Role of p120-catenin in cadherin trafficking

p120-catenin (p120) has emerged over the past several years as an important regulatory component of the cadherin adhesive complex. A core function of p120 in mammalian cells is to stabilize cadherins at the cell membrane by modulating cadherin membrane trafficking and degradation. In this way, p120 levels act as a set point mechanism that tunes cell-cell adhesive interactions. The primary control point for this regulatory activity appears to be at the level of cadherin internalization from the plasma membrane, although p120 may also impact other aspects of cadherin trafficking and turnover. In the following review, the general mechanisms of cadherin trafficking are discussed, and models for how p120 may influence cadherin membrane dynamics are presented. In one model, p120 may function as a "cap" to bind the cadherin cytoplasmic tail and prevent cadherin interactions with endocytic membrane trafficking machinery. Alternatively, p120 may stabilize cell junctions or regulate membrane trafficking machinery through interactions with small GTPases such as Rho A, Rac and Cdc42. Through these mechanisms p120 exerts influence over a wide range of biological processes that are dependent upon tight regulation of cell surface cadherin levels.     

Ankyrin-G Inhibits Endocytosis of Cadherin Dimers

Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Therefore, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. However, the mechanisms that control junctional plasticity are not fully understood. Vascular endothelial cadherin (VE-cadherin) is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with β- and α-catenin, which serve to increase adhesive strength. Furthermore, p120-catenin binds to the cytoplasmic tail of cadherin and stabilizes it at the plasma membrane. Here we report that induced cis dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. However, we find that ankyrin-G, a protein that links membrane proteins to the spectrin-actin cytoskeleton, associates with VE-cadherin and inhibits its endocytosis. Ankyrin-G inhibits VE-cadherin endocytosis independent of p120 binding. We propose a model in which ankyrin-G associates with and inhibits the endocytosis of VE-cadherin cis dimers. Our findings support a novel mechanism for regulation of VE-cadherin endocytosis through ankyrin association with cadherin engaged in lateral interactions.     

VE-cadherin: adhesion at arm's length

VE-cadherin was first identified in the early 1990s and quickly emerged as an important endothelial cell adhesion molecule. The past decade of research has revealed key roles for VE-cadherin in vascular permeability and in the morphogenic events associated with vascular remodeling. The details of how VE-cadherin functions in adhesion became apparent with structure-function analysis of the cadherin extracellular domain and with the identification of the catenins, a series of cytoplasmic proteins that bind to the cadherin tail and mediate interactions between cadherins and the cytoskeleton. Whereas early work focused on the armadillo family proteins -catenin and plakoglobin, more recent investigations have identified p120-catenin (p120^ctn) and a related group of armadillo family members as key binding partners for the cadherin tail. Furthermore, a series of new studies indicate a key role for p120^ctn in regulating cadherin membrane trafficking in mammalian cells. These recent studies place p120^ctn at the hub of a cadherin-catenin regulatory mechanism that controls cadherin plasma membrane levels in cells of both epithelial and endothelial origin. endothelial cell; cytoskeleton; -catenin; p120^ctn; cell adhesion; vascular endothelial cadherin     

Guilt by association: What p120-catenin has to hide

Members of the p120-catenin family associate with cadherins and regulate their stability at the plasma membrane. How p120-catenin limits cadherin endocytosis has long remained a mystery. In this issue, Nanes et al. (2012. J. Cell Biol. doi:10.1083/jcb.201205029) identify a conserved acidic motif within cadherins that acts as a physical platform for p120-catenin binding. However, in the absence of p120-catenin, the motif acts as an endocytic signal. These results provide new insight into p120-catenin’s role as guardian of intercellular junction dynamics.Adhesion receptors of the classical cadherin family have a major role in establishing tissue organization and maintaining tissue homeostasis (Gumbiner, 1996). Classical cadherins are transmembrane glycoproteins that use their extracellular domains to establish calcium-dependent trans homophilic interactions with cadherins in neighboring cells. To enhance adhesive strength, cadherin ectodomains oligomerize through lateral (cis) interactions, whereas their cytoplasmic domains anchor to the actomyosin cytoskeleton. The cytoplasmic domain of cadherins is highly conserved and binds to proteins called catenins. p120-catenin (p120) associates with the transmembrane adjacent domain (juxtamembrane; JMD) of the cadherin cytoplasmic tail, whereas β-catenin interacts with the more distal portion of cadherin’s cytoplasmic domain. β-Catenin in turn, binds α-catenin, which, through multiple interactions, both indirect and direct, can associate with the actin cytoskeleton (Perez-Moreno and Fuchs, 2006).Cellular rearrangements are orchestrated by dynamic assembly/disassembly of cadherin complexes. The process is fueled by endocytosis of cadherin complexes (Le et al., 1999; de Beco et al., 2009). Endocytosis can be stimulated by proteins that associate with cadherin–catenin complexes, including proteases that shed the cadherin ectodomains, and the ubiquitin ligase Hakai (Fujita et al., 2002). Cadherin internalization can be regulated by different pathways depending on the cellular context, involving clathrin-dependent and clathrin-independent mechanisms. These endocytic processes must be carefully regulated, as an untimely destabilization of cadherin-mediated adhesion can lead to alterations in tissue architecture and growth, features of several diseases, including cancers (Mosesson et al., 2008).In the past decade, p120 catenins (p120, ARVCF, δ-catenin, and p0071) have emerged as critical regulators of cadherin-mediated adhesion (Reynolds, 2007). p120, the founding family member, is a component of cadherin complexes (Reynolds et al., 1994), and its association with the cadherin JMD is important for retaining cadherins at the membrane (Ireton et al., 2002). Moreover, p120 loss causes rapid internalization of cadherins, followed by proteasomal and/or lysosomal-mediated degradation (Davis et al., 2003; Xiao et al., 2003a,b, 2005; Miyashita and Ozawa, 2007).Although these studies expose p120 as a master regulator of cadherin levels at the membrane, exactly how p120 governs cadherin endocytosis rates has remained unclear. Based upon experiments in which endocytic machinery components (clathrin, dynamin, and AP2) have been impaired (Chiasson et al., 2009) or cadherin endocytic motifs have been mutated (Hong et al., 2010; Troyanovsky et al., 2007), researchers have posited that p120 binding to cadherins may in some way prevent junctional complex endocytosis. In this issue, Nanes et al. add new molecular insights into the mechanism. The authors show that the VE-cadherin JMD functions as a bimodal platform for either p120 binding or endocytic signaling. Moreover, they identify a key conserved amino acid residue within the JMD, which, when mutated, blocks endocytosis without the need for p120.Recently, the cocrystallization of p120 bound to E-cadherin’s JMD has yielded insights into the essential residues of this binding interface (Ishiyama et al., 2010). Previous studies had attributed the core function of p120-cadherin to its ability to bind and mask a dileucine endocytic motif present in the JMD (Miyashita and Ozawa, 2007; Hong et al., 2010). The crystal structure showed that interactions between p120 and the JMD domain might be sufficient to sterically prevent accessibility of the dileucine cadherin endocytic motif to endocytic adaptors such as the AP2-clathrin adaptor, thereby placing this motif at the crux of the bimodal switch controlling the mutually exclusive binding of either p120 or the endocytic machinery.The affinity of p120 and AP2 for the JMD dileucine motif is similar, pointing toward the existence of a balanced regulation of cadherin endocytic rates and cadherin retention at the membrane. However, evaluating this balance in cellular contexts has not been possible because of the inability to uncouple p120 binding to the JMD and endocytosis. Nanes et al. (2012) have now overcome this hurdle. They first used a simulated model of the p120–E-cadherin crystal structure, which highlighted a conserved p120-binding region that is present in the JMD of both VE- and E-cadherin. However, the VE-cadherin JMD lacked endocytic dileucine and tyrosine residues present in E-cadherin, which are involved in clathrin internalization and Hakai-dependent ubiquitination, respectively.Because both types of adherens junctions undergo dynamic endocytic-based remodeling, the authors astutely realized that they might be able to exploit VE- and E-cadherin differences to unearth novel endocytic signals within the sequence that might be conserved among cadherins. To this end, the author first used mutant VE-cadherin chimeric proteins, consisting of the cytoplasmic domain of VE-cadherin fused to the extracellular domain of the IL-2 receptor, and internalization assays. They discovered that the core p120-binding region on its own was endocytosed, in a fashion similar to the full VE-cadherin cytoplasmic tail. This occurred in a clathrin-dependent manner, as previously observed in Kowalzcyk’s laboratory (Chiasson et al., 2009). Point mutagenesis identified some mutants no longer able to bind p120, which is consistent with previous findings (Thoreson et al., 2000). But the authors made an interesting finding: mutations in a conserved acidic motif (DEE) within the p120-core binding region of the JMD displayed loss of p120 binding and also blocked cadherin internalization (Fig. 1). Moreover, DEE mutant VE-cadherins localized stably at the membrane even in the absence of p120, although with an increased diffusion within the membrane. This increase in mobility suggests a reduction in cadherin lateral clustering, a process modulated by the binding of p120 to the JMD (Yap et al., 1998). Interestingly, in crystal structures, the E-cadherin JMD binding to p120 induced oligomerization of the complex (Ishiyama et al., 2010).Open in a separate windowFigure 1.Model of VE-cadherin stabilization at the cell membrane. (A) VE-cadherin binds to p120 and β-catenin. p120 associates with the juxtamembrane (JMD) domain of the cadherin cytoplasmic tail, whereas β-catenin binds to the more distal portion (catenin binding domain, CBD). Cadherin internalization is triggered by p120 dissociation, exposing a conserved endocytic factor recognition motif (DEE; 646–648) within the JMD. (B) When this motif is mutated in VE-cadherin, adherens junctions are resistant to endocytosis independent of p120 binding.These new tools now allow uncoupling of p120 binding from cadherin endocytosis, which will be instrumental in unraveling new p120 cadherin roles in cell adhesion. The VE-cadherin mutant that fails to bind to p-120 still coimmunoprecipitates with β-catenin. These findings are intriguing, given that overexpression of p120 can rescue the otherwise poor adhesive properties of cadherins mutant for β-catenin binding (Ohkubo and Ozawa, 1999). In addition, interactions between p120 and α-catenin at adherens junctions seem to contribute in preventing cadherin endocytosis (Troyanovsky et al., 2011). Given these collective results, it will be interesting in the future to measure the binding affinities of endocytosis-uncoupled VE-cadherin mutants for its binding partners.Overall, these data provide strong evidence that the JMD landing pad provides the nuts and bolts of the decision of whether an adherens junction remains at the cell surface or whether it is internalized. But who makes the decision? Recent results from Gumbiner’s group provide a possible clue. They show that cadherin activation stimulates the dephosphorylation of specific Ser/Thr residues within the N-terminal domain of p120, and this in turn stabilizes intercellular adhesion (Petrova et al., 2012).The new tools developed by Kowalczyk’s group (Nanes et al., 2012) will pave the way for researchers to dig further into the mechanism. In the current study, the authors use their newfound tools to analyze the consequences to cell migration when p120-JMD binding is uncoupled from endocytosis. In scratched monolayers of endothelial cells, cell migration was decreased. Importantly, when they examined the VE-cadherin mutant in which p120 binding was blocked but cadherin internalization could proceed normally, cell migration was largely normal. These findings indicate that the migration defects seen in the cells expressing the E-cadherin mutant are rooted in inhibition of endocytosis, rather than lack of p120 recruitment to junctions. They further suggest that endocytic trafficking of cadherins is necessary to transiently destabilize cell–cell contacts that otherwise impede migration. This notion is particularly intriguing given that when E-cadherins are stabilized at intercellular junctions, they can sequester proteins that are required for integrin-based migration (Livshits et al., 2012). Kowalczyk’s findings (Nanes et al., 2012) now suggest a means by which dynamic changes in intercellular adhesion can be achieved to trigger such downstream events.Although less well characterized, there are other regulatory circuits that might also be affected by transiently liberating p120 from intercellular junctions. Thus, for example, p120 enhances cadherin stability through its ability to interact with afadin and Rap1, thereby bridging connections with nectin intercellular junctions (Hoshino et al., 2005). Other direct and indirect p120 associates that might affect cadherin internalization include the endocytic adaptor Numb (Sato et al., 2011) and the signaling enzyme γ-secretase (Kiss et al., 2008). Additionally, p120 can also regulate Rac1 activity, which influences cadherin endocytosis in a clathrin-independent way (Akhtar and Hotchin, 2001). Thus, removing p120 or devising additional mutations to uncouple these interactions may be needed to fully unravel all the mysteries underlying p120’s power in governing intercellular adhesion in tissue development and maintenance (Davis and Reynolds, 2006; Elia et al., 2006; Perez-Moreno et al., 2006; Smalley-Freed et al., 2010; Marciano et al., 2011; Stairs et al., 2011; Chacon-Heszele et al., 2012; Kurley et al., 2012). That said, by dissecting p120’s web at the crossroads between intercellular junction stabilization and endocytosis, Kowalczyk and coworkers (Nanes et al., 2012) now illustrate the power of their approach and provide new insights into how similar strategies might ultimately enable this molecular crossword puzzle to be solved.     

Rab35 GTPase and cancer: Linking membrane trafficking to tumorigenesis

Rab35 is a small GTPase that is involved in many cellular processes, including membrane trafficking, cell polarity, lipid homeostasis, immunity, phagocytosis and cytokinesis. Recent studies showed that activating mutations confer Rab35 with oncogenic properties. Conversely, downregulation of Rab35 inverts apico‐basal cell polarity and promotes cell migration. Here we review Rab35’s known functions in membrane trafficking and signaling, cell division and cell migration in cancer cells and discuss the importance of Rab35‐dependent membrane trafficking in cancer progression.      

Protecting your tail: regulation of cadherin degradation by p120-catenin

Work in various model systems has yielded conflicting views of how p120-catenin participates in adherens junction assembly and regulation. A series of recent studies indicate that a core function of p120-catenin in mammalian cells is to regulate cadherin turnover by modulating the entry of cadherins into degradative endocytic pathways. By this mechanism, cellular levels of p120-catenin perform a 'rheostat' or 'set point' function that controls steady-state cadherin levels. These studies parallel a growing interest in the regulation of cadherin levels at the cell surface by membrane trafficking pathways. Collectively, the findings suggest exciting new roles for p120-catenin at the interface between cadherins and membrane trafficking machinery, and imply novel mechanisms by which p120-catenin may regulate cell adhesion and migration in the context of development and cancer.     

The p120 family of cell adhesion molecules

p120 is the prototypic member of the p120 subfamily of armadillo-related proteins that includes p0071, delta-catenin/NPRAP, ARVCF and the more distantly related plakophilins 1-3. Like armadillo, beta-catenin and plakoglobin these proteins are involved in mediating cell-cell adhesion. Besides their junctional localization they also reveal a cytoplasmic and nuclear localization. Non-cadherin-associated, cytoplasmic p120 functions in Rho signaling and regulation of cytoskeletal organization and actin dynamics. The nuclear function remains largely unsolved. Some characteristics seem to be shared by the various members of the family but it seems unlikely that p120-related proteins have solely redundant functions and compete for interactions with identical binding partners. Stabilization of cadherins at the membrane seems a common function of p120, p0071, delta-catenin and ARVCF but it is not yet known if and how these proteins confer distinct properties to cellular junctions. Moreover, p0071, NPRAP and ARVCF have a C-terminal PDZ-binding motif that is lacking in p120 pointing to distinct roles of these proteins. PDZ domains are found in a series of proteins involved in establishing cell polarity in epithelial cells. Thus, p120 proteins may not only be master regulators of cadherin abundance and activity but play additional roles in regulating cell polarity. This review focuses on the putative roles of p120 proteins in cell polarity.     

Endocytic activity of HIV‐1 Vpu: Phosphoserine‐dependent interactions with clathrin adaptors

HIV‐1 Vpu modulates cellular transmembrane proteins to optimize viral replication and provide immune‐evasion, triggering ubiquitin‐mediated degradation of some targets but also modulating endosomal trafficking to deplete them from the plasma membrane. Interactions between Vpu and the heterotetrameric clathrin adaptor protein (AP) complexes AP‐1 and AP‐2 have been described, yet the molecular basis and functional roles of such interactions are incompletely defined. To investigate the trafficking signals encoded by Vpu, we fused the cytoplasmic domain (CD) of Vpu to the extracellular and transmembrane domains of the CD8 α‐chain. CD8‐VpuCD was rapidly endocytosed in a clathrin‐ and AP‐2‐dependent manner. Multiple determinants within the Vpu CD contributed to endocytic activity, including phosphoserines of the β‐TrCP binding site and a leucine‐based ExxxLV motif. Using recombinant proteins, we confirmed ExxxLV‐dependent binding of the Vpu CD to the α/σ2 subunit hemicomplex of AP‐2 and showed that this is enhanced by serine‐phosphorylation. Remarkably, the Vpu CD also bound directly to the medium (μ) subunits of AP‐2 and AP‐1; this interaction was dependent on serine‐phosphorylation of Vpu and on basic residues in the μ subunits. We propose that the flexibility with which Vpu binds AP complexes broadens the range of cellular targets that it can misdirect to the virus' advantage.      

The Juxtamembrane Region of the Cadherin Cytoplasmic Tail Supports Lateral Clustering, Adhesive Strengthening, and Interaction with p120ctn

Cadherin cell–cell adhesion molecules form membrane-spanning molecular complexes that couple homophilic binding by the cadherin ectodomain to the actin cytoskeleton. A fundamental issue in cadherin biology is how this complex converts the weak intrinsic binding activity of the ectodomain into strong adhesion. Recently we demonstrated that cellular cadherins cluster in a ligand-dependent fashion when cells attached to substrata coated with the adhesive ectodomain of Xenopus C-cadherin (CEC1-5). Moreover, forced clustering of the ectodomain alone significantly strengthened adhesiveness (Yap, A.S., W.M. Brieher, M. Pruschy, and B.M. Gumbiner. Curr. Biol. 7:308–315). In this study we sought to identify the determinants of the cadherin cytoplasmic tail responsible for clustering activity. A deletion mutant of C-cadherin (CT669) that retained the juxtamembrane 94–amino acid region of the cytoplasmic tail, but not the β-catenin–binding domain, clustered upon attachment to substrata coated with CEC1-5. Like wild-type C-cadherin, this clustering was ligand dependent. In contrast, mutant molecules lacking either the complete cytoplasmic tail or just the juxtamembrane region did not cluster. The juxtamembrane region was itself sufficient to induce clustering when fused to a heterologous membrane-anchored protein, albeit in a ligand-independent fashion. The CT669 cadherin mutant also displayed significant adhesive activity when tested in laminar flow detachment assays and aggregation assays. Purification of proteins binding to the juxtamembrane region revealed that the major associated protein is p120^ctn. These findings identify the juxtamembrane region of the cadherin cytoplasmic tail as a functionally active region supporting cadherin clustering and adhesive strength and raise the possibility that p120^ctn is involved in clustering and cell adhesion.     

Rab‐mediated trafficking role in neurite formation

Neuronal cells are characterized by the presence of two confined domains, which are different in their cellular properties, biochemical functions and molecular identity. The generation of asymmetric domains in neurons should logically require specialized membrane trafficking to both promote neurite outgrowth and differential distribution of components. Members of the Rab family of small GTPases are key regulators of membrane trafficking involved in transport, tethering and docking of vesicles through their effectors. RabGTPases activity is coupled to the activity of guanine nucleotide exchange factors or GEFs, and GTPase‐activating proteins known as GAPs. Since the overall spatiotemporal distribution of GEFs, GAPs and Rabs governs trafficking through the secretory and endocytic pathways, affecting exocytosis, endocytosis and endosome recycling, it is likely that RabGTPases could have a major role in neurite outgrowth, elongation and polarization. In this review we summarize the evidence linking the functions of several RabGTPases to axonal and dendritic development in primary neurons, as well as neurite formation in neuronal cell lines. We focused on the role of RabGTPases from the trans‐Golgi network, early/late and recycling endosomes, as well as the function of some Rab effectors in neuritogenesis. Finally, we also discuss the participation of the ADP‐ribosylation factor 6, a member of the ArfGTPase family, in neurite formation since it seems to have an important cross‐talk with RabGTPases.

     

Making and breaking contacts: the cellular biology of cadherin regulation

Cadherin-mediated cell-cell interactions are dynamic processes, and cadherin function is tightly regulated in response to cellular context and signaling. Ultimately, cadherin regulation is likely to reflect the interplay between a range of fundamental cellular processes, including surface organization of receptors, cytoskeletal organization and cell trafficking, that are coordinated by signaling events. In this review we focus on recent advances in understanding how interplay with membrane trafficking and other cell-cell junctions can control cadherin function. The endocytosis of cadherins, and their post-internalization fate, influences surface expression and metabolic stability of these adhesion receptors. Similarly, at the surface, components of tight junctions provide a mode of cross-talk that regulates assembly of adherens junctions.     

Regulation of Cadherin Trafficking

Cadherins are a large family of cell–cell adhesion molecules that tether cytoskeletal networks of actin and intermediate filaments to the plasma membrane. This function of cadherins promotes tissue organization and integrity, as demonstrated by numerous disease states that are characterized by the loss of cadherin-based adhesion. However, plasticity in cell adhesion is often required in cellular processes such as tissue patterning during development and epithelial migration during wound healing. Recent work has revealed a pivotal role for various membrane trafficking pathways in regulating cellular transitions between quiescent adhesive states and more dynamic phenotypes. The regulation of cadherins by membrane trafficking is emerging as a key player in this balancing act, and studies are beginning to reveal how this process goes awry in the context of disease. This review summarizes the current understanding of how cadherins are routed and how the interface between cadherins and membrane trafficking pathways regulates cell surface adhesive potential. Particular emphasis is placed on the regulation of cadherin trafficking by catenins and the interplay between growth factor signaling pathways and cadherin endocytosis.     

Desmosomal cadherins utilize distinct kinesins for assembly into desmosomes

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

μ2‐Dependent endocytosis of N‐cadherin is regulated by β‐catenin to facilitate neurite outgrowth

Circuit formation in the brain requires neurite outgrowth throughout development to establish synaptic contacts with target cells. Active endocytosis of several adhesion molecules facilitates the dynamic exchange of these molecules at the surface and promotes neurite outgrowth in developing neurons. The endocytosis of N‐cadherin, a calcium‐dependent adhesion molecule, has been implicated in the regulation of neurite outgrowth, but the mechanism remains unclear. Here, we identified that a fraction of N‐cadherin internalizes through clathrin‐mediated endocytosis (CME). Two tyrosine‐based motifs in the cytoplasmic domain of N‐cadherin recognized by the μ2 subunit of the AP‐2 adaptor complex are responsible for CME of N‐cadherin. Moreover, β‐catenin, a core component of the N‐cadherin adhesion complex, inhibits N‐cadherin endocytosis by masking the 2 tyrosine‐based motifs. Removal of β‐catenin facilitates μ2 binding to N‐cadherin, thereby increasing clathrin‐mediated N‐cadherin endocytosis and neurite outgrowth without affecting the steady‐state level of surface N‐cadherin. These results identify and characterize the mechanism controlling N‐cadherin endocytosis through β‐catenin‐regulated μ2 binding to modulate neurite outgrowth.      

p120-catenin and β-catenin differentially regulate cadherin adhesive function

Vascular endothelial (VE)-cadherin, the major adherens junction adhesion molecule in endothelial cells, interacts with p120-catenin and β-catenin through its cytoplasmic tail. However, the specific functional contributions of the catenins to the establishment of strong adhesion are not fully understood. Here we use bioengineering approaches to identify the roles of cadherin–catenin interactions in promoting strong cellular adhesion and the ability of the cells to spread on an adhesive surface. Our results demonstrate that the domain of VE-cadherin that binds to β-catenin is required for the establishment of strong steady-state adhesion strength. Surprisingly, p120 binding to the cadherin tail had no effect on the strength of adhesion when the available adhesive area was limited. Instead, the binding of VE-cadherin to p120 regulates adhesive contact area in a Rac1-dependent manner. These findings reveal that p120 and β-catenin have distinct but complementary roles in strengthening cadherin-mediated adhesion.     

The regulatory or phosphorylation domain of p120 catenin controls E-cadherin dynamics at the plasma membrane

In contrast to growth factor-stimulated tyrosine phosphorylation of p120, its relatively constitutive serine/threonine phosphorylation is not well understood. Here we examined the role of serine/threonine phosphorylation of p120 in cadherin function. Expression of cadherins in cadherin-null cells converted them to an epithelial phenotype, induced p120 phosphorylation and localized it to sites of cell contact. Detergent solubility and immunofluorescence confirmed that phosphorylated p120 was at the plasma membrane. E-cadherin constructs incapable of traveling to the plasma membrane did not induce serine/threonine phosphorylation of p120, nor did cadherins constructs incapable of binding p120. However, an E-cadherin cytoplasmic domain construct artificially targeted to the plasma membrane did induce serine/threonine phosphorylation of p120, suggesting phosphorylation occurs independently of signals from cadherin dimerization and trafficking through the ER/Golgi. Solubility assays following calcium switch showed that p120 isoform 3A was more effective at stabilizing E-cadherin at the plasma membrane relative to isoform 4A. Since the major phosphorylation domain of p120 is included in isoform 3A but not 4A, we tested p120 mutated in the known phosphorylation sites in this domain and found that it was even less effective at stabilizing E-cadherin. These data suggest that serine/threonine phosphorylation of p120 influences the dynamics of E-cadherin in junctions.     

Multiple Roles of VARP in Endosomal Trafficking: Rabs,Retromer Components and R‐SNARE VAMP7 Meet on VARP

VARP (VPS9‐ankyrin‐repeat protein, also known as ANKRD27) was originally identified as an N‐terminal VPS9 (vacuolar protein sorting 9)‐domain‐containing protein that possesses guanine nucleotide exchange factor (GEF) activity toward small GTPase Rab21 and contains two ankyrin repeat (ANKR) domains in its central region. A number of VARP‐interacting molecules have been identified during the past five years, and considerable attention is now being directed to the multiple roles of VARP in endosomal trafficking. More specifically, VARP is now known to interact with three different types of key membrane trafficking regulators, i.e. small GTPase Rabs (Rab32, Rab38 and Rab40C), the retromer complex (a sorting nexin dimer, VPS26, VPS29 and VPS35) and R‐SNARE VAMP7. By binding to several of these molecules, VARP regulates endosomal trafficking, which underlies a variety of cellular events, including melanogenic enzyme trafficking to melanosomes, dendrite outgrowth of melanocytes, neurite outgrowth and retromer‐mediated endosome‐to‐plasma membrane sorting of transmembrane proteins.      

An intracellular domain with a novel sequence regulates cell surface expression and synaptic clustering of leucine‐rich repeat transmembrane proteins in hippocampal neurons

Leucine‐rich repeat transmembrane proteins (LRRTMs) are single‐spanning transmembrane proteins that belong to the family of synaptically localized adhesion molecules that play various roles in the formation, maturation, and function of synapses. LRRTMs are highly localized in the post‐synaptic density; however, the mechanisms and significance of LRRTM synaptic clustering remain unclear. Here, we focus on the intracellular domain of LRRTMs and investigate its role in cell surface expression and synaptic clustering. The deletion of 55–56 residues in the cytoplasmic tail caused significantly reduced synaptic clustering of LRRTM1–4 in rat hippocampal neurons, whereas it simultaneously resulted in augmented LRRTM1–2 cell surface expression. A series of deletions and further single amino acid substitutions in the intracellular domain of LRRTM2 demonstrated that a previously uncharacterized sequence at the region of ‐16 to ‐13 from the C‐terminus was responsible for efficient synaptic clustering and proper cell surface trafficking of LRRTMs. Furthermore, the clustering‐deficient LRRTM2 mutant lost the ability to promote the accumulation of post‐synaptic density protein‐95 (PSD‐95). These results suggest that trafficking to the cell surface and synaptic clustering of LRRTMs are regulated by a specific mechanism through this novel sequence in the intracellular domain that underlies post‐synaptic molecular assembly and maturation.

     

An acidic extracellular pH disrupts adherens junctions in HepG2 cells by Src kinases‐dependent modification of E‐cadherin

We have previously shown that culturing HepG2 cells in pH 6.6 culture medium increases the c‐Src‐dependent tyrosine phosphorylation of β‐catenin and induces disassembly of adherens junctions (AJs). Here, we investigated the upstream mechanism leading to this pH 6.6‐induced modification of E‐cadherin. In control cells cultured at pH 7.4, E‐cadherin staining was linear and continuous at cell–cell contact sites. Culturing cells at pH 6.6 was not cytotoxic, and resulted in weak and discontinuous junctional E‐cadherin staining, consistent with the decreased levels of E‐cadherin in membrane fractions. pH 6.6 treatment activated c‐Src and Fyn kinase and induced tyrosine phosphorylation of p120 catenin (p120ctn) and E‐cadherin. Inhibition of Src family kinases by PP2 attenuated the pH 6.6‐induced tyrosine phosphorylation of E‐cadherin and p120ctn, and prevented the loss of these proteins from AJs. In addition, E‐cadherin was bound to Hakai and ubiquitinated. Furthermore, pH 6.6‐induced detachment of E‐cadherin from AJs was blocked by pretreatment with MG132 or NH₄Cl, indicating the involvement of ubiquitin‐proteasomal/lysosomal degradation of E‐cadherin. An early loss of p120ctn prior to E‐cadherin detachment from AJs was noted, concomitant with a decreased association between p120ctn and E‐cadherin at pH 6.6. PP2 pretreatment prevented the dissociation of these two proteins. In conclusion, pH 6.6 activated Src kinases, resulting in tyrosine phosphorylation of E‐cadherin and p120ctn and a weakening of the association of E‐cadherin with p120ctn and contributing to the instability of E‐cadherin at AJs. J. Cell. Biochem. 108: 851–859, 2009. © 2009 Wiley‐Liss, Inc.