N-cadherin structure

Structures of the D1 fragment from N-cadherin have two dimer interfaces in common, suggesting that these interfaces may be biologically relevant. One of these interfaces is formed between molecules in an antiparallel pair, as if their C termini were pointing toward opposing cell surfaces, and it has been postulated that this interface corresponds to the adhesive interface between cadherins emanating from opposing cells. The second preserved interface, the "strand dimer," involves an exchange of the N-terminal beta strand between partner molecules, and features the complete intercalation of the side chain from the conserved residue tryptophan 2 into a conserved pocket in the hydrophobic core of the partner molecule. The strand dimer involves molecules arranged in a parallel manner, which first led to the suggestion that cadherins might protrude from the cell surface as dimers. To investigate the possible biological function of the lateral "strand dimer" observed in crystal structures of an N-cadherin D1 domain extracellular fragment, site-directed mutagenesis studies were undertaken of this molecule. Mutation of most residues important in the strand dimer interface abolishes the ability of N-cadherin to mediate cell adhesion. Mutation of an analogous central residue (Trp-2) in E-cadherin also abrogates the adhesive capacity of that molecule. The crystal structure of a Ca2+-complexed two-domain fragment from N-cadherin was also determined. This structure, like its E-cadherin counterpart, does not adopt the strand dimer conformation. This suggests the possibility that classical cadherins might stably exist in both dimeric and monomeric forms. Data from several laboratories imply that lateral dimerization or clustering of cadherins may increase their adhesivity. The strand dimer may play a role in this activation. The mutagenesis data presented here show that disruption of the strand dimer interface, while maintaining the structural integrity, expression, and localization of classical cadherins, can abrogate the ability of these molecules to function in cell adhesion. The crystal structures of the D1D2 fragments from both N- and E-cadherins, which are both monomeric, show that the strand dimer interface alone may be insufficient to hold cadherin ectodomains together as dimers. This suggests the possibility that other dimerization mechanisms may trigger activation of cadherin adhesivity through strand dimer formation, specifically, that inside-out signal transduction through cytoplasmic dimerization could activate cadherin adhesion. Thus, it seems likely that the adhesive properties of cadherins could depend on intracellular signaling pathways (Tamura, 1998). \

The mutant N-cadherin phenotype

To investigate the functions of N-cadherin in vivo, the gene encoding this adhesion protein was mutated in mice. Although N-cadherin is expressed at the time of gastrulation and neurulation, both neurulation and somitogenesis initiate apparently normally in homozygous mutant embryos. However, the resulting structures are often malformed. The somites of the mutant embryos are small, irregularly shaped, and less cohesive when compared with those of their wild-type littermates, and the epithelial organization of the somites is partially disrupted. Undulation of the neural tube is also observed in the mutant embryos. Homozygous mutant embryos die by day 10 of gestation. The mesodermal and endodermal cell layers of the yolk sac are separated in the mutants. The most dramatic cell adhesion defect is observed in the primitive heart: although myocardial tissue forms initially, the myocytes subsequently dissociate and the heart tube fails to develop normally. In vitro studies of cardiac myocytes derived from N-cadherin mutant embryos show that the cells can loosely aggregate and beat synchronously, demonstrating that electrical coupling can occur between N-cadherin-deficient cardiac myocytes. These results show that N-cadherin plays a critical role in early heart development as well as in other morphogenetic processes (Radice, 1997).

Inappropriate expression of N-cadherin by tumor cells derived from epithelial tissue results in conversion of the cell to a more fibroblast-like cell, with increased motility and invasion. The present study was designed to determine which domains of N-cadherin make it different from E-cadherin, with respect to altering cellular behavior, such as which domains are responsible for the epithelial to mesenchymal transition and increased cell motility and invasion. To address this question, chimeric cadherins were constructed, comprising of selected domains of E- and N-cadherin. The chimeras were transfected into epithelial cells to determine their effect on cell morphology and cellular behavior. A 69-amino acid portion of EC-4 of N-cadherin is necessary and sufficient to promote both an epithelial to mesenchymal transition in squamous epithelial cells and increased cell motility. Different cadherin family members promote different cellular behaviors (Kim, 2000).

It has been suggested that N-cadherin can interact with and activate fibroblast growth factor receptors (FGFR) in neurons and ovarian surface epithelial cells. To date, this interaction has not been substantiated by other labs. N-cadherin-mediated cell motility of breast cancer cells can be decreased by an inhibitor of the FGF-mediated signal transduction pathway. FGF causes a dramatic increase in motility in N-cadherin-expressing cells. The FGFRs contain a HAV sequence that has been proposed to interact with EC4 of N-cadherin. It is interesting to note that the 69-amino acid segment of N-cadherin includes the sequences proposed to interact with the FGFRs. The structure of a portion of FGFR1 bound to FGF2 has been determined. The histidine and valine side chains of the HAV sequence in FGFR1 are involved in intradomain contacts and, thus, appear to be unavailable for interacting with partner molecules. Thus, the precise role the FGFR plays in N-cadherin-dependent cell motility is still unknown and it is not clear at this time whether N-cadherin and the FGFR directly interact with one another (Kim, 2000).

Members of the cadherin family of cell adhesion molecules are thought to be crucial regulators of tissue patterning and organogenesis. During pancreatic ontogeny N-cadherin is initially expressed in the pancreatic mesenchyme and later in pancreatic endoderm. Analysis of N-cadherin-deficient mice has revealed that these mice suffer from selective agenesis of the dorsal pancreas. Further analysis demonstrates that the mechanism for the lack of a dorsal pancreas involves an essential function of N-cadherin as a survival factor in the dorsal pancreatic mesenchyme (Esni, 2001).

Genetic screens in zebrafish identified several loci that play essential roles in the patterning of retinal architecture. One of them, glass onion, encodes the N-cadherin gene. The glom117 mutant allele contains a substitution of the Trp2 residue known for its essential role in the adhesive properties of classic cadherins. Both the glom117 and pactm101b mutant N-cadherin alleles affect the polarity of the retinal neuroepithelial sheet and, unexpectedly, both result in cell-nonautonomous phenotypes in retinal patterning. The late onset of mutant N-cadherin phenotypes may be due to the ability of classic cadherins to substitute each other's function (Malicki, 2003).

E-cadherin (E-cad; cadherin 1) was conditionally substituted with N-cadherin (N-cad; cadherin 2) during intestine development by generating mice in which an Ncad cDNA was knocked into the Ecad locus. Mutant mice were born, demonstrating that N-cad can structurally replace E-cad and establish proper organ architecture. After birth, mutant mice gradually developed a mutant phenotype in both the small and large intestine and died at ~2-3 weeks of age, probably due to malnutrition during the transition to solid food. Molecular analysis revealed an extended domain of cells from the crypt into the villus region, with nuclear localization of β-catenin (β-cat; Ctnnb1) and enhanced expression of several β-cat target genes. In addition, the BMP signaling pathway was suppressed in the intestinal epithelium of the villi, suggesting that N-cad might interfere with BMP signaling in the intestinal epithelial cell layer. Interestingly, mutant mice developed severe dysplasia and clusters of cells with neoplastic features scattered along the crypt-villus axis in the small and large intestine. This experimental model indicates that, in the absence of E-cad, the sole expression of N-cad in an epithelial environment is sufficient to induce neoplastic transformations (Libusova, 2010).

Zebrafish neural tube morphogenesis requires Scribble-dependent oriented cell divisions

How control of subcellular events in single cells determines morphogenesis on the scale of the tissue is largely unresolved. The stereotyped cross-midline mitoses of progenitors in the zebrafish neural keel provide a unique experimental paradigm for defining the role and control of single-cell orientation for tissue-level morphogenesis in vivo. This study shows that the coordinated orientation of individual progenitor cell division in the neural keel is the cellular determinant required for morphogenesis into a neural tube epithelium with a single straight lumen. This study shows that Scribble is required for oriented cell division, and its function in this process is independent of canonical apicobasal and planar polarity pathways. A role is identified for Scribble in controlling clustering of β-catenin foci in dividing progenitors. Loss of either Scrib or N-cadherin results in abnormally oriented mitoses, reduced cross-midline cell divisions, and similar neural tube defects. It is proposed that Scribble-dependent nascent cell-cell adhesion clusters between neuroepithelial progenitors contribute to define orientation of their cell division. Finally, the data demonstrate that while oriented mitoses of individual cells determine neural tube architecture, the tissue can in turn feed back on its constituent cells to define their polarization and cell division orientation to ensure robust tissue morphogenesis (Žigman, 2011).

N- and E-cadherins in Xenopus are specifically required in the neural and non-neural ectoderm, respectively, for F-actin assembly and morphogenetic movements

Transmembrane cadherins are calcium-dependent intercellular adhesion molecules. Recently, they have also been shown to be sites of actin assembly during adhesive contact formation. However, the roles of actin assembly on transmembrane cadherins during development are not fully understood. This study shows, using the developing ectoderm of the Xenopus embryo as a model, that F-actin assembly is a primary function of both N-cadherin in the neural ectoderm and E-cadherin in the non-neural (epidermal) ectoderm, and that each cadherin is essential for the characteristic morphogenetic movements of these two tissues. However, depletion of N-cadherin and E-cadherin did not cause dissociation in these tissues at the neurula stage, probably owing to the expression of C-cadherin in each tissue. Depletion of each of these cadherins is not rescued by the other, nor by the expression of C-cadherin, which is expressed in both tissues. One possible reason for this is that each cadherin is expressed in a different domain of the cell membrane. These data indicate the combinatorial nature of cadherin function, the fact that N- and E-cadherin play primary roles in F-actin assembly in addition to roles in cell adhesion, and that this function is specific to individual cadherins. They also show how cell adhesion and motility can be combined in morphogenetic tissue movements that generate the form and shape of the embryonic organs (Nandadasa, 2009).

N-Cadherin and junctional dynamics

Neuroepithelial cells can generate neurons (nonepithelial cells). For chick and mouse embryos, the epithelial character of neuroepithelial cells was investigated in the context of neurogenesis by examining the presence of the molecular components of tight junctions during the transition from the neural plate to the neural tube. Immunoreactivity for occludin, a transmembrane protein specific to tight junctions, is detected at the apical end of the lateral membrane of neuroepithelial cells throughout the chick neural plate. During neural tube closure, occludin disappears from all neuroepithelial cells. Correspondingly, the addition of horseradish peroxidase to the apical side of the neuroepithelium by injection into the amniotic cavity of mouse embryos reveals the presence of functional tight junctions in the neural plate (Embryonic Day 8), but not the neural tube (Embryonic Day 9). In contrast to occludin, expression of ZO-1, a peripheral membrane protein of tight junctions, increases from the neural plate to the neural tube stage, also being confined to the apical end of the lateral neuroepithelial cell membrane. This localization coincides with that of N-cadherin, whose expression increases concomitantly with the disappearance of occludin. It is proposed that the loss of tight junctions from neuroepithelial cells reflects an overall decrease in their epithelial nature, which precedes the generation of neurons (Aaku-Saraste, 1996).

To test whether glycosyl phosphatidylinositol-linked T-cadherin is a component of cell junctions like classical cadherins, its distribution and targeting were examined in polarized epithelial cells. In vivo, T-cadherin is detected on the apical cell surface of the chick intestinal epithelium. In cultures of transfected Madin-Darby canine kidney cells, T-cadherin is also expressed apically, whereas classical N-cadherin resides basolaterally. Both cadherins are directly targeted to their respective membrane domains. Mutant proteins were expressed in Madin-Darby canine kidney cells to identify the regions responsible for differential cadherin localization. NDeltacyt, an N-cadherin cytoplasmic domain deletion mutant, is stably distributed basolaterally. This mutant is transported to both the apical and basolateral membrane compartments, followed by preferential removal from the apical surface. T-NDeltacyt, a T-cadherin mutant with the N-cadherin cytoplasmic domain deletion, is localized basolaterally, whereas N-TGPI, a GPI-anchored N-cadherin mutant, resides at the apical domain. The T-cadherin carboxyl-terminal 76 amino acids contain the apical targeting signal and include the signal for GPI anchor attachment. Basolateral localization of N-cadherin is achieved through targeting signals in the cytoplasmic domain. Thus, GPI-linked T-cadherin is not a component of cell junctions, consistent with a function as a recognition rather than a cell adhesion molecule (Koller, 1996).

N-cadherin is a transmembrane Ca2+-dependent glycoprotein that is part of adherens junctions. It functions with the cell adhesion N-terminal extracellular domain as a site of homophilic cell-cell contacts. The intracellular C-terminal domain provides via a catenin complex the interaction with the cytoskeleton. Ectopic expression of chicken N-cadherin in adult rat cardiomyocytes (ARC) in culture is obtained after microinjection into non-dividing cardiomyocytes; the exogenous protein colocalizes with the endogenous N-cadherin at the plasma membrane of the cell and form contact sites. A dominant negative chicken N-cadherin mutant was constructed by a large deletion of the extracellular domain. When expressed, this mutant inhibits the function of the endogenous rat N-cadherin, probably by competing for the catenin complex binding domain, which is essential for the formation of a stable cell-cell contact in ARC. The injected cells lose contact with neighbouring cells and retract; the connections of the gap junctions are pulled out as well. This can be avoided by another N-cadherin mutation, which, in addition to the N-terminal truncation, contains a deletion of the catenin binding domain. In the case of the truncated N-cadherin at the N terminus, the sarcomeric structure of the myofibrils of ARC is also affected. Myofibrils are the most vulnerable cytoskeletal structures affected by the overexpressed dominant negative N-cadherin mutation. Similar behaviour is shown when cardiomyocytes separate following Ca2+ depletion and when new cell-cell contacts are formed after Ca2+ replenishment. N-cadherin is thought to be the essential component for establishing new cell-cell contacts, which eventually lead to a new formation of intercalated disc-like structures in the cardiac cell culture (Hertig, 1996a).

The spatio-temporal appearance and distribution of proteins forming the intercalated disc were investigated in adult rat cardiomyocytes (ARC). The 'redifferentiation model' of ARC involves extensive remodelling of the plasma membrane and of the myofibrillar apparatus. It represents a valuable system to elucidate the formation of cell-cell contact between cardiomyocytes and to assess the mechanisms by which different proteins involved in the cell-cell adhesion process are sorted in a precise manner to the sites of function. The appearance of N-cadherin, the catenins and connexin-43 within newly formed adherens and gap junctions was studied. Here first evidence is provided for a formation of two distinct and separable N-cadherin/catenin complexes in cardiomyocytes. Both complexes are composed of N-cadherin and alpha-catenin; they bind to either beta-catenin or plakoglobin (homologs of Drosophila Armadillo) in a mutually exclusive manner. The two N-cadherin/catenin complexes are assumed to be functionally involved in the formation of cell-cell contacts in ARC; however, the differential appearance and localization of the two types of complexes may also point to a specific role during ARC differentiation. The newly synthesized beta-catenin containing complex is more abundant during the first stages in culture after ARC isolation, while the newly synthesized plakoglobin containing complex progressively accumulates during the morphological changes of ARC. ARC forms a tissue-like pattern in culture whereby the new cell-cell contacts can be dissolved through Ca2+ depletion. The presence of cAMP and replenishment of Ca2+ content in the culture medium not only allows reformation of cell-cell contacts but also affects the relative protein ratio between the two N-cadherin/catenin complexes, increasing the relative amount of newly synthesized beta-catenin over plakoglobin at a particular stage of ARC differentiation. The clustered N-cadherin/catenin complexes at the plasma membrane appear to be a prerequisite for the following gap junction formation; a temporal sequence for the appearance of adherens junction proteins and of gap junctions forming connexin-43 is suggested (Hertig, 1996b).

Cadherins are calcium-dependent, cell surface glycoproteins involved in cell-cell adhesion. To function in cell-cell adhesion, the transmembrane cadherin molecule must be associated with the cytoskeleton via cytoplasmic proteins known as catenins. Three catenins have been identified: alpha-catenin, beta-catenin, and gamma-catenin (also known as plakoglobin). The domain of the cadherin molecule important for its interaction with the catenins has been mapped to the COOH-terminal 70 amino acids, but less is known about regions of the catenins that allow them to associate with one another or with the cadherin molecule. Carboxyl-terminal deletions of plakoglobin were transfected into the human fibrosarcoma HT-1080 and immunofluorescence localization and co-immunoprecipitation were used to map the regions of plakoglobin that allow it to associate with N-cadherin and with alpha-catenin. Plakoglobin is an armadillo family member containing 13 weakly similar internal repeats. The alpha-catenin-binding region maps within the first repeat and the N-cadherin-binding region maps within repeats 7 and 8 (Sacco, 1995).

Cadherins are Ca(2+)-dependent, cell surface glycoproteins involved in cell-cell adhesion. Extracellularly, transmembrane cadherins such as E-, P-, and N-cadherin self-associate, while intracellularly they interact indirectly with the actin-based cytoskeleton. Several intracellular proteins (termed catenins, including alpha-catenin, beta-catenin, and plakoglobin) are tightly associated with these cadherins and serve to link them to the cytoskeleton. In fibroblasts, alpha-actinin, but not vinculin, colocalizes extensively with the N-cadherin/catenin complex. This is in contrast to epithelial cells where both cytoskeletal proteins colocalize extensively with E-cadherin and catenins. Alpha-actinin, but not vinculin, coimmunoprecipitates specifically with alpha- and beta-catenin from N- and E-cadherin-expressing cells, but only if alpha-catenin is present. Alpha-actinin coimmunoprecipitates with the N-cadherin/catenin complex in an actin-independent manner. Thus it is proposed that cadherin/catenin complexes are linked to the actin cytoskeleton via a direct association between alpha-actinin and alpha-catenin (Knudsen, 1995).

Phosphorylated tyrosine residues on beta-catenin are correlated with loss of cadherin function. Only nontyrosine phosphorylated beta-catenin is associated with N-cadherin in E10 chick retina tissue. A PTP1B-like tyrosine phosphatase associates with N-cadherin and may function as a regulatory switch controlling cadherin function by dephosphorylating beta-catenin, thereby maintaining cells in an adhesion-competent state. The PTP1B-like phosphatase is itself tyrosine phosphorylated. These direct binding experiments, with phosphorylated and dephosphorylated molecules, and the treatment of cells with tyrosine kinase inhibitors, indicate that the interaction of the PTP1B-like phosphatase with N-cadherin depends on its tyrosine phosphorylation. Concomitant with the tyrosine kinase inhibitor-induced loss of the PTP1B-like phosphatase from its association with N-cadherin, phosphorylated tyrosine residues are retained on beta-catenin; the association of N-cadherin with the actin containing cytoskeleton is lost, and N-cadherin-mediated cell adhesion is prevented. Tyrosine phosphatase inhibitors also result in the accumulation of phosphorylated tyrosine residues on beta-catenin, and loss of the association of N-cadherin with the actin-containing cytoskeleton; they prevent N-cadherin mediated adhesion, presumably by directly blocking the function of the PTP1B-like phosphatase. The binding of two cadherin specific ligands to the cell surface results in the accumulation of phosphorylated tyrosine residues on beta-catenin, uncoupling of N-cadherin from its association with the actin containing cytoskeleton, and loss of N-cadherin function. Ligand binding results in the absence of the PTP1B-like phosphatase from its association with N-cadherin as well as the loss of the tyrosine kinase and tyrosine phosphatase activities that otherwise co-precipitate with N-cadherin. This effect is similar to that observed with tyrosine kinase inhibitors, suggesting that the ligand interaction inhibits a tyrosine kinase, thereby preventing the phosphorylation of the PTP1B-like phosphatase, and its association with N-cadherin. Taken together these data indicate that a PTP1B-like tyrosine phosphatase can regulate N-cadherin function through its ability to dephosphorylate beta-catenin and that the association of the phosphatase with N-cadherin is regulated via the interaction of the GalNAcPTase with its proteoglycan ligand. In this manner, the GalNAcPTase-proteoglycan interaction may play a major role in morphogenetic cell and tissue interactions during development (Balsamo, 1996).

Intercellular communication mediated by gap junctions is important for tissue homeostasis in the avascular lens; extensive areas of gap junctions form between fiber cells during fiber cell differentiation and lens development. The role of the calcium-dependent cell adhesion molecule, N-cadherin, was examined in the process of gap junction formation between fiber cells. Lentoids, multicellular structures with characteristics of differentiated fiber cells, were isolated from embryonic chick lens cultures and subsequently paired to provide an in vitro model of fiber cell interactions. Gap junction formation between cells of paired lentoids was monitored by observing the lentoid-to-lentoid transfer of fluorescent dyes, either calcein or Lucifer yellow, over a time course of up to 48 hr. Dye transfer between lentoids is inhibited upon the addition to the medium of a monoclonal antibody specific for N-cadherin, and also by the reduction of extracellular calcium in the incubation medium. However, the addition of an antibody to a fiber-cell-specific integral membrane protein, MIP, does not change the time course nor extent of dye transfer between lentoids. These results, using cultured embryonic cells, extend those from previous studies with cell lines and transfected cells. It is concluded that cadherin interactions facilitate the formation of gap junctions between embryonic lens fiber cells, by the stabilization of membrane appositions and/or by the generation of an intracellular signal(s) (Frenzel, 1996).

Llgl1 connects cell polarity with cell-cell adhesion in embryonic neural stem cells

Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. This study reports that mice with embryonic neural stem-cell-specific deletion of Llgl1 Nestin-Cre/Llgl1fl/fl), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1fl/fl brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. This study shows that LLGL1 directly binds to and promotes internalization of N-cadherin (see Drosophila Cadherin-N), and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1 (see Drosophila aPKC), restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells (Jossin, 2017).

PAPC couples the segmentation clock to somite morphogenesis by regulating N-cadherin dependent adhesion

Vertebrate segmentation is characterized by the periodic formation of epithelial somites from the mesenchymal presomitic mesoderm (PSM). How the rhythmic signaling pulse delivered by the Segmentation Clock is translated into the periodic morphogenesis of somites remains poorly understood. This study focused on the role of Paraxial protocadherin (PAPC/Pcdh8) in this process. In chicken and mouse embryos, PAPC expression is tightly regulated by the Clock and Wavefront system in the posterior PSM. PAPC exhibits a striking complementary pattern to N-Cadherin (CDH2; see Drosophila Cadherin-N), marking the interface of the future somite boundary in the anterior PSM. Gain and loss of function of PAPC in chicken embryos disrupt somite segmentation by altering the CDH2-dependent epithelialization of PSM cells. These data suggest that clathrin-mediated endocytosis is increased in PAPC expressing cells, subsequently affecting CDH2 internalization in the anterior compartment of the future somite. This in turn generates a differential adhesion interface, allowing formation of the acellular fissure that defines the somite boundary. Thus periodic expression of PAPC in the anterior PSM triggers rhythmic endocytosis of CDH2, allowing for segmental de-adhesion and individualization of somites (Chal, 2017).

Signaling upstream of N-cadherin: Sphingosine 1-phosphate receptor regulation of N-cadherin mediates vascular stabilization

Vascular stabilization, a process by which nascent vessels are invested with mural cells, is important in angiogenesis. The molecular basis of vascular stabilization regulated by sphingosine 1-phosphate (S1P), a platelet-derived lipid mediator, is described. S1P1 receptor-dependent cell-surface trafficking and activation of the cell-cell adhesion molecule N-cadherin is essential for interactions between endothelial and mural cells. Endothelial cell S1P1/Gi/Rac pathway induces microtubule polymerization, resulting in trafficking of N-cadherin to polarized plasma membrane domains. S1P treatment modulates the phosphorylation of N-cadherin as well as p120-catenin and induces the formation of cadherin/catenin/actin complexes containing novel regulatory and trafficking factors. The net result of endothelial cell S1P1 receptor activation is the proper trafficking and strengthening of N-cadherin-dependent cell-cell adhesion with mural cells. Perturbation of N-cadherin expression with small interfering RNA profoundly attenuated vascular stabilization in vitro and in vivo. S1P-induced trafficking and activation of N-cadherin provides a novel mechanism for the stabilization of nascent blood vessels by mural cells and may be exploited to control angiogenesis and vascular diseases (Paik, 2004).

The S1P1 receptor (also known as EDG-1), a G-protein-coupled receptor (GPCR) for the bioactive lipid S1P, was originally cloned from vascular endothelial cells (EC) as an inducible gene. Previous work has implicated this receptor as a major regulator of EC function in vitro, including the regulation of cell survival, migration, and morphogenesis. In cultured EC, S1P interaction with its receptors S1P1 and S1P3 induced the small GTPase Rho- and Rac-dependent assembly of VE-cadherin-based adherens junctions. Global deletion of the S1p1 gene results in a fully penetrant embryonic lethality at embryonic days 12.5-14.5 (E12.5-E14.5) of gestation. The phenotype of the animals included aberrant mural cell ensheathment of the dorsal aorta and other vessels. In addition, tissue-specific knock-out of S1P1 in endothelial cells phenocopies the global knock-out phenotype, whereas VSMC-specific knock-out did not induce embryonic lethality. These data indicate that signaling of S1P1 in the endothelial cells regulates the mural cell coverage of nascent vessels in trans and that signaling in mural cells is dispensable for developmental vascular stabilization (Paik, 2004 and references therein).

This study defines two essential steps in the formation of S1P1-induced N-cadherin-based EC-mural cell junctions. The first step involves N-cadherin trafficking to the EC surface. S1P induced the activation of the small GTPase Rac, which is essential for proper trafficking of N-cadherin via the microtubule (MT) cytoskeleton network. The MT polymerization in response to S1P was followed by monitoring the dynamics of EB1-GFP, a MT tip-binding protein. The mechanism by which Rac1 stimulates MT polymerization is thought to involve the activation of the protein kinase p65PAK and phosphorylation of stathmin, a MT destabilizing protein (Paik, 2004).

Next, it was found that S1P increases N-cadherin-bead binding to cells even in the presence of nocodazole, suggesting that S1P1 may regulate the adhesive activity of N-cadherin. It has been proposed that anchoring of the cadherin C terminus to the actin cytoskeleton may stabilize cadherin-mediated adhesion. The GPCR S1P1 induces alpha-, ß-, and p120-catenin association with N-cadherin, which may further support cadherin-dependent adhesion. Moreover, clustering of cadherin through the juxtamembrane domain (JMD) may be regulated by S1P1 signaling. Requirement of JMD in the regulation of cadherin adhesive activity has been postulated. p120-catenin, originally identified as a Src substrate that binds to the JMD of cadherin, may influence the dimerization or clustering of cadherin. Interestingly, the angiogenic factor VEGF induces the decrease in serine/threonine phosphorylation but the increase in tyrosine phosphorylation of p120-catenin in EC. S1P regulates the phosphorylation of p120-catenin in a similar manner. Further, it has been shown that N-cadherin tyrosine phosphorylation is induced by S1P. Indeed, p120-catenin has been shown to be strongly tyrosine phosphorylated at early times of calcium-induced keratinocyte differentiation, which leads to increased interaction between p120-catenin and cadherin. The finding that the active signaling via the S1P1 GPCR is needed for N-cadherin activation/trafficking suggest a novel mechanism in the regulation of cell-cell adhesion and vascular stabilization (Paik, 2004).

Interaction of N-cadherin with beta catenin

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).

The relationship between adhesive interactions across the synaptic cleft and synaptic function has remained elusive. Ultrastructural studies have failed to detect changes in synaptic morphology following stimulation. There is the intriguing notion that synaptic activity alters junctional morphology, and that synaptic adhesion may be influenced by activity even after synaptic membranes have been stabilized and full synaptic function is acquired. At certain CNS synapses, pre- to post-synaptic adhesion is mediated at least in part by neural- (N-) cadherin. To begin an exploration of the relationship between synaptic junctional adhesion and synaptic function in the CNS, the stability, conformational states, and distribution of N-cadherin was measured following vigorous depolarization of hippocampal neurons in culture, induced by transient exposure to high K+, which maximizes the synaptic response, and other stimulation paradigms. Upon depolarization of hippocampal neurons in culture by K+ treatment, or application of NMDA or alpha-latrotoxin, synaptic N-cadherin dimerizes and becomes markedly protease resistant. These properties are indices of strong, stable, enhanced cadherin-mediated intercellular adhesion. N-cadherin retains protease resistance for at least 2 hr after recovery, while other surface molecules, including other cadherins, are completely degraded. The acquisition of protease resistance and dimerization of N-cadherin is not dependent on new protein synthesis, nor is it accompanied by internalization of N-cadherin. By immunocytochemistry, it has been found that high K+ selectively induces surface dispersion of N-cadherin, which, after recovery, returns to synaptic puncta. N-cadherin dispersion under K+ treatment parallels the rapid expansion of the presynaptic membrane consequent to the massive vesicle fusion that occurs with this type of depolarization. In contrast, with NMDA application, N-cadherin does not disperse but does acquire enhanced protease resistance and dimerizes. These data strongly suggest that synaptic adhesion is dynamically and locally controlled, and modulated by synaptic activity (Tanaka, 2000).

There is substantial evidence to support the view that lateral clustering and dimerization are critically important features in regulating cadherin-mediated intercellular adhesion. It is now generally accepted that a shift from monomer to dimer and cadherin clustering activates classic cadherins at the surface into an adhesively competent conformation. It therefore seemed reasonable to see if such a shift could be detected in the N-cadherin population in stimulated cultures. Experiments in epithelial cells have demonstrated that adhesively competent E-cadherin is found in a Triton X-100-insoluble fraction, rich in cytoskeletal elements, which anchor the cytoplasmically disposed domains of cell surface adhesion molecules. Using this approach, Triton X-100-soluble and -insoluble fractions were prepared from stimulated and unstimulated neuronal cultures. Although the majority of proteins are solubilized under these conditions, some Triton-insoluble material remains. This material was collected, pelleted, and treated with 8 M urea, a strong chaotropic agent that serves to disassociate cytoskeletal complexes. Upon electrophoresis, a prominent band (Mr ~230 kDa) is revealed with N-cadherin (EC1) antibodies that is faint in control cultures, but upon stimulation and recovery becomes three and four times more intense, respectively. This band, which has the Mr of an N-cadherin homodimer, can be competed out with purified recombinant N-cadherin. On excision of this 230 kDa band from the gel, and treatment with either heat or alkali, the 127 kDa N-cadherin monomer is generated. Additionally, in the urea-treated fraction, the 230 kDa N-cadherin form is readily detected with two different antibodies, one that recognizes the N-terminal domain, and the other that recognizes the C-terminal domain of N-cadherin. This shows that both the extracellular and intracellular segments of wild-type N-cadherin are intact. These data are consistent with the specific induction of homodimerization of N-cadherin by synaptic stimulation, a conformational change that is sustained during recovery, although the possibility of heterooligomerization with other molecules has not been completely ruled out. It is very likely that cadherin homodimerization represents a highly stable, protease-resistant form of N-cadherin that is very active in cell adhesion and anchored to the cytoskeleton. The finding that dimeric N-cadherin increases in mass amount upon synaptic stimulation strongly suggests a shift in the local synaptic cadherin population from monomer to dimer during the course of the stimulation experiment (Tanaka, 2000).

It is proposed that synaptic stimulation leads to an increase in adhesion at N-cadherin-mediated synaptic junctions. The net effect of increased adhesion between pre- and postsynaptic membranes; (i.e., increased molecular packing of strand-dimerized, adhesively engaged cadherins at synaptic puncta) may be to (1) subtly change the area of lateral association between pre- and post-synaptic membranes; (2) perhaps affect the diffusion rates of materials released in the cleft, or (3) modify, also subtly, the average distance between the pre- and postsynaptic membranes over a microdomain. This last concept is particularly intriguing because there is actually a range of measured diameters for CNS synaptic clefts from between 150 and 250 Å, the significance of which is unknown. Lastly, the observed change in cadherin conformation may, through cadherin-associated proteins, yield a reorganization of excitatory neurotransmitter receptors and/or signaling molecules such as those implicated in synaptic 'tagging'. In this regard, it is interesting that a relationship between N-cadherin and glutamatergic synapses has been noted in hippocampal neurons, where N-cadherin first appears in development at all synapses but rapidly becomes restricted solely to a subpopulation of excitatory synapses (Benson, 1998). It remains to be determined precisely how adhesive changes at the synapse are translated into effects on synaptic plasticity. Arguments could be presented that support either synaptic potentiation or depression as a consequence of strengthened synaptic adhesion (Tanaka, 2000).

Extracellular interactions between GluR2 and N-cadherin in spine regulation

Via its extracellular N-terminal domain (NTD), the AMPA receptor subunit GluR2 promotes the formation and growth of dendritic spines in cultured hippocampal neurons. The first N-terminal 92 amino acids of the extracellular domain are necessary and sufficient for GluR2's spine-promoting activity. Moreover, overexpression of this extracellular domain increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Biochemically, the NTD of GluR2 can interact directly with the cell adhesion molecule N-cadherin, in cis or in trans. N-cadherin-coated beads recruit GluR2 on the surface of hippocampal neurons, and N-cadherin immobilization decreases GluR2 lateral diffusion on the neuronal surface. RNAi knockdown of N-cadherin prevents the enhancing effect of GluR2 on spine morphogenesis and mEPSC frequency. These data indicate that in hippocampal neurons N-cadherin and GluR2 form a synaptic complex that stimulates presynaptic development and function as well as promoting dendritic spine formation (Saglietti, 2007).

Activity-regulated N-cadherin endocytosis

Enduring forms of synaptic plasticity are thought to require ongoing regulation of adhesion molecules, such as N-cadherin, at synaptic junctions. Little is known about the activity-regulated trafficking of adhesion molecules. This study demonstrates that surface N-cadherin undergoes a surprisingly high basal rate of internalization. Upon activation of NMDA receptors (NMDAR), the rate of N-cadherin endocytosis is significantly reduced, resulting in an accumulation of N-cadherin in the plasma membrane. β-catenin, an N-cadherin binding partner, is a primary regulator of N-cadherin endocytosis. Following NMDAR stimulation, β-catenin accumulates in spines and exhibits increased binding to N-cadherin. Overexpression of a mutant form of ß-catenin, Y654F, prevents the NMDAR-dependent regulation of N-cadherin internalization, resulting in stabilization of surface N-cadherin molecules. Furthermore, the stabilization of surface N-cadherin blocks NMDAR-dependent synaptic plasticity. These results indicate that NMDAR activity regulates N-cadherin endocytosis, providing a mechanistic link between structural plasticity and persistent changes in synaptic efficacy (Tai, 2007).

N-cadherin and neural development Evolutionary homologs continued: part 2/3 | part 3/3

Cadherin-N: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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