Historical Background: The Larger Cadherin Superfamily
Cadherins were originally discovered as mediators of specific cell-cell adhesive structures, often called adherens junctions (AJ) (Gumbiner 2005; van Roy and Berx 2008; van Roy 2013). The basic and well-documented model for this function involves two modes of interaction between the most amino-terminal ECs (called EC1): homophilic (between identical cadherin species) and homotypic (between identical cell types). Both cis (between neighboring cadherin molecules in the same plasma membrane) and trans interactions (between apposing cadherin molecules) have been described in considerable detail (van Roy and Berx 2008). Cell-cell adhesion mediated by classic cadherins is considered the basis of cell sorting out during both embryonic development and the morphogenesis of most organs (Gumbiner 2005; Halbleib and Nelson 2006; Niessen et al. 2011; van Roy 2013; Suzuki and Hirano 2016). Although the cadherin activity during these important processes might be explained by cadherins serving as cell-type specific molecular glues, it is increasingly clear that signaling by and to cadherins plays major roles. Indeed, cadherins are required not only for rigid, static cell-cell contacts, but also for regulation of dynamic morphogenetic cell movements by active remodeling of intercellular junctions and the associated cytoskeletal structures (Harris and Tepass 2010; Takeichi 2014).
From these studies, several hallmarks became clear: Ancient family members always have long ectodomains with numerous ECs, which during evolution became progressively shorter (Hulpiau and van Roy 2011). Current classic and desmosomal cadherins have 5 ECs, whereas protocadherins have 6 to 7 ECs (Fig. 2). This, together with the finding of many long cadherin-like proteins in unicellular choanoflagellates, indicates that the cadherin family members originally served in extracellular sensing and cell-cell recognition rather than in specific cell-cell adhesion.
Cadherins characteristically have an ectodomain that is largely or exclusively composed of ECs, but their TM domains and in particular their CDs are much more diverse (Fig. 2). Quite often, cadherins have their own subfamily-specific CD, often with one to several motifs. These CDs are conserved within the same subfamily, but apparently they are not related to each other and often not at all to the “standard” CD of classic cadherins. Cadherin-13, also called T-cadherin (truncated), stands out because it is the only known cadherin lacking a TM domain. Instead, it is linked to the cell surface by a glycosylphosphatidylinositol (GPI) anchor (reviewed by Berx and van Roy in Nelson and Fuchs 2010). Moreover, the recently resolved structure of its EC1 domain is unusual. The flamingo/CELSR cadherins have, besides an ectodomain of nine ECs, a 7-TM domain, which is exceptional for cadherins. At the outer side of the plasma membrane, the cadherin ectodomains are mainly involved in homophilic bonds (i.e.,between like molecules), linking neighboring cells in specialized junctions. Recent findings have revealed quite some variation in cadherin binding modes, deviating from the “classic” zipper-like structure of ligated E-cadherin arrays (Fig. 1b) (Shapiro in Suzuki and Hirano 2016). This seems to serve very well various molecule-specific functions.
Signaling by Classic Cadherin-Catenin Complexes
During both normal embryogenesis and pathological processes, such as invasive cancer, cadherin type switching is a frequent occurrence that forms the basis for the migration of particular cells and for generation of new structures with new functions (reviewed by van Roy 2014). A notable example is the dynamic but highly controlled behavioral change of cells derived from the neural crest.
On the one hand, junctional p120ctn stabilizes the cadherins, but on the other hand, cadherins counteract cytoplasmic p120ctn, which can modulate members of the Rho GTPase family (reviewed by Pieters et al. 2012a). Also, it has recently become clear that junctional αE-catenin can serve as a tension transducer for the CCC (Yonemura et al. 2010) (Fig. 1c). When an external force is applied to epithelial cells, for instance, during wound healing, actomyosin-driven tension induces an unfolded conformation in αE-catenin so that it can recruit vinculin to the CCC. This serves as a positive feedback because mechanical coupling between actomyosin networks and the CCC is reinforced in this way. At the same time, the force-dependent αE-catenin/vinculin interaction strengthens the intercellular junctions and contributes to tissue integrity despite the external force (Takeichi 2014; Lecuit and Yap 2015). On the other hand, αE-catenin dimers were shown to preferentially dissociate from the CCC and to locally block the binding of the actin nucleating and branching factor ARP2/3 complex (reviewed by Tan et al. in Suzuki and Hirano 2016) (Fig. 1b). At the same time, binding of αE-catenin to formin is promoted in a way that facilitates the formation of elongated F-actin fibers that can associate effectively with myosin-II (Fig. 1b and c). More recently, it was demonstrated that AJs interact also with microtubules, either the plus ends of microtubules via β-catenin or the minus ends via p120ctn, Plekha7 and Nezha (Camsap3) (reviewed by Harris and Tepass 2010; Takeichi 2014). Association of the AJ with microtubules facilitates junctional assembly and also allows the AJ to influence intracellular structuring. For instance, the orientation of both symmetric and asymmetric cell divisions are influenced by AJs (reviewed by Harris and Tepass 2010).
By sequestering β-catenin in the junctions, this important signaling molecule is prevented from entering the nucleus and activating Lef/Tcf-dependent gene activation ( β-catenin). Similarly, p120ctn in complex with classic cadherins cannot shuttle into the nucleus, where it would contribute to Wnt signaling by retrieving the transcriptional inhibitor Kaiso to the cytoplasm. In the case of E-cadherin, p120ctn binding to the JMD of classic cadherins shields both a di-leucine motif required in mammalian cells for clathrin-mediated endocytosis and a Tyr residue that, when phosphorylated, is recognized by Hakai, an E3 ubiquitin ligase (reviewed by Niessen et al. 2011) (see next section).
- 4.Native cadherin ligation, that is, the interaction in trans between cadherins across cell-cell boundaries, can result in two types of signaling: increased juxtacrine signaling by neighboring noncadherin molecules and a direct effect on cadherin-mediated signaling. To dissociate the first type of adhesion-dependent processes from the second type, researchers triggered cell-associated cadherins with, for instance, beads coated with recombinant cadherin ectodomains. Studies of this type have revealed a major role for small GTPases of the Rho family (Kooistra et al. 2007; Niessen et al. 2011; Pieters et al. 2012b; Ratheesh et al. in van Roy 2013). Although the effects seen are complex and much dependent on the cadherin type involved, as well as on cell type and cell activation status, several useful models have emerged. For instance, E-cadherin ligation induces local and transient activation of Rac1 and Cdc42 GTPases (Fig. 3). This might occur by activation of appropriate GEFs (guanine nucleotide exchange factors) such as Tiam1 and Vav2, and it appears to be dependent on association of p120ctn with cadherin. Two potential mediators of cadherin-induced GTPase activation are the protein tyrosine kinase c-Src and the lipid kinase PI3K, which are both stimulated upon E-cadherin ligation and lead to Tiam1 and Vav2 activation (Ratheesh et al. in van Roy 2013), while also Rap1 is involved. This is a Ras-like small GTPase, which includes Vav2 and Tiam1 among its numerous effectors (Kooistra et al. 2007).
Rap1 by itself can be activated upon cadherin ligation in at least four possible ways (Fig. 3). (a) The E-cadherin cytoplasmic domain transiently recruits the Rap-GEF C3G in nascent junctions. (b) Junctional β-catenin recruits MAGI scaffold proteins and PDZ-GEF1, and this macromolecular complex activates Rap1. (c) Nectins, which are immunoglobulin-like adhesion molecules, promote cadherin-dependent junction formation (reviewed by van Roy and Berx 2008) and also induce Rap1 activation by a process involving c-Src, Crk, and C3G (Hoshino et al. 2005). (d) Various RTKs can associate with classic cadherins (see below) and trigger signaling pathways leading to Rap1 activation. Interestingly, afadin (AF6), an actin-binding adaptor protein, couples the cytoplasmic domains of nectins to junctional p120ctn and α-catenin (Mandai et al. in van Roy 2013) (Fig. 3). Afadin has a Rap1 binding domain and is indeed recruited to nascent junctions through the action of Rap1. There, it strengthens the binding of p120ctn to E-cadherin what counteracts the endocytosis of unligated E-cadherin and hence contributes to maturation of cadherin-based and nectin-based junctions (Hoshino et al. 2005; Fujiwara et al. in Suzuki and Hirano 2016).
When and where are such cadherin-modulated signals important? Nascent epithelial junctions show a restricted membrane zone with active GTP-Rac1, recruitment of Arp2/3, and increased lamellipodial activity at the site of the expanding intercellular contacts (reviewed by Niessen et al. 2011). Subsequent expansion and completion of cell-cell adhesion is dependent on RhoA activation at the distal edges of the intercellular contacts, what locally induces actomyosin contractility. The cadherin-induced activation of Rac1 (and also Cdc42) can have different consequences, including actin nucleation through activation of WASP-WAVE family proteins and the Arp2/3 complex, local inhibition of RhoA by activation of p190RhoGAP, and inhibition of E-cadherin endocytosis through IQGAP1, which is an F-actin cross-linking protein and a downstream target of Rac and Cdc42 (Ratheesh et al. in van Roy 2013) (Fig. 3).
Interestingly, similar signaling molecules are at play also in neural synapses (reviewed by Brigidi and Bamji 2011). Postsynaptic spine morphology is indeed dependent on modulation of small GTPases by p120ctn, the related neural δ-catenin, and α-catenin. So, clustering of postsynaptic N-cadherin with associated p120ctn and α-catenin leads to recruitment of afadin, which in turn recruits the Rac-GEF kalirin-7, promoting Rac1-dependent widening of the spine head. Moreover, p120ctn and δ-catenin can inhibit RhoA, which regulates spine length and density. These findings collectively show that functional interaction of catenins, be it β-catenin, p120ctn or α-catenin, with small GTPases and actin remodeling proteins is a recurrent theme that has evident implications for correct tissue morphogenesis (reviewed by Harris and Tepass 2010).
Signaling by Posttranslational Modification of Classic Cadherins
Of the wide variety of possible posttranslational modifications that might affect and modulate classic cadherins and associated proteins, (de)phosphorylation and proteolytic processing have been studied in most detail. For instance, the cytoplasmic domains of classic cadherins as well as the associated armadillo proteins p120ctn and β-catenin are efficient substrates for various tyrosine kinases, both receptor tyrosine kinases (RTKs) and nonreceptor tyrosine kinases such as Src, Fyn, and Fer (reviewed by Niessen et al. 2011; Pieters et al. 2012b; Tan et al. in Suzuki and Hirano 2016). Tyrosine phosphorylation of cadherin and catenin is generally thought to perturb their functions, but there is ample evidence that weak or transient phosphorylation under physiological conditions stimulates cadherin-mediated adhesion. Ligation of E-cadherin stimulates the c-Src signaling pathway in a delicate biphasic way: weak signals are supportive of E-cadherin-based cell-cell contacts in a positive feedback (see also Fig. 3), whereas strong signals inhibit E-cadherin functionality in a negative feedback (reviewed by Niessen et al. 2011).
In the case of N-cadherin, the ectodomain has been demonstrated to interact molecularly and functionally with the FGF-receptor ectodomain to prevent FGFR internalization and allow sustained receptor activation and downstream signaling (reviewed by Berx and van Roy in Nelson and Fuchs 2010; van Roy 2014). In endothelial cells, VEGFR2 associates with the CD instead of the ectodomain of VE-cadherin (cadherin-5). This interaction prevents receptor internalization, but in this case also VEGFR2 receptor-induced signaling, as VE-cadherin (but not N-cadherin) recruits the tyrosine phosphatase Dep1. A similar attenuation occurs with respect to the FGFR1 receptor. This combined effect inhibits endothelial cell proliferation and migration and promotes in this way vascular stability, a regulatory process disturbed by N-cadherin upregulation in tumor-associated vasculature (Giampietro et al. 2012; reviewed by van Roy 2014). VE-cadherin is tyrosine phosphorylated in response to a wide spectrum of signals, including triggering by VEGF and various inflammatory mediators. This protein modification leads to weakening of endothelial junctions and is counteracted by the endothelial-specific transmembrane tyrosine phosphatase VE-PTP (reviewed by Dejana and Vestweber in van Roy 2013).
Generally, the cellular response to cadherin/catenin phosphorylation appears to be much influenced by the cadherin type, the cell type, and the microenvironment. Therefore, the following interactions serve as representative examples rather than as rules of thumb. In adherent cells expressing N-cadherin, the cadherin-associated p120ctn recruits the nonreceptor tyrosine kinase Fer, which activates the protein Tyr phosphatase PTP1B (PTPN1) when this is complexed to junctional β-catenin (Fig. 4b). This promotes junctional integrity (reviewed by Pieters et al. 2012b). Interestingly, a similar mechanism of junctional stabilization occurs in excitatory synapses, where junctional p120ctn at the presynaptic membrane recruits Fer, leading to activation of the Tyr phosphatase SHP2 (PTPN11), which in turn dephosphorylates β-catenin and promotes junctional integrity at synapses (Lee et al. 2008) (Fig. 4b). Further, synaptic functionality is promoted by various molecular interactions with junctional N-cadherin, β-catenin, and p120ctn homologs (reviewed by Brigidi and Bamji 2011).
Signaling by Nonclassic Cadherins
Compared to classic cadherins, knowledge on nonclassic and cadherin-related atypical cadherins is still sparse. Nevertheless, it points to multiple signaling functions.
While Fmi/CELSR is considered a core protein that mediates PCP at the cellular level, two other atypical cadherin proteins, Fat and Dachsous (Ds), are considered upstream or parallel signaling proteins for PCP, as they mediate PCP at a more global embryonic or organ level (Gray et al. 2011). In mammals, Fat4 is the true ortholog of fly Fat, and Dchs1 is the ortholog of Ds (Hulpiau and van Roy 2011). It is noteworthy that the cytoplasmic domain of FAT4 deviates completely from those of FAT1 to FAT3. Both Fat and Ds have very long ectodomains with numerous EC repeats, and they interact heterophilically with each other at cell junctions. While the ectodomains of classic cadherins adopt a calcium-dependent linear conformation, those of very long cadherin ectodomains seem to be bended due to the presence of several noncalcium binding linkers between ECs (Tsukasaki et al. 2014). Polarity across embryos/organs is mediated both by increased Ds expression at the proximal side and by increased Four-jointed (Fj) at the distal side (Sopko and McNeill 2009; Sharma and McNeill in van Roy 2013) (Fig. 7). Fj is a Golgi kinase that, intriguingly, phosphorylates the ectodomains of Fat and Ds, what modulates their mutual binding affinities. In fly, strong triggering of Fat by Ds leads to cis-dimerization of Fat and to phosphorylation of its C-terminus by the associated Dco (Disc Overgrown, the fly homolog of CK1ε) (Fig. 7, top). Evidence indicates that Dco activity promotes Fat activity in the Hippo pathway, possibly by recruiting Expanded, which is an upstream Hippo regulator, to the subapical membrane (Sharma and McNeill in van Roy 2013). The Hippo or Yap pathway is an emerging signaling network with critical importance for controlling organ size, and its derangement is being reported in an increasing number of human cancer types. Thus, importantly, actively signaling Fat not only mediates PCP but, at least in Drosophila, also activates the Hippo signaling pathway. Evidence for interaction between Fat4 and Hippo signaling exists in zebrafish, but not yet in mammals (Sharma and McNeill in van Roy 2013).
Signaling by Protocadherins
Genuine protocadherins comprise more than half of the cadherin species in mammals and have typical cytoplasmic domains completely different from those of classic cadherins. Reportedly, they have much weaker or even undetectable adhesion potential compared to classic cadherins. Consequently, they are believed to have a signaling function rather than an adhesive function (Kahr et al. in van Roy 2013; Hayashi and Takeichi 2015; Jontes in Suzuki and Hirano 2016). Analyses of protocadherins have been lagging behind those of classic cadherins, and until recently functional studies have been performed only on a few protocadherins and mostly in Xenopus. The cytoplasmic domain of arcadlin, which corresponds to the rat protocadherin-8 (Pcdh8), interacts in synaptic junctions with N-cadherin. Excitation of hippocampal neurons upregulates arcadlin, and this promotes N-cadherin internalization, which is accelerated by the homophilic interaction of arcadlin ectodomains and involves an associated MAPKKK named TAO2β. A similar observation of cross-inhibition involves the induction by activin of PAPC, a Pcdh8 homolog in Xenopus, what is linked to decreased adhesion activity of C-cadherin, a classic cadherin in frog. For several members of the so-called nonclustered δ2-protocadherin subfamily (comprising Pcdh8, Pcdh10, Pcdh17, Pcdh18, and Pcdh19), a specific cytoplasmic interaction with the actin-organizing complex Nap1/WAVE has been demonstrated. Indeed, a functional WIRS motif (WAVE regulatory complex interacting receptor sequence) has been identified in their respective CDs (Chen et al. 2014). At least several of these protocadherins are enriched at sites of cell-cell contact and recruit there the WAVE complex, which in turn recruits lamellipodin (Lpd) and the Ena/VASP regulators of actin polymerization. This leads to weakening of the classic cadherin-catenin complex and facilitation of cell membrane motility at cell-cell contacts (Fig. 1a). This molecular mechanism has been proposed to be instrumental in concerted axonal growth cone extension and guidance (Hayashi and Takeichi 2015; Jontes in Suzuki and Hirano 2016). One could therefore conclude that a general function of protocadherins is to negatively regulate the activity of classic cadherins. However, Pcdh19 in zebrafish acts synergistically with N-cadherin to control cell migration during anterior neurulation, and recent elegant studies in zebrafish have indicated subtle but significant functions of Pcdh19 in regulating neural proliferation, maintenance of cell contacts, neurite fasciculation, and arborization (Cooper et al. 2015; Jontes in Suzuki and Hirano 2016). It will thus be challenging to unravel in detail the underlying mechanisms of protocadherin functions. Meanwhile, mutations in nonclustered protocadherins genes have been identified as causing several neurodevelopmental disorders, and cancer-confined silencing of these genes has been often observed as well (Jontes in Suzuki and Hirano 2016) (van Roy 2014).
The so-called clustered protocadherins (cPcdhs) comprise extended groups of protocadherin-α, protocadherin-β, and protocadherin-γ genes, arranged in tandem on a single chromosomal site. While the ectodomains of each of the encoded cPcdh isoforms are unique, a large part of their cytoplasmic domains is shared among the clustered subfamily members. In neuronal populations, various regulatory mechanisms of cPcdh isoform expression lead to a multitude of different coexpression patterns allowing unique cell recognition properties. Thus, combinatorial expression and heteromultimeric complex formation of cPcdh isoforms has been found to be instrumental in achieving major effects on neuronal development: inhibition of apoptosis, specific synapse formation, and dendritic arborization on the basis of self-avoidance between dendrites of the same origin (Hirayma and Yagi in van Roy 2013; Hayashi and Takeichi 2015; Mah and Weiner in Suzuki and Hirano 2016). It is obvious that the ectodomain variability of cPcdhs plays a key role in these remarkable functions, while knowledge on the signaling by the CDs is still limited. Inhibitory association of these CDs with the tyrosine kinases PYK2 and FAK seems to be involved in, respectively, neuronal survival and dendritic arborization, but many details of signaling by cPcdhs await elucidation.
This essay deals with selected examples of intra- and cross-cellular signaling by members of the large cadherin superfamily. Much attention is paid to E-cadherin, which, as founder of the superfamily, is an extensively studied “classic” cadherin. As a mediator of specific intercellular junctions, both static and dynamic ones, it has a pivotal role in epithelial cell behavior, tissue formation, and suppression of cancer. Compelling evidence has been gathered that E-cadherin is much more than “molecular glue,” because it serves as a hub for several signaling pathways. On the one hand, association of the armadillo protein p120ctn with the juxtamembrane domain of classic cadherins, as well as activation of various small GTPases, contributes to junctional stability. On the other hand, cadherin ligation generates signals that contribute to cell differentiation and inhibit growth stimulatory signaling, in part by binding via its C-terminal domain the proto-oncogenic β-catenin. Important modulators of cadherin-associated signaling are cytoskeletal components, such as actomyosin, and several enzymes, in particular receptor tyrosine kinases and counteracting phosphatases. E-cadherin is also processed by several proteases to generate various fragments with signaling activity. The many other members of the superfamily include protocadherins, which may act negatively on classic cadherins in various ways. The flamingo/CELSR cadherins and the very extended Dachsous and Fat cadherins play important roles in the signaling towards planar cell polarity. In conclusion, signaling by cadherins is a rapidly growing research field with multiple proven or proposed links to mammalian pathologies. Recent investigations on cadherin-linked signaling raised many intriguing questions that might be addressed by contemporary multidisciplinary approaches.
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