Cadherin Tales: Regulation of Cadherin Function by Endocytic Membrane Trafficking Chantel M. Cadwell1, Wenji Su1,2, Andrew P. Kowalczyk1,3,4* Department of Cell Biology, Emory University School of Medicine, Atlanta GA, 30322, USA. 2Biochemistry, Cell, and Developmental Biology Graduate Training Program, Emory University, Atlanta GA, 30322, USA.

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1

3

Department of Dermatology, Emory University School of Medicine, Atlanta GA, 30322 USA

4

Winship Cancer Institute, Emory University, Atlanta GA, 30322

*Corresponding Author: Andrew Kowalczyk, [email protected]

Synopsis

Cellular rearrangements during development, wound healing, and metastasis require dynamic regulation of adhesion. Endocytic membrane trafficking has emerged as a fundamental mechanism by which cells confer plasticity to adhesive junctions. Recent studies indicate that the juxtamembrane domain of classical cadherins contains multiple endocytic motifs, or “switches”, that can be used by cellular membrane trafficking machinery to regulate adhesion. This review focuses on p120-catenin and other cadherin binding proteins, ubiquitin ligases, and growth factors as key modulators of cadherin membrane trafficking. Abstract Cadherins are the primary adhesion molecules in adherens junctions and desmosomes and play essential roles in embryonic development. Although significant progress has been made in understanding cadherin structure and function, we lack a clear vision of how cells confer plasticity upon adhesive junctions to allow for cellular rearrangements during development, wound healing, and metastasis. Endocytic membrane trafficking has emerged as a fundamental mechanism by which cells confer a dynamic state to adhesive junctions. Recent studies indicate that the juxtamembrane domain of classical cadherins contains multiple endocytic motifs, or “switches”, that can be used by cellular membrane trafficking machinery to regulate adhesion. The cadherin binding protein p120-catenin appears to be the master regulator of access to these switches, thereby controlling cadherin endocytosis and turnover. This review focuses on p120-catenin and other cadherin binding proteins, ubiquitin ligases, and growth factors as key modulators of cadherin membrane trafficking.

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Introduction Intercellular junctions mediate cell-cell adhesion and communication, and participate in the 1 establishment of cell polarity and tissue compartmentalization . Different types of cell-cell junctions have evolved to meet different tissue organizational requirements, to modulate cell proliferation and migration, and to withstand various types of osmotic and mechanical stresses. Cadherins are adhesion molecules that mediate strong cell-cell interactions that serve to integrate cells within epithelial sheets and tubules. The assembly of cadherins into large, electron dense macromolecular complexes that are coupled to the cytoskeleton is a design feature of adhesive junctions that contributes to tissue integrity. However, the dynamic cellular rearrangements that are observed during development and wound healing signify a plasticity to adhesive cell-cell contacts that is poorly understood. This review focuses on the role of membrane trafficking pathways, with an emphasis on cadherin endocytosis, as a means by which cells confer a dynamic nature to cell adhesion structures.

Molecular components of Adhesive Intercellular Junctions Adherens junctions and desmosomes are adhesive intercellular junctions that mechanically integrate adjacent cells within various types of tissues 2-5. As classically defined, the adherens junction is a circumferential belt of adhesive contact that is coupled to the actin cytoskeleton, while desmosomes are 6,7 spot-weld like contacts that are linked to intermediate filaments . Cadherins are the primary adhesion molecules at both adherens junctions and desmosomes. In the adherens junction, classical cadherins such as E-cadherin are linked to the actin cytoskeleton by catenins, a group of proteins that associate with the cadherin tail and form a scaffold for actin associations (Figure 1). β-catenin binds to the carboxyl terminal tail of classical cadherins and couples cadherins to α-catenin and other actin binding proteins. In contrast, p120catenin binds the juxtamembrane domain (JMD) of classical cadherins and plays a key role in modulating cadherin turnover (see below). A number of other proteins not depicted here also localize to adherens junctions and play additional roles in cadherin adhesion and signaling 8. The desmosome has an analogous 9 organization, where desmogleins and desmocollins mediate heterophillic adhesion and are coupled to the intermediate filament cytoskeleton through adaptor proteins such as plakoglobin and desmoplakin. Desmosomes play especially important roles in tissue integrity, as evidenced by the numerous epidermal and heart defects associated with desmosome dysfunction 10,11. As discussed below, both adherens junctions and desmosomes are modulated by endocytic trafficking pathways that regulate cadherin cell surface levels. Cadherin trafficking pathways A key mechanism for modulating intercellular adhesion is through the regulation of cadherin availability at the cell surface. In general, steady state levels of cadherin at the plasma membrane are determined by the rates of endocytosis and degradation, which decrease surface levels, and through synthesis of new protein and recycling, which serve to increase cadherin availability at the plasma membrane. Cadherins are retrieved from the cell surface through distinct mechanisms that can be categorized as clathrin dependent or clathrin independent endocytosis (Figure 2). Of these mechanisms, clathrin-mediated endocytosis is the best characterized. Studies have shown that E-, N-, and VE-cadherin, are targeted for clathrin-mediated endocytosis through the binding of clathrin adaptor proteins 12,13. Adaptor proteins recruit components of the endocytic machinery and cluster the targeted receptors into clathrin14,15 . Vesicles then bud off from the plasma membrane through a scission event mediated by the coated pits 14 GTPase dynamin . After endocytosis, internalized proteins can be recycled back to the plasma membrane, often in a polarized manner 15-19, or routed to the lysosome for degradation. In addition to clathrin-mediated internalization, cadherins undergo endocytosis independent of clathrin, though these processes are not well characterized. Clathrin independent endocytosis can occur

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through caveolin-mediated internalization, macropinocytosis, and in the case of the desmosomal cadherins, 12,14 (Figure 2). Although E-cadherin is typically internalized by through lipid raft mediated mechanisms clathrin-mediated mechanisms, a study by Lu and colleagues found that E-cadherin also undergoes 20 caveolin-mediated endocytosis in response to EGF in a human tumor cell line overexpressing EGFR . Furthermore, Bryant et al. reported that E-cadherin is co-internalized with p120 and β-catenin through 17 macropinocytosis in response to EGF in a breast carcinoma cell line . Similarly, cadherin-6B, a cadherin expressed in chick premigratory cranial neural crest cells, has been reported to undergo both clathrin21 dependent endocytosis and macropinocytosis . Further evidence for macropinocytosis of cadherin comes from Paterson and colleagues, who describe E-cadherin endocytosis that is both clathrin- and caveolinindependent. Interestingly, this endocytic pathway affects E-cadherin that is not engaged in trans interactions at the cell junction 22. Lastly, desmosomal cadherins are internalized through a lipid raft23,24 , and recent evidence suggests association of these cadherins with flottilins 25. mediated pathway Collectively, these observations indicate that various cadherins can be internalized through different endocytic pathways under different circumstances. It will be important to determine how the choice of endocytic machinery impacts cadherin function and the biological contexts in which different trafficking decisions modulate cell fate and behavior. p120-catenin: master guardian of cadherin endocytosis p120-catenin (p120) has emerged as an essential regulator of cadherin trafficking. p120 was originally identified as a major Src substrate, and subsequently as an armadillo family protein that binds to the juxtamembrane domain of classical cadherin cytoplasmic tails 26-28. Unlike β-catenin, p120 does not appear to play a direct role in linking cadherins to the actin cytoskeleton. A breakthrough in understanding how p120 regulates cadherins came from p120 null cells 29 and siRNA approaches, which revealed that 30,31 . In the absence of p120, classical p120 regulates cadherin turnover by regulating cadherin endocytosis cadherins are degraded through an endo-lysosomal degradation pathway. A series of tissue specific knock out studies confirmed that p120 regulates cadherin levels in vivo 32-35. Thus, p120 functions as a master regulator of cadherin cell surface levels in a variety of mammalian tissues. Structural studies of the E-cadherin-p120-catenin complex revealed that the cadherin JMD contains two types of p120 binding interfaces. The core p120-binding region is a highly conserved series of residues 36,37 . NMR studies revealed that p120 also interacts with membrane that represents a static binding interface proximal residues of E-cadherin in a more dynamic interface that varies considerably between classical cadherins 36 (Figure 3). In the case of VE-cadherin, this interaction prevents the association of the cadherin 38 tail with clathrin and clathrin adaptor complexes, thereby preventing cadherin endocytosis . These observations led to the hypothesis that the JMDs of classical cadherins contain endocytic signals that drive cadherin internalization, and that p120 binding to the cadherin tail acts as a brake on cadherin endocytosis by masking these internalization motifs 31,36,39. This model is outlined in detail below. Cadherin endocytic motifs The regulation of cadherins by p120-catenin has been examined in both vertebrate and invertebrate model organisms. In flies and worms, p120 appears to play a supportive but not essential role in 13 regulating cadherin turnover . However, the majority of data derived from mammalian systems support a model in which the cadherin JMD harbors multiple types of endocytic signals, and that p120 functions as a guardian that controls access of endocytic adaptors to these motifs (Figure 3), thereby regulating cadherin 12 endocytosis . Indeed, the observation that the JMD of the classical cadherins can drive endocytosis when fused to the extracellular and transmembrane domains of the IL2-receptor supports this model 31. Furthermore, p120 binding to these receptors is necessary to block endocytosis of these chimeric receptors. Although p120 is known to inhibit Rho A 40,41, this GTPase regulatory activity is apparently independent of 38 the ability of p120 to inhibit endocytosis . For example, p120 mutants that are able to bind to the cadherin tail but which are unable to inhibit Rho A block the entry of VE-cadherin into clathrin and AP-2 enriched membranes, demonstrating that regulation of cadherin endocytosis is mechanistically independent of p120 regulation of RhoA 38. These findings suggest that the cadherin tail harbors endocytic activity and that p120 physically occupies the cadherin tail to inhibit internalization. Further support for the idea that p120 binding physically occupies an endocytic signal comes from structural studies and from mutational analysis of endocytic motifs in the classical cadherin JMD 36. In the case of VE-cadherin, gain and loss of function approaches demonstrated that the core p120-binding region contains a unique endocytic motif comprising three acidic residues (DEE) 42. Based on homology and structural analysis of the E-cadherin-p120 complex, the DEE motif is physically occupied by p120 binding to the cadherin tail 36. In addition, when residues adjacent to the DEE sequence are mutated to disrupt p120

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binding, cadherin endocytosis is increased 21,42,43. Lastly, p120 mutants that are unable to bind to the 42 cadherin tail are likewise unable to block cadherin endocytosis . Collectively, these studies demonstrate that the cadherin JMD is a dual function motif that mediates p120 binding or, when p120 dissociates, 44 mediates cadherin endocytosis . In addition to VE-cadherin, the core p120-binding domain of E-cadherin, N-cadherin and Drosophila 42 E-cadherin can mediate endocytosis . However, in the case of mammalian E-cadherin, mutation of the DEE sequence only modestly inhibits internalization 42. Rather, it appears that a dileucine motif is the 43,45 . This motif in E-cadherin is membrane proximal to the core dominant driver of E-cadherin endocytosis p120-binding site in a region identified by NMR studies to be part of a dynamic p120-binding interface 36. Again, these studies suggest that p120 binding prevents E-cadherin endocytosis by occupying the cadherin tail and blocking access of clathrin adaptors, such as AP-2, to the LL motif. There are other examples of endocytic motifs in the JMD of classical cadherins. For example, the dileucine motif identified in E-cadherin is conserved in several other cadherins, including cadherin-6B (Figure 3). Interestingly, in contrast to E-cadherin, the LL motif in cadherin-6B is not required for endocytosis 21 . Nonetheless, mutation of the p120-binding site in cadherin-6B increases endocytosis, again suggesting that p120 binding to this cadherin JMD prevents internalization 21,36,43. The identity of the dominant endocytic motif in cadherin-6B has not yet been determined. However, cadherin-6B harbors an “NDE” sequence rather than the DEE motif identified in VE-cadherin (Figure 3). Therefore, it is possible that these acidic residues in the core p120-binding site of cadherin-6B function as an endocytic signal, but this possibility has not yet been tested. In addition to the DEE and dileucine motifs, a recent study has exposed a new endocytic motif that is also potentially subject to regulation by p120. A mesenchymal cadherin that is expressed in osteoblasts (cadherin-11) directly interacts with clathrin through a unique sequence, VFEEE, in the juxtamembrane 46 domain of the cadherin tail . Mutation of this sequence inhibits endocytosis of the cadherin in PC3mm2 cells, a highly metastatic cell prostate tumor cell line 46. The VFEEE sequence is similar to the LxEx(D/E) 47 clathrin-binding box sequence found in the c-terminal tail of arrestin3 . Interestingly, sequence alignments reveal that the LxEx(D/E) motif (Figure 3) may also be present in N- and P- cadherin, although it is not yet clear if clathrin binds to and mediates endocytosis of these other cadherins. Importantly, the VFEEE sequence resides in the region of the cadherin tail that is involved in dynamic interactions with p120, and this motif presumably is also masked when p120 is bound to the cadherin. It will be of interest to determine if this clathrin-binding motif in cadherin-11 can mediate internalization of the cadherin independently of the putative acidic endocytic motif (DDE) in the core p120-binding region of this cadherin. Ubiquitination of the cadherin tail In addition to endocytic motifs that are constitutively active, ubiquitination is a posttranslational modification recognized as an important signal for internalization and degradation of a wide range of cell surface receptors, including cadherins 48. The first ubiquitin ligase to be identified that targets cadherins for ubiquitin modification is an E3 ligase termed Hakai. Hakai is a c-Cbl-like protein that binds to and ubiquitinates E-cadherin, inducing internalization of the cadherin in MDCK cells 49. Hartstock and Nelson report that mutation of lysine 5 (K5) in a truncated E-cadherin mutant, containing only the JMD and targeted to the mitochondria, blocked degradation in response to Hakai 50. These data indicate that Hakai ubiquitinates and targets E-cadherin for degradation through posttranslational modification of the cadherin JMD. Hakai mediated ubiquitination of E-cadherin is dependent upon phosphorylation of two tyrosine residues in the E-cadherin JMD that are required for the ligase to bind to the cadherin tail. These tyrosine residues are not fully conserved in other classical cadherins, indicating that various cadherins may be targeted selectively by different E3 ligases. For example, VE-cadherin is targeted by K5, an E3 ligase expressed by human herpesvirus-8 (HHV-8) 51. HHV-8 causes Kaposi’s sarcoma, an angioproliferative neoplasm, which is associated with increased vascular permeability. Work from our lab indicates that K5 targets VE-cadherin for ubiquitination and down-regulation through an endo-lysosomal pathway (Nanes et al., submitted). Interestingly, we found that K5 does not target E-cadherin, an observation that further demonstrates selectivity among E3 ligases for different cadherins. Furthermore, K5 does not require the DEE motif that is needed for constitutive endocytosis of VE-cadherin. Instead, two membrane proximal lysine residues (K626, K633) are required for K5-induced ubiquitination and internalization of VE-cadherin. These lysine residues are located within the region of the cadherin tail associated with dynamic interactions with p120, indicating possible regulation by p120 binding to the cadherin tail. In support of this idea, p120 over-expression protects VE-cadherin from K5-mediated endocytosis and degradation (Nanes et al.,

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submitted). These findings indicate that distinct motifs in the JMD of VE-cadherin regulate constitutive (eg. DEE motif) and inducible (ubiquitin-mediated) internalization. Additionally, this model serves as another example of the involvement of p120 in the regulation of cadherin endocytosis, most likely through physically masking ubiquitination sites in the cadherin JMD. A corollary hypothesis is that ubiquitination of the cadherin JMD may sterically hinder p120 binding. This idea is supported by the fact that VE-cadherin does not colocalize with p120 in endocytic compartments in cells expressing the K5 E3 ligase (Nanes et al., submitted). Similarly, mutation of a lysine residue in E-cadherin that is required for ubiquitination by Hakai 50 increased p120 binding to the cadherin JMD . The dynamics and reciprocity of the interactions between p120 and ubiquitin modification of the cadherin JMD require further study to fully understand the hierarchy of this regulation and how it contributes to biology and pathophysiology. The K5 ubiquitin ligase that targets VE-cadherin is structurally related to a family of mammalian 52 ligases termed membrane-associated RING-CH (MARCH) proteins . These E3 ligases are a family of transmembrane ubiquitin ligases that are involved in the regulation of various cell surface proteins. Very little is currently known about the expression patterns, subcellular localization and substrate specificity of MARCH family ligases. One study in zebrafish found that overexpression of MARCH8, originally named cMIR, resulted in E-cadherin ubiquitination and down regulation of surface levels of E-cadherin 53. We have also observed that several MARCH family genes target VE-cadherin for degradation (unpublished results), but the biological context for this regulation is not currently known. The data above indicate that the JMD of classical cadherins harbors a variety of endocytic motifs that can be regulated by p120-catenin binding to the cadherin tail. It is likely that other proteins that bind to the cadherin JMD will have similar or opposing roles. One example that has emerged already is the spectrinactin cytoskeletal adaptor protein, ankyrin-G 30,54. Ankyrin-G binds to E-cadherin and retains it at the lateral 30 membrane of polarized MDCK cells and, in cooperation with clathrin, establishes apical-lateral polarity . Moreover, a study from Jenkins et al. reports micron-sized domains consisting of an underlying membrane skeleton comprised of ankyrin-G and its partner β-spectrin that inhibit endocytosis of E-cadherin through the 54 exclusion of clathrin and clathrin dependent cargo in epithelial lateral membranes . In addition, we recently reported that ankyrin-G inhibits endocytosis of VE-cadherin and stabilizes localization of the cadherin at cellcell junctions in vascular endothelial cells 55. Given the size of Ankyrin-G isoforms, it is unlikely that both Ankyrin-G and p120 occupy the cadherin tail at the same time. These findings raise the interesting possibility that distinct cadherin complexes stabilized by various JMD binding partners differentially influence the architecture of different plasma membrane domains. The data above support a model in which mammalian p120 acts as a master guardian of cadherin endocytosis by physically masking endocytic motifs or residues important for posttranslational modification (Figure 3). However, this model does not seem to extend to non-mammalian model systems. In fact, multiple studies have found that p120 is dispensable in Drosophila and C. elegans 56-58. However, a recent report from Bulgakova and Brown demonstrates that Drosophila p120-catenin is required for embryonic epithelial morphogenic movement in the fly and, rather than inhibiting cadherin internalization, facilitates endocytosis 59 of the E-cadherin-Bazooka subcomplex . Additionally, in contrast to Hakai-mediated endocytosis of Ecadherin in mammalian cells, it appears that Drosophila Hakai does not play a major role in the downregulation of DE-cadherin, though the ligase is required for epithelial integrity and endoderm and mesoderm 60 morphogenesis . Drosophila Hakai and DE-cadherin were found to colocalize in endosomal compartments that differ from known Rab5, Rab7, or Rab11 positive vesicles 60. Surprisingly, the association of Drosophila Hakai does not require the cytoplasmic tail of DE-cadherin. More studies are needed to elucidate the specific function of Hakai association with DE-cadherin during Drosophila embryogenesis and to clarify how p120 regulates cadherin trafficking and cell adhesion in non-mammalian model systems. Endocytic adaptors Endocytic signals are amino-acid motifs that mediate endocytosis by specifying interactions with endocytic adaptor complexes. One of the most important clathrin adapters is the AP-2 complex. AP-2 binds cargo molecules, clathrin, and phospholipids at the plasma membrane, and is thus a key mediator of clathrin-mediated endocytosis. Typically, AP-2 recognizes cargo proteins harboring tyrosine or dileucine 15,61 . A subunit of AP-2 was found to co-immunoprecipitate with E-cadherin 62 and, as based motifs mentioned above, mutation of the dileucine motif in the E-cadherin JMD prevents endocytosis. Furthermore, AP-2 mediated internalization of E-cadherin requires p120 dissociation 43, consistent with the model that p120 regulates cadherin-dependent endocytosis by physically blocking endocytic motifs. As discussed above, the dileucine motif is not present in VE-cadherin or Drosophila DE-cadherin (Figure 3). Yet, AP-2 coimmunoprecipitates and colocalizes with the VE-cadherin tail and undergoes clathrin, dynamin, and AP-2 dependent internalization 38. Similarly, DE-cadherin is internalized through a clathrin and AP-2 dependent

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mechanism during the establishment of planar cell polarity in Drosophila germ band extension 63. Additional studies are needed to understand when AP-2 mediated endocytosis is critical for cadherin regulation, and to fully characterize motifs in the cadherin tail responsible for AP-2 binding. Other endocytic adaptor molecules, including AP-1B, Dab2, and Numb are implicated in cadherin regulation to establish and maintain cell polarity. The adaptor protein Numb interacts with the E-cadherinp120 complex to regulate cadherin endocytosis and maintain apicobasal polarity in epithelial cells 62. Sato and colleagues find that in the absence Numb, E-cadherin and p120 are mislocalized from the basolateral to 62 the apical membrane of polarized MDCKII cells . Interestingly, they report a mechanism for p120 regulation of cadherin internalization that does not involve p120 dissociation from the cadherin. Rather, they find that Numb interacts directly with p120 bound to the cadherin tail to induce endocytosis 62. Down-regulation of Disabled-2 (Dab2), another adaptor protein associated with clathrin-mediated endocytosis, results in the loss of apicobasal polarity in murine embryonic endoderm and an accumulation of cell surface E-cadherin 64. Though it was not directly assessed, it is likely that the increased surface E-cadherin is due to decreased internalization caused by loss of Dab2 function. The region of the cadherin tail that associates with Dab2 is not known. In addition to endocytosis, clathrin adaptors play key roles in recycling. Ling et al. found that the adaptor protein AP-1B mediates recycling of E-cadherin to the basolateral membrane of polarized MDCK cells. AP-1B associates with the cadherin indirectly through association with a lipid kinase (PIPK1γ) that 65 binds to the catenin-binding domain of the cadherin tail . Interestingly, a mutation in the cadherin tail associated with gastric cancer prevents PIPK1γ binding to the cadherin tail, and thereby inhibits cadherin interactions with clathrin adaptors. The result is deficient cadherin trafficking to the membrane and inhibition of adherens junction assembly. This work highlights the need to identify clathrin adaptors and other cadherin-binding partners that mediate trafficking to and from the plasma membrane. Regulation of cadherin trafficking by other armadillo family proteins The p120 family includes other armadillo proteins that regulate cadherin endocytosis, including ARVCF, δ-catenin, and p0071 28,66. Like p120, these proteins colocalize with cadherins at adherens junctions and can stabilize cadherins at the plasma membrane. p0071 localizes to both adherens junctions and desmosomes and exhibits sequence homology to both p120 and the desmosomal protein plakophilin-1 28 . p0071 also inhibits endocytosis of VE-cadherin 39. Mutation of residues adjacent to the DEE endocytic motif in the core p120-binding region of VE-cadherin disrupts p0071 binding to the cadherin, suggesting that p0071 inhibits endocytosis in a manner similar to p120 67. In addition, δ-catenin and ARVCF can substitute for p120 in stabilizing E-cadherin at the plasma membrane in mammalian cells 68. Indeed, in Xenopus the 69 loss of ARVCF results in a phenotype similar to the loss of p120 . Depletion of either protein results in disrupted gastrulation and axial elongation. Importantly, exogenous expression of either protein rescues the depletion of the other, indicating overlapping roles during vertebrate development 69. As mentioned above, p120 is essential for vertebrate life but appears dispensable in invertebrate organisms 56-58. Interestingly, the p120 subfamily of armadillo proteins expanded dramatically with the evolutionary appearance of vertebrate 70 organisms . The precise reason for the expansion of this family of genes is not known, but it may reflect a necessity for additional levels of control over cadherin trafficking during embryonic patterning of vertebrate tissues. Several studies suggest that β-catenin may play a role in cadherin trafficking to the plasma membrane as well as retrieval of the cadherin from the cell surface via endocytic pathways. β-catenin binds to newly synthesized cadherin in the endoplasmic reticulum and traffics to the plasma membrane in 71 association with the cadherin . This interaction is thought to protect the cadherin from proteolytic 72 degradation and ensure its delivery to the cell surface . β-catenin binding to the cadherin tail and subsequent endocytosis are modulated by posttranslational modification of the β-catenin binding domain. Phosphorylation of E-cadherin by casein kinase I (CK1) on a serine in the catenin-binding domain was found to weaken the interaction between β-cadherin and the cadherin tail, resulting in increased cadherin endocytosis 73,74. These authors also found that inhibition of casein kinase I (CK1) resulted in stable cadherin based cell contacts, and that overexpression of CK1 resulted in disrupted contacts 73. In a separate study, McEwen and colleagues found that phosphorylation of the β-catenin binding domain of E-cadherin stabilized β-catenin interactions with the cadherin tail and prevented cadherin turnover 75. In addition, NMDA receptor activation in hippocampal neurons enhances β-catenin binding to N-cadherin and decreases N76 cadherin endocytosis, again suggesting that β-catenin binding can inhibit cadherin endocytosis . These authors also showed that overexpression of a β-catenin phosphorylation-deficient mutant that exhibits increased association with N-cadherin inhibits internalization of the cadherin independent of NMDA receptor stimulation 76. However, at least one study suggests that β-catenin may promote cadherin internalization.

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Sharma and colleagues revealed that β-catenin mediates macropinocytosis of N-cadherin in NIH 3T3 77 fibroblasts . Although a clearly defined model for how β-catenin binding regulates cadherin trafficking has not yet emerged, it is likely that this catenin influences cadherin endocytosis and junction stability through mechanisms that transcend its role as a cytoskeletal adaptor protein. Likewise, even less is known about how α-catenin might contribute to cadherin trafficking, although a role for this cadherin associated protein is certainly conceivable. Growth Factor Regulation of cadherin trafficking A number of growth factor signaling pathways are associated with regulation of cadherin endocytosis, including vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and epithelial growth factor (EGF). Often, the relationship between the growth factor and cadherin regulation is bidirectional. Growth factors can influence cadherin endocytosis, while cadherin endocytosis can modulate growth factor signaling. This two-way regulation indicates that adherens junctions are not only acted upon by growth factors, but can also actively tune cell signaling in response to soluble extracellular cues. VEGF is a proangiogenic factor that stimulates the growth of new blood vessels, and is an 78,79 . important modulator of vascular permeability through its effects on endothelial intercellular junctions Gavard and Gutkind demonstrated that stimulation of VE-cadherin endocytosis is one mechanism by which VEGF disrupts endothelial junctions. VEGF induces Src-dependent phosphorylation of serine 665 (S665) in the JMD of the cadherin tail, which functions to recruit β-arrestin to drive clathrin-mediated endocytosis of VE-cadherin 80. Furthermore, regulation of VE-cadherin and VEGF is bidirectional. For example, association of VE-cadherin with the VEGF receptor (VEGFR) retains the VEGFR at the cell surface and inhibits downstream MAPK signaling 81. This process occurs through a mechanism that requires β-catenin and the density-enhanced phosphatase-1 (DEP-1), which dephosphorylates the VEGFR at cell-cell junctions 82. This regulation of VEGFR2 signaling through interactions with VE-cadherin appears to be a mechanism by which VE-cadherin modulates proliferative VEGF signaling during angiogenesis. During development and wound healing, growth factors are often expressed in a carefully choreographed sequence or in combinations that allow for fine-tuning of various signaling pathways. For example, VEGF-mediated internalization of VE-cadherin is antagonized by angiopoietin-1. Angiopoetin-1 is 83 a proangiogenic factor that stabilizes developing blood vessels by inhibiting Src activation by VEGF . Interestingly, FGF also plays a role in stabilizing endothelial adherens junctions and counter-acting VEGF. However, FGF regulates cadherin stability by regulating the availability of a phosphatase, SHP2. SHP2 reduces levels of VE-cadherin tyrosine phosphorylation (Y658) within the JMD, and thereby stabilizes p120 binding to the cadherin tail 84,85. In addition to angiogenesis, the FGF family of growth factors are involved in embryonic patterning and the coordination of cellular morphogenic movements during development 86. In MCF-7 cells, a breast cancer cell line, the activation of FGF receptor 1 (FGFR1) by FGF results in the co-internalization of FGFR1 and E-cadherin and subsequent translocation of FGFR1 to the nucleus 87. Interestingly, overexpression of either E-cadherin or p120 blocks internalization of both the cadherin and FGF1, resulting in disrupted FGF87 induced signaling, and inhibition of FGFR1 nuclear translocation . In addition, inhibiting E-cadherin 87 internalization attenuates MAPK activation by FGF . Interestingly, Suyama and colleagues found that Ncadherin inhibits FGFR-1 internalization in MCF-7 cells, which results in prolonged expression of the receptor on the cell surface. This sustained surface expression lead to persistent MAPK signaling and promoted tumor cell invasion 88. Differential regulation of FGF signaling by E-cadherin or N-cadherin may provide a mechanism that controls the balance between epithelial and mesenchymal cell phenotypes. The EGFR signaling pathway is involved in regulating cell proliferation, differentiation, and motility, processes that require the dynamic regulation of cell adhesion. Indeed, cadherin endocytosis is also regulated through EGF signaling. As discussed above, EGF has been shown to induce both caveolindependent endocytosis of E-cadherin 20 and macropinocytosis of E-cadherin junctional complexes 17. Further studies are needed to fully understand the significance of E-cadherin endocytosis through different pathways in response to EGF signaling, and how these events are coupled to tumor cell phenotypes. In addition to classical cadherins, EGF signaling can also regulate endocytosis of desmosomal cadherins. Klessner and colleagues found that EGF signaling, together with metalloprotease-mediated cleavage, 89 regulates Desmoglein 2 (Dsg2) endocytosis and desmosomal adhesion . Not surprisingly, regulation of EGFR and E-cadherin appears to be bidirectional. In tumor cells, mutations in the extracellular domain of Ecadherin reduce internalization of EGFR in response to EGF stimulation, resulting in sustained activation of the growth factor receptor 90. EGF regulation of cadherin endocytosis is not restricted to tumor cells. A recent study from Song et al. demonstrates that EGF expression regulates endosomal E-cadherin trafficking during zebrafish epiboly movements. In this study, the loss of Pou5f1, a transcription factor important for the

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regulation of pluripotency during embryonic development, was found to impair E-cadherin intracellular 91 trafficking though the regulation of EGF expression . The data presented suggest that Pou5f1 dependent EGF expression controls E-cadherin endocytosis and initiates the transition from the stationary blastula state to the motile gastrula state. While this study highlights the relationship between EGF and E-cadherin during development, the data may be relevant to certain cancers as well. These numerous examples of growth factor mediated control of cadherin endocytosis and bidirectional regulation underscore the central role of the adhesion and signaling network during development and disease. Cadherin recycling and the recycling pathway Cadherins that enter the endocytic pathway are not always fated for lysosomal degradation, but rather, a pool of internalized cadherin can also be recycled back to the plasma membrane. In fact, whether the cadherin is degraded or recycled can determine the amount and location of the cadherin at the cell surface. Little is known about the molecular mechanisms that govern cadherin recycling after internalization, though studies have begun to elucidate important components. For example, members of the exocyst complex, sec5, sec6, and sec15 are required for DE-cadherin recycling in Drosophila. Loss of these components in Drosophila epithelial cells results in accumulation of DE-cadherin in enlarged recycling vesicles and inhibition of DE-cadherin delivery to the membrane 92. In addition, sec3 colocalizes with a distinct subset of exocyst complexes that are found specifically at desmosomes and is required for 93 desmosome assembly in MDCK cells . In mammalian epithelial cells, the scaffold protein PASL1 regulates E-cadherin trafficking. Depletion of PALS1 results in the mislocalization of the exocyst complex, an accumulation of cytoplasmic E-cadherin puncta, and inefficient delivery of E-cadherin to the cell surface 94. The recycling pathway can participate in the delivery of newly synthesized cadherins to the plasma membrane. Lock and Stow found that in polarized epithelial cells, newly synthesized E-cadherin does not travel directly from the Golgi complex to the plasma membrane, but instead, the cadherin is transited 18 through Rab11 positive endosomes . Rab11 is a small GTPase that plays critical roles in post-Golgi trafficking and the recycling of a variety of receptors, including adhesion molecules 95. E-cadherin is targeted to the basolateral membrane of polarized cells, and this localization is dependent upon the dileucine motif in the E-cadherin tail that is also important for clathrin-mediated endocytosis 96. Importantly, Rab11 mutants disrupt delivery of wild-type E-cadherin to the plasma membrane, while mutations in the E-cadherin dileucine motif important for clathrin-mediated endocytosis and targeting to the basolateral membrane lead to mis-sorting of the cadherin to the apical membrane. This finding suggests that Rab11 recycling endosomes are an intermediate compartment for post-Golgi trafficking and are involved in E-cadherin 18 exocytosis specifically to the basolateral membrane . In addition, a recent study found that VE-cadherin recycling and endothelial barrier function is regulated by Rab11 97, further highlighting the important role for this GTPase in modulating classical cadherin availability at cell-cell junctions in a variety of cell types. A recent study in Drosophila follicular epithelium demonstrated that three distinct pathways mediate DE-cadherin targeting to the apicolateral membrane 19. Two of these pathways involve trafficking of newly synthesized DE-cadherin to the zonula adherens (ZA) of Drosophila follicular epithelium. One pathway is dependent upon Rab11 and targets newly synthesized cadherin directly to the apicolateral membrane. An alternate pathway targets newly synthesized DE-cadherin to the lateral membrane. This pool of DE-cadherin is then retrieved from the lateral cell surface, recycled, and trafficked selectively to the apicolateral 19 membrane in a mechanism dependent upon RabX1 . The third pathway involves the transport of DEcadherin from the lateral membrane to the ZA through cadherin flow within the plasma membrane, independent of Rab11 and RabX1 19. Future studies will be needed to elucidate the precise purpose of such complex regulation. An intriguing possibility is that epithelial cell shape changes can be regulated by differentially employing pathways that control DE-cadherin delivery to different plasma membrane sites, thereby fine-tuning membrane content and architecture. Concluding comments: Studies over the past 15 years have identified some of the basic membrane trafficking pathways that handle cadherin delivery to and from the cell surface. p120-catenin has emerged as the master guardian of plasma membrane cadherin levels by protecting cadherin tails from endocytic machinery. A series of amino-acid motifs have been identified in the cadherin JMD that drive cadherin internalization, and in some cases, the endocytic adaptors that mediate cadherin endocytosis by these motifs have been revealed. As a field, we are now left with the challenge to understand how these effectors of cadherin cell surface levels are regulated, and to determine the biological contexts in which their influence is most important. For example, it is likely that processes requiring cell migration such as angiogenesis, wound healing, and tumor metastasis rely on the plasticity of cell-cell adhesion that is conferred by endocytic

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trafficking of cadherins. This possibility is supported by the observation that mutations in the DEE endocytic 42 motif in VE-cadherin inhibit endothelial cell migration . The identification and mutation of cadherin endocytic motifs will allow us to determine how these endocytic signals in various cadherins contribute to adhesion, migration, and patterning of a variety of tissues. Likewise, the identification of ubiquitin ligases that target cadherins will open new avenues to understand how these modifiers of cadherin trafficking influence cell adhesion in different biological contexts. Thus, the study of cadherin trafficking is now fertile ground for the discovery of new and fundamental regulatory mechanisms that modulate cell adhesion during development and disease.

Acknowledgements: We would like to thank members of the Kowalczyk and Mattheyses labs for insightful discussions during preparation of this manuscript. In particular, we are grateful to Dr. Cynthia Myers for her critical feedback and helpful suggestions. The Kowalczyk lab is supported by grants from the National Institutes of Health (R01AR048266 and R01AR050501).

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Fiigure Legends s

Fiigure 1. Molec cular compone ents of adhesive intercellul ar junctions. IIn adherens jun nctions, the cadherin cy ytoplasmic tail binds to p120-catenin and β-catenin. β-cate enin binds direcct to α-catenin, an actin bindin ng prrotein that coup ples the comple ex to actin filam ments. In the de esmosome, the e cytoplasmic ttails of desmossomal ca adherins (Desm moglein and De esmocollin) bind to plakoglobiin. Desmoplakiin associates w with the desmo osomal ca adherin comple ex through plak koglobin and lin nks the comple ex to intermedia ate filaments. P Plakophilin clussters de esmoplakin com mplexes and enhances interm mediate filamen nt association w with the desmo osomal comple ex.

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Fiigure 2. Overv view of cadherrin trafficking pathways. Ne ewly synthesize ed classical cad dherins are exp ported fro om the Golgi to o the plasma membrane m eithe er directly or th rough Rab11 p positive endoso omes (A). Classsical ca adherins are internalized through multiple pa athways includ ding clathrin-me ediated endocyytosis (B), cave eolinmediated m endoc cytosis (C), or macropinocytos m sis (D). Interna lized cadherin is either degra aded in the lyso osome orr recycled back k to the plasma a membrane. Desmosomal D ca adherins are intternalized through a lipid raft mediated m pathway (E).

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Fiigure 3. p120-catenin is a master m guardia an of cadherin n endocytosis.. (A) p120-cate enin (p120) bind ds to th he juxtamembra ane domain (JM MD) of the cadherin tail and p physically blockks endocytic m motifs from adap ptor prrotein binding and a posttransla ational modifica ations that drive e endocytosis. Dissociation o of p120 expose es th hese motifs in the cadherin taiil, resulting in cadherin c interna alization. Expa anded view of the JMD, illustra ated on n the right, dep picts endocytic motifs or important residues in the static or dynamic regio on of p120 binding. (B B) Sequence allignment of the e JMDs of class sical cadherins . Green; conse erved DEE sequence required d for VE-cadherin end docytosis, brow wn; conserved dileucine motiff required for E E-cadherin endo ocytosis, red; lyysine re esidues identifie ed as putative ubiquitination sites, s yellow; cllathrin-binding motif, blue; ph hosphorylation site (s serine 665) imp portant for β-arrrestin driven en ndocytosis of V VE-cadherin. O Open boxes; potential endocyttic motifs. m

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