rac activity were necessary for productive adhesive contacts to form following initial homophilic ligation. We conclude that E-cadherin is a cellular receptor that is activated upon homophilic ligation to signal through PI 3-kinase and Rac. We propose that a key function of these cadherin-activated signals is to control adhesive contacts, probably via regulation of the actin cytoskeleton, which ultimately serves to mediate adhesive cell-cell recognition.