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Eph signaling: a structural view Juha-Pekka Himanen and Dimitar B. Nikolov Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA

Eph receptors, the largest subfamily of receptor tyrosine kinases, and their ephrin ligands are important mediators of cell–cell communication regulating cell attachment, shape and mobility. Both Ephs and ephrins are membrane-bound and their interactions at sites of cell– cell contact initiate unique bidirectional signaling cascades, with information transduced in both the receptor-expressing and the ligand-expressing cells. Recent structural and biophysical studies summarized in this review reveal unique molecular features not previously observed in any other receptor–ligand families and explain many of the biochemical and signaling properties of Ephs and ephrins. Of particular importance is the insight into how approximation of ligandexpressing and receptor-expressing cells could lead to the formation and activation of highly ordered signaling centers at cell–cell interfaces. The vast majority of cell surface receptor-mediated signal transduction systems are involved in unidirectional communication. However, at least two groups of interacting membrane-anchored proteins, the Eph and ephrin families [1], send information bidirectionally – that is, into both interacting cells. Eph receptors, first shown to be important regulators of axon path-finding and neuronal cell migration [2,3], are now known to have roles in controlling a diverse array of other cell–cell interactions, including those of vascular endothelial cells [4–6] and specialized epithelia [7–10]. Several plasma-membrane-attached ligands termed ephrins bind Eph-family receptor tyrosine kinases (RTKs) and activate their tyrosine kinase catalytic domains [11–13]. Concomitant with activation of Eph kinases and transduction of the typical forward signal into the receptor-bearing cell, the ligand–receptor interaction also leads to transduction of a reverse signal into the ephrin-bearing cell [14]. The Ephs and the ephrins are both divided into two subclasses – A and B – based on their affinities for each other and on sequence conservation [15] (http://cbweb.med.harvard.edu/ eph-nomenclature). In general, the nine different EphA RTKs (EphA1–EphA9) bind promiscuously to, and are activated by, six A-ephrins (ephrinA1–ephrinA6), and the EphB subclass receptors (EphB1–EphB6 and, in some cases, EphA4) interact with three different B-ephrins (ephrinB1–ephrinB3). The membrane attachment of both Ephs and ephrins provides a mechanism by which their interaction occurs only at sites of cell–cell contact. This leads to the multimerization of both molecules to distinct clusters within their respective plasma membranes, resulting in the formation of discrete signaling centers restricted to zones of Corresponding author: Dimitar B. Nikolov ([email protected]).

cell–cell and axon–cell contact [16,17]. Most RTKs activate signaling pathways that ultimately target transcription patterns in the nucleus, which regulate cell proliferation and/or differentiation. By contrast, the Ephs and ephrins regulate cell migration, repulsion, adhesion or attachment to the extracellular matrix. The signaling cascades that they initiate, therefore, ultimately converge on targets such as integrins and small Rho-family GTPases. The past several years have witnessed major advances in our understanding of the structure and the molecular mechanisms of action of Eph receptors and ephrins, and these are the subjects of this review. The Eph ligand-binding domain resembles carbohydrate-binding proteins Like all RTKs, the Eph receptors are type-I transmembrane proteins (Fig. 1). Their extracellular region contains a highly conserved N-terminal domain that is both necessary and sufficient for ligand recognition and binding [18], followed by a cysteine-rich region and two fibronectintype III repeats, which might be involved in receptor– receptor dimerization interactions [19] and/or interactions

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Fig. 1. Activation of the bidirectional Eph –ephrin signaling pathway. Upon binding, Ephs and ephrins form a 2:2 circular heterotetramer causing the phosphorylation of their cytoplasmic domains. The receptor-binding domains of the B-class ephrins are shown in red, the ligand-binding domains of the Ephs in blue, and the kinase and sterile-a-motif (SAM) domains in green. All the other structural elements are indicated in gray. The phosphate groups are purple. Class A ephrins have a similar overall structure but they are attached to the cell via a glycosylphosphatidylinositol (GPI) linker and lack the transmembrane region and the cytoplasmic domain. Abbreviations: PDZ, PSD95/Dlg/ZO1 (PDZ)-binding motif; JM, juxtamembrane region.

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with other proteins (e.g. NMDA receptors [20]). The Eph intracellular side contains a juxtamembrane region, a conserved kinase domain, a sterile-a-motif (SAM) domain, and a PSD95/Dlg/ZO1 (PDZ)-binding motif [21]. X-ray crystallographic studies reveal that the ligandbinding domain of the mouse EphB2 receptor has a compact globular structure (Fig. 2a) with a b-sandwich ‘jellyroll’ folding topology [22]. The b-strands are connected by loops of varying length, including a long, wellordered loop (H– I), which packs against the concave b-sheet, and two partially disordered loops that protrude from the middle of the convex b-sheet. These loops, indicated with red and orange on Fig. 2a, respectively, play central roles in ligand recognition and binding. The Eph ligand-binding domain is unique to this RTK family and shares no significant sequence or precise structural homology with other known proteins. Nevertheless, similar folding topology is observed in some carbohydratebinding proteins [22–24]. This finding suggested that the homology in molecular architecture might include the location of the ligand-binding site. Indeed, it is now known that the EphB2 tetramerization surface region (or ‘lowaffinity’ ligand-interaction region) is localized to the concave b-sheet around the H–I loop, in a position similar to the location of the carbohydrate-binding site in lectins. Furthermore, the only sequence feature that is conserved within, but differs between, the two Eph receptor subclasses is the length of the H–I loop. This observation, as well as structurebased mutagenesis experiments demonstrating that a

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Fig. 2. Structures of the extracellular domains of EphB2 and ephrinB2. (a) The ligand-binding domain of EphB2. The N and C termini of the molecule are labeled. The two b-sheets are colored blue and green; the class-specificity loop (H– I) is orange and the loops involved in the high-affinity heterodimer Eph –ephrin interactions are red. Disulfide bonds are shown as gray balls and sticks. (b) The extracellular receptor-binding domain of ephrinB2. Disulfide bonds are shown in gray and the Eph-binding loop (G– H) is in blue. (c) The ephrin dimer observed in ephrinB2 crystals in the absence of Eph receptor. (d) The EphB2-–phrinB2 tetramer. Eph receptors are blue and ephrins are red. The high-affinity dimerization interfaces are indicated by arrows. http://tins.trends.com

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chimeric EphB2 receptor with an EphA3 H–I loop recognizes both A- and B-class receptors [22], has prompted the loop to be named the ‘class-specificity loop’. Another intriguing possibility is that the carbohydrate moieties at the putative ephrin glycosylation sites might be directly involved in the ligand–receptor interactions. Although there is no direct evidence for this, it is interesting that, in the crystal structure of the EphB2–ephrinB2 complex [25], a conserved ephrin glycosylation site (asparagine at position 39 in ephrinB2) is located near the tetramerization ligand– receptor interface and the H–I loop. Because bacterially produced non-glycosylated ephrinB2 binds its cognate receptors with high affinity and specificity [22], carbohydrate-mediated interactions are unlikely to occur at the ‘high-affinity’ ligand–receptor dimerization interface [22] (see section on Eph-receptor–ephrin recognition), but they could play a role at ‘lower-affinity’ interfaces, mediating ligand–receptor tetramerization and/or clustering. This issue warrants further investigation by studying, for example, whether ephrin mutations affecting Asn39 would affect Eph–ephrin signaling. Ephrins are structurally similar to plant nodulins and photocyanins Ephrins are characterized by the presence of a unique Nterminal receptor-binding domain (RBD) (Fig. 1), which is separated from the membrane via a linker of , 40 amino acids. A-ephrins are attached to the cell via a glycosylphosphatidylinositol (GPI) linkage. B-ephrins possess a transmembrane region and a short, but highly conserved, 80 amino acid cytoplasmic domain, which includes a C-terminal PDZ-binding motif. Recent X-ray crystallographic studies have revealed that the ephrin RBD has a globular b-barrel structure (Fig. 2b) with a Greek key folding topology [25,26]. Interestingly, in crystals of uncomplexed ephrinB2, the molecule forms homodimers by burying the hydrophobic surface regions around the G–H loop (Fig. 2c). Because this same loop is involved in receptor binding, it is likely that the ephrin molecules exhibit significant rearrangement when their homodimers are displaced following interaction with the Eph receptors. As expected, the membrane-proximal linker is completely disordered [26] and does not seem to contribute to the receptor–ligand interaction [25]. The primary sequences of ephrins have no similarities with that of any other protein and the ephrin structure is, indeed, novel for a signaling molecule. Unexpectedly, however, the ephrins share a significant structural homology with the cupredoxin – phytocyanin family of Cu2þ-binding proteins. In addition, there is a plant nodulin subfamily that shares sequence similarity with the phytocyanins, indicating that nodulin proteins are also structurally similar to the ephrins. Interestingly, the nodulins are extracellular signaling proteins that can be GPI-anchored to the cell surface. The significance of the ephrin – nodulin structural homology is, as yet, unclear but could point to a more widely phylogenetically conserved role of this receptor– ligand structural motif [27]. The structure of the cytoplasmic domain of human ephrinB2 in solution is now also available [28]. This reveals that the 48 N-terminal ephrin residues are unstructured and

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prone to aggregation. By contrast, the highly conserved 33 C-terminal residues form a well-packed hairpin structure followed by a more flexible PDZ-binding tail. Eph-receptor –ephrin recognition The first step in the initiation of Eph-mediated signaling is the recognition and binding of Eph receptors and ligands located on closely opposed cell surfaces. Biophysical studies indicate that, in solution, isolated extracellular Eph and ephrin domains interact with each other via a multiple-step process. Initially, they form high-affinity heterodimers that are the predominant form of the complex throughout a large concentration range [29,30]. Dimer pairs can then tetramerize with a much lower KD to form 2:2 heterotetramers which, at very high concentrations, can form higher-order oligomers. Functional unidirectional signaling complexes are usually trimers composed of two receptors and one ligand, because this is sufficient to bring two intracellular signaling domains (e.g. kinase domains) together. By contrast, a functional bidirectional signaling complex is expected to be at least tetrameric, with two molecules initiating signaling in each direction. Crystallographic studies of the EphB2 – ephrinB2 complex [25] indeed reveal a tetrameric complex forming a ring-like structure, in which each receptor interacts with two ligands and each ligand with two receptors (Fig. 2d). One of the two distinct ligand –receptor interfaces is extensive and is presumably responsible for the initial 1:1 high-affinity binding. The second interface is smaller and is suggested to be responsible for the assembly of the dimers into functional tetrameric 2:2 complexes. In the planar structure of the tetramer, the molecules are arranged so that the C-termini of both ligands are located on one side, and the C-termini of the receptors on the other. This molecular architecture allows the membraneassociated ephrins and Eph receptors to interact between the surfaces of adjacent cells. Comparison of the structures of the bound and free molecules indicates that their recognition proceeds via an induced-fit mechanism (or ligand-induced receptor folding). More specifically, the ligand-receptor dimerization interface centers on the long G–H loop of ephrinB2, which is inserted into a channel on the surface of EphB2. The EphB2 loops forming the sides of this channel are unstructured in the unbound receptor but fold upon ligand binding to generate an extensive interaction surface that is complementary to the ligand G–H loop. The thermodynamic driving force for complex formation derives from the preference of the hydrophobic G–H loop to be buried in a hydrophobic environment away from the polar solvent. The secondarystructure rearrangements are strictly localized to the interaction interface and, therefore, downstream signaling is most likely to be triggered in the cytoplasmic sides, not through the conformational changes in the interacting ligands and receptors, but through their translocational rearrangements and repositioning relative to each other. However, it is not yet clear whether, or how, the other extracellular domains and regions of Ephs and ephrins, which are not visualized in the current crystal structures, participate in signal-initiating interactions. The structure of the EphB2–ephrinB2 complex also http://tins.trends.com

yields insight into the molecular basis for the observed Eph– ephrin subclass specificity. In particular, several key interacting side-chain pairs at the ligand–receptor dimerization interface are composed of bulky polar residues (conserved in B-subclass ligands), positioned against small polar residues (conserved in B-subclass receptors). In A-subclass members, the corresponding side chains are either hydrophobic or polar but with switched positions of the bulky and the small side chains. As a result, ligand–receptor combinations containing mixed subclasses would have either two bulky residues facing each other or polar residues facing hydrophobic ones and would, therefore, be energetically unfavorable. At the tetramerization interface, the H–I classspecificity loop further ensures that not only the Eph– ephrin dimers, but also their tetramers, contain molecules from only a single subclass. It is yet to be established whether Eph–ephrin signaling clusters on cells expressing multiple ligands or receptors from the same subclass can contain more than one type of ligand and/or receptor. Receptor activation The activity of RTKs is tightly regulated because it controls crucial events, including cell proliferation, differentiation and death. Activation of all RTKs follows some general rules [31– 34]. Ligand binding serves to bring together two catalytically repressed kinase domains and to hold them in an orientation favoring phosphorylation in trans. One of the monomers consequently phosphorylates regulatory sequences on the other monomer, leading to the activation of its catalytic domain. The active kinase can then phosphorylate other molecules, including the kinase domains of neighboring receptors, initiating the downstream signaling cascade. The most general mechanism for stimulating kinase activity involves the phosphorylation of the activation loop within the kinase domain, which in its non-phosphorylated form blocks the kinase active site. The structural basis for this activation has been well established [35] and reviewed [36 –38]. In addition, it was recently suggested that in many RTKs, including the Eph, Kit, Flt, plateletderived growth-factor b (PDGFb and TrkB receptors, the juxtamembrane region is also involved in regulation of the kinase activity [1,39]. The crystal structure of the intracellular region of EphB2 containing both the kinase and juxtamembrane domains reveals the molecular basis for this type of receptor activation [40]. The unphosphorylated juxtamembrane region forms a well-ordered, mostly helical structure (Fig. 3a, blue) that interacts intimately with the N-terminal lobe of the kinase (Fig. 3a, orange) and weakly with the C-terminal lobe (Fig. 3a, green). The interactions cause the distortion of a key a helix, leading to the displacement of a catalytic glutamate residue away from the active site and to kinase inactivation. In addition, the activation loop is prevented from attaining its active conformation. Phosphorylation of the juxtamembrane tyrosine residues (Fig. 3a, red) would lead to kinase activation because steric and electrostatic forces would push the phosphorylated juxtamembrane region away from the kinase, relieving the structural constraints that distort the active site. In addition, upon phosphorylation, the solventexposed juxtamembrane region becomes available for

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Fig. 3. Structures of the intracellular domains of Eph receptors. (a) The EphB2 kinase domain, auto-inhibited by the non-phosphorylated juxtamembrane region. The C-terminal kinase lobe is green and the N-terminal lobe is orange. The two regulatory tyrosine residues in the blue juxtamembrane region are shown in red. The ATP location in the active site is indicated in gray. (b) Structure of the monomeric sterile-a-motif (SAM) domain of EphA4.

interaction with signaling proteins containing phosphotyrosine-binding motifs. This model is fully consistent with earlier experiments, in which mutations changing the two juxtamembrane tyrosine residues to glutamic acid residues did not cause loss in catalytic activity but abolished SH2 binding [41,42]. It is interesting that the juxtamembrane region of the type-I transforming-growth-factor-b (TGFb-receptor serine/threonine kinase plays a similar role, elucidated by the crystal structures of the molecule in its phosphorylated [43] and non-phosphorylated [44] states. In this case, the non-phosphorylated juxtamembrane region acts together with another protein, FK506-binding protein (FKBP12), to suppress the kinase activity – again via a distortion of the active site. Juxtamembrane serine/threonine phosphorylation by the type II TGFb receptor creates a docking site for the Smad2 protein, which displaces FKBP12, initiating TGFb signaling. Although the precise structural details of kinase regulation of the Eph and the TGFb receptors are different, they illustrate an emerging general property of receptor juxtamembrane regions to perform a double duty during signaling. In their unphosphorylated state they repress the kinase catalytic activity and, upon phosphorylation, they serve as docking sites for downstream signaling proteins [39]. Mechanism of Eph-mediated signaling The recent structural studies of Eph receptors, ephrins and their complex suggest a likely mechanism for initiation of bidirectional signaling [22,25,26,45] (Fig. 1): Prior to cell– cell contact, Ephs and ephrins are loosely pre-clustered at the cell surface, probably in cholesterol-rich lipid rafts [46]. Unbound ephrins can form low-affinity homodimers. Upon cell–cell contact, ligands and receptors bind each other with 1:1 stoichiometry and nanomolar affinity, via an extensive heterodimerization interface. Eph–ephrin heterodimerization creates complementary interaction surfaces, which join dimer pairs into the tetrameric complexes shown in Fig. 2d, thereby allowing the receptor kinase domains to http://tins.trends.com

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phosphorylate each other and to initiate forward signaling. Importantly, the disruption of ephrin–ephrin homodimers and the Eph–ephrin tetramer formation also results in a repositioning of the B-ephrin transmembrane and cytoplasmic domains, converting them from an inactive to an active configuration. This allows for tyrosine phosphorylation of the ephrin cytoplasmic tail by an as-yet-unidentified kinase and for the initiation of reverse signaling. The tetramers are further arranged into the higher-order clusters observed in vivo at the sites of cell–cell contact. It should be noted that, although Eph–ephrin tetramerization is likely to be sufficient for receptor phosphorylation, the formation of ligand–receptor clusters is absolutely essential for physiological signaling. Moreover the size of the aggregates could define the nature of the generated signals [47,48]. Other Eph and ephrin regions are probably also involved in the final positioning of the bidirectional signaling complexes, including the extracellular cysteinerich linker [19] and intracellular SAM domain of the receptors [49,50], as well as the C-terminal PDZ-domainbinding sites of both receptors and ligands [51– 55]. The Eph SAM domain is of particular interest for the clustering process because in many other proteins it mediates protein – protein interactions, including homoand hetero-oligomerization. All Eph receptors contain a conserved SAM domain but, surprisingly, removal of this domain does not appear to disrupt Eph signaling [47]. Several recent crystallographic [49,50,56] and nuclear magnetic resonance (NMR) [57] studies have revealed that the , 70 amino acid domain has a compact helical structure (Fig. 3b). However, the implications of this finding for the precise function of the SAM domain during Eph signaling are not straightforward because the different structures reveal different modes of homotypic interaction, including dimerization [49], multimerization [50] or no interaction at all [56]. Furthermore, biophysical studies document that isolated SAM domains are mostly monomeric in solution, and form low-affinity dimers only at very high concentrations (0.5 mM or more, which can be relevant in densely packed Eph signaling clusters) [58]. It is well established that Eph receptors accomplish their principal function of axon guidance by monitoring and responding to ephrin gradients. The formation of Eph – ephrin clusters might indeed be necessary for the high sensitivity and wide dynamic range of the response to graded ligand densities as observed, for example, with the bacterial chemotaxis receptors [59]. It is interesting that a dynamic model for the clustering of chemotaxis receptors suggests that in the clusters the receptors adopt an arrangement in which they interact with different neighboring receptors via their cytoplasmic and periplasmic domains [59]. It is intriguing, therefore, to speculate that in the Eph– ephrin clusters the cytoplasmic (kinase or SAM) domains of an activated receptor could also intimately interact with the intracellular domains of receptors from neighboring tetramers. The Eph–ephrin high-affinity interactions resulting in stable complexes provided, until recently, a dilemma about how these molecules mediate repulsive cell–cell interactions. An elegant recent study by Flanagan and colleagues [60] reveals that ephrinA2 colocalizes with ADAM10

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(a membrane-bound disintegrin and metalloprotease also known as Kuzbanian). The study suggests a general mechanism in which, after a ligand- and a receptor-expressing cell interact, ADAM10 is activated and cleaves the Eph-bound ephrins from the cell surface, allowing the ligand–receptor complex to be internalized and degraded in the receptorexpressing cell. Consequently, the mechanical adhesion of the two cells is broken and they can move apart. EphrinA2 is cleaved by the protease domain of ADAM10 in the membrane-proximal linker region but the formation of a stable ADAM10– ephrin complex involves the ephrin RBD and the ADAM10 disintegrin and/or cysteine-rich domains. Interestingly, a 15 amino acid peptide derived from the ephrin RBD causes a concentration-dependent, synergistic activation of ADAM10-mediated ephrinA2 cleavage in the presence of the EphA3 receptor. Because the peptide was discovered as part of a putative motif conserved in other ADAM10 substrates, it was suggested that it might directly bind to, and activate, the protease [26,60]. The structures of free and Eph-bound ephrins reveal that this peptide corresponds to a region around the G– H loop, which is directly involved both in ephrin homodimerization and in receptor binding, and is unlikely to be exposed for direct interactions with ADAM10. An alternative model, based on the assumption that ephrin homodimers inhibit ADAM10 cleavage while Eph – ephrin complexes activate it, can be proposed that could also explain the observation that the peptide-induced cleavage of ephrins is suppressed at high peptide concentrations. At relatively low concentrations, the peptide would target the ephrin homodimerization interface causing a partial monomerization of the cleavage-resistant ephrin homodimers and their subsequent shedding. At high concentrations, the peptide would target the Eph – ephrin interface, causing dissociation of their complexes and inhibition of ephrin shedding. Finally, the ability of Ephs and ephrins to form ordered multimeric assemblies is at the heart of an emerging architectural role they might play, which is distinct from their direct signaling role. More specifically, Eph receptors and ephrins can create stable membrane-associated platforms for the architectural organization of various cellular structures. The best-studied example is their role in the formation, organization and function of some excitatory CNS synapses [61]. Specifically, it has been shown that EphB2 receptors associate with NMDA receptors, controlling both the density of the NMDA receptor in postsynaptic clusters and the number of postsynaptic release sites [20,62,63]. Consequently, lack of EphB2 impairs long-term potentiation (LTP) and long-term depression (LTD) [64,65]. Interestingly, based on the known Eph – ephrin complex structure and assuming that the other extracellular regions of the molecules adopt relatively extended conformations, the distance between the membranes of two interacting cells can be estimated at , 20 nM – which corresponds to the size of the synaptic cleft. Conclusions and perspectives Eph and ephrin signaling is crucial for the development and maintenance of many tissues and organs, and our understanding of Eph and ephrin function and mechanism http://tins.trends.com

of action is constantly growing. Recent structural and biophysical studies of the extracellular and cytoplasmic functional domains of these molecules reveal features not previously seen in any other receptor– ligand families and explain many of their unique biochemical and signaling properties. Further structures of the ephrin cytoplasmic domain in complex with its kinase and other binding partners are necessary to elucidate the mechanism of reverse signaling. In addition, structural biology methods can be used to study the highly ordered multi-protein assemblies constructed around the Eph – ephrin complexes as they interact with other molecules, such as metalloproteases, neurotransmitter receptors, and adaptor and scaffold proteins. It is now clear that Ephs and ephrins are involved not only in early developmental processes, but also in the function of the adult organism [1], and that the ability to modulate their signaling could have important medical applications. In the adult brain, for example, EphB signaling regulates neural stem cell migration and possibly proliferation and, therefore, Eph agonists could have therapeutic potential [66]. Increasing evidence also implicates Eph family proteins in angiogenesis and cancer [67]. The structure of the EphB2– ephrinB2 complex reveals a potential drug-binding target [25] and computational structure-based screens of small molecule databases can now be performed for Eph antagonists. Peptides based on the inserted ephrin loop could also potentially disrupt Eph–ephrin complex formation, inhibiting Eph signaling. Finally, the availability of high-resolution structural information should allow structure-based design of ligands and receptors with altered affinities and specificities [22] which, in light of the overlapping expression patterns of ligands and receptors with similar specificities, should serve as important tools for understanding the precise functional roles of individual family members. Acknowledgements We thank Marina Himanen for help with figure preparation. D.B.N is a PEW fellow and a Bressler Scholar. Our research on axon guidance molecules is funded by the NIH, The Christopher Reeve Paralysis Foundation, and the New York State Spinal Cord Injury Research Program.

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