Table of contents

Other Drosophila cadherins

Mutations in the dachsous gene of Drosophila lead to striking defects in the morphogenesis of the thorax, legs, and wings. The dachsous gene has been cloned and shown to encode a huge transmembrane protein that is a member of the cadherin superfamily, similar to the fat gene. Both Dachsous and Fat proteins contain large tandem arrays of cadherin domains--27 and 34, respectively--as compared with 4 cadherin domains in classic vertebrate cadherins. In addition, Dachsous and Fat each has a cytoplasmic domain with sequence similarity to the cytoplasmic beta-catenin-binding domain of classic vertebrate cadherins. The dachsous gene is expressed in the ectoderm of embryos, whereas its expression in larvae is restricted to imaginal discs and specific regions of the brain (Clark, 1995).

Mutations in the recessive Drosophila tumor gene l(2)gl affect growth and structural properties of neural tissues and imaginal discs during larval development. Anti-l(2)gl sera show cross-reaction to a mouse protein that is localized at cell-cell contact sites in tissue culture cells. Moreover, amino acid sequence homology to deduced amino acid sequences of members of the vertebrate cadherin cell-adhesion molecule family suggests that the l(2)gl gene product may have properties of a cell-adhesion molecule (Klambt, 1989).

Recessive lethal mutations in the fat locus of Drosophila cause hyperplastic, tumor-like overgrowth of larval imaginal discs, defects in differentiation and morphogenesis, and death during the pupal stage. The fat locus encodes a novel member of the cadherin gene superfamily: an enormous transmembrane protein of over 5000 amino acids with a putative signal sequence, 34 tandem cadherin domains, four EGF-like repeats, a transmembrane domain, and a novel cytoplasmic domain. This novel member of the cadherin gene superfamily functions as a tumor suppressor gene and is required for correct morphogenesis (Mahoney, 1991).

Dictyostelium Cadherins

The cell adhesion molecule DdCAD-1 regulates morphogenesis through differential spatiotemporal expression in Dictyostelium discoideum

During development of Dictyostelium, multiple cell types are formed and undergo a coordinated series of morphogenetic movements guided by their adhesive properties and other cellular factors. DdCAD-1 is a unique homophilic cell adhesion molecule encoded by the cadA gene. It is synthesized in the cytoplasm and transported to the plasma membrane by contractile vacuoles. In chimeras developed on soil plates, DdCAD-1-expressing cells showed greater propensity to develop into spores than did cadA-null cells. When development was performed on non-nutrient agar, wild-type cells sorted from the cadA-null cells and moved to the anterior zone. They differentiated mostly into stalk cells and eventually died, whereas the cadA-null cells survived as spores. To assess the role of DdCAD-1 in this novel behavior of wild-type and mutant cells, cadA-null cells were rescued by the ectopic expression of DdCAD-1-GFP. Morphological studies have revealed major spatiotemporal changes in the subcellular distribution of DdCAD-1 during development. Whereas DdCAD-1 becomes internalized in most cells in the post-aggregation stages, it is prominent in the contact regions of anterior cells. Cell sorting is also restored in cadA- slugs by exogenous recombinant DdCAD-1. Remarkably, DdCAD-1 remains on the surface of anterior cells, whereas it is internalized in the posterior cells. Additionally, DdCAD-1-expressing cells migrate slower than cadA- cells and sort to the anterior region of chimeric slugs. These results show that DdCAD-1 influences the sorting behavior of cells in slugs by its differential distribution on the prestalk and prespore cells (Sriskanthadevan, 2011).

Invertebrate cadherins

The Caenorhabditis elegans embryo provides a model system for studying how cells move and change shape to generate body and tissue morphologies. At hatching, the outermost cellular layer of the body consists of a monolayer of 85 epithelial cells, called hypodermal cells, that are linked together by adherens junctions. During embryogenesis, hypodermal cells are involved in two distinct processes that transform the initially ellipsoidal mass of embryonic cells into a long, thin worm; these processes are called body enclosure and body elongation. The hypodermal cells are born on the dorsal surface of the embryo. As the hypodermal cells develop adherens junction connections, they begin to spread as a sheet across the embryo until the contralateral edges of the sheet meet at the ventral midline. In the anterior of the embryo, ventral hypodermal cells on the periphery of the spreading sheet develop filopodial extensions that may function to draw the contralateral edges of the sheet together. In the posterior of the embryo, the contralateral edges appear to be drawn together by a purse-string-like contraction that completes the enclosure process (Costa, 1998).

In several respects, these processes are similar to epithelial cell movements described in a variety of systems, such as wound healing in vertebrates and dorsal closure in Drosophila. At the completion of body enclosure in C. elegans, the apical surfaces of the hypodermal cells resemble rectangles that are elongated along the circumferential contour of the embryo's body. These apical surfaces begin to change shape, constricting along the circumferential contour of the body and elongating along the anterior-posterior (longitudinal) axis. The coordinate changes in the shapes of the hypodermal cells appear to cause the body to decrease in circumference and to elongate about fourfold along its longitudinal axis. Each hypodermal cell contains an array of actin filament bundles in its apical cortex; these bundles are oriented parallel to the circumferential contour of the body, and are here termed circumferential filament bundles (CFBs). The CFBs appear to connect with the adherens junction that encircles each hypodermal cell. Microtubules in the dorsal and ventral hypodermal cells are aligned parallel to the CFBs. However, few if any microtubules appear to contact the adherens junction; most instead terminate before, or run parallel to, the junction. The parallel filament bundles bridge two opposing sides of each hypodermal cell, apparently connecting to the subapical adherens junction. Contraction of the filament bundles has been proposed as the force that elongates the embryo; the bundles become shorter and thicker during elongation and drugs that disrupt actin filament organization prevent elongation. Apical constriction of cells has been shown in other systems to drive the invagination of epithelial sheets -- because of the closed, cylindrical geometry of the hypodermal sheet in C. elegans, an analogous apical constriction might instead drive body elongation (Costa, 1998 and references).

Mutants defective in morphogenesis have been isolated that identify three genes required for both cell migration during body enclosure and cell shape change during body elongation. Analyses of hmp-1, hmp-2, and hmr-1 mutants suggest that products of these genes anchor contractile actin filament bundles at the adherens junctions between hypodermal cells and, thereby, transmit the force of bundle contraction into cell shape change. The protein products of all three genes localize to hypodermal adherens junctions in embryos. The sequences of the predicted HMP-1, HMP-2, and HMR-1 proteins are related to the cell adhesion proteins alpha-catenin, beta-catenin/Armadillo, and classical cadherin, respectively. This putative catenin-cadherin system is not essential for general cell adhesion in the C. elegans embryo, but rather mediates specific aspects of morphogenetic cell shape change and cytoskeletal organization (Costa, 1998).

In flies and vertebrates, Armadillo/beta-catenin forms a complex with Tcf/Lef-1 transcription factors, serving as an essential co-activator to mediate Wnt signaling. It also associates with cadherins to mediate adhesion. In Caenorhabditis elegans, three putative beta-catenin homologs have been identified: WRM-1, BAR-1 and HMP-2. WRM-1 and the Tcf homolog POP-1 mediate Wnt signaling by a mechanism that has challenged current views of the Wnt pathway. BAR-1 is the only beta-catenin homolog that interacts directly with POP-1. BAR-1 mediates Wnt signaling by forming a BAR-1/POP-1 bipartite transcription factor that activates expression of Wnt target genes such as the Hox gene mab-5. HMP-2 is the only beta-catenin homologue that interacts with the single cadherin of C. elegans, HMR-1. It is concluded that a canonical Wnt pathway exists in C. elegans. Furthermore, this analysis shows that the functions of C. elegans beta-catenins in adhesion and in signaling are performed by separate proteins (Korswagen, 2000).

HMP-2 is the only C. elegans beta-catenin that interacts with the single classical cadherin, HMR-1. This interaction agrees with the hmp-2 mutant phenotype and with the colocalization of HMP-2 with HMR-1 and the alpha-catenin HMP-1 in adherens junctions. It is concluded that HMP-2 functions specifically in adhesion. Vertebrates express a second beta-catenin-like protein, Plakoglobin, which is part of the desmosomal adhesion complex. It is unclear whether Plakoglobin functions specifically in adhesion or also has a role in Wnt signaling. The functions of Armadillo and beta-catenin in Wnt signaling and adhesion are physically separable. It is unclear whether Armadillo/beta-catenin in adherens junctions may directly or indirectly affect the cytoplasmic signaling pool. Mutations in cadherins have been identified in many epithelial tumors. An attractive explanation for the oncogenic potential of cadherin mutations is the release of beta-catenin from adherens junctions, which in turn can interact with Tcf transcription factors to activate the expression of Wnt target genes. These data indicate that, at least in C. elegans, the adhesion and signaling pools of beta-catenin are separate entities (Korswagen, 2000).

A sea urchin cadherin, termed LvG-cadherin (for Lytechinus variegatus Goliath-cadherin) has been cloned. The deduced amino acid sequence predicts that it is a transmembrane protein with an apparent relative molecular mass of 303 kDa. The cytoplasmic domain shows significant sequence identity to that of vertebrate classic cadherins. However, the extracellular domain is distinguished from its vertebrate counterparts by both an increased number of cadherin-specific repeats (13) and the presence of four EGF-like repeats proximal to the transmembrane domain. In order to gain insight into the regulation of cadherin function during morphogenesis, the sea urchin embryo was used as a model system to study the regulation of cadherin localization during epithelial-mesenchymal conversion and convergent-extension movements. Polyclonal antibodies raised against the cytoplasmic domain of the cloned sea urchin cadherin recognize three major polypeptides of M(r) 320, 140, and 125 kDa and specifically stain adherens junctions (and to a lesser extent, lateral membrane domains in all epithelial tissues of the embryo). Analysis of embryos during gastrulation demonstrates that changes in cadherin localization are observed in cells undergoing an epithelial-mesenchymal conversion. Ingression of primary mesenchyme cells is accompanied by the rapid loss of junctional cadherin staining and the coincident accumulation of cadherin in intracellular organelles. These data are consistent with the idea that the de-adhesion of mesenchymal cells from neighboring epithelial cells involves the regulated endocytosis of cell surface cadherin molecules. Conversely, neither cadherin abundance nor localization is altered in cells of the gut, which undergo convergent-extension movements during the formation of the archenteron. This observation indicates that these movements do not require the loss of junctional cadherin molecules. Instead, the necessary balance between adhesion and motility may be achieved by regulating the expression of different subtypes of cadherin molecules or modifying interactions between cadherins and catenins, proteins that bind the cytoplasmic domain of cadherin and are necessary for cadherin adhesive function. Taken together, these data are consistent with the hypothesis that the sea urchin possesses several cadherins, including a novel member of the cadherin family, and that the dynamic regulation of cadherin localization plays a role in epithelial to mesenchymal conversions during gastrulation (Miller, 1997).

cdh-3, a cadherin superfamily member of C. elegans is expressed in developing epithelial cells, but also in a number of neuroectodermal cells that extend processes along some of these epithelial cells. A loss-of-function mutation affects the morphogenesis of a single cell, hyp10, which forms the tip of the nematode tail. The lack of detectable defects associated with the other cells expressing cdh-3 reporter constructs hints at the existence of other genes that can compensate for cdh-3 loss of function (Pettitt, 1996).

The dramatic cell-shape changes necessary to form a multicellular organism require cell-cell junctions to be both pliable and strong. The zonula occludens (ZO) subfamily of membrane-associated guanylate kinases (MAGUKs) are scaffolding molecules thought to regulate cell-cell adhesion, but there is little known about their roles in vivo. To elucidate the functional role of ZO proteins in a living embryo, the sole C. elegans ZO family member, ZOO-1, was characterized. ZOO-1 localizes with the cadherin-catenin complex during development, and its junctional recruitment requires the transmembrane proteins HMR-1/E-cadherin and VAB-9/claudin, but surprisingly, not HMP-1/alpha-catenin or HMP-2/beta-catenin. zoo-1 knockdown results in lethality during elongation, resulting in the rupture of epidermal cell-cell junctions under stress and failure of epidermal sheet sealing at the ventral midline. Consistent with a role in recruiting actin to the junction in parallel to the cadherin-catenin complex, zoo-1 loss of function reduces the dynamic recruitment of actin to junctions and enhances the severity of actin filament defects in hypomorphic alleles of hmp-1 and hmp-2. These results show that ZOO-1 cooperates with the cadherin-catenin complex to dynamically regulate strong junctional anchorage to the actin cytoskeleton during morphogenesis (Lockwood, 2008).

Non-junctional E-Cadherin clusters regulate the actomyosin cortex in the C. elegans zygote

Classical cadherins (see Drosophila Shotgun) are well known for their essential function in mediating cell-cell adhesion via their extra-cellular cadherin domains and intra-cellular connections to the actin cytoskeleton. There is evidence, however, of adhesion-independent cadherin clusters existing outside of cell-cell junctions. What function, if any, these clusters have is not known. HMR-1, the sole classical cadherin in Caenorhabditis elegans, plays essential roles during gastrulation, blastomere polarity establishment, and epidermal morphogenesis. To elucidate the physiological roles of non-junctional cadherin, HMR-1 was analyzed in the C. elegans zygote, which is devoid of neighbors. Non-junctional clusters of HMR-1 form during the one-cell polarization stage and associate with F-actin at the cortex during episodes of cortical flow. Non-junctional HMR-1 clusters downregulate RHO-1 (see Drosophila Rho1) activity and inhibit accumulation of non-muscle myosin II (NMY-2; see Drosophila Zipper ) at the anterior cortex. HMR-1 clusters were found to impede cortical flows and play a role in preserving the integrity of the actomyosin cortex, preventing it from splitting in two. Importantly, an inverse relationship was uncovered between the amount of HMR-1 at the cell surface and the rate of cytokinesis. The effect of HMR-1 clusters on cytokinesis is independent of their effect on NMY-2 levels, and is also independent of their extra-cellular domains. Thus, in addition to their canonical role in inter-cellular adhesion, HMR-1 clusters regulate RHO-1 activity and NMY-2 level at the cell surface, reinforce the stability of the actomyosin cortex, and resist its movement to influence cell-shape dynamics (Padmanabhan, 2016).

Vertebrate E-cadherin protein interactions

The E-cadherin/catenin complex, an organizer of epithelial structure and function, is disturbed in invasive cancer. The HAV (histidine alanine valine) sequence in the first extracellular domain of E-cadherin is crucial for homophilic interactions between cadherins. Specific peptides containing a single HAV sequence interfere with the functions of the E-cadherin/catenin complex. Cells either expressing or not expressing specific cadherins were challenged with both cadherin and noncadherin peptides comprising a central HAV sequence. Specific E-cadherin peptides inhibit cell aggregation, disturb the epithelial morphotype and are able to stimulate invasion of cells expressing E-cadherins. Conditioned medium, containing E-cadherin fragments, also stimulate invasion in contrast to conditioned medium from which the E-cadherin fragments are removed. These studies show that E-cadherin functions are inhibited by homologous proteolytic HAV-containing fragments that are released in an autocrine manner and subsequently inhibit the E-cadherin/catenin complex. In this way such cadherin fragments may induce and support cancer invasion (No, 1999).

The effect of N-cadherin, and its free or membrane-anchored cytoplasmic domain, was studied to determine the level and localization of beta-catenin and to assess its ability to induce lymphocyte enhancer-binding factor 1 (LEF-1)-responsive transactivation. These cadherin derivatives form complexes with beta-catenin and protect it from degradation. N-cadherin directs beta-catenin into adherens junctions, and the chimeric protein induces diffuse distribution of beta-catenin along the membrane whereas the cytoplasmic domain of N-cadherin colocalizes with beta-catenin in the nucleus. Cotransfection of beta-catenin and LEF-1 into Chinese hamster ovary cells induces transactivation of a LEF-1 reporter, which is blocked by the N-cadherin-derived molecules. Expression of N-cadherin and an interleukin 2 receptor/cadherin chimera in SW480 cells relocates beta-catenin from the nucleus to the plasma membrane and reduces transactivation. The cytoplasmic tails of N- or E-cadherin colocalize with beta-catenin in the nucleus, and suppress the constitutive LEF-1-mediated transactivation, by blocking beta-catenin-LEF-1 interaction. Moreover, the 72 C-terminal amino acids of N-cadherin stabilize beta-catenin and reduce its transactivation potential. These results indicate that beta-catenin binding to the cadherin cytoplasmic tail either in the membrane, or in the nucleus, can inhibit beta-catenin degradation and efficiently block its transactivation capacity (Sadot, 1998).

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 the cell adhesive activity of E-cadherin; this failure in adhesive activity has been ascribed to the failure of the deletion mutants to associate with catenins. Based on the present results, this concept needs revision. Leukemia cells (K562) expressing E-cadherin with COOH-terminal deletion of 37 or 71 amino acid residues show 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) show E-cadherin-dependent aggregation. Thus, deletion of the membrane-proximal region results in activation of the nonfunctional E-cadherin polypeptides. However, these cells do not show compaction. Chemical cross-linking reveals 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 (Ozawa, 1998).

The E-cadherin/catenin complex regulates Ca++-dependent cell-cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein, GP2CAD1, which comprises the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, is efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 result in delivery of less than 10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, greater than 90% of each mutant protein is retained in the ER. None of these mutants form a strong interaction with beta-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks beta-catenin binding is also localized intracellularly. Thus, beta-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, it is suggested that beta-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane (Chen, 1999).

Electron microscopy of a recombinant E-cadherin ectodomain pentamerized by the assembly domain of cartilage oligomeric matrix protein (ECADCOMP) has been used to analyze the role of cis-dimerization and trans-interaction in the homophilic association of this cell adhesion molecule. The Ca2+ dependency of both interactions was investigated. Low Ca2+ concentrations (50 muM) stabilize the rod-like structure of E-cadherin. At medium Ca2+ concentration (500 muM), two adjacent ectodomains in a pentamer form cis-dimers. At high Ca2+ concentration (>1 mM), two cis-dimers from different pentamers form a trans-interaction. The X-ray structure of an N-terminal domain pair of E-cadherin reveals two molecules per asymmetric unit in an intertwisted X-shaped arrangement with closest contacts in the Ca2+-binding region between domains 1 and 2. Contrary to previous data, Trp2 is docked in the hydrophobic cavity of its own molecule, and is therefore not involved in cis-dimerization of two molecules. This is supported further by W2A and A80I (a residue involved in the hydrophobic cavity surrounding Trp2) mutations in ECADCOMP, which both lead to abrogation of the trans- but not the cis-interaction. Structural and biochemical data suggest a link between Ca2+ binding in the millimolar range and Trp2 docking, both events being essential for the trans-association (Pertz, 1999).

The cadherin/catenin complex mediates Ca2+-dependent cell-cell interactions that are essential for normal developmental processes. It has been proposed that sorting of cells during embryonic development is due, at least in part, to expression of different cadherin family members or to expression of differing levels of a single family member. Expression of dominant-negative cadherins has been used experimentally to decrease cell-cell interactions in whole organisms and in cultured cells. In this study, the mechanism of action of extracellular domain-deleted dominant-negative cadherin are elucidated, showing that it is not cadherin isotype-specific, and that it must be membrane-associated but the orientation within the membrane does not matter. In addition, membrane-targeted cytoplasmic domain cadherin with the catenin-binding domain deleted does not function as a dominant-negative cadherin. Expression of extracellular domain-deleted dominant-negative cadherin results in down-regulation of endogenous cadherins, which presumably contribute to the non-adhesive phenotype (Nieman, 1999).

beta-catenin is a multifunctional protein found in three cell compartments: the plasma membrane, the cytoplasm and the nucleus. The cell has developed elaborate ways of regulating the level and localization of beta-catenin to assure its specific function in each compartment. One aspect of this regulation is inherent in the structural organization of beta-catenin itself; most of its protein-interacting motifs overlap so that interaction with one partner can block binding of another at the same time. Using recombinant proteins, it was found that E-cadherin and lymphocyte-enhancer factor-1 (LEF-1) form mutually exclusive complexes with beta-catenin; the association of beta-catenin with LEF-1 is competed out by the E-cadherin cytoplasmic domain. Similarly, LEF-1 and adenomatous polyposis coli (APC) form separate, mutually exclusive complexes with beta-catenin. In Wnt-1-transfected C57MG cells, free beta-catenin accumulates and is able to associate with LEF-1. The absence of E-cadherin in E-cadherin minus embryonic stem (ES) cells also leads to an accumulation of free beta-catenin and its association with LEF-1, thereby mimicking Wnt signaling. beta-catenin/LEF-1-mediated transactivation in these cells is antagonized by transient expression of wild-type E-cadherin, but not of E-cadherin lacking the beta-catenin binding site. The potent ability of E-cadherin to recruit beta-catenin to the cell membrane and prevent its nuclear localization and transactivation has also been demonstrated using SW480 colon carcinoma cells (Orsulic, 1999).

M-cadherin, a calcium-dependent intercellular adhesion molecule, is expressed in skeletal muscle cells. Its pattern of expression, both in vivo and in cell culture as well as functional studies, has implied that M-cadherin is important for skeletal muscle development, in particular the fusion of myoblasts into myotubes. M-cadherin forms complexes with the catenins in skeletal muscle cells similar to E-cadherin in epithelial cells. This suggests that the muscle-specific function of the M-cadherin catenin complex might be mediated by additional interactions with yet unidentified cellular components, especially cytoskeletal elements. These include the microtubules that also have been implicated in the fusion process of myoblasts. Evidence is presented that the M-cadherin catenin complex interacts with microtubules in myogenic cells by using three independent experimental approaches. (1) Analysis by laser scan microscopy reveals that the destruction of microtubules by nocodazole leads to an altered cell surface distribution of M-cadherin in differentiating myogenic cells. In contrast, disruption of actin filaments has little effect on the surface distribution of M-cadherin. (2) M-cadherin antibodies coimmunoprecipitate tubulin from extracts of nocodazole-treated myogenic cells but not of nocodazole-treated epithelial cells ectopically expressing M-cadherin. On the contrary, tubulin antibodies coimmunoprecipitate M-cadherin from extracts of nocodazole-treated myogenic cells but not of nocodazole-treated M-cadherin-expressing epithelial cells. (3) M-cadherin and the catenins, but not a panel of control proteins, are copolymerized with tubulin from myogenic cell extracts even after repeated cycles of assembly and disassemly of tubulin. Moreover, neither M-cadherin nor E-cadherin can be found in a complex with microtubules in epithelial cells ectopically expressing M-cadherin. These data are consistent with the idea that the interaction of M-cadherin with microtubules might be essential to keep the myoblasts aligned during fusion, a process in which both M-cadherin and microtubules have been implicated (Kaufmann, 1999).

Beta-catenin, a member of the Armadillo repeat protein family, binds directly to the cytoplasmic domain of E-cadherin, linking it via alpha-catenin to the actin cytoskeleton. A 30-amino acid region within the cytoplasmic domain of E-cadherin, conserved among all classical cadherins, has been shown to be essential for beta-catenin binding. This region harbors several putative casein kinase II (CKII) and glycogen synthase kinase-3beta (GSK-3beta) phosphorylation sites and is highly phosphorylated. In vitro this region is indeed phosphorylated by CKII and GSK-3beta, which results in an increased binding of beta-catenin to E-cadherin. Additionally, in mouse NIH3T3 fibroblasts expression of E-cadherin with mutations in putative CKII sites results in reduced cell-cell contacts. Thus, phosphorylation of the E-cadherin cytoplasmic domain by CKII and GSK-3beta appears to modulate the affinity between beta-catenin and E-cadherin, ultimately modifying the strength of cell-cell adhesion (Lickert, 2000).

Protein phosphatase 2A (PP2A) plays central roles in development, cell growth and transformation. Inactivation of the murine gene encoding the PP2A catalytic subunit Calpha by gene targeting generates a lethal embryonic phenotype. No mesoderm is formed in Calpha minus embryos. During normal early embryonic development Calpha is predominantly present at the plasma membrane whereas the highly homologous isoform Cbeta is localized to the cytoplasm and nuclei, suggesting the inability of Cbeta to compensate for vital functions of Calpha in Calpha minusembryos. In addition, PP2A is found in a complex containing the PP2A substrates E-cadherin and beta-catenin. In Calpha minus embryos, E-cadherin and beta-catenin are redistributed from the plasma membrane to the cytosol. Cytosolic concentrations of beta-catenin are low. These results suggest that Calpha is required for stabilization of E-cadherin/ beta-catenin complexes at the plasma membrane (Gotz, 2000).

These data suggest that PP2A is involved in wnt signaling. One likely explanation is that PP2A containing the Calpha subunit binds to and stabilizes the E-cadherin/beta-catenin complex within the plasma membrane. PP2A forms a complex with beta-catenin and E-cadherin. It is likely that PP2A regulates the phosphorylation status of E-cadherin and beta-catenin at the plasma membrane. In the cytosol, the activity of PP2A may not be high enough to offset GSK3 beta-mediated phosphorylation of beta-catenin. Wnt signaling increases cytoplasmic concentrations of PP2A by releasing it from the plasma membrane into the cytosol. Increased concentrations of PP2A effectively offset the GSK3 beta-mediated phosphorylation of beta-catenin thus preventing its degradation, and promoting its translocation to the nucleus with subsequent activation of wnt target genes. In the Calpha knockout situation both E-cadherin and beta-catenin are no longer stabilized in the plasma membrane. It is likely that both are phosphorylated and translocated to the cytoplasm. These data show that cytoplasmic E-cadherin is stable, and suggest that the associated beta-catenin is targeted for degradation resulting in reduced beta-catenin levels. It is proposed that in the absence of PP2A Calpha, wnt signaling releases beta-catenin from the axin/APC/GSK3 beta complex; beta-catenin associates with the highly abundant cytoplasmic E-cadherin before it reaches the nucleus, where it is phosphorylated and becomes degraded. Consequently, no target genes of wnt signaling, including Brachyury or goosecoid, are transcribed and embryonic development halts (Gotz, 2000).

This model is consistent with the finding that Calpha is mainly plasma membrane-associated in the inner cell mass, but this association becomes lost during differentiation. This model would also explain why the wnt target gene T-brachyury is negatively regulated by E-cadherin: T-brachyury mRNA levels are high in E-cad minus embryonic stem cells (ES) cells, but absent in wild-type ES cells. Basal cytoplasmic levels of non-phosphorylated beta-catenin may be quenched by cytoplasmic or membrane-bound E-cadherin, preventing transcription of T-brachyury in the absence of a wnt signal. In E-cad minus cells, the quencher is missing, allowing low but significant amounts of non-phosphorylated beta-catenin to be translocated into the nucleus, and to activate transcription of T-brachyury. This is consistent with the structural organization of beta-catenin itself; most of its protein-interacting motifs overlap so that interaction with one partner can block binding of another at the same time. Using recombinant proteins, it has been found that E-cadherin and lymphocyte-enhancer factor-1 (LEF-1), which targets beta-catenin to the nucleus, form mutually exclusive complexes with beta-catenin. This model is also consistent with the known association of PP2A with axin. Axin is a negative regulator of embryonic axis formation in vertebrates (Gotz, 2000).

A new cell-cell adhesion system has been found at cadherin-based cell-cell adherens junctions (AJs) consisting of at least nectin and l-afadin. Nectin is a Ca2+-independent homophilic immunoglobulin-like adhesion molecule, and l-afadin is an actin filament-binding protein that connects the cytoplasmic region of nectin to the actin cytoskeleton. Both the trans-interaction of nectin and the interaction of nectin with l-afadin are necessary for their colocalization with E-cadherin and catenins at AJs. The mechanism of interaction between these two cell-cell adhesion systems at AJs has been examined by the use of alpha-catenin-deficient F9 cell lines and cadherin-deficient L cell lines stably expressing their various components. Nectin and E-cadherin are colocalized through l-afadin and the COOH-terminal half of alpha-catenin at AJs. Nectin trans-interacts independently of E-cadherin, and the complex of E-cadherin and alpha- and ß-catenins is recruited to nectin-based cell-cell adhesion sites through l-afadin without the trans-interaction of E-cadherin. These results indicate that nectin and cadherin interact through their cytoplasmic domain-associated proteins and suggest that these two cell-cell adhesion systems cooperatively organize cell-cell AJs (Tachibana, 2000).

At least two models are proposed for the formation of cell-cell AJs. One model is that nectin and E-cadherin independently form the respective trans-interactions and the nectin/afadin and cadherin/catenin systems recruit each other to form compact cell-cell AJs. The other model is that nectin first forms a trans-interaction that recruits, through l-afadin, the cadherin-catenin system in which E-cadherin does not trans-interact, followed by the trans-interaction of E-cadherin at nectin-based cell-cell adhesion sites, finally leading to the formation of compact cell-cell AJs. It is currently unknown which is the case, but it is likely that the nectin/afadin system plays a key role in the organization of cell-cell AJs in cooperation with the cadherin-catenin system (Tachibana, 2000).

By the use of epithelial cells it has been shown in afadin (-/-) mice and afadin (-/-) embryoid bodies that not only cadherin-based AJs, but also claudin/occludin-based TJs are impaired in these mutant cells. Claudin and occludin are Ca2+-independent homophilic cell adhesion molecules at TJs, of which cytoplasmic domains interact with ZO-1, -2, and -3. ZO-1 and -2 are F-actin-binding proteins that connect the cytoplasmic regions of claudin and occludin to the actin cytoskeleton. Behavior of nectin and l-afadin is different from that of E-cadherin and similar to that of ZO-1 during the formation of TJs in cultured MDCK cells. These results suggest that the nectin/afadin system plays a key role in proper organization of not only cadherin-based cell-cell AJs, but also claudin/occludin-based TJs. It remains unknown how the nectin/afadin system organizes TJs properly, but l-afadin may also indirectly connect nectin to the component of TJs through an unidentified factor. It is of crucial importance to clarify the molecular linkages among these three different cell-cell adhesion systems (Tachibana, 2000).

As a component of adherens junctions and the Wnt signaling pathway, ß-catenin binds cadherins, Tcf family transcription factors, and the tumor suppressor APC. The crystal structures of both unphosphorylated and phosphorylated E-cadherin cytoplasmic domain complexed with the arm repeat region of ß-catenin has been determined. The interaction spans all 12 arm repeats, and features quasi-independent binding regions that include helices that interact with both ends of the arm repeat domain and an extended stretch of 14 residues that closely resembles a portion of XTcf-3. Phosphorylation of E-cadherin results in interactions with a hydrophobic patch of ß-catenin that mimics the binding of an amphipathic XTcf-3 helix. APC contains sequences homologous to the phosphorylated region of cadherin, and is likely to bind similarly (Huber, 2001).

The cadherin cytoplasmic domain is not structured in the absence of ß-catenin, and binds in an extended conformation that forms a large interface with ß-catenin. This mode of binding may allow for degrees of regulation that would be impossible for an interaction involving a well-structured ligand. The surface of such a ligand is presented on a relatively rigid scaffold, and local changes can therefore affect the entire interface between the two proteins. In contrast, the interface between a ligand that is unstructured in its unbound state and its partner can be altered locally without affecting the rest of the interface. In this manner, posttranslational modifications like phosphorylation can modulate the interaction in a graded fashion rather than serving as a simple on/off switch (Huber, 2001).

The armadillo repeat domain architecture complements the binding of extended polypeptides by providing a large surface-to-volume ratio and elongated interaction surface. Multiple, quasi-independent interactions provide the possibility of having a minimal 'core' binding region while allowing other interactions to be more dynamic. Separate binding regions can be regulated independently, enabling combinatorial regulation of the interaction and the integration of multiple input signals. In the case of E-cadherin, the extended region III appears to be absolutely required for the interaction with ß-catenin, since destabilizing the interface by mutation of either ß-catenin Lys435 or Lys312 to glutamate destroys binding. In contrast, the enhanced binding of phosphorylated E-cadherin (region IV) or the decreased binding of E-cadherin upon phosphorylation of ß-catenin Tyr654 (region II) demonstrate that the overall affinity can be modulated without completely eliminating the interaction of these two proteins. An extended interface may also make the system somewhat resistant to mutations; for example, none of 18 ß-catenin alanine mutations eliminates binding to E-cadherin (J. von Kries and W. Birchmeier, personal communication to Huber, 2001). Combined, these features create a robust interface subject to regulation; this is likely to be important in determining the mechanical properties and dynamics of subcellular assemblies such as the adherens junction (Huber, 2001).

E-cadherin controls a wide array of cellular behaviors including cell-cell adhesion, differentiation and tissue development. Presenilin-1 (PS1), a protein involved in Alzheimer's disease, controls a gamma-secretase-like cleavage of E-cadherin. This cleavage is stimulated by apoptosis or calcium influx and occurs between human E-cadherin residues Leu731 and Arg732 at the membrane-cytoplasm interface. The PS1/gamma-secretase system cleaves both the full-length E-cadherin and a transmembrane C-terminal fragment, derived from a metalloproteinase cleavage after the E-cadherin ectodomain residue Pro700. The PS1/gamma-secretase cleavage dissociates E-cadherins, beta-catenin and alpha-catenin from the cytoskeleton, thus promoting disassembly of the E-cadherin-catenin adhesion complex. Furthermore, this cleavage releases the cytoplasmic E-cadherin to the cytosol and increases the levels of soluble beta- and alpha-catenins. Thus, the PS1/gamma-secretase system stimulates disassembly of the E-cadherin-catenin complex and increases the cytosolic pool of beta-catenin, a key regulator of the Wnt signaling pathway (Marambaud, 2002).

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, a functional cadherin ligand (hE/Fc) has been used to directly test whether E-cadherin ligation regulates phosphatidylinositol 3-kinase (PI 3-kinase) and Rac signaling. 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 involves an early phase, which is PI 3-kinase-independent, and a later amplification phase, which is inhibited by wortmannin. PI 3-kinase and Rac activity are necessary for productive adhesive contacts to form following initial homophilic ligation. It is concluded that E-cadherin is a cellular receptor that is activated upon homophilic ligation to signal through PI 3-kinase and Rac. It is proposed 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 (Kovacs, 2002a).

Cadherin cell adhesion molecules are major determinants of tissue patterning thatfunction in cooperation with the actin cytoskeleton. In the context of stable adhesion, cadherin/catenin complexes are often envisaged to passively scaffold onto cortical actin filaments. However, cadherins also form dynamic adhesive contacts during wound healing and morphogenesis. Actin polymerization has been proposed to drive cell surfaces together in these processes, although F-actin reorganization also occurs as cell contacts mature. The interaction between cadherins and actin is therefore likely to depend on the functional state of adhesion. The relationship between cadherin homophilic binding and cytoskeletal activity has been analyzed during early cadherin adhesive contacts. Dissecting the specific effect of cadherin ligation alone on actin regulation is difficult in native cell-cell contacts, due to the range of juxtacrine signals that can arise when two cell surfaces adhere. Therefore homophilic ligation was activated using a specific functional recombinant protein. This study provides the first evidence that E-cadherin associates with the Arp2/3 complex actin nucleator (see Drosophila WASp, an Arp2/2 interacting protein) and demonstrates that cadherin binding can exert an active, instructive influence on cells to mark sites for actin assembly at the cell surface (Kovacs, 2002b).

It is increasingly evident that dynamic cell-cell contacts entail a complex interaction between cadherins and actin. Two important novel aspects to this interaction have been identified. (1) Rather than passively responding to changes in the cytoskeleton, E-cadherin adhesion can, in fact, regulate the cytoskeleton by marking sites for actin assembly to occur at the cell surface. In the context of a planar adhesion assay, it is suggested that such cadherin-directed actin assembly manifests in the formation of broad lamellipodia. Equivalent processes in native cell-cell contacts may include the rapid actin reorganization that occurs when migrating cells first touch one another and the extension of cell surfaces over one another seen in compaction and in convergent-extension morphogenetic movements. Indeed, cadherin-directed actin assembly provides an attractive mechanism for cells to move upon one another, as exemplified by DE-cadherin-dependent border cell migration in the Drosophila egg chamber. (2) This instructive influence involves a physical association between the cadherin/catenin adhesion complex and the Arp2/3 complex. Therefore, rather than solely binding to preformed actin filaments, classical cadherins can also associate with and regulate the actin assembly machinery itself. This does not, of course, exclude roles for other forms of actin reorganization (e.g., filament bundling) in cadherin-actin interactions. Future studies will provide insight into the precise mechanisms that couple the cadherin/catenin complex to the Arp2/3 complex (Kovacs, 2002b and references therein).

Finally, a striking feature of these findings was the stringent spatial restriction of actin assembly, specifically to nascent cadherin contacts at the margins of contact zones. Similarly, the pool of p34Arc associating with E-cadherin remains relatively constant as cells adhered, suggesting tight regulation of the association between these complexes. This stringency likely reflects underlying constraints on cadherin signaling to the actin cytoskeleton. In particular, it has been recently found that E-cadherin ligation directly activates the Rac GTPase, which was essential for cells to form cadherin-based lamellipodia. Of note, Rac was recruited to nascent contacts at the leading edges of cadherin-based lamellipodia. It is therefore postulated that cadherin ligation marks sites for cell surface extension by locally activating Rac, which, in turn, stimulates cadherin-associated Arp2/3 to drive actin assembly. This is consistent both with evidence that Arp2/3 requires signal-mediated stimulation to achieve full activity and that Rac plays a key role in cadherin function. Indeed, several potential pathways have been identified that signal to Arp2/3, and attempts are being made to identify the Rac-dependent effector(s) that activate the Arp2/3 complex upon E-cadherin ligation. Elucidating the molecular mechanisms responsible for cadherin-directed actin assembly and its regulation is likely to yield new insights into the role of classical cadherins in cellular recognition and morphogenesis (Kovacs, 2002b and references therein).

E-cadherin controls a wide array of cellular behaviors including cell-cell adhesion, differentiation and tissue development. Presenilin-1 (PS1), a protein involved in Alzheimer's disease, controls a gamma-secretase-like cleavage of E-cadherin. This cleavage is stimulated by apoptosis or calcium influx and occurs between human E-cadherin residues Leu731 and Arg732 at the membrane-cytoplasm interface. The PS1/gamma-secretase system cleaves both the full-length E-cadherin and a transmembrane C-terminal fragment, derived from a metalloproteinase cleavage after the E-cadherin ectodomain residue Pro700. The PS1/gamma-secretase cleavage dissociates E-cadherins, ß-catenin and alpha-catenin from the cytoskeleton, thus promoting disassembly of the E-cadherin-catenin adhesion complex. Furthermore, this cleavage releases the cytoplasmic E-cadherin to the cytosol and increases the levels of soluble ß- and alpha-catenins. Thus, the PS1/gamma-secretase system stimulates disassembly of the E-cadherin-catenin complex and increases the cytosolic pool of ß-catenin, a key regulator of the Wnt signaling pathway (Marambaud, 2002).

ß-Catenin and p120 (see Drosophila Adherens junction protein p120) bind through their Armadillo repeat domains to the catenin binding domain and juxtamembrane domains of cadherins, respectively. Compared with ß-catenin, p120 is rather loosely associated with the cadherin complex, a property that may be important for its mechanism of action. Indirect evidence has suggested that p120-catenin (p120) can both positively and negatively affect cadherin adhesiveness. The p120 gene is mutated in SW48 cells, and the cadherin adhesion system is impaired as a direct consequence of p120 insufficiency. Restoring normal levels of p120 caused a striking reversion from poorly differentiated to cobblestone-like epithelial morphology, indicating a crucial role for p120 in reactivation of E-cadherin function. The rescue efficiency is enhanced by increased levels of p120, and reduced by the presence of the phosphorylation domain, a region previously postulated to confer negative regulation. Surprisingly, the rescue is associated with substantially increased levels of E-cadherin. E-cadherin mRNA levels are unaffected by p120 expression, but E-cadherin half-life is more than doubled. Direct p120-E-cadherin interaction is crucial, since p120 deletion analysis reveals a perfect correlation between E-cadherin binding and rescue of epithelial morphology. Interestingly, the epithelial morphology can also be rescued by forced expression of either WT E-cadherin or a p120-uncoupled mutant. Thus, the effects of uncoupling p120 from E-cadherin can be at least partially overcome by artificially maintaining high levels of cadherin expression. These data reveal a cooperative interaction between p120 and E-cadherin and a novel role for p120 that is likely indispensable in normal cells (Ireton, 2002).

Interaction of Fer with the β-catenin and the cadherin complex

β-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and alpha-catenin. Interaction of β-catenin with alpha-catenin is regulated by the phosphorylation of β-catenin Tyr-142. This residue can be phosphorylated in vitro by Fer or Fyn tyrosine kinases (see Drosophila Fps oncogene analog). Transfection of these kinases to epithelial cells disrupts the association between both catenins. Whether these kinases are involved in the regulation of this interaction by K-ras was examined. Stable transfectants of the K-ras oncogene in intestinal epithelial IEC18 cells were generated which show little alpha-catenin-β-catenin association with respect to control clones; this effect is accompanied by increased Tyr-142 phosphorylation and activation of Fer and Fyn kinases. As reported for Fer, Fyn kinase is constitutively bound to p120 catenin; expression of K-ras induces the phosphorylation of p120 catenin on tyrosine residues increasing its affinity for E-cadherin and, consequently, promotes the association of Fyn with the adherens junction complex. Yes tyrosine kinase also binds to p120 catenin but only upon activation, and stimulates Fer and Fyn tyrosine kinases. These results indicate that p120 catenin acts as a docking protein facilitating the activation of Fer/Fyn tyrosine kinases by Yes and demonstrate the role of these p120 catenin-associated kinases in the regulation of β-catenin-alpha-catenin interaction (Piedra, 2003).

The function of Type 1, classic cadherins depends on their association with the actin cytoskeleton, a connection mediated by alpha- and β-catenin. The phosphorylation state of β-catenin is crucial for its association with cadherin and thus the association of cadherin with the cytoskeleton. The phosphorylation of β-catenin is regulated by the combined activities of the tyrosine kinase Fer and the tyrosine phosphatase PTP1B. Fer phosphorylates PTP1B at tyrosine 152, regulating its binding to cadherin and the continuous dephosphorylation of β-catenin at tyrosine 654. Fer interacts with cadherin indirectly, through p120ctn. The interaction domains of Fer and p120ctn and peptides corresponding to these sequences release Fer from p120ctn in vitro and in live cells, resulting in loss of cadherin-associated PTP1B, an increase in the pool of tyrosine phosphorylated β-catenin and loss of cadherin adhesion function. The effect of the peptides is lost when a β-catenin mutant with a substitution at tyrosine 654 is introduced into cells. Thus, Fer phosphorylates PTP1B at tyrosine 152 enabling it to bind to the cytoplasmic domain of cadherin, where it maintains β-catenin in a dephosphorylated state. Cultured fibroblasts from mouse embryos targeted with a kinase-inactivating ferD743R mutation have lost cadherin-associated PTP1B and β-catenin, as well as localization of cadherin and β-catenin in areas of cell-cell contacts. Expression of wild-type Fer or culture in epidermal growth factor restores the cadherin complex and localization at cell-cell contacts (Xu, 2004).

Cadherin structure and subcellular distribution

E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface. Targeting information in the cytoplasmic tail of E-cadherin was analyzed by utilizing chimeras of E-cadherin fused to the ectodomain of the interleukin-2alpha (IL-2alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK(1) epithelial cells. Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin. Truncation mutants unable to bind beta-catenin were correctly targeted, showing, contrary to current understanding, that beta-catenin is not required for basolateral trafficking. These results also provide evidence that dileucine-mediated targeting is maintained in LLC-PK(1) cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line. These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane (Miranda, 2001).

E-cadherin plays an essential role in cell polarity and cell-cell adhesion; however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. The post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) was traced in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, DeltaS1-E-cadherin, colocalize with Rab11 and fuse with the recycling endosome before diverging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical DeltaS1-E-cadherin was unaffected. This study thus demonstrates a novel pathway for Rab11 dependent, dileucine-mediated, mu1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion (Lock, 2005).

E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane-bound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. This study examined the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN (Lock, 2005).

The Ca2+-independent immunoglobulin-like molecule nectin first forms cell-cell adhesion and then assembles cadherin at nectin-based cell-cell adhesion sites, resulting in the formation of adherens junctions (AJs). Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. This study examined the roles and modes of action of nectin and afadin in the formation of AJs in cultured MDCK cells. The trans-interaction of nectin assembled E-cadherin, which associates with p120(ctn), beta-catenin, and alpha-catenin at the nectin-based cell-cell adhesion sites in an afadin-independent manner. However, the assembled E-cadherin showed weak cell-cell adhesion activity and might be the non-trans-interacting form. This assembly is mediated by the IQGAP1-dependent actin cytoskeleton, which is organized by Cdc42 and Rac small G proteins that are activated by the action of trans-interacting nectin through c-Src and Rap1 small G protein in an afadin-independent manner. However, Rap1 binds to afadin, and this Rap1-afadin complex then interacts with p120(ctn) associated with non-trans-interacting E-cadherin, thereby causing the trans-interaction of E-cadherin. Thus, nectin regulates the assembly and cell-cell adhesion activity of E-cadherin through afadin, nectin signaling, and p120(ctn) for the formation of AJs in Madin-Darby canine kidney cells (Sato, 2006).

Cadherin expression and function in cultured cells

Human embryonal carcinoma (EC) cells typically require high cell densities to maintain their characteristic phenotype; they are generally subject to differentiation when cultured at low cell densities, marked by changes in morphology and expression of the surface antigen, SSEA-1. To test whether cadherin mediated cell-to-cell adhesion may be responsible for maintaining an EC phenotype it was ascertained that human EC cells generally express E- and P-cadherins, and are subject to cadherin mediated, Ca2+ dependent aggregation. However, in the NTERA2 human EC cell line, inhibition of cadherin mediated adhesion by culture in low levels of Ca2+ do not result in the changes typically seen under low cell density conditions. Low Ca2+ levels also do not affect the pattern of differentiation in these cells following induction with retinoic acid. Therefore, cadherin-mediated cell adhesion does not appear to play a role in maintaining an EC phenotype. However, culture at both low cell density and in the absence of Ca2+ does result in changes in the patterns of cadherin expression, suggesting a feedback regulatory effect of cell-to-cell adhesion. Further, lithium (which inhibits the cytoplasmic kinase GSK3beta and hence influences beta-catenin levels) does cause differentiation of NTERA2 cells. However, consideration of the phenotype of the resultant cells suggests that this effect may be because of lithium mimicking activation of a Wnt signaling pathway, rather than an effect on signaling consequent upon cadherin mediated cell to cell adhesion (Giesberts, 1999).

It is widely held that segregation of tissues expressing different cadherins results from cadherin-subtype-specific binding specificities. This belief is based largely upon assays in which cells expressing different cadherin subtypes aggregate separately when shaken in suspension. In various combinations of L cells expressing NCAM, E-, P-, N-, R-, or B-cadherin, coaggregation occurs when shear forces are low or absent but can be selectively inhibited by high shear forces. Cells expressing P- vs E-cadherin coaggregate and then demixe, one population enveloping the other completely. To distinguish whether this demixing is due to differences in cadherin affinities or expression levels, the latter were varied systematically. Cells expressing either cadherin at a lower level become the enveloping layer, as predicted by the Differential Adhesion Hypothesis. However, when cadherin expression levels are equalized, cells expressing P- vs E-cadherin remains intermixed. In this combination, 'homocadherin' (E-E; P-P) and 'heterocadherin' (E-P) adhesions must therefore be of similar strength. Cells expressing R- vs B-cadherin coaggregate but demix to produce configurations of incomplete envelopment. This signifies that R- to B-cadherin adhesions must be weaker than either 'homocadherin' adhesion. Together, cadherin quantity and affinity are factors that control tissue segregation and assembly through specification of the relative intensities of mature cell-cell adhesions (Duguay, 2003).

Table of contents

shotgun: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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