The ins and outs of E-cadherin trafficking

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The ins and outs of E-cadherin trafficking David M. Bryant and Jennifer L. Stow Institute for Molecular Bioscience and School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072, Australia

One way of controlling the activity of E-cadherin – a protein that is, simultaneously, a major cell-adhesion molecule, a powerful tumour suppressor, a determinant of cell polarity and a partner to the potent catenin signalling molecules – is to keep it on the move. During the past two decades, many insights into the fundamental role of E-cadherin in these processes have been garnered. Studies during the past five years have begun to reveal the importance of intracellular trafficking as a means of regulating the functions of E-cadherin. E-cadherin is trafficked to and from the cell surface by exocytic and multiple endocytic pathways. In this article, we survey the vesicle-trafficking machinery that is responsible for the sorting, transport, actin association and vesicle targeting of E-cadherin to regulate its movement and function during growth and development and, possibly, in cancer. Moving membrane receptors, transporters and channels onto and off of the cell surface by vesicular transport is a recognized mechanism for regulating their functions [1–3]. Such mechanisms rely on the fact that exocytic and endocytic trafficking at the cell surface can be exquisitely regulated both in time and in space, and tightly coupled to changing cellular requirements: evidenced most clearly in studies at the mammalian synapse [4]. It has become increasingly apparent that cells can regulate adhesion using a similar strategy – by moving adhesion-competent proteins such as integrins [5] and cadherins onto and off of the cell surface. E-cadherin, which is the prototypical member of the classic cadherin family, is a major component of the adherens junction (AJ), at which it provides cell–cell adhesion through Ca2C-dependent, homophilic binding between molecules on adjacent epithelial cells [6]. At the AJ, E-cadherin is bound to catenins. b-Catenin attaches to the C-terminal region of E-cadherin and then to a-catenin, through which the complex is linked to the actin cytoskeleton [7] (Figure 1). The Src substrate p120ctn binds to a site on the juxtamembrane domain of the E-cadherin cytoplasmic tail [8]. Varied roles in modulating the adhesive functions of cadherins have been attributed to p120ctn and its emerging roles in cadherin trafficking are discussed later. The function of the cadherin–catenin complex at AJs is modulated Corresponding author: Jennifer L. Stow ([email protected]).

by dimerization, phosphorylation and ubiquitination, in addition to the individual roles of the catenins (for review, see Ref. [9]). However, E-cadherin is not always at the AJ – it also spends variable amounts of time in vesicles trafficking to and from the cell surface. Therefore, cadherin-based adhesion is not a static state but, instead, is a dynamic equilibrium between cadherin complexes at the AJ and those in intracellular vesicles and compartments. The trafficking of E-cadherin necessarily involves two types of dynamic molecular interactions. The first reflects changes in the composition of the cadherin–catenin

Adherens junction

Early or sorting endosome

Recycling endosome

TGN

Late endosome or MVB

Lysosome

Golgi complex

β-Catenin p120ctn α-Catenin

E-cadherin Actin TRENDS in Cell Biology

Figure 1. Exocytic and endocytic trafficking of E-cadherin. Newly synthesized E-cadherin is trafficked from the trans Golgi network (TGN) to the cell surface for incorporation, together with catenins, into adherens-junction complexes. Surface E-cadherin can be endocytosed, during which it encounters a variety of known (unbroken arrows) or proposed (broken arrows) trafficking steps that recycle it back to the cell surface or target it for degradation. Abbreviation: MVB, multivesicular body.

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complex itself, whereas the second involves the engagement of vesicle-trafficking machinery. A growing body of data is emerging on both counts, providing new insights into both the sequential nature of catenin function in trafficking and the precise pathways that are traversed by cadherins through the cell. The trafficking machinery contains a vast array of molecules that execute the sorting, loading, transport and delivery of cargo such as E-cadherin in vesicular carriers for exocytic or endocytic trafficking. These molecules include members of several G-protein families and their regulators, coats, adaptors, lipid kinases, SNAREs [SNAP (synaptosomal-associated protein) receptors], cytoskeletal motors and coiled-coil proteins [4,10]. Table 1 contains a compendium of trafficking-machinery proteins that have been implicated in E-cadherin trafficking. This list is, undoubtedly, incomplete because many aspects of cadherin trafficking are yet to be elucidated. Although some of the additional machinery that is required for E-cadherin trafficking can be anticipated, based on the current understanding of trafficking pathways, the main challenge will be to determine how specific parts of the machinery regulate cadherin availability and function under different physiological circumstances. In this article, we summarize the growing body of evidence regarding the mechanisms and pathways of E-cadherin trafficking both to and from the cell surface. We also provide a framework for understanding the endocytosis of this important molecule in different physiological situations.

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Exocytosis: sorting and delivery in polarized cells During trafficking to and from the cell surface, cadherin–catenin complexes are dynamically assembled and disassembled. b-Catenin binds to E-cadherin early in the biosynthetic pathway and, generally, the two proteins are trafficked and sorted as a complex for delivery to the epithelial-cell surface in Madin–Darby canine kidney (MDCK) cells [11,12]. By contrast, there is evidence that a-catenin [13] and p120ctn [12] do not join the complex until it is near or at the basolateral cell surface, which is consistent with the established roles of a-catenin and p120ctn at the AJ [9]. Conversely, N-cadherin, which is the classical neuronal cadherin, seems to associate earlier with p120ctn, before furin cleavage of the cadherin propeptide and delivery to the plasma membrane [14–16]. The difference in temporal assembly between the E-cadherin–catenin and N-cadherin–catenin complexes is yet to be resolved but it might reflect bona fide variations in the processing of these complexes or cell-type differences. The levels of synthesis and exocytic trafficking of E-cadherin are modulated and influenced by the status of epithelial cells, correlating with morphogenesis or adhesion requirements (Figure 2). The trans Golgi network (TGN) offers the first major site for differential regulation or sorting of newly synthesized E-cadherin. Because E-cadherin is intimately involved in the establishment and maintenance of epithelial-cell polarity, it must be sorted and delivered itself in a polarized manner to the lateral cell surface. The cytoplasmic tail of

Table 1. Proteins implicated specifically in the trafficking of E-cadherina Component

Function

Proposed role in E-cadherin trafficking

Refs

Exocytosis b-catenin Dileucine motif BIG2 Furin FAM Kinesin EZRIN Sec6/8

Cadherin–catenin complex Sorting signal ARF–GEF Proprotein convertase Deubiquitination Molecular motor Actin–membrane linker protein Exocyst complex

Coupled at ER; facilitates, but is not essential for, trafficking Basolateral sorting at TGN Sorting and vesicle budding at TGN Cleavage of pro-sequence Post-Golgi trafficking Trafficking of cadherin to plasma membrane (via p120ctn) Regulation of delivery to plasma membrane Docking of vesicles at apicolateral junctions

[11,17] [11,12,17] [23] [16] [26] [14,61] [68] [69]

Endocytosis Hakai p120ctn Clathrin Dynamin Rab5 ARF6

Ubiquitin ligase Cadherin–catenin complex Coated-pit formation Pinchase GTPase GTPase

[44] [47,48] [34,70] [35,36,71] [33,40] [35,71]

RTKs IQGAP HDlg Rac1 RhoA Cdc42 PKC

Signalling kinase Scaffolding protein Scaffolding protein GTPase GTPase GTPase Signalling kinase

Ubiquitination of complex for endocytosis Stabilization at the plasma membrane Invagination of membrane to form endosome Pinching off of membrane invagination to form endosome Formation of early endosomes Regulation of endosome formation; signalling to actin to facilitate endocytosis Signalling for phosphorylation, ubiquitination and/or degradation Regulation of link with cytoskeleton Stabilization at cell–cell contacts Stabilization at plasma membrane; attachment to actin Stabilization at plasma membrane; attachment to actin Stabilization at plasma membrane; attachment to actin Sends signals for actin rearrangements needed for recycling

Other Presenilin-1 Polycystin-1 Src

Unknown Unknown Non-receptor kinase

Trafficking out of Golgi; proteolytic cleavage at membrane Trafficking out of Golgi; stabilization of complex at membrane Regulates stability and assembly of cell–cell contacts

[24,25] [74] [75]

a

[45] [72] [27] [73] [73] [73] [70]

Abbreviations: ARF, ADP-ribosylation factor; ER, endoplasmic reticulum; FAM, Fat facets in mouse; GEF, guanine-nucleotide-exchange factor; hDlg, human Discs large; PKC, protein kinase C; RTKs, receptor tyrosine kinases; TGN, trans Golgi network.

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(b) Exocytosis Endocytosis (c) (a)

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Figure 2. The relationship between E-cadherin trafficking and epithelial morphogenesis. (a) In preconfluent or non-adherent cells, E-cadherin is synthesized in abundance and is trafficked constantly to and from the membrane by unregulated pathways (with little effect) before productive cell–cell adhesion occurs. (b) In mature polarized epithelia, the trafficking of E-cadherin is minimized. Surface E-cadherin is incorporated stably into adherens junctions, which requires only small amounts of E-cadherin synthesis and leaves only a small pool of E-cadherin to recycle at the surface. (c) More-profound loss of adhesion accompanies cells that undergo epithelial-to-mesenchymal transition or during tumourigenesis, in which E-cadherin might be endocytosed for downregulation or degradation, with no new synthesis to replace it. Consequently, cadherin-based adhesion and cell polarity are lost. Exocytosis is represented by blue spheres and endocytosis is represented by red spheres.

E-cadherin contains several putative sorting signals. Amongst these, there is a highly conserved dileucine motif that is found in the juxtamembrane region of most classic cadherins [17], with the interesting exception of VE-cadherin (which must be alternately sorted). The dileucine motif is required for the sorting and basolateral trafficking of E-cadherin [12,17]. In the absence of this motif, the E-cadherin–b-catenin complex is missorted, which results in the disruption of cell polarity and morphology. Typically, dileucine sorting signals are recognized by specific adaptor complexes at the TGN or at the plasma membrane to assemble clathrin-coated vesicles for basolateral or endocytic trafficking, respectively [18]. The nature of the specific adaptor complex that recognizes the cadherin dileucine motif is not yet known, although AP-1 is a candidate [19]. AP-1 and GGA adaptors are recruited to the TGN membranes by activated forms of the GTPase ADP-ribosylation factor (ARF) and ARF-exchange factors such as BIG2 [20–22]. The recent identification of a disease-causing mutation in the gene that encodes BIG2 indicates a role for this exchange factor in the sorting of Ecadherin [23]. Mutations in the gene encoding BIG2 manifest as pathologies that result from defects in cadherin-mediated neural-precursor proliferation and neuronal migration [23]. The proper sorting of E-cadherin and the efficient formation of vesicles are crucial steps for maintaining cadherin trafficking and, thus, cellular function in cell lines and tissues. A variety of other molecules has been shown to associate with or affect cadherin–catenin complexes during trafficking. The interrelated roles of actin and several GTPases in E-cadherin trafficking are discussed in Box 1. There is early evidence to implicate a variety of other proteins in the trafficking of cadherins but, as yet, there is no coordinating theme for the roles of these proteins. For example, the overexpression of dominant negative forms of presenilin-1, which is a g-secretase component, seems to block the exit of cadherin complexes from the endoplasmic reticulum and disrupt cell–cell adhesion [24]. Furthermore, presenilin-1 also interacts www.sciencedirect.com

Box 1. Actin, GTPases and the cadherin connection in trafficking A variety of GTPases function, in part, at the busy interface of exocytic and endocytic trafficking of E-cadherin to coordinate the attachment and detachment of the cadherin–catenin complex to actin (for review, see Ref. [73]). For stable incorporation into the adherens junctions (AJs), E-cadherin is anchored to the actin cytoskeleton [76] by a process that involves both Rho family GTPases and their accessory proteins (e.g. exchange factors). Rac1, RhoA and Cdc42 have each been implicated in controlling the stability of E-cadherin attachment to actin at the AJ. This attachment to actin, through catenins, is dynamic and can be modulated to allow for either stabilization or further trafficking. Rac1 can regulate the endocytosis of E-cadherin [36] and, in concert with the actin-scaffolding protein EZRIN, it regulates the trafficking of E-cadherin to the membrane during epithelial morphogenesis [68]. Exchange factors such as Tiam-1 [77] can also regulate the endocytic process by controlling the activation of their cognate small G proteins (e.g. Rac1). The Rac and Cdc42 effector protein IQGAP mediates attachment to actin by regulating the link between a- and b-catenin [72]. IQGAP can also destabilize this connection, thus enabling endocytosis to occur [78]. The signalling to actin by Protein kinase C regulates the endocytosis of VE-cadherin [79] and the trafficking of E-cadherin both to and from the membrane during recycling [70]. Thus, multiple interactions between E-cadherin– catenin complexes and actin are not only important for the stability of junctions but are also needed for trafficking. Actin, actin-binding proteins, actin-based motors and Rho GTPases also function as part of the trafficking machinery at the cell surface and in trafficking from the trans Golgi network [80]. GTPases of other subfamilies have also been implicated in E-cadherin trafficking. The expression of mutant forms of Rab5 [40] or ADP-ribosylation factor (ARF)6 [35,71] can block the endocytosis of E-cadherin. Furthermore, ARF6 works at multiple levels, regulating Src-induced or growth-factor-induced clathrin-mediated E-cadherin endocytosis by recruitment of the dynamin-regulating kinase Nm23-H1 [71]. ARF6 can also reduce the levels of active Rac1, ultimately leading to the disruption of cell–cell junctions and to migration. [71]. Rab5 regulates early-endosome formation and it is interesting to note that the recently identified guanine-nucleotideexchange factor Alsin seems to interact with both Rab5 and Rac1, thus providing further molecular links between endocytosis and actin remodelling [81].

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with E-cadherin to regulate the proteolytic cleavage of the cadherin–catenin complex and the disassembly of AJs [25]. The deubiquitinating enzyme Fat facets in mouse (FAM) localizes to the Golgi complex in some epithelial cells and associates with the E-cadherin–b-catenin complex in a manner that suggests its involvement in trafficking to the plasma membrane [26]. The human homologue of the Discs large protein (hDlg) binds to E-cadherin and functions as a molecular scaffold that links E-cadherin to phosphatidylinositol 3-kinase (PtdIns 3K) signalling [27]. Disruption of this interaction results in the redistribution of E-cadherin from AJs to intracellular locations. hDlg might regulate the stability of the E-cadherin–actin association at the junction, ultimately affecting both exocytosis and endocytosis. There are many additional proteins that associate with E-cadherin at AJs but, because these proteins have not yet had specific roles in trafficking described for them, they are not discussed herein. The vesicles that transport basolateral-membrane proteins from the TGN to the cell surface dock and fuse at specific sites on the apicolateral membrane of polarized epithelial cells [28]. The Sec6/8 complex [29] in mammalian cells and its counterpart in budding yeast, the multigene exocyst complex [30], delineate sites for vesicle docking on the plasma membrane. In epithelial cells, Sec6/8 complexes are concentrated at the apical junctional zone of the lateral membrane by forming a series of sequential interactions with junctional proteins, including E-cadherin [31]. Thus, E-cadherin has dual roles as both cargo and organizer, which helps to guarantee its own delivery to the appropriate sites on the lateral membrane. A syntaxin-4– SNARE complex on the lateral membrane [32] is also likely to be involved in the fusion of E-cadherin-containing vesicles with the plasma membrane. Endocytosis of surface cadherin: many means to many ends E-cadherin leads a dynamic existence, even after it is delivered to the cell surface and incorporated into AJs. In recent years, a host of studies using different cadherins and cell systems have highlighted the prevalence and functional importance of surface-cadherin internalization. Accordingly, many potential regulators of this trafficking and several possible routes of endocytosis have been identified, including both clathrin-dependent [33,34] and clathrin-independent pathways [35], and even caveolae [36,37]. To verify the nature of the vesicles that are used

for cadherin trafficking by these pathways, definitive studies such as immunolabelling at an ultrastructural level are needed. This is particularly true in the case of caveolae, which are, ultimately, defined only as morphological entities [38]: generally, biochemical markers such as caveolin-1 are not specific to caveolae. It is probable that E-cadherin can use many pathways for its internalization from the cell surface. These many means could, ultimately, lead to many ends, including the recycling of E-cadherin back to the cell surface or its transient sequestration inside the cell (by sorting or recycling endosomes) and/or routeing for eventual degradation (by late endosomes and lysosomes) (Figure 1). Superimposed onto these membrane pathways are physiological conditions that initiate the internalization of E-cadherin. Table 2 summarizes a series of possible routes for E-cadherin endocytosis and the physiological scenarios that are associated with them. It will be important to determine the vesicular pathways and endocytic compartments that are involved in E-cadherin endocytosis as they relate to specific physiological stimuli or developmental events. Experimentally, the internalization of surface E-cadherin is commonly induced using the Ca2Cswitch technique. The reduction of extracellular Ca2C disrupts cell–cell contacts and induces the internalization of E-cadherin en masse as intact hemi-junctions [39] into special compartments that also contain the surface proteins syntaxin 4 and NaC/KC-ATPase [34]. Although this method provides a useful experimental tool [33,36,40], it is not readily reconciled with any of the physiological scenarios described in Table 2. It will be interesting to determine how and where the decisions are made about the fate of E-cadherin. A strong preliminary speculation is that the early or sorting endosome is a crucial central site for decision making that is related to cadherin function. Cadherins that are internalized by several routes could, and apparently do, pass through Rab5- and EEA1-positive early endosomes [33,40]. The early endosome has a slightly acidic environment, a tubular morphology and many attendant proteins to customize it for functions such as the dissociation of receptors and ligands, the targeting of proteins for recycling or degradation, and the recruitment and activation of signalling cascades [3]. All of these functions are used in one or more scenarios of cadherin trafficking. It is tempting to speculate that the decision to recycle or degrade E-cadherin is enacted, at some level, by the endosome-retention machinery. Members of the Sorting nexin (SNX) protein family that is present from yeast to

Table 2. Possible routes and scenarios for E-cadherin endocytosis Routea

Characteristics

Effect on adhesion

Refs

1 2

Unregulated internalization of E-cadherin (e.g. in sparse, non-adherent cells) Variable recycling capacity (e.g. during morphogenesis or in mature epithelia)

[35] [33]

3

Signal-mediated endocytosis of E-cadherin (e.g. with RTKs in morphogenesis)

4

Degradation of E-cadherin (e.g. by transcriptional downregulation)

None ‘Fine tuning’ or major temporary downregulation Temporary or permanent downregulation by recycling or degradative pathways Permanent loss of adhesion

a

[40,42,75] [49,50]

These routes can be viewed as ‘working definitions’ of endocytosis in the context of different physiological situations. Each route might involve the uptake of E-cadherin by one or more types of vesicle (e.g. clathrin coated or non-clathrin coated). The division of routes 2, 3 and 4 is arbitrary because they relate to overlapping scenarios but all relate (in ways that influence adhesion) to regulated, stimulus-coupled or cognate forms of endocytosis. This is in contrast to the unregulated, bulk uptake of membrane and Ecadherin (route 1) that is, nonetheless, governed by specific trafficking machinery [e.g. ADP-ribosylation factor (ARF)6] [35].

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humans are ubiquitous and are involved in various aspects of endocytic trafficking. One such member, SNX1, localizes to early endosomes, in which it can bind to and influence the relative amounts of epidermal growth factor (EGF) receptor and other receptors that are targeted for recycling or degradation (for review, see Ref. [41]). Thus, through the activities of the early endosome, cadherin-based cell–cell adhesion and catenin functions could be downregulated to varying degrees, either in a cognate fashion during morphogenesis or inadvertently in metastasis. E-cadherin endocytosis in morphogenesis and development To regulate their adhesive potential, epithelial cells at different stages of development or in morphogenesis are required to balance the relative levels of exocytic and endocytic trafficking of E-cadherin (Figure 2). The different routes for E-cadherin endocytosis (Table 2) are also relevant in this context because they provide the opportunity for intracellular trafficking with or without influencing adhesion. An important technical point that is relevant to the staining or biochemical detection of E-cadherin in vesicles is that intracellular vesicles containing E-cadherin can belong to either exocytic or endocytic pathways (Figure 2). This is particularly evident in preconfluent cells [Table 2 (route 1)]. The careful dissection and quantification of both exocytic and endocytic traffic might shed further light on how cadherin-based adhesion is regulated jointly by the delivery and turnover of E-cadherin at the plasma membrane throughout development and in cancer. The presence of an ongoing pathway for the recycling of E-cadherin at the surface provides a ready-made mechanism for cells to modulate cell adhesion dynamically. For example, in confluent, polarized MDCK cells, a small amount of surface E-cadherin recycles to and from the plasma membrane, even in the absence of exogenous experimental manipulation [33]. This provides a variable capacity for adhesive cells to fine tune their adhesion and morphology [Table 2 (route 2)]. For example, stimulation with growth factors or epithelial-to-mesenchymal transition (EMT) [42] might upregulate the same or a related endocytosis pathway that results in recycling or other fates for internalized cadherins. This represents a signalmediated route of internalization for this cadherin [Table 2 (route 3)]. The fate of endocytosed E-cadherin reflects the need for either transient or permanent downregulation of E-cadherin-based adhesion: for example, in wound healing or metastasis, respectively. Signalling by tyrosine kinases such as Src or by several receptor tyrosine kinases (RTKs) regulates the expression, function and trafficking of E-cadherin. Tyrosine phosphorylation of the E-cadherin–catenin complex results in the endocytosis of E-cadherin and the disruption of cell–cell adhesion [43]. Fujita et al. [44] identified Hakai, which is a c-Cbl-like ubiquitin ligase that binds to tyrosine residues in the juxtamembrane tail domain of E-cadherin. Hakai ubiquitinates E-cadherin in response to hepatocyte growth factor (HGF) signalling to induce E-cadherin endocytosis, thereby reducing www.sciencedirect.com

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cell–cell adhesion and facilitating cell motility [44]. Hakai-induced ubiquitination might be either a general regulator of E-cadherin endocytosis or a regulator that operates in specific circumstances such as at the behest of RTKs. The binding sites for Hakai and p120ctn are closely apposed in the intracellular juxtamembrane domain of E-cadherin and, accordingly, p120ctn is displaced by the binding of Hakai to E-cadherin before endocytosis [44]. In a recent study, Hunter and colleagues [37] proposed an integrated model of malignant transformation that involves E-cadherin endocytosis, EGF and b-catenin signalling, and caveolin-1 function. The authors reported that chronic EGF stimulation of epidermoid carcinoma cells initiates a complex interplay between the transcriptional repression and the downregulation of E-cadherin. The resulting EMT-like progression requires the endocytosis of surface E-cadherin and the involvement of caveolin-1 [37]. Several other recent studies have suggested a more intimate association between RTKs and the E-cadherin– catenin complex. The physical interaction of RTKs with the E-cadherin–catenin complex, partly in concert with b-catenin, has been reported for c-Met, the insulinlike growth factor (IGF)-1 receptor and the EGF receptor (for review, see Ref. [45]). This potentially more direct mechanism for the regulation of adhesion and morphogenesis might involve the co-endocytosis of cadherin and RTK [40]. However, E-cadherin is not alone in its heterophilic interactions with RTKs. The vascular endothelial growth factor (VEGF) receptor 2 associates with VE-cadherin [45] and the association of different members of the fibroblast growth factor (FGF) receptor family with N-cadherin also seems to regulate retention of the complex at the plasma membrane [46]. It will be of interest to determine how the coupling of RTKs to the cadherin– catenin complex might influence the intracellular localization of these proteins and, furthermore, regulate the response of cells to the growth factors themselves. Although the previously mentioned routes of internalization might involve the sequestration of E-cadherin in endosomal compartments, other routes might exist for the directed degradation of E-cadherin to allow for morphogenetic movements and development [Table 2 (route 4)]. In some situations, E-cadherin is delivered to late endosomes and lysosomes for degradation. Recent studies have indicated a role for lysosomes [47] and, possibly, the proteasome [48] in the degradation of cadherins, although the trafficking of cadherin through the later endocytic compartments is yet to be fully mapped. The investigation of E-cadherin regulation at the genomic level has emphasized the key role of E-cadherin adhesive function during both development and malignant transformation. The binding of various transcription factors, perhaps most prominently members of the Snail superfamily, to the E-cadherin promoter represses the transcription of E-cadherin [49–52]. Accordingly, members of the Snail superfamily have emerged as being key regulators in the EMT process, which requires the disruption of E-cadherin adhesive function at the membrane both in cell culture and in vivo [53] (for review, see

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Ref. [54]). However, the fate of the cadherin that is already engaged in cell–cell adhesive contacts is not fully understood in these systems. Furthermore, although the ectopic expression of these repressors alone can adequately halt the transcription of E-cadherin, this is not enough for the total loss of E-cadherin in cells [51,55]. This caveat further emphasizes the requirement for the endocytosis of surface cadherin, concomitant with its transcriptional repression, to disrupt its adhesive function and, presumably, to target the cadherin for degradation. Although several different growth factors can induce the expression of Snail, Slug or SIP1, it is the dynamin-dependent endocytosis of surface cadherin that is required for morphogenetic movements to occur [54,56]. Thus, it will be of interest to determine the importance of intracellular trafficking of cadherins during development. Abuse and misuse of E-cadherin endocytosis The multiple routes of internalization for E-cadherin provide the ability to control adhesion carefully, although the aberrant regulation of these trafficking pathways can also have pathophysiological consequences. Most human metastases arise from epithelial cells and require the disruption of E-cadherin adhesive function for malignant transformation (for review, see Ref. [45]). Furthermore, although several mechanisms have been identified at the genomic level for the disruption of E-cadherin function, the endocytic trafficking of E-cadherin could also be a mechanism for the attenuation of this powerful tumour suppressor protein (Table 2). The endocytosis of E-cadherin also provides an entry portal for pathogens. Two surface proteins on the bacterium Listeria monocytogenes hijack the E-cadherinrecycling machinery to enable internalization of the bacterium from the lumen of the gut into epithelial cells. The Internalin A and Internalin B proteins on the surface of L. monocytogenes bind to E-cadherin and c-Met, respectively, to induce actin-dependent membrane ruffling and, ultimately, internalization (for review, see Ref. [57]). The bacterium Helicobacter pylori, whose effector protein CagA also activates the c-Met receptor, can use a cellscattering-dependent route of entry [58]. Consequently, understanding the endocytic trafficking of E-cadherin has implications for pathogen entry into epithelia, in addition to both development and cancer. p120ctn: motor, anchor and life preserver In a series of recent publications, new mechanisms have emerged for the regulation of E-cadherin exocytosis, endocytosis and turnover through the actions of p120ctn as a partner for a microtubule motor, an anchor and a life preserver, respectively, for E-cadherin [14,47,48]. It is difficult to classify p120ctn as a regulator of a particular internalization route and, furthermore, it might have differing roles in each of the internalization pathways previously described (Table 2). In mammalian cell lines, the levels of p120ctn are crucial for determining the levels of classic cadherins [47,48]. The knockdown of p120ctn using small interfering RNAs (siRNAs) reduces the levels of cadherin expression and cell adhesion, hence the lifepreserver role for this catenin [48]. The reconstitution of www.sciencedirect.com

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p120ctn (or its family members) in p120ctn-mutant cell lines restores both E-cadherin levels and cell–cell adhesion [59]. The rheostatic effect of p120ctn is exerted partly by the regulation of E-cadherin endocytosis, presumably for degradation. Xiao et al. [47] showed that the overexpression of p120ctn blocks both the endocytosis and the downregulation of the related VE-cadherin, ostensibly through lysosomal compartments. Several studies have revealed a potential direct role for p120ctn as a microtubule-motor-associated protein for the vesicular transport of cadherins [14,60,61]. The binding of p120ctn to kinesin heavy chains helps to regulate the microtubule-driven trafficking of N-cadherin to the plasma membrane [14]. Microtubule association also regulates the targeting of p120ctn and its ability to activate Rho GTPases [60,61]. Thus, one of the roles of p120ctn is as a molecular anchor – attaching cadherins to microtubules or stabilizing E-cadherin complexes at AJs, at which it limits the release of cadherins for endocytosis. Furthermore, interplay between p120ctn and Rho GTPases seems to regulate adhesion and motility in a variety of cell types [62]. Therefore, an extended role for p120ctn in E-cadherin trafficking might involve signalling to Rho GTPases that regulate endocytosis themselves (Box 1). The signalling to p120ctn from upstream kinases also seems to regulate the modulatory role of p120ctn in E-cadherin function and turnover. The signalling of both Src and growth factors [e.g. HGF, EGF and plateletderived growth factor (PDGF)] results in p120ctn phosphorylation at several sites [63,64], with Src and RTKs acting either independently or in concert to regulate adhesion [65]. Understanding how differential signalling both to and from p120ctn controls E-cadherin endocytosis is a key issue to be resolved. Several studies have shown that p120ctn is not, at least in some routes, internalized with E-cadherin [47,48]. Curiously, the fate of the other catenins (a- and b-catenin) during endocytosis has not yet been investigated in detail. Observations suggest that some or all of the b-catenin is removed from E-cadherin during endocytosis [33,37] but this important issue has not yet been resolved. The endocytosis of E-cadherin into a series of compartments that have different environments provides the opportunity to disassemble the cadherin– catenin complex dynamically. Coincidentally, this provides opportunities for the independent signalling or functioning of catenins and of E-cadherin itself. Concluding remarks In this article, we have summarized the divergent studies that have provided evidence of E-cadherin trafficking and that have started to piece together the puzzle of how this adhesion molecule is regulated and managed throughout the life of an epithelial cell. Important pieces of the puzzle still outstanding include the full identification of the vesicles that transport E-cadherin to and from the cell surface and the nature of the cytoskeletal associations that enable the dynamic anchoring or movement of cadherin–catenin complexes. The near future should hold important revelations for a broader understanding of cadherin trafficking. Does cadherin endocytosis regulate the functions and signalling of catenins? Indeed,

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endocytic trafficking might be important in the regulation of the balance between the roles of b-catenin in adhesion and transcription (i.e. cadherin-bound versus cytoplasmic or nuclear pools of b-catenin) [66]. In addition, future studies must address the potential cell-type and cadherinsubtype differences in the post-Golgi trafficking of newly synthesized cadherins. In epithelial cells, there are different routes to the basolateral surface, including traffic via intervening endosomes. In MDCK cells, there is direct trafficking from the TGN to the lateral membrane but, as in other epithelia [67], traffic that is moving towards the basolateral surface might be transported via intervening endosomes. Similarly, the working descriptions of the possible routes for E-cadherin endocytosis in Table 2 lend themselves to future refinement or redefinition in different developmental, physiological and pathological contexts. Finally, future studies might reveal new functions for E-cadherin during its trafficking or after its delivery to intracellular compartments.

Acknowledgements We thank members of the Stow laboratory and Rob Parton for lively discussions and reading of the manuscript. We apologize to those authors whose work was not cited directly owing to space limitations. D.M.B. is supported by an Australian Postgraduate Award scholarship and J.L.S. is supported by a fellowship and research grants from the National Health and Medical Research Council of Australia.

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