CADHERINS AS MODULATORS OF CELLULAR PHENOTYPE

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1 Annu. Rev. Cell Dev. Biol : doi: /annurev.cellbio Copyright c 2003 by Annual Reviews. All rights reserved First published online as a Review in Advance on June 20, 2003 CADHERINS AS MODULATORS OF CELLULAR PHENOTYPE Margaret J. Wheelock and Keith R. Johnson Department of Oral Biology, College of Dentistry and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska ; mwheelock@unmc.edu Key Words catenin, signaling, adhesion, junctions Abstract Cadherins are transmembrane glycoproteins that mediate calciumdependent cell-cell adhesion. The cadherin family is large and diverse, and proteins are considered to be members of this family if they have one or more cadherin repeats in their extracellular domain. Cadherin family members are the transmembrane components of a number of cellular junctions, including adherens junctions, desmosomes, cardiac junctions, endothelial junctions, and synaptic junctions. Cadherin function is critical in normal development, and alterations in cadherin function have been implicated in tumorigenesis. The strength of cadherin interactions can be regulated by a number of proteins, including the catenins, which serve to link the cadherin to the cytoskeleton. Cadherins have been implicated in a number of signaling pathways that regulate cellular behavior, and it is becoming increasingly clear that integration of information received from cell-cell signaling, cell-matrix signaling, and growth factor signaling determines ultimate cellular phenotype and behavior. CONTENTS INTRODUCTION THE CADHERIN SUPERFAMILY The Extracellular Domain The Transmembrane and Cytoplasmic Domains CADHERINS IN CELLULAR JUNCTIONS Adherens Junctions Desmosomes Intercalated Disc Endothelial Junctions Synaptic Junctions CADHERINS IN TISSUE DEVELOPMENT AND INTEGRITY Cadherins Are Important in the Establishment of Cellular Polarity Cadherins Mediate the Sorting of Cells in Embryonic Tissues CADHERINS IN CANCER E-Cadherin as a Tumor Suppressor Disruption of the E-Cadherin/Catenin Complex Expression of Inappropriate Cadherins /03/ $

2 208 WHEELOCK JOHNSON SIGNALING THROUGH CADHERINS The wnt Pathway Signaling Through Rho GTPases Signaling Through Receptor Tyrosine Kinases SUMMARY AND PERSPECTIVES INTRODUCTION Cadherins constitute a large family of glycoproteins comprised of an extracellular domain responsible for cell-cell interactions, a transmembrane domain, and a cytoplasmic domain that frequently is linked to the cytoskeleton. Cadherins play a key role in calcium-dependent cell-cell interactions and function not only to establish tight cell-cell adhesion but also to define adhesive specificities of cells. In addition, they have been implicated in a number of signaling pathways that regulate cellular behavior. In development, the expression of each family member is spatiotemporally regulated so as to be correlated with morphogenetic events where cell adhesion or segregation is involved. Because of the importance of cadherins to cell recognition, adhesion, sorting, and signaling, disruption of cadherin function has significant implications in disease states, including cancer. More than 100 proteins that can be placed into the cadherin super family have been identified. To thoroughly review this large family of proteins is a challenge that cannot be met in one article. Thus, where appropriate, we refer the reader to recent reviews covering specific aspects of cadherin function. THE CADHERIN SUPERFAMILY The cadherin family of proteins can be divided into subfamilies based on molecular characteristics. These subgroups include Type I cadherins, Type II cadherins, desmosomal cadherins, protocadherins, and seven-pass transmembrane or Flamingo cadherins. In addition, cadherin family members have been identified that do not fit into a defined subfamily. These include FAT, Daschsous, T-cadherin, Ksp-cadherin, LI-cadherin, and the RET proto-oncoprotein, to name a few. Excellent reviews have been published recently that provide detailed descriptions of the phylogenic and genomic organization of the cadherin superfamily (Angst et al. 2001, Gallin 1998, Nollet et al. 2000, Yagi & Takeichi 2000). Proteins are designated as members of the broadly defined cadherin family if they have one or more cadherin repeats. A cadherin repeat is an independently folding sequence of approximately 110 amino acids that contains motifs with the conserved sequences DRE, DXNDNAPXF, and DXD (Oda et al. 1994, Takeichi 1990). Calcium is essential for cadherin adhesive function and for protection against protease digestion (Grunwald et al. 1981; Hyafil et al. 1981; Takeichi 1977, 1988; Yoshida & Takeichi 1982). Structural studies have shown this is because calcium ions bind to specific residues in each cadherin repeat to ensure its proper folding and to confer rigidity upon the extracellular domain (Koch

3 CADHERINS 209 et al. 1999). The name cadherin was coined by Takeichi and stems from calciumdependent cell-cell adhesion system (Yoshida-Noro et al. 1984). The Extracellular Domain Type I and Type II cadherins have five extracellular (EC) repeats, and Type I can be distinguished from Type II by the presence of a histidine, alanine, valine (HAV) tripeptide within the most N-terminal extracellular repeat (EC1). Type I and Type II cadherins promote mainly, but not exclusively, homotypic interactions, and EC1 is essential for these interactions. Evidence supporting this idea comes from studies demonstrating that antibodies produced against the extracellular domain of cadherins inhibit cell-cell adhesion and that the binding sites for many of these antibodies map to EC1. In addition, cadherin molecules with a deletion in EC1 fail to mediate cell-cell interactions. Evidence for involvement of the HAV sequence in Type I cadherins comes from mutational studies and from studies showing that synthetic peptides containing a HAV sequence inhibit cadherin-mediated adhesion (Blaschuk et al. 1995; Johnson et al. 1999; Knudsen et al. 1998; Takeichi 1990; Wheelock et al. 1996, 2001). Structural studies of EC1 or EC1 plus EC2 from various Type I cadherins have shown that the HAV tripeptide and surrounding amino acids mediate cadherin self-association through interactions with a separate set of amino acids within EC1 of the interacting cadherin on the adjacent cell (Nagar et al. 1996, Overduin et al. 1995, Shapiro et al. 1995). Recently the entire extracellular domain of the Type I cadherin, Xenopus C-cadherin, was crystallized and the structure solved. The adhesive face that is proposed to mediate the interactions between two cells is a twofold symmetric interaction defined by a conserved tryptophan side chain in cadherin EC1 from one cell that inserts into a hydrophobic pocket in EC1 from an adjacent cell (Boggon et al. 2002). Exceptions to homotypic interactions have been noted in both Type I and Type II cadherins. For example, the Type I cadherins, N-cadherin and R-cadherin, interact with one another to form trans dimers as well as cis dimers (Shan et al. 2000). In addition, Xenopus B-cadherin has been shown to form functional dimers with chicken E-cadherin (Murphy-Erdosh et al. 1995). Shimoyama et al. transfected cadherinnegative L-cells with a variety of Type II cadherins and showed that these cadherins mediated strong homotypic interactions, but also mediated heterotypic interactions, depending upon the particular combinations tested (Shimoyama et al. 2000). The desmosomal cadherins, desmoglein and desmocollin, have been best studied in human and mouse. Three genes encode desmoglein (desmogleins 1 3), and three genes encode desmocollin (desmocollins 1 3). Desmocollins each have five EC domains. Desmoglein 1 has four EC domains, whereas desmogleins 2 and 3 each have five EC domains. Similar to the specificity of the Type I and II cadherins, the specificity for protein-protein interactions in the extracellular domain of the desmosomal cadherins lies within EC1, and adhesion is dependent upon calcium ions. In vivo studies using transfected cells have indicated that the desmosomal cadherins interact in a heterotypic manner (Garrod et al. 2002). That is, desmoglein interacts with desmocollin, and cells must express at least one desmoglein isoform

4 210 WHEELOCK JOHNSON and one desmocollin isoform to assemble a desmosome. However, a recent in vitro study by the Magee laboratory used purified recombinant EC1-2 of desmoglein 2 and EC1-2 of desmocollin 2 in biophysical studies to demonstrate an interaction between these two peptides (Syed et al. 2002). These authors used sedimentationequilibrium and BIAcore experiments to show that desmocollin displays both homotypic and heterotypic interactions, whereas desmoglein participates only in heterotypic interactions. Since structural data on the extracellular domains of the desmosomal cadherins have not yet been presented, the specific molecular and structural interactions necessary to promote cell-cell adhesion are not known. The protocadherins were identified by Suzuki s laboratory when they used degenerate PCR primers directed against the extracellular domain of Type I cadherins to identify new family members in rat brain (Sano et al. 1993). They identified a diverse subfamily of molecules distantly related to Type I cadherins, which they termed protocadherins. Phylogenetic analysis has excluded some cadherins previously considered to be protocadherins, e.g., Flamingo, FAT, DN-cadherin and Daschsous, from this subgroup (Nollet et al. 2000). Protocadherins typically have 6 or 7 extracellular cadherin repeats, but family members have been identified that have as few as 4 or as many as 11 extracellular cadherin repeats (Frank & Kemler 2002, Nollet et al. 2000, Yagi & Takeichi 2000). Standard aggregation assays using expression of transfected proteins into cadherin-negative L-cells were used to show that some protocadherins can mediate homotypic calcium-dependent cell-cell interactions, even if they do not evoke the strong cell-cell interactions mediated by Type I or Type II cadherins (Obata et al. 1995, Sano et al. 1993, Telo et al. 1998, Yamagata et al. 1999). An unusual subfamily of cadherins, termed flamingo cadherins, was first identified in Drosophila. Flamingo (formerly called starry night) is one of a group of proteins that regulates planar cell polarity in Drosophila (Chae et al. 1999). This group of planar polarity proteins also includes wingless (or wnt), armadillo (the prototype of the family to which β-catenin and plakoglobin belong), frizzled, and disheveled. The extracellular domains of the flamingo cadherins have eight or nine cadherin repeats, as well as other structural motifs, including the flamingo box, cysteine-rich regions, laminin A globular domains, and EGF-like motifs (Usui et al. 1999). Drosophila flamingo has nine extracellular cadherin repeats; its mouse homologue (mouse flamingo 1) has eight cadherin repeats (Usui et al. 1999); and a recently identified human homologue (EGFL2) has nine cadherin repeats (Vincent et al. 2000). Transfection of Drosophila S2 cells, which exhibit very weak endogenous cell aggregation, with Drosophila flamingo resulted in cells that robustly aggregated, demonstrating that flamingo cadherins can mediate calcium-dependent cell-cell adhesion (Usui et al. 1999). The Transmembrane and Cytoplasmic Domains Most, but not all, cadherins are Type I single-pass transmembrane proteins. Exceptions include T-cadherin (also known as H-cadherin), which is linked to the plasma

5 CADHERINS 211 membrane through a glycosylphosphatidylinositol lipid anchor (Lee 1996, Ranscht & Dours-Zimmermann 1991), and the flamingo cadherins, which are seven-pass transmembrane proteins (Chae et al. 1999, Usui et al. 1999). The majority of Type I and Type II cadherins are transmembrane components of adherens-type junctions and, as such, have a cytoplasmic domain that interacts with catenins, which in turn link the cadherin to the actin cytoskeleton and facilitate clustering into the junctional structure. The desmosomal cadherins make up the transmembrane component of a separate cell-cell junction, the desmosome, and are associated with plakoglobin and desmoplakin, which link the cadherins to the intermediate filament cytoskeleton (Green & Gaudry 2000). Some cadherins such as Ksp cadherin and LI-cadherin have very short cytoplasmic domains and are not likely to be associated with the cytoskeleton. Likewise, T-cadherin (or H-cadherin), which is linked to the plasma membrane through a lipid tail, is not associated with the cytoskeleton. The cytoplasmic domains of protocadherins are quite diverse and are unrelated to those of Type I or II cadherins. Moreover, there is little evidence that they bind to cytoskeleton-associated proteins (Nollet et al. 2000, Sano et al. 1993, Telo et al. 1998). However, protocadherins do localize to regions of cell-cell contact and can influence cellular signaling pathways (Frank & Kemler 2002, Yagi & Takeichi 2000). For example, members of one subfamily of protocadherins, the cadherin-related neuronal receptors (CNR cadherins), are found within the synaptic junctions in the brain and were first identified because their cytoplasmic domains interacted with the tyrosine kinase Fyn. It has been proposed that the CNR cadherins are involved in generating specific neuronal connections. The genomic organization of the CNR cadherins is similar to that of immunoglobulin genes, and there is evidence of class switching that could give rise to a large number of diverse transcripts (Wu & Maniatis 1999). The flamingo cadherins, which span the plasma membrane seven times, share homology with G protein coupled receptors and, as such, are likely to be involved in a number of cellular signaling pathways (Chae et al. 1999, Usui et al. 1999). These proteins do not bind catenins or other known cytoskeleton-associated proteins and thus are not associated with the cytoskeleton via mechanisms employed by Type I, Type II, or desmosomal cadherins. They do, however, localize to cell-cell borders in a polarized manner and appear to regulate cell polarity in Drosophila wing hair cells (Chae et al. 1999, Usui et al. 1999). Figure 1 presents representative examples of various types of cadherins. CADHERINS IN CELLULAR JUNCTIONS Cadherins were first identified as the proteins responsible for calcium-dependent cell-cell interactions. As the molecules involved in these interactions were characterized, it became clear that cadherins were the transmembrane component of cellular structures that had been previously documented as cell-cell junctions at the microscopic level.

6 212 WHEELOCK JOHNSON Adherens Junctions The adherens junction is a cellular structure found near the apical surface of polarized epithelial cells. E-cadherin is typically the cadherin found in the adherens junction, although other Type I and Type II cadherins are found in similar structures in various cell types. For example, squamous epithelial cells express both E-cadherin and P-cadherin, and each of these cadherins is found within adherens junctions (Johnson et al. 1993). In culture, normal fibroblasts and some cell lines derived from fibrosarcomas form junctions that structurally resemble adherens junctions but contain N-cadherin or P-cadherin rather than E-cadherin (Ko et al. 2000, Sacco et al. 1995, Yonemura et al. 1995). At the cytoplasmic face of the junction, β-catenin or plakoglobin interacts directly with a core region of 30 amino acids within the C terminus of the cadherin cytoplasmic domain (Jou et al. 1995, Stappert & Kemler 1994). Beta-catenin and plakoglobin are both members of the Armadillo family of proteins and share about 65% identity (Fouquet et al. 1992). Although these two proteins likely play very different roles in cellular signaling pathways (Zhurinsky et al. 2000), it is thought that they can directly substitute for one another as structural components of the adherens junction (Butz et al. 1992, Knudsen & Wheelock 1992, Peifer et al. 1992). Beta-catenin and plakoglobin consist of N- and C-terminal tails flanking a central armadillo repeat domain composed of 12 armadillo repeats, of approximately 42 amino acids each, that mediates binding to the cadherin cytoplasmic domain. The three-dimensional structure of the armadillo repeat region of β-catenin shows that each armadillo repeat is made up of three α helices that pack against one another to form a super helix featuring a positively charged groove (Huber et al. 1997). When the cytoplasmic domain of E-cadherin was cocrystallized with the armadillo repeat region of β-catenin, Huber & Weis showed that the interaction face spanned the entire length of the armadillo repeats of β-catenin and involved the C-terminal 100 residues of the cadherin cytoplasmic domain (Huber & Weis 2001). The N-terminal portion of both β-catenin and plakoglobin interacts with α- catenin, which links the cadherin to the cytoskeleton (Aberle et al. 1994, Hulsken et al. 1994, Jou et al. 1995, Pokutta & Weis 2000, Rubinfeld et al. 1995, Sacco et al. 1995). Studies from a number of laboratories showed that cells either lacking α-catenin or sustaining mutations in α-catenin did not form the tight cell-cell contacts typical of adherens junctions (Hirano et al. 1992, Nagafuchi et al. 1994, Nagafuchi & Tsukita 1994, Watabe et al. 1994). Further data from Nagafuchi & Tsukita showed that both the N terminus and the C terminus of α-catenin participated in connecting the cadherin complex to the cytoskeleton (Nagafuchi et al. 1994). Alpha-catenin interacts with the actin cytoskeleton both indirectly through actin-binding proteins such as α-actinin, vinculin and ZO1, and directly through interactions with actin filaments (Knudsen et al. 1995, Nieset et al. 1997, Rimm et al. 1995, Watabe-Uchida et al. 1998, Weiss et al. 1998). Thus the cell-cell adherens junction is a plasma membrane structure composed of transmembrane cadherins associated directly with either β-catenin or plakoglobin, which in turn, associate with

7 CADHERINS 213 α-catenin. Alpha-catenin mediates the interaction between the cadherin-catenin complex and the actin cytoskeleton. In addition to β-catenin and plakoglobin, another catenin, p120 catenin, binds to the cytoplasmic domain of Type I and Type II cadherins. p120 catenin is a member of a subgroup of armadillo family members that have been shown to be localized in various cellular junctions (Anastasiadis & Reynolds 2000). p120 catenin was initially identified as a Src substrate and subsequently shown to interact with the highly conserved juxtamembrane domain of cadherins (Reynolds et al. 1989, 1992, 1994). Beta-catenin and plakoglobin bind to the C terminus of the cadherin cytoplasmic tail in the so-called catenin-binding domain, and their binding is independent of p120 catenin binding. Unlike β-catenin and plakoglobin, p120 catenin does not appear to have a structural role in the junctional complex. Rather, it is thought to regulate, both positively and negatively, cadherin adhesive activity (Thoreson et al. 2000, Yap et al. 1998). How p120 catenin regulates cadherin adhesive activity is not yet understood, and the answer could be quite complicated because there are numerous p120 catenin splice variants and a number of closely related family members (Anastasiadis & Reynolds 2000). Similar proteins include ARVCF (armadillo repeat gene-deleted in Velo-Cardio-Facial syndrome), δ-catenin, and p0071 (Anastasiadis & Reynolds 2000). These proteins are also localized to adherens junctions and may function in a manner similar to p120 catenin. Lateral dimers seen in the crystal structure of EC1 of N-cadherin prompted the proposal that cadherins cluster into junctional complexes via cis interactions between cadherins on the surface of the same cell (Shapiro et al. 1995). Although the exact mechanism of lateral dimerization has been controversial, both biological and structural studies support this hypothesis, and it is generally accepted (Boggon et al. 2002). Co-immunoprecipitation experiments demonstrated that cadherins form dimers in calcium concentrations that are too low to support adhesive (or trans) dimers (Chitaev & Troyanovsky 1998, Klingelhofer et al. 2002). Gumbiner s laboratory used a unique protein oligomerization system to show that when cadherins are present on the cell surface in a cluster, they promote stronger adhesion than when they are unclustered (Yap et al. 1997). This laboratory also showed that the juxtamembrane domain (corresponding to the binding site for p120 catenin) is essential for clustering of the cadherin, suggesting that this may be one role that p120 catenin plays in the adherens junction (Yap et al. 1998). Figure 2 presents a cartoon of an adherens junction. Desmosomes Desmosomes are prominent cellular structures especially abundant in tissues that experience mechanical stress. Structurally, the desmosome is similar to the adherens junction. That is, it is composed of a transmembrane cadherin and proteins that link the cadherin to the cytoskeleton, in this case, the intermediate filament cytoskeleton. The desmosomal cadherins interact in a heterotypic manner; thus

8 214 WHEELOCK JOHNSON each desmosome must have at least one desmocollin and one desmoglein. The cytoplasmic domains of desmogleins and desmocollins have been shown to bind directly to plakoglobin (Korman et al. 1989; Mathur et al. 1994; Troyanovsky et al. 1994a,b). In addition, the domains on plakoglobin that interact with the desmosomal cadherins have been identified (Chitaev et al. 1996, Wahl et al. 2000, Witcher et al. 1996). Plakoglobin, in turn, interacts with desmoplakin, which interacts with the keratin intermediate filament cytoskeleton of epithelial cells (Bornslaeger et al. 1996, Kouklis et al. 1994, Kowalczyk et al. 1997). Desmosomes also contain a member of the p120 catenin subfamily of armadillo proteins termed plakophilins. As with p120 catenin in the adherens junction, the precise role plakophilins play in the desmosome is not yet elucidated. Excellent reviews describing the assembly and function of desmosomes have recently been published and the reader is referred to them for more information about these structures (Green & Gaudry 2000, Ishii & Green 2001). Figure 2 presents a cartoon of a desmosome. In stratified squamous epithelial tissues, desmoglein and desmocollin are expressed in a differentiation-specific manner. Desmoglein 1 and desmocollin 1 appear to be the major desmosomal cadherins in the epidermis where they are most prominent in the upper layers. Desmoglein 3 is expressed at high levels in the basal layers, and its expression decreases as the cells differentiate (Jensen & Wheelock 1996). In addition, squamous epithelial tissues such as the oral mucosa do not show the same expression pattern as does the epidermis (Garrod et al. 2002). Thus expression of desmosomal cadherins is not only differentiation-specific within a single tissue but is also tissue specific. Desmoglein 2 and desmocollin 2 are ubiquitously expressed in all tissues that make desmosomes, whether they are squamous epithelial tissues or simple epithelial tissues. However, even simple epithelia have differential expression of plakophilin family members (Borrmann et al. 2000). Therefore, it has been suggested that desmosomal cadherins may play a role in directing differentiation in epithelial tissues (Borrmann et al. 2000, North et al. 1996). Intercalated Disc In cardiac muscle, structural integrity is maintained by a unique junctional complex termed the intercalated disc, which consists of three separate junctions: the adherens junction, the desmosome, and the gap junction (Severs 1990). N-cadherin is the transmembrane protein found in the cardiac adherens junction, which is the site of attachment of the myofibrils and enables transmission of contractile force. The desmosome interacts with desmin intermediate filaments to provide structural support, and the gap junction provides intercellular communication. Desmoglein 2 and desmocollin 2 are the desmosomal cadherins in the intercalated disc. Transgenic mouse studies have shown that N-cadherin in the adherens junction of the heart plays a critical role in maintaining cardiac function (Ferreira-Cornwell et al. 2002, Luo et al. 2001). In addition, targeted disruption of the plakoglobin gene was lethal at day of embryonic development, and the defect was shown to

9 CADHERINS 215 be in cardiac cell-cell adhesion, indicating that the desmosome in the intercalated disc is essential for cardiac function (Ruiz et al. 1996). Endothelial Junctions Endothelial cells also form a unique type of cadherin-containing cell-cell junction. These cells express approximately equal levels of N-cadherin (a Type I cadherin) and VE-cadherin (an endothelial cell specific Type II cadherin). VE-cadherin is localized to junctions, whereas N-cadherin is diffusely localized on the surface of these cells (Navarro et al. 1998). The role of N-cadherin in endothelial cells has not yet been elucidated, but it is quite clear that this cadherin is not a component of the endothelial cell junctional complex. VE-cadherin forms complexes with β-catenin, plakoglobin, and p120 catenin and likely establishes connections to the actin cytoskeleton that resemble typical adherens junctions (Lampugnani et al. 1995). Similar to epithelial cells, endothelial cells have junctions that are connected to the intermediate filament cytoskeleton. Unlike epithelial cells, endothelial cell intermediate filaments are composed of vimentin rather than keratin. Also, unlike epithelial cells, endothelial cells do not express any desmosomal cadherins. VEcadherin that is associated with β-catenin is restricted to typical α-catenin/actin interactions. However, when VE-cadherin is associated with plakoglobin, it can either connect to the actin cytoskeleton through α-catenin, forming a typical adherens junction, or it can associate with vimentin filaments through interactions with desmoplakin (Kowalczyk et al. 1998). This second type of junction has been termed a complexus adherens (Lampugnani & Dejana 1997). VE-cadherin is endothelial cell specific and forms a unique association with both actin filaments and vimentin intermediate filaments. A diagram of an endothelial cell junction is presented in Figure 2. Synaptic Junctions Chemical synapses are specialized junctions between cells in the nervous system that occur between a presynaptic nerve terminal and a postsynaptic target cell. The cells are electrically isolated from one another by the narrow synaptic cleft. At the active zone, the presynaptic cell releases neurotransmitter in precisely the position where the receptors on the target cell are located. Type I cadherins (E-cadherin and N-cadherin) and catenins are localized at regions just outside the active zone and mediate cell-cell adhesion between the presynaptic cell and the postsynaptic cell (Uchida et al. 1996). A sub-class of protocadherins, termed CNRs, are also found in synaptic junctions; however, they are within the active zone (Yamagata et al. 1999). It has been suggested that the CNR protocadherins play a role in synaptic cleft signaling, in addition to cellular adhesion, because they form a molecular complex with Fyn tyrosine kinase within the active zone (Kohmura et al. 1998). Thus, it is likely that Type I cadherins form a junction structurally similar to the adherens junction that serves to isolate the synaptic cleft and CNR protocadherins function within the cleft. Synaptic junctions and the role

10 216 WHEELOCK JOHNSON cadherins play in these junctions have been recently reviewed (Bruses 2000, Goda 2002, Yagi & Takeichi 2000). CADHERINS IN TISSUE DEVELOPMENT AND INTEGRITY The formation of tissues during embryogenesis requires the coordination of cellular processes, including polarization, aggregation, segregation, and migration. Each of these processes depends on the expression of specific adhesion proteins. The different cadherin family members are expressed in specific spatiotemporal patterns during the development of an embryo, which is consistent with the idea that cadherins play a role in the regulation of morphogenesis and tissue formation. Experimental data, both in vitro and in vivo, support this concept. Cadherins Are Important in the Establishment of Cellular Polarity A critical event in early vertebrate development is the polarized assembly of junctional proteins at the apical surface of cells, which facilitates compaction. Cultured MDCK cells have provided an excellent model system to study the role cadherins play in establishing cell polarity. Epithelial cell polarity is maintained, in part, because apical and basolateral proteins are sorted into distinct vesicles in the trans Golgi network and are targeted to their respective membrane compartments. Epithelial cells receive molecular cues via cell-cell contact that is mediated by E-cadherin. The association of E-cadherin with underlying cytoskeletal elements results in targeting of synthetic vesicles to the appropriate membrane (Yeaman et al. 1999). The importance of cell-cell adhesion to the targeted secretion of proteins at the apical surface of MDCK cells was first demonstrated by depleting calcium in the culture medium (Vega-Salas et al. 1987), and the central role of E-cadherin in this process was demonstrated by Nelson s laboratory (McNeill et al. 1990). These authors transfected non-polar L-cell fibroblasts with E-cadherin and showed that the transfected cells redistributed Na +,K + -ATPase and fodrin from a diffuse pattern to regions of cell-cell contact that resembled those seen in polarized epithelial cells. Establishment of cellular polarity occurs in the earliest stages of vertebrate development and is mediated by E-cadherin. As expected, targeted disruption of the gene encoding E-cadherin in mice is lethal very early in development, which complicates investigations into the role of cadherin-mediated cellular polarity in vertebrate development (Larue et al. 1994, Riethmacher et al. 1995). However, well-designed studies in Drosophila and Caenorhabditis elegans have established a role for cellular polarity in invertebrate development. Drosophila and C. elegans express cadherin family members in tissue-specific patterns, similar to those seen in vertebrates. Genetic screens have identified proteins that are required for the

11 CADHERINS 217 proper localization of proteins in a polarized fashion in a number of cell types, and these proteins include not only cadherins but also scaffolding proteins and signaling molecules. For example, an atypical protein kinase C, PKC-3, in a complex with its binding partner (PAR-3/ASIP), is required for establishment of the embryonic anterior-posterior axis in C. elegans. This complex functions at the single-cell stage to correctly localize proteins within the membrane, ensuring appropriate protein distribution at the first asymmetric cell division (Ohno 2001). Ohno and colleagues showed that the mammalian homologue of PAR-3/ASIP is localized at junctional complexes in cultured MDCK cells and that disrupting the function of this complex using a dominant-negative form of PKC-3 resulted in disruption of junctional complexes and a non-polarized localization of Na +,K + -ATPase (Suzuki et al. 2001). Excellent reviews by Wedlich and Ohno discuss the importance of cell polarity to invertebrate development and speculate on the implications of these findings to vertebrate development (Ohno 2001, Wedlich 2002). Cadherins Mediate the Sorting of Cells in Embryonic Tissues The experimental sorting of intermixed embryonic cells into their respective tissues was demonstrated almost 50 years ago (Townes & Holtfreter 1955). Since that time, it has become clear that cadherins constitute a major cellular adhesion/recognition system that confers upon cells the ability to sort from one another. For example, Takeichi s laboratory showed that cadherin-negative L-cells transfected with E-cadherin segregated from L-cells transfected with P-cadherin when these two populations of cells were mixed together in an aggregation assay (Nose et al. 1988). In addition, Steinberg & Takeichi showed that cells expressing differing levels of the same cadherin sorted from one another (Steinberg & Takeichi 1994). Takeichi hypothesized that during embryogenesis, cadherins mediate the sorting of groups of cells into specific tissues (Takeichi 1988). The complex expression patterns that have emerged as numerous cadherin family members have been identified lend support to this hypothesis. The general idea is that when a group of cells separates from an existing cell layer, the segregating cells are expressing a qualitatively or quantitatively altered set of cadherin family members. This may mean they turn off some cadherin genes and/or turn on other cadherin genes (Takeichi 1990). Since the inception of this idea, a number of embryonic systems have been carefully examined and the data support this hypothesis. The Radice laboratory used N-cadherin / embryonic stem cells to generate chimeric mouse embryos composed of wild-type and N-cadherin-deficient cells. A total knock out of N-cadherin is lethal in mice at a very early stage owing to severe cardiac defects (Radice et al. 1997). Thus only chimeric mice that had very few N-cadherin-deficient cells in the myocardium survived long enough for the interactions between N-cadherin-negative and N- cadherin-positive cells to be examined. Interestingly, N-cadherin-deficient cells sorted from their surrounding cells in brain and heart, indicating that changes in a single cadherin family member could mediate sorting of otherwise apparently identical cells within an embryo (Kostetskii et al. 2001).

12 218 WHEELOCK JOHNSON The migration of mouse melanocyte precursor cells (melanoblasts) from the neural crest to the dermis, epidermis, and hair follicle illustrates the role of cadherins in the sorting of cells to form a complex tissue that is made up of several distinct cell types. An elegant study by Nishimura et al. (1999) showed that melanoblasts are negative for both E-cadherin and P-cadherin as they migrate into the dermis. Upon entry into the epidermis, there is a 200-fold increase in the expression of E-cadherin, whereas P-cadherin remains low. As the cells migrate further, three distinct populations are established: (a) melanoblasts that are E-cadherin negative /P-cadherin negative and remain in the dermis where they are compatible with the surrounding connective tissue; (b) melanoblasts that are E-cadherin high /P-cadherin low and reside within the epidermis surrounded by keratinocytes that express high levels of E-cadherin and low levels of P-cadherin; and (c) melanoblasts that are E-cadherin negative /P-cadherin high that are found in the hair follicle. P-cadherin is highly expressed by the basal cells of the epidermis and on the hair matrix. Nishimura et al. (1999) proposed that changes in expression of E-cadherin and P-cadherin in the melanoblast leads to differential interactions with the surrounding cells and serves to guide the cells as they migrate to their final destination. Compartmentalization in development is a morphogenetic strategy to pattern the embryo and maintain body structure. For example, in the vertebrate central nervous system the neural tube is divided into several units that serve to restrict the movement of neuroepithelial cells. Restricting cells into their appropriate compartments during development may play a role in providing the basic framework for neural circuits (Figdor & Stern 1993). Combinatorial expression of cadherins subdivides the embryonic brain into functional subunits. A Type II cadherin, cadherin-6, is expressed on the lateral ganglionic eminence, and a Type I cadherin, cadherin-4, is expressed on the future cerebral cortex. Thus, cadherin-6 and cadherin-4 delineate these two neighboring brain subdivisions (Redies & Takeichi 1996). To show that these two cadherins are important in maintaining the boundary between the lateral ganglionic eminence and the cerebral cortex, Inoue et al. (2001) used electroporation to ectopically express cadherin-6 in cells at the boundary. The cells were sorted into the cadherin-6-rich lateral ganglionic eminence. Likewise, when they electroporated cadherin-4, the cells were sorted into the cerebral cortex (Inoue et al. 2001). To show that expression of a specific cadherin within a subdivision of the brain is critical for the ability of the electroporated cells to sort into the proper compartment, Inoue et al. (2001) engineered cadherin-6 null mice and showed that preferential sorting of cadherin-6 electroporated cells was totally abolished. This study provided an elegant in vivo demonstration of the role of cadherins in cell sorting in the brain and provided evidence that differential expression of cadherin-4 and cadherin-6 is necessary to maintain the boundary between the lateral ganglionic eminence and the cerebral cortex. Cadherins of each subtype are highly expressed within the central nervous system, which led Yagi & Takeichi (2000) to propose that the cadherin superfamily plays an important role in establishing and maintaining the complex neural networks that are found within the brain.

13 CADHERINS 219 The above-mentioned studies are but a small fraction of the experimental data implicating cadherins in the complex cell sorting events that occur when an organism is developing and forming tissues. Most investigators in the field accept the idea that cadherins mediate sorting of cells both in vitro and in vivo. However, the molecular mechanisms underlying this activity are not fully understood. It is tempting to assume that the intrinsic binding specificity of the cadherin is responsible for both junction formation and cell sorting. However, cell sorting may be a complex activity that involves not only specific protein-protein interactions at the cell surface but also downstream signaling events. Niessen & Gumbiner (2000) recently developed a unique assay designed to address the role of cadherin adhesive activity in the cell sorting process. These authors used an adhesion flow assay, in which purified cadherin extracellular domain is immobilized on the surface of a capillary tube. Cells expressing various cadherin family members are then allowed to attach to the substrate for a specified length of time, with increasing flow rates then applied. Surprisingly, these authors found that Type I cadherins show a wide range of adhesive properties that was not expected on the basis of their cell sorting properties. For example, in their system, CHO cells transfected with human N-cadherin efficiently sorted from CHO cells transfected with human E-cadherin, as shown by previous studies. However, in their adhesion flow assay, CHO cells transfected with human N-cadherin and CHO cells transfected with human E-cadherin adhered equally well to immobilized extracellular domain of human E-cadherin. These studies suggest that, at least in some cases, cells expressing distinct cadherins cannot aggregate with one another even though the cadherins they express do physically interact, which implicates events downstream of cell-cell interactions in cell sorting. CADHERINS IN CANCER E-Cadherin as a Tumor Suppressor The majority of studies implicating cadherins in tumorigenesis have focused on E-cadherin because it is the major cadherin expressed by epithelial cells, which are the origin of most human cancers. In vitro and in vivo studies showed that inhibiting E-cadherin activity with function-perturbing antibodies changed normal epithelial cells into invasive cells. In addition, invasive E-cadherin-negative carcinoma cells were converted to non-invasive cells by exogenous expression of E-cadherin. Moreover, an important in vivo study showed that downregulation of E-cadherin activity using a dominant-negative form of E-cadherin in a mouse model system for pancreatic cancer (the Rip1Tag2 mouse) resulted in transition of a well-differentiated pancreatic β-cell adenoma to an invasive carcinoma (Perl et al. 1998). These authors further showed that crossing the Rip1Tag2 mice with mice that maintain expression of E-cadherin in the β-tumor cells resulted in arrest at the adenoma stage. It has been well established that E-cadherin functions as a tumor suppressor, and a number of reviews have dealt with this in detail. The

14 220 WHEELOCK JOHNSON reader is referred to these for a more extensive discussion (Behrens 1999, Guilford 1999, Thiery 2002, Wheelock et al. 2001). Downregulation of E-cadherin function can occur via several mechanisms, including gene mutations. Missense mutations, splice site mutations, and truncation mutations have all been reported in the E-cadherin gene and such mutations frequently occur in combination with loss of heterozygosity of the wild-type allele (Berx et al. 1998). Despite the number of mutations that have been reported in the E-cadherin gene, the majority of carcinomas appear to lack mutations in this gene. Nonetheless, immunohistochemical studies show that loss of E-cadherin is common to many tumors, and it is thought that tumor cells must downregulate the activity of E-cadherin in order to invade surrounding tissues (Hajra & Fearon 2002). Thus in most cases, the mechanism for loss of E-cadherin does not involve irreversible, genetic alterations. Aside from mutations in the gene, there are several mechanisms whereby tumor cells can decrease the activity of E-cadherin, including promoter methylation, transcriptional repression, and posttranslational modification of the cadherin/catenin complex. Hypermethylation of CpG islands, which is a mechanism for gene silencing in normal cells and can occur during the process of tumorigenesis, has been demonstrated in the E-cadherin promoter in human carcinomas, including breast, prostate, bladder, colon, and oral cancers (Chang et al. 2002, Graff et al. 2000, Kanazawa et al. 2002, Nass et al. 2000, Ribeiro-Filho et al. 2002). Graff et al. (2000) recently showed that methylation of the E-cadherin promoter in invasive ductal carcinoma of the breast begins early in tumorigenesis, prior to the invasive stage. They further showed that promoter methylation is dynamic, heterogeneous, and unstable. These authors proposed that methylation of the E-cadherin promoter, as well as those of other genes relevant to tumorigenesis, may facilitate the dynamic phenotypic heterogeneity that drives metastatic progression (Graff et al. 2000). Epithelial to mesenchymal transitions, in which epithelial cells are converted to motile, fibroblast-like cells, are regular events during normal embryonic development. Similar events are seen as epithelial cells progress through the stages of carcinogenesis. A hallmark of epithelial-to-mesenchymal transition is decreased expression of E-cadherin, and it has been hypothesized that transcriptional repressors that control the expression of E-cadherin in development may also regulate the expression of E-cadherin during tumorigenesis (Thiery 2002). Such repressors include snail, slug, SIP1, and E2A. Batlle et al. (2000) showed that snail binds to 3 E-boxes within the promoter of human E-cadherin and represses its transcription. In addition, transfection of antisense snail mrna was able to restore E-cadherin expression in a pancreatic tumor cell line (Batlle et al. 2000). Cano et al. (2000) extended these studies to show that transfection of snail into MDCK cells resulted in loss of E-cadherin and induction of an invasive phenotype. They further showed that tumors induced in nude mice were invasive when the injected cells expressed snail and that these invasive tumors showed a complete lack of E-cadherin expression (Cano et al. 2000). Hajra et al. (2002) showed that snail and

15 CADHERINS 221 the closely related protein, slug, could repress the expression of E-cadherin when transfected into human breast cancer cell lines, and that expression of slug, but not snail, was closely correlated with repression of the E-cadherin gene in vivo. These authors suggested that slug is likely to function as an E-cadherin repressor in the progression of breast cancer. SIP1, whose specificity partly overlaps with that of snail, is a Smad-interacting zinc finger protein that binds to the E-boxes in the E-cadherin promoter. Studies from the van Roy laboratory showed that SIP1 silences E-cadherin expression in much the same manner as snail and that expression of SIP1 in MDCK cells also produced invasive cells with decreased expression of E-cadherin. They further showed that E-cadherin expression was inversely correlated with SIP1 expression in a number of carcinoma-derived cell lines (Comijn et al. 2001). Another transcriptional repressor, the E2A gene product, E12/E47, has been shown to bind to the E-box region of the mouse E-cadherin promoter and repress E-cadherin expression (Perez-Moreno et al. 2001). Similar to expression by snail and SIP1, the expression of the E2A gene is inversely correlated with that of E-cadherin in invasive human carcinomas. The above studies indicate that downregulation of E-cadherin expression in tumors can be achieved by a variety of different mechanisms. Disruption of the E-Cadherin/Catenin Complex An alternative mechanism for inactivating the adhesive function of E-cadherin in tumor cells is to disrupt the connection between the cadherin and the cytoskeleton. For example, mutations in β-catenin that disrupt its binding to α-catenin result in a non-adhesive phenotype. In addition, mutations in the gene that encodes α- catenin effectively inactivate E-cadherin function by not allowing the cadherin complex to associate with the cytoskeleton. Moreover, cells deficient in catenins display normal adhesiveness when transfected with cdnas encoding functional catenins. The Fuchs laboratory used mice to ablate the gene encoding α-catenin in order to examine the effect of disrupting the connection between E-cadherin and the cytoskeleton (Vasioukhin et al. 2001). Because α-catenin is required for early development, these authors targeted ablation of the gene to the surface epithelia using the K-14 promoter, which is not expressed until embryonic day The α-catenin null epidermis showed hyperproliferation, suprabasal mitoses, and characteristics of squamous cell carcinoma in situ, a precancerous skin condition. Molecular analysis of keratinocytes derived from these mice showed that the hyperproliferative phenotype was due to increased Ras and MAPK activity (Vasioukhin et al. 2001). In addition to mutations that disrupt the connection of E-cadherin to the cytoskeleton, the adhesive strength of E-cadherin can be altered during tumorigenesis by posttranslational modification. E-cadherin harbors a number of serines and threonines within the β-catenin binding domain that are putative phosphorylation sites for casein kinase I and II and glycogen synthase kinase-3β phosphorylation. Lickert et al. (2000) showed that phosphorylation of these sites serves to modulate

16 222 WHEELOCK JOHNSON the affinity of E-cadherin for β-catenin and thus determines the strength of the resulting cell-cell interactions. In vitro studies from Kemler s laboratory suggest that serine/threonine phosphorylation of β-catenin may also regulate its affinity for α-catenin, which would impact cell adhesion by modulating the association of the cadherin/catenin complex with the cytoskeleton (Bek & Kemler 2002). It has been recognized for some time that Src-transformed epithelial cells display decreased cell-cell adhesion. Although the cadherins are poor substrates for tyrosine kinases, β-catenin, plakoglobin, and p120 catenin are highly phosphorylated on tyrosine in Src-transformed epithelial cells. Roura et al. (1999) used recombinant Src to phosphorylate β-catenin in vitro and showed that phosphorylation on tyrosine 654 reduced the affinity of β-catenin for the cytoplasmic domain of E-cadherin. They further showed that tyrosine 654 is phosphorylated under conditions in which adherens junctions are disrupted, suggesting that the decreased affinity of phosphorylated β-catenin for E-cadherin has important in vivo consequences. Expression of Inappropriate Cadherins Finally, studies from our laboratory and others have shown that expression of an inappropriate cadherin in epithelial cells is yet another way that tumor cells can alter their adhesive function (Hazan et al. 1997, Islam et al. 1996, Nieman et al. 1999, Pishvaian et al. 1999). In some cases, this may be due to downregulation of E-cadherin upon expression of the inappropriate cadherin (Islam et al. 1996). In other cases, mesenchymal cadherins can have a direct and dominant influence on the phenotype of epithelial cells, despite their continued expression of E-cadherin. Expression of N-cadherin or cadherin-11 by oral squamous epithelial cells or breast epithelial cells produced cells that were more motile and more invasive in in vitro assays and more metastatic in vivo (Hazan et al. 2000, Nieman et al. 1999, Pishvaian et al. 1999). Using chimeras between E-cadherin and N-cadherin, our laboratory showed that 80 amino acids within extracellular domain four of N-cadherin were necessary and sufficient to transform nonmotile squamous epithelial cells into highly motile invasive cells (Kim et al. 2000). In addition, we presented evidence that N-cadherin s effect on cell motility was mediated through the FGF receptor signaling pathway (Nieman et al. 1999). The Hazan laboratory recently showed that N-cadherin interacts directly with Ig domains one and two in the extracellular domain of the FGF receptor-1 and that this interaction prevents receptor internalization, resulting in increased receptor expression at the cell surface and enhanced downstream signaling (Suyama et al. 2002). Cavallaro et al. (2001) presented further data indicating that N-cadherin also interacts with FGF receptor-4, and in this case, the interaction is mediated by N-CAM, a cell-cell adhesion molecule of the Ig superfamily. In summary, regardless of the mechanism, disrupting the function of the E- cadherin/catenin complex in epithelial cells favors the formation of invasive tumorigenic cells.

17 CADHERINS 223 SIGNALING THROUGH CADHERINS Cells obtain information from their environment to modulate their behavior during normal embryonic development and during abnormal processes such as tumorigenesis. Such signals can be initiated by growth factors, cytokines, and other soluble signaling molecules found in the circulation and in the interstitial compartment. Alternatively, signaling cascades can be activated through interactions of cells with the surrounding extracellular matrix or with one another. Sometimes, an interaction with matrix components or with other cells stimulates the modulation of, or promotes cross talk with, well-established signaling pathways. Two excellent articles discuss the role of cell-matrix interactions mediated by integrins in cell signaling (Juliano 2002, Miranti & Brugge 2002). Integrins can initiate signals directly through focal adhesion kinase and the MAP kinase pathway; they can signal to the cytoskeleton via Rho GTPases and can modulate signals the cell obtains from receptor tyrosine kinases, G protein coupled receptors, and cytokine receptors. It is our hypothesis that cells obtain information not only through contacts with the extracellular matrix, but also through contacts with one another, and it is the integration of signals from these two sources that regulates cellular behavior. Conceptually the life of a cadherin junction can be separated into three stages: formation, maintenance, and disassembly. Junction formation has distinct phases; initial contacts are made, then grow and mature, and eventually form clusters of cadherins and associated molecules that can be recognized ultrastructurally as junctions. In some cases, recruitment of cadherins from intracellular stores may follow the initial cell-cell contact (Mary et al. 2002). Because junctions are not static but dynamic, perturbations in the rate of addition or subtraction of components could have significant effects on whether junctions persist, grow, or disappear. Internalization and recycling of cadherins can play a role in junction maintenance (Le et al. 2002). Disassembly could result from attrition or from a large-scale breaking of contacts, for example by proteolysis. There appear to be cell-type-specific differences in how junctions are initiated and maintained. Thus polarized epithelial cells, squamous epithelial cells, fibroblasts, myoblasts, and other cells may not share identical mechanisms. In addition, there may be cadherin-specific differences in the way junctions form, persist, and disappear. Thus, the investigation into signals downstream of cadherins promises to be both complex and stimulating. Several model systems have been established to investigate signals immediately downstream of cadherin engagement. One relatively clean system involves plating isolated, cadherin-expressing cells on a substrate containing the corresponding recombinant cadherin extracellular domain. The pathways activated as the cells cadherins engage the substrate are determined by examining the recruitment of molecules to the leading, spreading edges of the cells. Another model involves a calcium switch. Because cadherins require approximately 200 nm calcium for activity, the cadherins on monolayers of cells can be activated by an abrupt elevation

18 224 WHEELOCK JOHNSON of the concentration of extracellular calcium. Cultures incubated in the presence of antibodies that block cadherin function serve as controls for the general effects of elevating extracellular calcium. Recent studies using these and other model systems have begun to identify mechanisms whereby cadherins can activate and/or modulate cellular signaling pathways, and we will briefly review some of these here. The wnt Pathway Probably the best-studied signaling pathway that involves cadherins is the β-catenin/wnt pathway. The wnt or wingless pathway plays a major role in developmental processes in vertebrates and invertebrates. Wnt is an extracellular matrix-associated growth factor that interacts with its receptor, a member of the frizzled family, to initiate a signal transduction pathway that stimulates synthesis of proteins involved in cell growth, such as cyclin D1 and myc (reviewed in Conacci-Sorrell et al. 2002). β-catenin s role in this pathway is to bind to members of the TCF/LEF family of transcription factors and function as a co-activator. In the absence of a wnt signal, β-catenin that is not bound to cadherin in an adherens junction is ubiquitinated and degraded by the proteosomal pathway. When wnt binds to its receptor, β-catenin degradation is inhibited, allowing it to accumulate in the nucleus, where it binds to TCF/LEF. The wnt pathway is tightly regulated during development, but its regulation may be disrupted during tumorigenesis. Because the binding of β-catenin to cadherins and to TCF/LEF is mutually exclusive, one way to regulate this pathway is through cadherin binding (Simcha et al. 2001). The Gumbiner laboratory demonstrated this in the colon carcinoma cell line, SW480, that over-expresses β-catenin owing to a mutation in APC, which results in disruption of normal β-catenin degradation (Gottardi et al. 2001). When they transfected E-cadherin constructs into SW480 cells, β-catenin/tcf signaling was inhibited and cell growth was suppressed. Interestingly, this activity was adhesion independent. That is, to be active in signaling and growth suppression, the constructs had to retain the β-catenin-binding site but did not have to retain adhesive activity. These authors concluded that only a fraction of the total β-catenin was available for signaling and that this pool could be decreased by over-expressing E-cadherin. Thus there is strong evidence that cadherins can modulate the wnt signal transduction pathway. Signaling Through Rho GTPases Recent studies have begun to tease apart the connection between cadherin function and Rho-family GTPases, and these studies have been recently reviewed (Braga 2002, Fukata & Kaibuchi 2001, Yap & Kovacs 2003). While the roles Rho family GTPases play in the various stages of a junction s life are being revealed, they have been best characterized in the early stages of junction formation soon after cells are plated on cadherin substrates or make contact following elevation of

19 CADHERINS 225 extracellular calcium. Rac has generally been reported as being activated following cadherin engagement. In different systems, either PI3 kinase or EGF receptor has been reported to be upstream of Rac. The case for Rho is less clear as it is activated in some model systems but not others. An interesting role for p120 catenin as a RhoGDI has been suggested by Reynolds and colleagues (see Anastasiadis et al. 2000, Anastasiadis & Reynolds 2001). Alternatively, p120 catenin may function as an activator of Vav2 as suggested by Burridge and collaborators (Noren et al. 2000). The current idea is that activation of Rho GTPases through interactions with p120 catenin plays a role in clustering of cadherins, which is an early event in the formation of a junction. We refer the reader to the more complete reviews for a description of the potential GEFs and downstream effectors of these GTPases. Clearly, this is an emerging area of signaling that involves cadherins. Signaling Through Receptor Tyrosine Kinases Cadherins have been reported to be intimately involved with several receptor tyrosine kinases. The Walsh and Doherty laboratories have extensively characterized the role of N-cadherin in neurite extension (Doherty et al. 2000, Skaper et al. 2001). The theme from their work is that N-cadherin signals through the fibroblast growth factor receptor. In their model, N-cadherin facilitates dimerization of the FGF receptor to initiate the signaling pathway, and as such, the signal is growth factor independent. A connection between the epidermal growth factor receptor and E-cadherin also has been reported by several investigators (Hoschuetzky et al. 1994, Pece & Gutkind 2000). A recent study found that E-cadherin can induce ligandindependent activation of the EGF receptor and subsequent activation of MAP kinase (Pece & Gutkind 2000). It is interesting to note that both the N-cadherindependent FGF receptor signaling involved in neurite extension and the E-cadherindependent EGF receptor signaling are ligand independent, suggesting that cadherins play a significant role in controlling cell fate through regulation of signaling pathways. The Dejana laboratory has demonstrated a link between VE-cadherin and signaling through the VEGF receptor (Carmeliet et al. 1999, Zanetti et al. 2002). VEGF is required as a survival factor for endothelial cells. In mice lacking VEcadherin, the VEGF signal is not relayed. These authors showed that the VEGF receptor is found in a complex with VE-cadherin, β-catenin, and PI-3 kinase. In this case, the association between VE-cadherin and VEGF receptor was ligand dependent. In contrast to the studies mentioned earlier, VE-cadherin exerted a negative, rather than positive, effect on growth factor signaling. A recent study suggests that the VEGF receptor and the adherens junction work in concert to transduce the shear stress signal that results as blood flows through the endothelial lining of vessels (Shay-Salit et al. 2002).

20 226 WHEELOCK JOHNSON Studies from our laboratory and the Hazan laboratory suggest that N-cadherin may influence tumor cell behavior through interactions with the FGF receptor. Epithelial cells typically express E-cadherin. However, in some aggressive tumors, the cells have turned on expression of N-cadherin. These tumors tend to be more invasive than N-cadherin-negative tumors (Hazan et al. 2000, Kim et al. 2000, Nieman et al. 1999, Suyama et al. 2002). Our laboratory showed that inhibitors of FGF receptor signaling reduced N-cadherin-mediated invasion, thus implicating N-cadherin in activation of FGF receptor signaling, analogous to that shown by Doherty et al. (2002) and Skaper et al. (2001) in neurite extension. We further determined that extracellular domain four of N-cadherin was necessary and sufficient for this activity and proposed that it may interact directly with the FGF receptor (Kim et al. 2000). Hazan s laboratory recently showed that N-cadherin is also involved in ligand-dependent FGF receptor signaling (Suyama et al. 2002). This study showed that N-cadherin interactions with the FGF receptor prolong signaling by stabilizing the growth factor receptor through interactions in the extracellular domains of the two proteins. Thus expression of N-cadherin by tumor cells can influence cellular behavior via ligand-dependent and ligand-independent interactions with the FGF receptor. SUMMARY AND PERSPECTIVES It is now clear that cadherin expression and function have profound effects on cellular phenotype and behavior. Cadherin expression patterns are critical to the developing embryo, not only to form cell-cell junctions, but also to promote cell sorting and cell-signaling events that regulate development. In addition, cadherin function and its perturbation are significant during tumorigenesis. Mutations that decrease the strength of cadherin-mediated adhesion can promote tumor invasion. In addition, it is becoming increasingly clear that cellular context plays a critical role in determining the consequences of cadherin expression. For example, N-cadherin promotes strong cell-cell adhesion in cardiac muscle as a component of the intercalated disc. However, when tumor cells of epithelial origin express N-cadherin, it promotes decreased adhesion and increased motility and invasion. Investigations into signaling pathways downstream of cadherins are in their infancy. As we learn more about the pathways downstream of specific cadherins, we will begin to better understand the differences between cadherins and how their expression can confer diverse cellular phenotypes. ACKNOWLEDGMENTS The authors thank Dr. Karen Knudsen, Lankenau Institute for Medical Research, and members of the Wheelock/Johnson laboratory for critically reviewing this manuscript. The Wheelock/Johnson laboratory is supported by grants GM51188 and DE12308 from the National Institutes of Health.

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29 CADHERINS 235 Zanetti A, Lampugnani MG, Balconi G, Breviario F, Corada M, et al Vascular endothelial growth factor induces SHC association with vascular endothelial cadherin: a potential feedback mechanism to control vascular endothelial growth factor receptor- 2 signaling. Arterioscler. Thromb. Vasc. Biol. 22: Zhurinsky J, Shtutman M, Ben-Ze ev A Plakoglobin and beta-catenin: protein interactions, regulation and biological roles. J. Cell Sci. 113(Pt. 18):

30 CADHERINS C-1 Figure 1 Comparison of the structure of various cadherin family members. Cadherins typically include a signal sequence and a propeptide. The cadherin extracellular domain is divided into cadherin repeats, which are numbered from the N terminus. Type I and Type II cadherins typically have 5 extracellular repeats, a juxtamembrane domain that binds p120 catenin, and a catenin-binding domain that interacts with -catenin. T-cadherin (also called H-cadherin) does not have a transmembrane domain but is linked to the membrane by a lipid tail. LI cadherin is unusual in that it has two inserts in the extracellular domain that disrupt the cadherin repeats. Desmoglein 1 is unusual because it has only 4 extracellular repeats; desmogleins 2 and 3 each have 5 extracellular repeats. Desmocollin is unusual because it is alternatively spliced in the cytoplasmic domain to produce type a and type b splice variants. CNR protocadherins are unique because they have a binding site for Fyn in the cytoplasmic domain. FAT is an unusual cadherin because it has 34 extracellular repeats. Flamingo is an unusual cadherin because it crosses the plasma membrane seven times and likely binds G proteins. The Ret proto oncoprotein is a member of the cadherin superfamily because it has 2 cadherin repeats in its extracellular domain.

31 C-2 WHEELOCK JOHNSON See legend on next page