N-cadherin Complex Research Articles

Distinct intramolecular interactions regulate autoinhibition of vinculin binding in αT-catenin and αE-catenin

α-Catenin binds directly to β-catenin and connects the cadherin–catenin complex to the actin cytoskeleton. Tension regulates α-catenin conformation. Actomyosin-generated force stretches the middle (M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. It is not known whether the intramolecular interactions that regulate epithelial (αE)-catenin binding are conserved across the α-catenin family. Here, we describe the biochemical properties of testes (αT)-catenin, an α-catenin isoform critical for cardiac function and how intramolecular interactions regulate vinculin-binding autoinhibition. Isothermal titration calorimetry showed that αT-catenin binds the β-catenin–N-cadherin complex with a similar low nanomolar affinity to that of αE-catenin. Limited proteolysis revealed that the αT-catenin M-region adopts a more open conformation than αE-catenin. The αT-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated αT-catenin M-region is not autoinhibited and thereby distinct from αE-catenin. However, the αT-catenin head (N- and M-regions) binds vinculin 1000-fold more weakly (low micromolar affinity), indicating that the N-terminus regulates the M-region binding to vinculin. In cells, αT-catenin recruitment of vinculin to cell–cell contacts requires the actin-binding domain and actomyosin-generated tension, indicating that force regulates vinculin binding. Together, our results show that the αT-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of αT-catenin allow it to function as a scaffold for building specific adhesion complexes.

Angiomotin like-1 is a novel component of the N-cadherin complex affecting endothelial/pericyte interaction in normal and tumor angiogenesis.

Transmission of mechanical force via cell junctions is an important component that molds cells into shapes consistent with proper organ function. Of particular interest are the cadherin transmembrane proteins, which play an essential role in connecting cell junctions to the intra-cellular cytoskeleton. Understanding how these biomechanical complexes orchestrate intrinsic and extrinsic forces is important for our understanding of the underlying mechanisms driving morphogenesis. We have previously identified the Amot protein family, which are scaffold proteins that integrate polarity, junctional, and cytoskeletal cues to modulate cellular shape in endothelial as well as epithelial cells. In this report, we show that AmotL1 is a novel partner of the N-cadherin protein complex. We studied the role of AmotL1 in normal retinal as well as tumor angiogenesis using inducible endothelial-specific knock-out mice. We show that AmotL1 is essential for normal establishment of vascular networks in the post-natal mouse retina as well as in a transgenic breast cancer model. The observed phenotypes were consistent with a non-autonomous pericyte defect. We show that AmotL1 forms a complex with N-cadherin present on both endothelial cells and pericytes. We propose that AmotL1 is an essential effector of the N-cadherin mediated endothelial/pericyte junctional complex.

N-Cadherin Dependent Collective Cell Invasion of Prostate Cancer Cells Is Regulated by the N-Terminus of α-Catenin

Cancer cell invasion is the critical first step of metastasis, yet, little is known about how cancer cells invade and initiate metastasis in a complex extracellular matrix. Using a cell line from bone metastasis of prostate cancer (PC3), we analyzed how prostate cancer cells migrate in a physiologically relevant 3D Matrigel. We found that PC3 cells migrated more efficiently as multi-cellular clusters than isolated single cells, suggesting that the presence of cell-cell adhesion improves 3D cell migration. Perturbation of N-cadherin function by transfection of either the N-cadherin cytoplasmic domain or shRNA specific to N-cadherin abolished collective cell migration. Interestingly, PC3 cells do not express α-catenin, an actin binding protein in the cadherin complex. When the full-length α-catenin was re-introduced, the phenotype of PC3 cells reverted back to a more epithelial phenotype with a decreased cell migration rate in 3D Matrigel. Interestingly, we found that the N-terminal half of α-catenin was sufficient to suppress invasive phenotype. Taken together, these data suggest that the formation of N-cadherin junctions promotes 3D cell migration of prostate cancer cells, and this is partly due to an aberrant regulation of the N-cadherin complex in the absence of α-catenin.

Lysophosphatidic acid modulates the association of PTP1B with N-cadherin/catenin complex in SKOV3 ovarian cancer cells

LPA (lysophosphatidic acid) is a natural phospholipid that plays important roles in promoting cancer cell proliferation, invasion and metastases. We previously reported that LPA induces ovarian cancer cell dispersal and disruption of AJ (adherens junction) through the activation of SFK (Src family kinases). In this study, we have investigated the regulatory mechanisms during the early phase of LPA-induced cell dispersal. An in vitro model of the ovarian cancer cell line SKOV3 for cell dispersal was used. LPA induces rapid AJ disruption by increasing the internalization of N-cadherin-β-catenin. By using immunoprecipitations, LPA was shown to induce increased tyrosine phosphorylation of β-catenin and alter the balance of β-catenin-bound SFK and PTP1B (phosphotyrosine phosphatase 1B). The altered balance of tyrosine kinase/phosphatase correlated with a concomitant disintegration of the β-catenin-α-catenin, but not the β-catenin-N-cadherin complex. This disintegration of β-catenin from α-catenin and the cell dispersal caused by LPA can be rescued by blocking SFK activity with the chemical inhibitor, PP2. More importantly, PP2 also restores the level of PTP1B bound to β-catenin. We propose that LPA signalling alters AJ stability by changing the dynamics of tyrosine kinase/phosphatase bound to AJ proteins. This work provides further understanding of the early signalling events regulating ovarian cancer cell dispersal and AJ disruption induced by LPA.

Linkage of N-cadherin to multiple cytoskeletal elements revealed by a proteomic approach in hippocampal neurons

The CNS synapse is an adhesive junction differentiated for chemical neurotransmission and is equipped with presynaptic vesicles and postsynaptic neurotransmitter receptors. Cell adhesion molecule cadherins not only maintain connections between pre- and postsynaptic membranes but also modulate the efficacy of synaptic transmission. Although the components of the cadherin-mediated adhesive apparatus have been studied extensively in various cell systems, the complete picture of these components, particularly at the synaptic junction, remains elusive. Here, we describe the proteomic assortment of the N-cadherin-mediated synaptic adhesion apparatus in cultured hippocampal neurons. N-cadherin immunoprecipitated from Triton X-100-solubilized neuronal extract contained equal amounts of β- and α-catenins, as well as F-actin-related membrane anchor proteins such as integrins bridged with α-actinin-4, and Na+/K+-ATPase bridged with spectrins. A close relative of β-catenin, plakoglobin, and its binding partner, desmoplakin, were also found, suggesting that a subset of the N-cadherin-mediated adhesive apparatus also anchors intermediate filaments. Moreover, dynein heavy chain and LEK1/CENPF/mitosin were found. This suggests that internalized pools of N-cadherin in trafficking vesicles are conveyed by dynein motors on microtubules. In addition, ARVCF and NPRAP/neurojungin/δ2-catenin, but not p120ctn/δ1-catenin or plakophilins-1, -2, -3, -4 (p0071), were found, suggesting other possible bridges to microtubules. Finally, synaptic stimulation by membrane depolarization resulted in an increased 93-kDa band, which corresponded to proteolytically truncated β-catenin. The integration of three different classes of cytoskeletal systems found in the synaptic N-cadherin complex may imply a dynamic switching of adhesive scaffolds in response to synaptic activity.

Merlin-Deficient Human Tumors Show Loss of Contact Inhibition and Activation of Wnt/β-Catenin Signaling Linked to the PDGFR/Src and Rac/PAK Pathways

Neurofibromatosis type 2 (NF2) is an inherited predisposition cancer syndrome characterized by the development of multiple benign tumors in the nervous system including schwannomas, meningiomas, and ependymomas. Using a disease model comprising primary human schwannoma cells, we previously demonstrated that adherens junctions (AJs) are impaired in schwannoma cells because of a ubiquitous, upregulated Rac activity. However, the mechanism by which loss of contact inhibition leads to proliferation remains obscure in merlin-deficient tumors. In this study, we show that proliferative Wnt/β-catenin signaling is elevated as active β-catenin (dephosphorylated at serine 37 and threoine 41) localizes to the nucleus and the Wnt targets genes c-myc and cyclin D1 are upregulated in confluent human schwannoma cells. We demonstrate that Rac effector p21-activated kinase 2 (PAK2) is essential for the activation of Wnt/β-catenin signaling because depletion of PAK2 suppressed active β-catenin, c-myc, and cyclin D1. Most importantly, the link between the loss of the AJ complex and the increased proliferation in human schwannoma cells is connected by Src and platelet-derived growth factor receptor–induced tyrosine 654 phosphorylation on β-catenin and associated with degradation of N-cadherin. We also demonstrate that active merlin maintains β-catenin and N-cadherin complex at the plasma membrane through direct regulation. Finally, we demonstrate that phosphorylation of tyrosine 654 is critical for the increased proliferation in human schwannoma cells because overexpression of a Y654F mutant β-catenin reduces hyperproliferation of schwannoma cells. We suggest a model that these pathways are coordinated and relevant for proliferation in merlin-deficient tumors.

Slit Has Complex Effects

Developing axons interact with cells and substrate encountered en route to their target through adhesion molecules such as N-cadherin. The repulsive guidance cue Slit acts through the Robo receptor to inhibit N-cadherin-mediated adhesion--and thereby neurite outgrowth--through the phosphorylation-dependent dissociation of β-catenin from N-cadherin. This occurs in conjunction with the formation of a multimeric complex that includes not only Robo and N-cadherin but also the Abl tyrosine kinase. Rhee et al. found that, after treatment with Slit, Abl and Robo immunoprecipitated from chick neural retina cells together with the adaptor protein Cables (for Cdk5 and Abl enzyme substrate). As the name implies, Cables is also a substrate for Cdk5, and Slit stimulated an increase in the amount of Cdk5 that immunoprecipitated with Abl and with cadherin, whereas it elicited a decrease in cadherin association with the Cdk5 regulatory subunit p35. p35 interacts with β-catenin, and disruption of the Cdk5-Cables interaction inhibited the Slit-induced loss of β-catenin and p35 from the N-cadherin complex, as well as inhibiting association of β-catenin with Cables. In vitro binding assays indicated that Cables bound to β-catenin and Abl, but not to N-cadherin. Further, Cables and p35 competed with each other for binding to β-catenin, and Cables knockdown led to an increase in the association of N-cadherin and β-catenin and a "flattened" phenotype in cells expressing a constitutively active form of Robo. Mutational analysis indicated that Abl phosphorylated β-catenin on tyrosine 489 (Y489), inhibiting the β-catenin-N-cadherin interaction. Y489-phosphorylated β-catenin translocated to the nucleus, where it interacted with the transcription factor Tcf and activated the transcription of a target gene reporter. Thus, Cables appears to play a key role in the formation of the Robo-N-cadherin complex, and release of β-catenin from N-cadherin allows Slit not only to coordinate axonal guidance and adhesion but also to mediate changes in gene transcription. J. Rhee, T. Buchan, L. Zukerberg, J. Lilien, J. Balsamo, Cables links Robo-bound Abl kinase to N-cadherin-bound β-catenin to mediate Slit-induced modulation of adhesion and transcription. Nat. Cell Biol. 9 , 883-892 (2007). [PubMed]

N-Cadherin Signaling in Synapse Formation and Neuronal Physiology

Neural cadherin (N-cadherin) is an adhesion receptor that is localized in abundance at neuronto- neuron synapses. N-cadherin contains an extracellular domain that binds to other cadherins on juxtaposed cell membranes, a single-pass transmembrane region, and a cytoplasmic tail that interacts with various proteins, including catenins, kinases, phosphatases, and presenilin 1. N-cadherin contributes to the structural and functional organization of the synaptic complex by ensuring the adhesion between synaptic membranes and organizing the underlying actin cytoskeleton. Additionally, recent findings have shown that N-cadherin may participate in synaptic physiology by regulating calcium influx through voltage-activated calcium currents. The diverse activities of N-cadherin stem from its ability to operate as both an adhesion molecule that links cytoskeletons across cell membranes and a ligand-activated homophilic receptor capable of initiating intracellular signaling. An important mechanism of cadherin signaling is the regulation of small Rho guanosine triphosphatase activity that affects cytoskeleton dynamics and calcium influx. Because both the regulation of cadherin adhesive activity and cadherin-mediated signaling are affected by the binding of molecules to the intracellular domain, changes in the composition of the N-cadherin complex are central to the regulation of cadherin-mediated functions. This article focuses on the roles that N-cadherin might play at the level of the synapse through its effect on adhesion and signaling in the proximity of the synaptic junction.

Assembly of the N-cadherin complex during synapse formation involves uncoupling of p120-catenin and association with presenilin 1

N-cadherin is an adhesion receptor that participates in both interaction between immature pre- and postsynaptic neurons and in the stabilization and function of matured neuron–neuron synapses. To better understand how the N-cadherin complex contributes to synapse formation, we examined its distribution and composition during synapse formation in the chick ciliary neurons. It was found that at early phases of synaptogenesis, N-cadherin is distributed in small clusters on the cell surface and primarily associates with p120-catenin and β-catenin. In contrast, as synaptic contacts matured, larger N-cadherin clusters were found localized adjacent to the active zone and associated with PS1 and γ-catenin, while p120- and β-catenin were dispersed among other cell regions, including axons. As it is known that PS1 binds γ-catenin and that uncoupled p120-catenin can alter the cytoskeleton via its effect on Rho GTPases, these changes in the molecular composition of the N-cadherin complex (represented by the uncoupling of p120-catenin and association with PS1) may correspond to distinct functional states of the complex involved in synaptic maturation.

Activation of the repulsive receptor Roundabout inhibits N-cadherin-mediated cell adhesion.

The formation of axon trajectories requires integration of local adhesive interactions with directional information from attractive and repulsive cues. Here, we show that these two types of information are functionally integrated; activation of the transmembrane receptor Roundabout (Robo) by its ligand, the secreted repulsive guidance cue Slit, inactivates N-cadherin-mediated adhesion. Loss of N-cadherin-mediated adhesion is accompanied by tyrosine phosphorylation of beta-catenin and its loss from the N-cadherin complex, concomitant with the formation of a supramolecular complex containing Robo, Abelson (Abl) kinase and N-cadherin. Local formation of such a receptor complex is an ideal mechanism to steer the growth cone while still allowing adhesion and growth in other directions.

Localization of the novel Xin protein to the adherens junction complex in cardiac and skeletal muscle during development.

Previously, we demonstrated that chick embryos treated with antisense oligonucleotides against a striated muscle-specific Xin exhibit abnormal cardiac morphogenesis (Wang et al. [1999] Development 126:1281-1294); therefore, we surmised a role for Xin in cardiac development. Herein, we examine the developmental expression of Xin through immunofluorescent staining of whole-mount mouse embryos and frozen heart sections. Xin expression is first observed within the heart tube of embryonic day 8.0 (E8.0) mice, exhibiting a peripheral localization within the cardiomyocytes. Colocalization of Xin with both beta-catenin and N-cadherin is observed throughout embryogenesis and into adulthood. Additionally, Xin is found associated with beta-catenin within the N-cadherin complex in embryonic chick hearts by coimmunoprecipitation. Xin is detected earlier than vinculin in the developing heart and colocalizes with vinculin at the intercalated disc but not at the sarcolemma within embryonic and postnatal hearts. At E10.0, Xin is also detected in the developing somites and later in the myotendon junction of skeletal muscle but not within the costameric regions of muscle. In cultured C2C12 myotubes, the Xin protein is found in many speckled and filamentous structures, coincident with tropomyosin in the stress fibers. Additionally, Xin is enriched in the regions of cell-cell contacts. These data demonstrate that Xin is one of the components at the adherens junction of cardiac muscle, and its counterpart in skeletal muscle, the myotendon junction. Furthermore, temporal and spatial expressions of Xin in relation to intercalated disc proteins and thin filament proteins suggest roles for Xin in the formation of cell-cell contacts and possibly in myofibrillogenesis.