Cadherins Mediate Cell-Cell Adhesions in Adherens Junctions and Desmosomes

The primary CAMs in adherens junctions and desmosomes belong to the cadherin family. In vertebrates, this protein superfamily of more than a hundred members can be grouped into at least six subfamilies, including classical cadherins and desmosomal cadherins, which we will describe below. The diversity of cadherins arises from the presence of multiple cadherin genes and alternative RNA splicing. It is not surprising that there are many different types of cadherins in vertebrates. Many different types of cells in the widely diverse tissues of these animals use cadherins to mediate adhesion and communication, the detailed requirements for which may differ for different types of cells and tissues. Members of the cadherin superfamily can also control cell morphology, such as the assembly and tight packing of microvilli on the apical surfaces of some epithelial cells (see Figures 20-10a and 20-11a). The brain expresses the largest number of different cadherins, presumably owing to the necessity of forming many specific cell-cell contacts to help establish its complex wiring pattern. Invertebrates, however, are able to function with fewer than 20 cadherins.

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Classical Cadherins The classical cadherins include E-, N-, and P-cadherins, named for the type of tissue in which they were initially identified (epithelial, neural, and placental, respectively). E- and N-cadherins are the most widely expressed, particularly during early differentiation. Sheets of polarized epithelial cells, such as those that line the small intestine or kidney tubules, contain abundant E-cadherin along their lateral surfaces. Although E-cadherin is concentrated in adherens junctions, it is present throughout the lateral surfaces, where it is thought to link adjacent cell membranes.

The results of experiments with L cells, a line of cultured mouse fibroblasts, demonstrated that E-cadherins preferentially mediate homophilic interactions. L cells express no cadherins and adhere poorly to each other and to other types of cells. When the E-cadherin gene was introduced into L cells, the cells were found to adhere preferentially to other cells expressing E-cadherin (Figure 20-12). These engineered cadherin-expressing L cells formed epithelium-like aggregates with one another and with epithelial cells isolated from lungs. Although most E-cadherins exhibit primarily homophilic binding, some mediate heterophilic interactions.

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EXPERIMENTAL FIGURE 20-12 E-cadherin mediates Ca2+-dependent adhesion of L cells. Under standard cell culture conditions, in the presence of calcium in the extracellular fluid, L cells do not aggregate into sheets (left). Introduction of a gene that causes the expression of E-cadherin in these cells results in their aggregation into epithelium-like clumps in the presence of calcium (center), but not in its absence (right). Bar, 60 µm.
[©1998 Adams, C. L. et al., J. Cell Biol. 142:1105–119. doi: 10.1083/jcb.142.4.1105; Figure 1E.]

The adhesiveness of cadherins depends on the presence of extracellular Ca2+; it is this property (calcium adhering) that gave rise to their name. For example, the adhesion of L cells expressing E-cadherin is prevented when the cells are bathed in a solution that is low in Ca2+ (see Figure 20-12). Some adhesion molecules require some minimal amount of Ca2+ in the extracellular fluid to function properly, whereas others, such as IgCAMs, are Ca2+ independent.

The role of E-cadherin in adhesion can also be demonstrated by experiments with cultured epithelial cells called Madin-Darby canine kidney (MDCK) cells (see Figure 4-4). A green fluorescent protein–labeled form of E-cadherin has been used in these cells to show that clusters of E-cadherin mediate the initial attachment of the cells and the subsequent zippering of the cells into sheets (Figure 20-13). In this experimental system, the addition of an antibody that binds to E-cadherin, preventing its homophilic interactions, blocks the Ca2+-dependent attachment of MDCK cells to one another and the subsequent formation of intercellular adherens junctions.

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EXPERIMENTAL FIGURE 20-13 E-cadherin mediates adhesive connections in cultured MDCK epithelial cells. An E-cadherin gene fused to green fluorescent protein (GFP) was introduced into cultured MDCK cells. The cells were then mixed together in a calcium-containing medium, and the distribution of fluorescent E-cadherin was visualized over time (shown in hours). Clusters of E-cadherin mediate the initial attachment and subsequent zippering up of the epithelial cells and the formation of junctions (bicellular junctions are where two cells join and appear as lines; tricellular junctions are the sites of intersection of three cells).
[©1998 Adams, C. L. et al., J. Cell Biol. 142:1105–119. doi: 10.1083/jcb.142.4.1105; Figure 2B.]

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Each classical cadherin molecule contains a single transmembrane domain, a relatively short C-terminal cytosolic domain, and five extracellular “cadherin” domains (called EC1–EC5) (see Figure 20-2). The extracellular domains are necessary for Ca2+ binding and cadherin-mediated cell-cell adhesion. Classical cadherin–mediated adhesion entails both cis lateral clustering (intracellular) and trans adhesive (intercellular) molecular interactions (see Figures 20-3 and 20-14a-c). The binding of three Ca2+ at each of the sites located between the cadherin repeats (see Figures 20-2 and 20-14a) stabilizes the elongated and curved structure of the extracellular domain. As we shall see shortly, the curved structure of cadherin’s extracellular domain is necessary for the proper molecular complementarity that stabilizes cis and trans binding between cadherin molecules. The cis and trans interactions of cadherins, together with their interactions with cytoplasmic adapter and cytoskeletal molecules, permit the zippering up of cadherins into adhesive arrays. Binding of the EC1 domain of one cadherin molecule to the EC1 domain of another on the adjacent cell is responsible for trans binding (Figure 20-14; see also Figure 20-3). Although the dissociation constant (Kd) for EC1–EC1 homophilic binding measured using isolated domains in solution is on the order of 10−5–10−4 mol/L (relatively weak, or low-affinity, binding), the multiple low-affinity interactions in arrays of intact cadherin molecules on adjacent cells sum to produce a very tight intercellular adhesion.

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FIGURE 20-14 Intercellular and intracellular interactions of classical cadherins in typical adherens junctions. (a)The exoplasmic cadherin domains [EC1-EC5, see ovals in part (b)] of E-cadherins at adherens junctions on adjacent cells are clustered by homophilic cis and trans interactions. The Ca2+-dependent elongated and curved structure of cadherin’s extracellular domains is necessary for stable cis and trans interactions. Sites representing individual cis and trans interactions are highlighted by dashed circles. (b) EC1-EC2 cis interaction: The binding of an EC1 domain of one cadherin to an EC2 domain of an adjacent cadherin on the same cell is responsible for cis interactions. In panels (b) and (c) the structure of each extracellular cadherin domain determined by X-ray crystallography is represented using a ribbon diagram and is highlighted by an oval. (c) EC1-EC1 trans interaction: Two views rotated by 90° of the trans binding of an EC1 domain of one cadherin to an EC1 domain of a cadherin on the adjacent cell. Only the EC1 and a portion of the EC2 domains of two trans interacting cadherins are shown. The left view shows the relative orientations of the main axes of the oval-shaped EC1 domains. The right view shows how a small segment of polypeptide at the N-terminus of each of the two EC1 domains [highlighted in yellow (cell 1) and blue (cell 2)] swings out and replaces the equivalent segment from its binding partner (strand swap, dashed oval). The strand swap places the side chain of a tryptophan residue on each of the segments into a binding pocket on the adjacent EC1 domain – an interaction that substantially stabilizes the trans binding. (d) The cytosolic domains of the E-cadherins bind directly or indirectly to multiple adapter proteins (e.g., β-catenin), which both connect the junctions to actin filaments (F-actin) of the cytoskeleton and participate in intracellular signaling pathways. Somewhat different sets of adapter proteins are illustrated in the two cells to emphasize that a variety of adapters can interact with adherens junctions. Some of these adapters, such as ZO-1, can interact with several different CAMs. See V. Vasioukhin and E. Fuchs, 2001, Curr. Opin. Cell Biol. 13:76 and J. Brasch, O. J. Harrison, B. Honig, and L. Shapiro, 2012, Trends Cell Biol. 22:299.
[Data from O. J. Harrison et al., 2011, Structure 19:244–256, PDB ID 3q2w.]

Determination of the structures of the extracellular domains of cadherins, together with analyses of the structures and binding properties of many mutants of the key binding domains, have provided a clear picture of the cis and trans interactions that underlie classical cadherin–mediated cell adhesion. The key features of cadherin cis and trans binding interactions are (1) the calcium-dependent curvature of the five extracellular cadherin domains that permits proper relative orientations of the EC1 and EC2 domains (see Figures 20-2 and 20-14); (2) for cis interactions, the binding of one side of an EC1 domain to a complementary surface on the EC2 domain of an adjacent molecule on the same membrane (see Figures 20-2 and 20-14); and (3) for trans interactions, the binding of a different surface of the EC1 domain to an EC1 domain from a cadherin molecule on the adjacent cell. The trans EC1–EC1 binding is stabilized when a small segment of the protein at the N-terminus of each of the two EC1 domains swings out and replaces the equivalent segment from its binding partner (strand swap; see Figure 20-14).

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EXPERIMENTAL FIGURE 20-15 E-cadherin activity is lost during the epithelial-to-mesenchymal transition and during cancer progression. A protein called Snail that suppresses the expression of E-cadherin is associated with the epithelial-to-mesenchymal transition (EMT). (a) Normal epithelial MDCK cells grown in culture. (b) Expression of the snail gene in MDCK cells causes them to undergo an EMT. (c) Distribution of E-cadherin detected by immunohistochemical staining (dark brown) in thin sections of tissue from a patient with hereditary diffuse gastric cancer. E-cadherin is seen at the intercellular borders of normal stomach gastric gland epithelial cells (right); no E-cadherin is seen at the borders of underlying invasive carcinoma cells.
[Panels (a) and (b) republished with permission of Elsevier, from Martinez Arias, M., “Epithelial mesenchymal interactions in cancer and development,” Cell, 2001, 105:4, 425–431; permission conveyed through the Copyright Clearance Center, Inc. Panel (c) republished with permission of John Wiley & Sons, Inc., from Carneiro, F., et al., “Model of the early development of diffuse gastric cancer in Ecadherin mutation carriers and its implications for patient screening,” J. Pathol., 2004, 203(2):681–7.]

The C-terminal cytosolic domain of classical cadherins is linked to the actin cytoskeleton by adapter proteins (see Figure 20-14d). These linkages are essential for strong adhesion, as a moderate increase in tension generated by the actin cytoskeleton induces the formation of larger clusters of cadherins and stronger intercellular adhesion. Some of the increased cadherin-mediated adhesion that accompanies increased force applied by the actin cytoskeleton appears to be mediated by one of the adapter proteins, α-catenin, a mechanosensor that links cadherin to actin filaments (see Figure 20-14d) and changes shape (stretches out) when subjected to force. This stretching uncovers additional binding sites for other adapter molecules on the α-catenin. Disruption of the interactions between classical cadherins and α-catenin or β-catenin—another common adapter protein that links classical cadherins to actin filaments (see Figure 20-14d)—dramatically reduces cadherin-mediated cell-cell adhesion. This disruption occurs spontaneously in tumor cells, which sometimes fail to express α-catenin, and can be induced experimentally by depleting the cytosolic pool of accessible β-catenin. The cytosolic domains of cadherins also interact with intracellular signaling molecules such as p120-catenin. Interestingly, β-catenin plays a dual role: it not only mediates cytoskeletal attachment, but also serves as a signaling molecule, translocating to the nucleus and altering gene transcription in the Wnt signaling pathway (see Figure 16-30).

Classical cadherins play a critical role during tissue differentiation. Each classical cadherin has a characteristic tissue distribution. In the course of differentiation, the amounts or types of cell-surface cadherins change, affecting many aspects of cell-cell adhesion, cell migration, and cell division. For instance, the normal reorganization of tissues during morphogenesis is often accompanied by the conversion of nonmotile epithelial cells into motile cells, called mesenchymal cells, that are precursors for other tissues. This epithelial-to-mesenchymal transition (EMT) is associated with a reduction in the expression of E-cadherin (Figure 20-15a, b). The EMT is also associated with pathology, as in the conversion of epithelial cells into malignant carcinoma cells. For example, certain ductal breast tumors and hereditary diffuse gastric cancer (Figure 20-15c) characteristically involve a loss of E-cadherin activity. It is well known that animal cell-cell contact can inhibit cell proliferation. During tissue development, once dividing epithelial cells have formed a well-defined, tightly bound epithelium, they have no need for further cell division unless they are damaged or receive a signal to undergo the EMT. It is now clear that one mechanism used to inhibit proliferation of epithelial cells in epithelia is E-cadherin- and catenin-mediated regulation of the Hippo pathway that controls cell proliferation (see Chapter 19).

The firm epithelial cell-cell adhesions mediated by cadherins in adherens junctions permit the formation of a second class of intercellular junctions in epithelia—tight junctions, to which we will turn shortly.

Infection with rhinoviruses (RV) is the most frequent cause of the common cold, and infection with virulent class C rhinoviruses (RV-C) can cause more severe illnesses, including exacerbation of asthma. To enter cells and replicate, RV-C must bind to cell-surface receptors. Recent studies have identified a cadherin-family member called CDHR3, which is highly expressed in epithelial cells in the human airway, as a receptor for RV-C. Pathogens such as RV-C often evolve to co-opt proteins that have normal functions in their target (host) tissues. Genetic studies have shown that a naturally occurring mutation in humans that changes a cysteine to tyrosine (C → Y) in the EC5 domain of CDHR3 is associated with increased wheezing illnesses and hospitalizations for childhood asthma. In cultured cells, this C → Y mutation increases the cell-surface expression of CDHR3 and the binding and replication of RV-C. Treatments that disrupt the RV-C/cadherin (CDHR3) interaction have the potential to prevent or treat respiratory diseases caused by RV-C.

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Desmosomal Cadherins Desmosomes (Figure 20-16) contain two specialized cadherins, desmoglein and desmocollin, whose cytosolic domains are distinct from those in the classical cadherins. The cytosolic domains of desmosomal cadherins bind to adapter proteins such as plakoglobin (similar in structure to β-catenin) and plakophilins, and these bind to a member of the plakin family of adapters, called desmoplakin. These adapters form the thick cytoplasmic plaques that are characteristic of desmosomes. The desmoplakins directly mediate plaque binding to intermediate filaments.

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FIGURE 20-16 Desmosomes. (a) Model of a desmosome between epithelial cells with attachments to the sides of intermediate filaments. The key CAMs in desmosomes are the desmosomal cadherins desmoglein and desmocollin. Adapter proteins bound to the cytoplasmic domains of these cadherins include plakoglobin, desmoplakins, and plakophilins. See B. M. Gumbiner, 1993, Neuron 11:551, and D. R. Garrod, 1993, Curr. Opin. Cell Biol. 5:30. (b) Electron micrograph of a thin section of a desmosome connecting two cultured differentiated human keratinocytes. Bundles of intermediate filaments radiate from the two darkly staining cytoplasmic plaques that line the inner surface of the adjacent plasma membranes. Inset: Electron microscopic tomograph of a desmosome linking two human epidermal cells (plasma membranes, pink; desmosomal cadherins, blue; bar, 35 nm).
[Part (b) republished by permission of Nature, from Al-Amoudi, A., et al., “The molecular architecture of cadherins in native epidermal desmosomes,” Nature, 2007, 450:832–837; permission conveyed through the Copyright Clearance Center, Inc.]

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The cadherin desmoglein was identified through studies of an unusual but revealing skin disease called pemphigus vulgaris, an autoimmune disease. Patients with autoimmune disorders synthesize self-attacking, or “auto,” antibodies that bind to a normal body protein. In pemphigus vulgaris, the auto-antibodies disrupt adhesion between epithelial cells, causing blisters of the skin and mucous membranes. The predominant auto-antibodies in patients were shown to be specific for desmoglein; indeed, the addition of such antibodies to normal skin induces the formation of blisters and disruption of cell adhesion.