Dynamic interplay between adhesive and lateral E-cadherin dimers

E-cadherin, an adhesive transmembrane protein of epithelial adherens junctions, forms two types of detergent-resistant dimers: adhesive dimers consisting of cadherin molecules derived from two neighboring cells and lateral dimers incorporating cadherins of the same cell. Both dimers depend on the integrity of the same residue, Trp156. While the relative amounts of these complexes are not certain, we show here that in epithelial A-431 cells, adhesive dimers may be a prevalent form. Inactivation of the calcium-binding sites, located between successive cadherin ectodomains, drastically reduced the amount of adhesive dimers and concomitantly increased the amount of lateral dimers. A similar interdependence of adhesive and lateral dimers was observed in digitonin-permeabilized cells. In these cells, adhesive dimers immediately disassembled after lowering the Ca2+ concentration below 0.1 mM. The disappearance of adhesive dimers was counterbalanced by an increase in Trp156-dependent lateral dimers. Increasing the calcium concentration to a normal level rapidly restored the original balance between adhesive and lateral dimers. We also present evidence that E-cadherin dimers in vivo have a short lifetime. These observations suggest that cadherin-mediated adhesion is based on the dynamic cycling of E-cadherin between monomeric and adhesive dimer states.     

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.     

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.     

Lateral dimerization of the E-cadherin extracellular domain is necessary but not sufficient for adhesive activity

Cadherins are transmembrane glycoproteins involved in Ca(2+)-dependent cell-cell adhesion. Using L cells coexpressing E-cadherin constructs with different epitope tags, we examined the lateral dimerization of E-cadherin and its adhesive activity by co-immunoprecipitation and aggregation assays, respectively. Although the transmembrane domain is required for dimerization, tail-less constructs possessing the transmembrane domain of either N-cadherin or CD45 show dimerization and are active in aggregation assays. Two mutant constructs having either of two amino acid substitutions, W2A or substitutions that disrupt the recognition sequence for endoproteolytic enzymes involved in removal of the precursor segment, cannot form dimers and are inactive in aggregation. These monomeric proteins, like their wild-type dimerizing counterparts, retain their Ca(2+)-dependent resistance to trypsin digestion, suggesting that dimerization per se does not induce a large conformational change. Two other constructs, having either an amino acid substitution, D134A, or a C-terminal deletion of 70 amino acid residues, retain the ability to associate laterally but are inactive in aggregation assays. Staurosporine treatment of cells expressing the latter construct increases aggregation but does not increase the extent of lateral dimerization. Thus, lateral dimerization is necessary, but not sufficient for adhesive activity.     

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.     

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.     

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.     

Crystal structure of the M-fragment of alpha-catenin: implications for modulation of cell adhesion.

The cytoskeletal protein alpha-catenin, which shares structural similarity with vinculin, is required for cadherin-mediated cell adhesion, and functions to modulate cell adhesive strength and to link the cadherins to the actin-based cytoskeleton. Here we describe the crystal structure of a region of alpha-catenin (residues 377-633) termed the M-fragment. The M-fragment is composed of a tandem repeat of two antiparallel four-helix bundles of virtually identical architectures that are related in structure to the dimerization domain of alpha-catenin and the tail region of vinculin. These results suggest that alpha-catenin is composed of repeating antiparallel helical domains. The region of alpha-catenin previously defined as an adhesion modulation domain corresponds to the C-terminal four-helix bundle of the M-fragment, and in the crystal lattice these domains exist as dimers. Evidence for dimerization of the M-fragment of alpha-catenin in solution was detected by chemical cross-linking experiments. The tendency of the adhesion modulation domain to form dimers may explain its biological activity of promoting cell-cell adhesiveness by inducing lateral dimerization of the associated cadherin molecule.     

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.     

Analysis of heterophilic and homophilic interactions of cadherins using the c-Jun/c-Fos dimerization domains

Cadherin-mediated cell-cell adhesion is initiated by cis dimerization of cadherin ectodomains at the cell surface followed by an antiparallel trans interaction of dimers on opposing cells. To resolve open questions concerning the molecular details and specificity of cis and trans interactions, ectodomains of E- and P-cadherin were analyzed by chemical cross-linking and by electron microscopy. At the high intrinsic concentration created by artificial oligomerization the N-terminal cadherin (CAD)-domain of P-cadherin are forming ring-like cis dimers. At 2 mm Ca(2+)-associated rings involving two cis dimers indicate trans contacts in electron micrographs. cis and trans interactions were further analyzed by heterodimerization of the ectodomains of E-cadherin (ECAD) and P-cadherin (PCAD) through the leucine zipper domains of c-Jun and c-Fos. ECADJun/ECADFos dimers predominantly form ring-like cis dimers at 1 mm Ca(2+) and double-ringed trans contacts above 2 mm Ca(2+). The Ca(2+)-dependent tetrameric trans contacts of ECADJun/ECADFos dimers are also detectable after chemical cross-linking. Only cis contacts but no trans interactions are observed for heterodimers of ECADFos and the Trp-2 to Ala mutant ECADW2AJun arguing for a decisive role of Trp-2 in trans but not cis interaction. Neither cis nor trans interaction was found for heterodimers of ECADJun and PCADFos suggesting that specificity for homophilic interactions already exists at the level of cis dimerization.     

The Membrane-proximal Region of the E-Cadherin Cytoplasmic Domain Prevents Dimerization and Negatively Regulates Adhesion Activity

Cadherins are transmembrane glycoproteins involved in Ca²⁺-dependent cell–cell adhesion. Deletion of the COOH-terminal residues of the E-cadherin cytoplasmic domain has been shown to abolish its cell adhesive activity, which has been ascribed to the failure of the deletion mutants to associate with catenins. Based on our present results, this concept needs revision. As was reported previously, leukemia cells (K562) expressing E-cadherin with COOH-terminal deletion of 37 or 71 amino acid residues showed almost no aggregation. Cells expressing E-cadherin with further deletion of 144 or 151 amino acid residues, which eliminates the membrane-proximal region of the cytoplasmic domain, showed E-cadherin–dependent aggregation. Thus, deletion of the membrane-proximal region results in activation of the nonfunctional E-cadherin polypeptides. However, these cells did not show compaction. Chemical cross-linking revealed that the activated E-cadherin polypeptides can be cross-linked to a dimer on the surface of cells, whereas the inactive polypeptides, as well as the wild-type E-cadherin polypeptide containing the membrane-proximal region, can not. Therefore, the membrane-proximal region participates in regulation of the adhesive activity by preventing lateral dimerization of the extracellular domain.     

Characterization of a region of the measles virus hemagglutinin sufficient for its dimerization

Attachment of measles virus (MV) to its cellular receptor is mediated by the viral envelope glycoprotein hemagglutinin (H). H exists at the viral surface as a disulfide-linked dimer which may associate into a tetramer. We aimed to define regions of H essential for its homo-oligomerization. To delineate these more precisely, we have generated a series of H ectodomain truncation mutants and studied their abilities to form both homotypic complexes and heterotypic complexes with full-length H. We define a "minimal unit" which is sufficient for MV H dimerization as that encompassing residues 1 to 151. This unit forms both homodimers and heterodimers with full-length H protein, although neither is transported to the cell surface even in the presence of other MV proteins. We show that cysteine residues at positions 139 and 154 are both critical in mediating covalent dimerization, not only of the truncated H mutants but also of full-length MV H protein. Even those cysteine mutants unable to form covalent intermolecular interactions are biologically active, mediating the formation of syncytia, albeit at a reduced rate. We demonstrate that this impaired capacity to mediate cell-to-cell fusion is based mainly on a reduced transport rate of the mutant molecules to the cell surface, indicating a role for covalent intermolecular interactions in efficient transport of MV H dimers to the cell surface.     

Ksp-cadherin is a functional cell-cell adhesion molecule related to LI-cadherin

Ksp- and LI-cadherin are structurally homologous proteins coexpressed with E-cadherin in renal and intestinal epithelia, respectively. Whereas LI-cadherin has been shown to mediate Ca2+-dependent homotypic cell-cell adhesion independent of stable interactions with the cytoskeleton, little is known about the physiological role of Ksp-cadherin. To analyze its potential adhesive and morphoregulatory functions, we expressed murine Ksp-cadherin in CHO cells. In this report, we show that Ksp-cadherin induces homotypic and Ca2+-dependent cell-cell adhesion that can be specifically blocked with antibodies raised against the cadherin repeats EC1 and EC2. Ksp-cadherin mediates about the same quantitative adhesive effect (aggregation index) as LI- and E-cadherin. However, the cellular phenotype induced by Ksp-cadherin resembles more closely that of LI- than E-cadherin. This could reflect our observation, that Ksp-cadherin, as well as LI-cadherin, does not directly interact with beta-catenin. In conclusion, both cadherins are thus not only structurally but also functionally related and may share other functions within their respective epithelia.     

Rac is a dominant regulator of cadherin-directed actin assembly that is activated by adhesive ligation independently of Tiam1

Classic cadherins function as adhesion-activated cell signaling receptors. On adhesive ligation, cadherins induce signaling cascades leading to actin cytoskeletal reorganization that is imperative for cadherin function. In particular, cadherin ligation activates actin assembly by the actin-related protein (Arp)2/3 complex, a process that critically affects the ability of cells to form and extend cadherin-based contacts. However, the signaling pathway(s) that activate Arp2/3 downstream of cadherin adhesion remain poorly understood. In this report we focused on the Rho family GTPases Rac and Cdc42, which can signal to Arp2/3. We found that homophilic engagement of E-cadherin simultaneously activates both Rac1 and Cdc42. However, by comparing the impact of dominant-negative Rac1 and Cdc42 mutants, we show that Rac1 is the dominant regulator of cadherin-directed actin assembly and homophilic contact formation. To pursue upstream elements of the Rac1 signaling pathway, we focused on the potential contribution of Tiam1 to cadherin-activated Rac signaling. We found that Tiam1 or the closely-related Tiam2/STEF1 was recruited to cell-cell contacts in an E-cadherin-dependent fashion. Moreover, a dominant-negative Tiam1 mutant perturbed cell spreading on cadherin-coated substrata. However, disruption of Tiam1 activity with dominant-negative mutants or RNA interference did not affect the ability of E-cadherin ligation to activate Rac1. We conclude that Rac1 critically influences cadherin-directed actin assembly as part of a signaling pathway independent of Tiam1. actin cytoskeleton; Cdc42; E-cadherin     

Ankyrin-G Inhibits Endocytosis of Cadherin Dimers

Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Therefore, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. However, the mechanisms that control junctional plasticity are not fully understood. Vascular endothelial cadherin (VE-cadherin) is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with β- and α-catenin, which serve to increase adhesive strength. Furthermore, p120-catenin binds to the cytoplasmic tail of cadherin and stabilizes it at the plasma membrane. Here we report that induced cis dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. However, we find that ankyrin-G, a protein that links membrane proteins to the spectrin-actin cytoskeleton, associates with VE-cadherin and inhibits its endocytosis. Ankyrin-G inhibits VE-cadherin endocytosis independent of p120 binding. We propose a model in which ankyrin-G associates with and inhibits the endocytosis of VE-cadherin cis dimers. Our findings support a novel mechanism for regulation of VE-cadherin endocytosis through ankyrin association with cadherin engaged in lateral interactions.     

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.     

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.     

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.     

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.     

Lateral dimerization is required for the homophilic binding activity of C-cadherin

Regulation of cadherin-mediated adhesion can occur rapidly at the cell surface. To understand the mechanism underlying cadherin regulation, it is essential to elucidate the homophilic binding mechanism that underlies all cadherin-mediated functions. Therefore, we have investigated the structural and functional properties of the extracellular segment of Xenopus C-cadherin using a purified, recombinant protein (CEC 1-5). CEC 1-5 supported adhesion of CHO cells expressing C-cadherin. The extracellular segment was also capable of mediating aggregation of microspheres. Chemical cross-linking and gel filtration revealed that CEC 1-5 formed dimers in the presence as well as absence of calcium. Analysis of the functional activity of purified dimers and monomers demonstrated that dimers retained substantially greater homophilic binding activity than monomers. These results demonstrate that lateral dimerization is necessary for homophilic binding between cadherin extracellular segments and suggest multiple potential mechanisms for the regulation of cadherin activity. Since the extracellular segment alone possessed significant homophilic binding activity, the adhesive activity of the extracellular segment in a cellular context was analyzed. The adhesion of CHO cells expressing a truncated version of C-cadherin lacking the cytoplasmic tail was compared to cells expressing the wild-type C-cadherin using a laminar flow assay on substrates coated with CEC 1-5. CHO cells expressing the truncated C-cadherin were able to attach to CEC 1-5 and to resist detachment by low shear forces, demonstrating that tailless C-cadherin can mediate basic, weak adhesion of CHO cells. However, cells expressing the truncated C-cadherin did not exhibit the complete adhesive activity of cells expressing wild-type C-cadherin. Cells expressing wild-type C-cadherin remained attached to CEC 1-5 at high shear forces, while cells expressing the tailless C-cadherin did not adhere well at high shear forces. These results suggest that there may be two states of cadherin-mediated adhesion. The first, relatively weak state can be mediated through interactions between the extracellular segments alone. The second strong adhesive state is critically dependent on the cytoplasmic tail.