Membrane-impermeable cross-linking provides evidence for homophilic, isoform-specific binding of desmosomal cadherins in epithelial cells

Desmosomes and adherens junctions are cadherin-based protein complexes responsible for cell-cell adhesion of epithelial cells. Type 1 cadherins of adherens junctions show specific homophilic adhesion that plays a major role in developmental tissue segregation. The desmosomal cadherins, desmocollin and desmoglein, occur as several different isoforms with overlapping expression in some tissues where different isoforms are located in the same desmosomes. Although adhesive binding of desmosomal cadherins has been investigated in a variety of ways, their interaction in desmosome-forming epithelial cells has not been studied. Here, using extracellular homobifunctional cross-linking, we provide evidence for homophilic and isoform-specific binding between the Dsc2, Dsc3, Dsg2, and Dsg3 isoforms in HaCaT keratinocytes and show that it represents trans interaction. Furthermore, the cross-linked adducts are present in the detergent-insoluble fraction, and electron microscopy shows that extracellular cross-linking probably occurs in desmosomes. We found no evidence for either heterophilic or cis interaction, but neither can be completely excluded by our data. Mutation of amino acid residues Trp-2 and Ala-80 that are important for trans interaction in classical cadherin adhesive binding abolished Dsc2 binding, indicating that these residues are also involved in desmosomal adhesion. These interactions of desmosomal cadherins may be of key importance for their ordered arrangement within desmosomes that we believe is essential for desmosomal adhesive strength and the maintenance of tissue integrity.     

Desmocollin 3-mediated Binding Is Crucial for Keratinocyte Cohesion and Is Impaired in Pemphigus

Desmocollin (Dsc) 1–3 and desmoglein (Dsg) 1–4, transmembrane proteins of the cadherin family, form the adhesive core of desmosomes. Here we provide evidence that Dsc3 homo- and heterophilic trans-interaction is crucial for epidermal integrity. Single molecule atomic force microscopy (AFM) revealed homophilic trans-interaction of Dsc3. Dsc3 displayed heterophilic interaction with Dsg1 but not with Dsg3. A monoclonal antibody targeted against the extracellular domain reduced homophilic and heterophilic binding as measured by AFM, caused intraepidermal blistering in a model of human skin, and a loss of intercellular adhesion in cultured keratinocytes. Because autoantibodies against Dsg1 are associated with skin blistering in pemphigus, we characterized the role of Dsc3 binding for pemphigus pathogenesis. In contrast to AFM experiments, laser tweezer trapping revealed that pemphigus autoantibodies reduced binding of Dsc3-coated beads to the keratinocyte cell surface. These data indicate that loss of heterophilic Dsc3/Dsg1 binding may contribute to pemphigus skin blistering.Desmogleins (Dsg)² and desmocollins (Dsc) are members of the Ca²⁺-dependent cadherin family of adhesion molecules that extend with their outer domains into the extracellular core of desmosomes. Desmosomal cadherins include four Dsg (Dsg1–4) and three Dsc3 isoforms (Dsc1–3) (1, 2). Desmosomal cadherins share a common domain organization with five N-terminally located extracellular subdomains (EC1–5). The membrane-distal EC1 domain is thought to contain the adhesive interface necessary for trans-interaction as could be concluded from structural analysis and blocking studies using peptides and antibodies (3–5). By establishing trans- and cis-interacting adhesive complexes, desmosomal cadherins participate in providing mechanical strength to stratified epithelia (6). In human epidermis Dsg1 and Dsc1 expression decreases from the outermost granular layer toward deeper layers, whereas Dsg3 and Dsc3 are primarily found in the basal layer and display an inverse expression gradient (7, 8). In contrast to classical cadherins present in adherens junctions that primarily undergo homophilic trans-interaction, desmosomal cadherins are generally believed to mediate both homo- and heterophilic binding (9). Recently, an important role of Dsc3 for integrity of murine epidermis was demonstrated in animals with conditional epidermal Dsc3 deficiency that suffered from severe intraepidermal blister formation (10) comparable with the phenotype of the autoimmune bullous skin disease pemphigus vulgaris (PV) (11). PV is associated with antibodies (Abs) against Dsg3, in part combined with Abs targeting Dsg1, whereas Dsg1 Abs alone are associated with pemphigus foliaceus (PF). However, PV and PF sera usually do not contain autoantibodies targeting Dsc3 (12). In view of the apparently important role of Dsc3 in epidermal adhesion, we addressed whether Dsg1 and Dsg3 might heterophilically interact with Dsc3 and whether Abs in pemphigus might interfere with such type of interaction.     

Desmosomal cadherins utilize distinct kinesins for assembly into desmosomes

The desmosomal cadherins, desmogleins (Dsgs) and desmocollins (Dscs), comprise the adhesive core of intercellular junctions known as desmosomes. Although these adhesion molecules are known to be critical for tissue integrity, mechanisms that coordinate their trafficking into intercellular junctions to regulate their proper ratio and distribution are unknown. We demonstrate that Dsg2 and Dsc2 both exhibit microtubule-dependent transport in epithelial cells but use distinct motors to traffic to the plasma membrane. Functional interference with kinesin-1 blocked Dsg2 transport, resulting in the assembly of Dsg2-deficient junctions with minimal impact on distribution of Dsc2 or desmosomal plaque components. In contrast, inhibiting kinesin-2 prevented Dsc2 movement and decreased its plasma membrane accumulation without affecting Dsg2 trafficking. Either kinesin-1 or -2 deficiency weakened intercellular adhesion, despite the maintenance of adherens junctions and other desmosome components at the plasma membrane. Differential regulation of desmosomal cadherin transport could provide a mechanism to tailor adhesion strength during tissue morphogenesis and remodeling.     

Genomic organization of mouse desmocollin genes reveals evolutionary conservation.

Desmosomal cadherins are a family of calcium regulated proteins involved in the formation of desmosomes, a type of cell junction important in maintaining cell adhesion and tissue stability. The desmosomal plaque consists of members of the desmosomal cadherin, plakin and armadillo family of proteins. Desmosomal cadherins are transmembrane glycoproteins that interact with desmosomal cadherins of the adjacent cells via their extracellular repeat domains and are divided in two subfamilies, the desmogleins (Dsg) and the desmocollins (Dsc). On the cytoplasmic side, the cadherins connect to the intermediate filament (IF) network indirectly by interacting with plakin and armadillo proteins. Here, we report the elucidation of the genomic structure of two mouse desmocollin genes, Dsc2 and Dsc3. Interestingly, at the genomic level, desmocollins show a higher degree of similarity to the classical cadherins, such as E-cadherin, than to the desmogleins.     

Direct Ca2+-dependent Heterophilic Interaction between Desmosomal Cadherins, Desmoglein and Desmocollin, Contributes to Cell–Cell Adhesion

Human fibrosarcoma cells, HT-1080, feature extensive adherens junctions, lack mature desmosomes, and express a single known desmosomal protein, Desmoglein 2 (Dsg2). Transfection of these cells with bovine Desmocollin 1a (Dsc1a) caused dramatic changes in the subcellular distribution of endogenous Dsg2. Both cadherins clustered in the areas of the adherens junctions, whereas only a minor portion of Dsg2 was seen in these areas in the parental cells. Deletion mapping showed that intact extracellular cadherin-like repeats of Dsc1a (Arg¹-Thr¹⁷⁰) are required for the translocation of Dsg2. Deletion of the intracellular C-domain that mediates the interaction of Dsc1a with plakoglobin, or the CSI region that is involved in the binding to desmoplakin, had no effect. Coimmunoprecipitation experiments of cell lysates stably expressing Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca²⁺-dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca²⁺-dependent manner. The contribution of the Dsg/ Dsc interaction to cell–cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell–cell adhesion.     

The binding of plakoglobin to desmosomal cadherins: patterns of binding sites and topogenic potential

Plakoglobin is the only protein that occurs in the cytoplasmic plaques of all known adhering junctions and has been shown to be crucially involved in the formation and maintenance of desmosomes anchoring intermediate-sized filaments (IFs) by its interaction with the desmosomal cadherins, desmoglein (Dsg), and desmocollin (Dsc). This topogenic importance of plakoglobin is now directly shown in living cells as well as in binding assays in vitro. We show that, in transfected human A-431 carcinoma cells, a chimeric protein combining the vesicle-forming transmembrane glycoprotein synaptophysin, with the complete human plakoglobin sequence, is sorted to small vesicles many of which associate with desmosomal plaques and their attached IFs. Immunoprecipitation experiments have further revealed that the chimeric plakoglobin-containing transmembrane molecules of these vesicles are tightly bound to Dsg and Dsc but not to endogenous plakoglobin, thus demonstrating that the binding of plakoglobin to desmosomal cadherins does not require its soluble state and is strong enough to attach large structures such as vesicles to desmosomes. To identify the binding domains and the mechanisms involved in the interaction of plakoglobin with desmosomal cadherins, we have developed direct binding assays in vitro in which plakoglobin or parts thereof, produced by recombinant DNA technology in E. coli, are exposed to molecules containing the "C- domains" of several cadherins. These assays have shown that plakoglobin associates most tightly with the C-domain of Dsg, to a lesser degree with that of Dsc and only weakly with the C-domain of E-cadherin. Three separate segments of plakoglobin containing various numbers of the so- called arm repeats exhibit distinct binding to the desmosomal cadherins comparable in strength to that of the entire molecule. The binding pattern of plakoglobin segments in vitro is compared with that in vivo. Paradoxically, in vitro some internal plakoglobin fragments bind even better to the C-domain of E-cadherin than the entire molecule, indicating that elements exist in native plakoglobin that interfere with the interaction of this protein with its various cadherin partners.     

Coordinated expression of desmoglein 1 and desmocollin 1 regulates intercellular adhesion

Desmoglein 1 (Dsg1) is a component of desmosomes present in the upper epidermis and can be targeted by autoimmune antibodies or bacterial toxins, resulting in skin blistering diseases. These defects in tissue integrity are believed to result from compromised desmosomal adhesion; yet, previous attempts to directly test the adhesive roles of desmosomal cadherins using normally non-adherent L cells have yielded mixed results. Here, two complementary approaches were used to better resolve the molecular determinants for Dsg1-mediated adhesion: (1) a tetracycline-inducible system was used to modulate the levels of Dsg1 expressed in L cell lines containing desmocollin 1 (Dsc1) and plakoglobin (PG) and (2) a retroviral gene delivery system was used to introduce Dsg1 into normal human epidermal keratinocytes (NHEK). By increasing Dsg1 expression relative to Dsc1 and PG, we were able to demonstrate that the ratio of Dsg1:Dsc1 is a critical determinant of desmosomal adhesion in fibroblasts. The distribution of Dsg1 was organized at areas of cell-cell contact in the multicellular aggregates that formed in these suspension cultures. Similarly, the introduction of Dsg1 into NHEKs was capable of increasing the aggregation of single cell suspensions and further enhanced the adhesive strength of intact epithelial sheets. Endogenous Dsc1 levels were also increased in NHEKs containing Dsg1, providing further support for the coordination of these two desmosomal cadherins in regulating adhesive structures. These Dsg1-mediated effects on intercellular adhesion were directly related to the presence of an intact extracellular domain as ETA, a toxin that specifically cleaves this desmosomal cadherin, inhibited adhesion in both fibroblasts and keratinocytes. Collectively, these observations demonstrate that Dsg1 promotes the formation of intercellular adhesion complexes and suggest that the relative level of Dsg and Dsc expressed at the cell surface regulates this adhesive process.     

Desmoglein-2: a novel regulator of apoptosis in the intestinal epithelium

Intestinal epithelial intercellular junctions regulate barrier properties, and they have been linked to epithelial differentiation and programmed cell death (apoptosis). However, mechanisms regulating these processes are poorly defined. Desmosomes are critical elements of intercellular junctions; they are punctate structures made up of transmembrane desmosomal cadherins termed desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) that affiliate with the underlying intermediate filaments via linker proteins to provide mechanical strength to epithelia. In the present study, we generated an antibody, AH12.2, that recognizes Dsg2. We show that Dsg2 but not another desmosomal cadherin, Dsc2, is cleaved by cysteine proteases during the onset of intestinal epithelial cell (IEC) apoptosis. Small interfering RNA-mediated down-regulation of Dsg2 protected epithelial cells from apoptosis. Moreover, we report that a C-terminal fragment of Dsg2 regulates apoptosis and Dsg2 protein levels. Our studies highlight a novel mechanism by which Dsg2 regulates IEC apoptosis driven by cysteine proteases during physiological differentiation and inflammation.     

Regulation of intestinal epithelial intercellular adhesion and barrier function by desmosomal cadherin desmocollin-2

The role of desmosomal cadherin desmocollin-2 (Dsc2) in regulating barrier function in intestinal epithelial cells (IECs) is not well understood. Here, we report the consequences of silencing Dsc2 on IEC barrier function in vivo using mice with inducible intestinal–epithelial-specific Dsc2 knockdown (KD) (Dsc2^ERΔIEC). While the small intestinal gross architecture was maintained, loss of epithelial Dsc2 influenced desmosomal plaque structure, which was smaller in size and had increased intermembrane space between adjacent epithelial cells. Functional analysis revealed that loss of Dsc2 increased intestinal permeability in vivo, supporting a role for Dsc2 in the regulation of intestinal epithelial barrier function. These results were corroborated in model human IECs in which Dsc2 KD resulted in decreased cell–cell adhesion and impaired barrier function. It is noteworthy that Dsc2 KD cells exhibited delayed recruitment of desmoglein-2 (Dsg2) to the plasma membrane after calcium switch-induced intercellular junction reassembly, while E-cadherin accumulation was unaffected. Mechanistically, loss of Dsc2 increased desmoplakin (DP I/II) protein expression and promoted intermediate filament interaction with DP I/II and was associated with enhanced tension on desmosomes as measured by a Dsg2-tension sensor. In conclusion, we provide new insights on Dsc2 regulation of mechanical tension, adhesion, and barrier function in IECs.     

Identification of the plakoglobin-binding domain in desmoglein and its role in plaque assembly and intermediate filament anchorage

The carboxyterminal cytoplasmic portions (tails) of desmosomal cadherins of both the desmoglein (Dsg) and desmocollin type are integral components of the desmosomal plaque and are involved in desmosome assembly and the anchorage of intermediate-sized filaments. When additional Dsg tails were introduced by cDNA transfection into cultured human epithelial cells, in the form of chimeras with the aminoterminal membrane insertion domain of rat connexin32 (Co32), the resulting stably transfected cells showed a dominant-negative defect specific for desmosomal junctions: despite the continual presence of all desmosomal proteins, the endogenous desmosomes disappeared and the formation of Co32-Dsg chimeric gap junctions was inhibited. Using cell transfection in combination with immunoprecipitation techniques, we have examined a series of deletion mutants of the Dsg1 tail in Co32-Dsg chimeras. We show that upon removal of the last 262 amino acids the truncated Dsg tail still effects the binding of plakoglobin but not of detectable amounts of any catenin and induces the dominant-negative phenotype. However, further truncation or excision of the next 41 amino acids, which correspond to the highly conserved carboxyterminus of the C-domain in other cadherins, abolishes plakoglobin binding and allows desmosomes to reform. Therefore, we conclude that this short segment provides a plakoglobin-binding site and is important for plaque assembly and the specific anchorage of either actin filaments in adherens junctions or IFs in desmosomes.     

EGFR and ADAMs cooperate to regulate shedding and endocytic trafficking of the desmosomal cadherin desmoglein 2

Regulation of classic cadherins plays a critical role in tissue remodeling during development and cancer; however, less attention has been paid to the importance of desmosomal cadherins. We previously showed that EGFR inhibition results in accumulation of the desmosomal cadherin, desmoglein 2 (Dsg2), at cell-cell interfaces accompanied by inhibition of matrix metalloprotease (MMP)-dependent shedding of the Dsg2 ectodomain and tyrosine phosphorylation of its cytoplasmic domain. Here, we show that EGFR inhibition stabilizes Dsg2 at intercellular junctions by interfering with its accumulation in an internalized cytoplasmic pool. Furthermore, MMP inhibition and ADAM17 RNAi, blocked shedding and depleted internalized Dsg2, but less so E-cadherin, in highly invasive SCC68 cells. ADAM9 and 15 silencing also impaired Dsg2 processing, supporting the idea that this desmosomal cadherin can be regulated by multiple ADAM family members. In contrast, ADAM10 siRNA enhanced accumulation of a 100-kDa Dsg2 cleavage product and internalized pool of Dsg2. Although both MMP and EGFR inhibition increased intercellular adhesive strength in control cells, the response to MMP-inhibition was Dsg2-dependent. These data support a role for endocytic trafficking in regulating desmosomal cadherin turnover and function and raise the possibility that internalization and regulation of desmosomal and classic cadherin function can be uncoupled mechanistically.     

Desmosomal cadherins in zebrafish epiboly and gastrulation

Background  

The desmosomal cadherins (DCs), desmocollin (Dsc) and desmoglein (Dsg), are the adhesion molecules of desmosomes, intercellular adhesive junctions of epithelia and cardiac muscle. Both the DCs and desmosomes have demonstrably essential roles in mammalian development. In order to initiate their study in a more tractable developmental system we have characterised zebrafish DCs and examined their roles in early zebrafish development.     

The C-terminal unique region of desmoglein 2 inhibits its internalization via tail–tail interactions

Desmosomal cadherins, desmogleins (Dsgs) and desmocollins, make up the adhesive core of intercellular junctions called desmosomes. A critical determinant of epithelial adhesive strength is the level and organization of desmosomal cadherins on the cell surface. The Dsg subclass of desmosomal cadherins contains a C-terminal unique region (Dsg unique region [DUR]) with unknown function. In this paper, we show that the DUR of Dsg2 stabilized Dsg2 at the cell surface by inhibiting its internalization and promoted strong intercellular adhesion. DUR also facilitated Dsg tail–tail interactions. Forced dimerization of a Dsg2 tail lacking the DUR led to decreased internalization, supporting the conclusion that these two functions of the DUR are mechanistically linked. We also show that a Dsg2 mutant, V977fsX1006, identified in arrhythmogenic right ventricular cardiomyopathy patients, led to a loss of Dsg2 tail self-association and underwent rapid endocytosis in cardiac muscle cells. Our observations illustrate a new mechanism desmosomal cadherins use to control their surface levels, a key factor in determining their adhesion and signaling roles.     

Assembly of connexin43 into gap junctions is regulated differentially by E-cadherin and N-cadherin in rat liver epithelial cells

Cadherins have been thought to facilitate the assembly of connexins (Cxs) into gap junctions (GJs) by enhancing cell-cell contact, however the molecular mechanisms involved in this process have remained unexplored. We examined the assembly of GJs composed of Cx43 in isogenic clones derived from immortalized and nontransformed rat liver epithelial cells that expressed either epithelial cadherin (E-Cad), which curbs the malignant behavior of tumor cells, or neuronal cadherin (N-Cad), which augments the invasive and motile behavior of tumor cells. We found that N-cad expression attenuated the assembly of Cx43 into GJs, whereas E-Cad expression facilitated the assembly. The expression of N-Cad inhibited GJ assembly by causing endocytosis of Cx43 via a nonclathrin-dependent pathway. Knock down of N-Cad by ShRNA restored GJ assembly. When both cadherins were simultaneously expressed in the same cell type, GJ assembly and disassembly occurred concurrently. Our findings demonstrate that E-Cad and N-Cad have opposite effects on the assembly of Cx43 into GJs in rat liver epithelial cells. These findings imply that GJ assembly and disassembly are the down-stream targets of the signaling initiated by E-Cad and N-Cad, respectively, and may provide one possible explanation for the disparate role played by these cadherins in regulating cell motility and invasion during tumor progression and invasion.     

Desmoglein 1–dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis

Dsg1 (desmoglein 1) is a member of the cadherin family of Ca²⁺-dependent cell adhesion molecules that is first expressed in the epidermis as keratinocytes transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports keratinocyte differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting keratinocyte differentiation. Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1). Instead, Dsg1 was required for suppression of epidermal growth factor receptor–Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program. In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.     

Defining the Interactions Between Intermediate Filaments and Desmosomes

Desmoplakin (DP), plakoglobin (PG), and plakophilin 1 (PP1) are desmosomal components lacking a transmembrane domain, thus making them candidate linker proteins for connecting intermediate filaments and desmosomes. Using deletion and site-directed mutagenesis, we show that remarkably, removal of ~1% of DP's sequence obliterates its ability to associate with desmosomes. Conversely, when linked to a foreign protein, as few as 86 NH₂-terminal DP residues are sufficient to target to desmosomes efficiently. In in vitro overlay assays, the DP head specifically associates with itself and with desmocollin 1a (Dsc1a). In similar overlay assays, PP1 binds to DP and Dsc1a, and to a lesser extent, desmoglein 1 (Dsg1), while PG binds to Dsg1 and more weakly to Dsc1a and DP. Interestingly, like DP, PG and PP1 associate with epidermal keratins, although PG is considerably weaker in its ability to do so. As judged by overlay assays, the amino terminal head domain of type II keratins appears to have a special importance in establishing these connections. Taken together, our findings provide new insights into the complexities of the links between desmosomes and intermediate filaments (IFs). Our results suggest a model whereby at desmosome sites within dividing epidermal cells, DP and PG anchor to desmosomal cadherins and to each other, forming an ordered array of nontransmembrane proteins that then bind to keratin IFs. As epidermal cells differentiate, PP1 is added as a molecular reinforcement to the plaque, enhancing anchorage to IFs and accounting at least partially for the increase in numbers and stability of desmosomes in suprabasal cells.     

Desmoglein 2 Is Less Important than Desmoglein 3 for Keratinocyte Cohesion

Desmosomes provide intercellular adhesive strength required for integrity of epithelial and some non-epithelial tissues. Within the epidermis, the cadherin-type adhesion molecules desmoglein (Dsg) 1–4 and desmocollin (Dsc) 1–3 build the adhesive core of desmosomes. In keratinocytes, several isoforms of these proteins are co-expressed. However, the contribution of specific isoforms to overall cell cohesion is unclear. Therefore, in this study we investigated the roles of Dsg2 and Dsg3, the latter of which is known to be essential for keratinocyte adhesion based on its autoantibody-induced loss of function in the autoimmune blistering skin disease pemphigus vulgaris (PV). The pathogenic PV antibody AK23, targeting the Dsg3 adhesive domain, led to profound loss of cell cohesion in human keratinocytes as revealed by the dispase-based dissociation assays. In contrast, an antibody against Dsg2 had no effect on cell cohesion although the Dsg2 antibody was demonstrated to interfere with Dsg2 transinteraction by single molecule atomic force microscopy and was effective to reduce cell cohesion in intestinal epithelial Caco-2 cells which express Dsg2 as the only Dsg isoform. To substantiate these findings, siRNA-mediated silencing of Dsg2 or Dsg3 was performed in keratinocytes. In contrast to Dsg3-depleted cells, Dsg2 knockdown reduced cell cohesion only under conditions of increased shear. These experiments indicate that specific desmosomal cadherins contribute differently to keratinocyte cohesion and that Dsg2 compared to Dsg3 is less important in this context.     

Cross-Talk between Adherens Junctions and Desmosomes Depends on Plakoglobin

Squamous epithelial cells have both adherens junctions and desmosomes. The ability of these cells to organize the desmosomal proteins into a functional structure depends upon their ability first to organize an adherens junction. Since the adherens junction and the desmosome are separate structures with different molecular make up, it is not immediately obvious why formation of an adherens junction is a prerequisite for the formation of a desmosome. The adherens junction is composed of a transmembrane classical cadherin (E-cadherin and/or P-cadherin in squamous epithelial cells) linked to either β-catenin or plakoglobin, which is linked to α-catenin, which is linked to the actin cytoskeleton. The desmosome is composed of transmembrane proteins of the broad cadherin family (desmogleins and desmocollins) that are linked to the intermediate filament cytoskeleton, presumably through plakoglobin and desmoplakin. To begin to study the role of adherens junctions in the assembly of desmosomes, we produced an epithelial cell line that does not express classical cadherins and hence is unable to organize desmosomes, even though it retains the requisite desmosomal components. Transfection of E-cadherin and/or P-cadherin into this cell line did not restore the ability to organize desmosomes; however, overexpression of plakoglobin, along with E-cadherin, did permit desmosome organization. These data suggest that plakoglobin, which is the only known common component to both adherens junctions and desmosomes, must be linked to E-cadherin in the adherens junction before the cell can begin to assemble desmosomal components at regions of cell–cell contact. Although adherens junctions can form in the absence of plakoglobin, making use only of β-catenin, such junctions cannot support the formation of desmosomes. Thus, we speculate that plakoglobin plays a signaling role in desmosome organization.Squamous epithelial cells typically contain two prominent types of cell–cell junctions: the adherens junction and the desmosome. The adherens junction is an intercellular adhesion complex that is composed of a transmembrane protein (a classical cadherin) and numerous cytoplasmic proteins (α-catenin, β-catenin and plakoglobin, vinculin and α-actinin; for reviews see Takeichi, 1990; Geiger and Ayalon, 1992). The cadherins are directly responsible for adhesive interactions via a Ca²⁺-dependent, homotypic mechanism, i.e., in the presence of sufficient Ca²⁺, cadherin on one cell binds to an identical molecule on an adjacent cell. The desmosome, also an intercellular adhesion complex, is composed of at least two different transmembrane proteins (desmoglein and desmocollin) as well as several cytoplasmic proteins, including desmoplakins and plakoglobin (Koch and Franke, 1994). The transmembrane components of the desmosome are members of the broadly defined cadherin family and also require Ca²⁺ for adhesive activity. However, decisive experimental evidence for homophilic or heterophilic interactions between desmosomal cadherins via their extracellular domains has not yet been presented (Koch and Franke, 1994; Kowalczyk et al., 1996). While members of the cadherin family constitute the transmembrane portion of both adherens junctions and desmosomes, the different classes of cadherins are linked to different cytoskeletal elements by the cytoplasmic components of each junction. Specifically, the classical cadherins are linked to actin filaments and the desmosomal cadherins to intermediate filaments.The organization of the proteins within the adherens junction is well understood (for reviews see Kemler, 1993; Cowin, 1994; Wheelock et al., 1996). Specifically, the intracellular domain of cadherin interacts directly with either plakoglobin or β-catenin, which in turns binds to α-catenin (Jou et al., 1995; Sacco et al., 1995). α-Catenin interacts with α-actinin and actin filaments, thereby linking the cadherin/ catenin complex to the cytoskeleton (Knudsen et al., 1995; Rimm et al., 1995). Cadherin/catenin complexes include either plakoglobin or β-catenin but not both (Näthke et al., 1994). The importance of the classical cadherins to the formation of adherens junctions and desmosomes has been demonstrated. Keratinocytes maintained in medium with low Ca²⁺ (i.e., 30 μM) grow as a monolayer and do not exhibit adherens junctions or desmosomes; however, elevation of Ca²⁺ concentration induces the rapid formation of adherens junctions followed by the formation of desmosomes (Hennings et al., 1980; Tsao et al., 1982; Boyce and Ham, 1983; Hennings and Holbrook, 1983; O''Keefe et al., 1987; Wheelock and Jensen, 1992; Hodivala and Watt, 1994; Lewis et al., 1994). Simultaneous blocking with functionperturbing antibodies against the two classical cadherins (E- and P-cadherin) found in keratinocytes inhibits not only Ca²⁺-induced adherens junction formation but also severely limits desmosome formation (Lewis et al., 1994; Jensen et al., 1996). Consistent with these findings, expression of a dominant-negative cadherin by keratinocytes results in decreased E-cadherin expression and delayed assembly of desmosomes (Fujimori and Takeicki, 1993; Amagai, et al., 1995). These data suggest some form of cross-talk between the proteins of the adherens junction and those of the desmosome. One candidate protein that might mediate such cross-talk is plakoglobin, since it is the only known common component of both junctions.Plakoglobin is found to be associated with the cytoplasmic domains of both the classical cadherins and the desmosomal cadherins. Despite the high degree of identity between plakoglobin and β-catenin (65% at the amino acid level; Fouquet et al., 1992), β-catenin only associates with the classical cadherins and not with the desmosomal cadherins. In the adherens junction, plakoglobin and β-catenin have at least one common function, i.e., the linking of cadherin to α-catenin and thus to actin. However, there is emerging evidence that other functions of these two proteins are not identical. For example, in a study by Navarro et al. (1993), E-cadherin transfected into a spindle cell carcinoma was shown to associate with α- and β-catenin, but not with the low levels of endogenous plakoglobin. The transfected cells did not revert to a more epithelial morphology in spite of the presence of functional E-cadherin, and the authors suggested that the lack of plakoglobin may have prevented such morphological reversion.In the present study, we have tested the hypothesis that plakoglobin, through its interaction with E- or P-cadherin, serves as a regulatory molecule for desmosome organization. Even though plakoglobin is not an essential structural component of the adherens junction (Sacco et al., 1995), our data indicate that plakoglobin can function as a regulator of desmosome formation only when it is associated with a classical cadherin. Thus, we propose that plakoglobin has at least two functions: (a) as a structural component of the adherens junction and the desmosome and (b) as a signaling molecule that regulates communication between the adherens junction and the desmosome.     

Interactions of Plakoglobin and ��-Catenin with Desmosomal Cadherins: BASIS OF SELECTIVE EXCLUSION OF ��- AND ��-CATENIN FROM DESMOSOMES*

Plakoglobin and β-catenin are homologous armadillo repeat proteins found in adherens junctions, where they interact with the cytoplasmic domain of classical cadherins and with α-catenin. Plakoglobin, but normally not β-catenin, is also a structural constituent of desmosomes, where it binds to the cytoplasmic domains of the desmosomal cadherins, desmogleins and desmocollins. Here, we report structural, biophysical, and biochemical studies aimed at understanding the molecular basis of selective exclusion of β-catenin and α-catenin from desmosomes. The crystal structure of the plakoglobin armadillo domain bound to phosphorylated E-cadherin shows virtually identical interactions to those observed between β-catenin and E-cadherin. Trypsin sensitivity experiments indicate that the plakoglobin arm domain by itself is more flexible than that of β-catenin. Binding of plakoglobin and β-catenin to the intracellular regions of E-cadherin, desmoglein1, and desmocollin1 was measured by isothermal titration calorimetry. Plakoglobin and β-catenin bind strongly and with similar thermodynamic parameters to E-cadherin. In contrast, β-catenin binds to desmoglein-1 more weakly than does plakoglobin. β-Catenin and plakoglobin bind with similar weak affinities to desmocollin-1. Full affinity binding of desmoglein-1 requires sequences C-terminal to the region homologous to the catenin-binding domain of classical cadherins. Although pulldown assays suggest that the presence of N- and C-terminal β-catenin “tails” that flank the armadillo repeat region reduces the affinity for desmosomal cadherins, calorimetric measurements show no significant effects of the tails on binding to the cadherins. Using purified proteins, we show that desmosomal cadherins and α-catenin compete directly for binding to plakoglobin, consistent with the absence of α-catenin in desmosomes.