Eph, a Protein Family Coming of Age: More Confusion, Insight, or Complexity? Martin Lackmann and Andrew W. Boyd (15 April 2008)

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1 Eph, a Protein Family Coming of Age: More Confusion, Insight, or Complexity? Martin Lackmann and Andrew W. Boyd (15 April 2008) Science Signaling 1 (15), re2. [DOI: /stke.115re2] The following resources related to this article are available online at This information is current as of 16 April Article Tools Visit the online version of this article to access the personalization and article tools: References Glossary Permissions This article cites 200 articles, 66 of which can be accessed for free: Look up definitions for abbreviations and terms found in this article: Obtain information about reproducing this article: Science Signaling (ISSN ) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue, NW, Washington, DC Copyright 2008 by the American Association for the Advancement of Science; all rights reserved.

2 CELL BIOLOGY Eph, a Protein Family Coming of Age: More Confusion, Insight, or Complexity? Martin Lackmann 1 * and Andrew W. Boyd 2 Published 15 April 2008 Since the mid-1980s, Eph receptors have evolved from being regarded as orphan receptors with unknown functions and ligands to becoming one of the most complex global positioning systems that regulates cell traffic in multicellular organisms. During this time, there has been an exponentially growing interest in Ephs and ephrin ligands, coinciding with important advances in the way biological function is interrogated through mapping of genomes and manipulation of genes. As a result, many of the original concepts that used to define Eph signaling and function went overboard. Clearly, the need for progress in understanding Eph-ephrin biology and the underlying molecular principles involved has been compelling. Many cell-positioning programs during normal and oncogenic development in particular, the patterning of skeletal, vascular, and nervous systems are modulated in some way by Eph-ephrin function. Undeniably, the complexity of the underlying signaling networks is considerable, and it seems probable that systems biology approaches are required to further improve our understanding of Eph function. Features of a Young Receptor Tyrosine Kinase Family It was in 1987, in the lead-up to the human genome project when homology cloning was the buzz term, that a previously unrecognized receptor tyrosine kinase (RTK) gene was discovered in a hepatoma cell line (1). It became apparent very quickly that Eph was the founding member of the most populous RTK family. The genes that encode Ephs and their ephrin ligands are present throughout the animal kingdom and have an origin that possibly predates the parazoan-eumetazoan bifurcation (2). An exponentially growing interest in these proteins over the past two decades (Fig. 1) leaves us today with an intriguingly complex picture that characterizes this protein family. Conservation of both the structure and function of Eph and ephrin gene products throughout evolution (3) contrasts with the dramatic increase in the number of members of each family in vertebrates. Considering signaling by RTKs as one of the universal concepts of cell-cell communication (4), it is tempting to speculate that the expansion of the Ephs to the largest of all 1 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia. 2 Leukaemia Foundation Laboratory, Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, 4029, Australia. *Corresponding author. , martin. lackmann@med.monash.edu.au RTK families in vertebrates reflects their role as a critical cell-positioning system, such that the increasing number of discrete receptors was essential for the evolution of the complex vertebrate body plan. In this context, it is interesting to consider that the cell-positioning function of Eph started with a single, primordial Caenorhabditis Cumulative number of PubMed listings elegans Eph receptor VAB-1 (5), which interacts with not one but four ephrins (EFN 1 to EFN 4) (6) to control kinasedependent and kinase-independent tasks by promoting cell-cell repulsion (7) or adhesion (8) in different cell types and during different stages of embryogenesis. The expansion in the numbers of Ephs and ephrins in vertebrates (to 16 Ephs and 9 ephrins) exceeds, particularly in the case of the receptors, what one would expect from the two putative genome duplications that occurred during the evolution of the vertebrate body plan. In bony fish in particular, zebrafish (Danio rerio), in which Fig. 1. The scientific interest in Eph-ephrin biology as measured by the number of publications. The graph illustrates the cumulative number of publications that appear in a PubMed search with ephrin OR Eph receptor as a search term, starting from the first citation in 1987 (1). Eph and ephrin expression and function have been studied in detail only some of the duplicated genes that arose from a further genome duplication have persisted. One explanation for this is that in these cases each of the duplicated genes has taken on a complementary role, and the sum of these preserved all of the functions of the ancestral gene (9). A large body of research in a range of vertebrates over the past two decades suggests that the concept of exploiting various Eph-ephrin combinations in different cellular contexts to modulate motile cell behavior has been a very successful mechanism that was consequently conserved and expanded during vertebrate evolution. Thus, the 16 vertebrate Ephs and 9 vertebrate ephrins, many of which regulate important cellular interactions, are involved in guiding organ development and patterning of the vascular, skeletal, and nervous systems. The glycosylphosphatidylinositol (GPI) anchored ephrin proteins expanded into a subfamily of six type A ephrins, which is complemented by a smaller subfamily of three type I transmembrane proteins known as type B ephrins. Based on structural features in their ligand-binding domains and their ephrin-binding preferences (10), Ephs are classified into 10 EphA and 6 EphB receptors, which preferentially bind to the type A and type B ephrins, respectively (Fig. 2). It is now emerging that this distinction may be oversimplified, because several Ephs are activated by both type A and type B ephrin ligands (11, 12), albeit at higher concentra- Page 1

3 Eph forward signal PTP Src PDZ SAM kinase Y EphA 1-10 Y JxM Y FN E E C EphA4 LBD A ephrin A5 LBD GPI ephrin A 1-6 Ephrin reverse signal EphB 1-5 EphB 6* FN C B TM ephrin B 1-3 endocytosis Cell rounding: RhoA Rac p-fak p-paxillin Repulsion ADAM10 EphA ephrin B EphB ephrin A transendocytosis transendocytosis Y Cell spreading: RhoA Rac p-fak p-cas p-paxillin Adhesion ADAM10 Fig. 2. Structural and functional features of Ephs and ephrins. Structural modules of EphA receptors (EphA1 to EphA10) include the N-terminal ligand-binding domain [(LBD): EphA, green; EphB, blue], cysteine-rich domain (C), EGF-like motif (E), fibronectin-type III motifs (FN), regulatory juxtamembrane domains (JxM) containing two tyrosine (Y) phosphorylation/sh2 domain binding sites, kinase domain, sterile-alpha-motif interaction domain (SAM), and PDZ-binding motif. The EphB6 kinase domain is catalytically inactive (EphB6*). Type A ephrins (ephrin A1 to ephrin A6) are tethered to the plasma membrane via GPI anchors, type B ephrins (ephrin B1 to ephrin B3) are transmembrane (TM) proteins containing SH2-docking sites and PDZ-binding motifs. Eph/ephrin interactions within the A and B subfamilies are indicated by solid arrows, whereas those across Eph/ephrin subfamilies specified by broken arrows. Eph forward and ephrin reverse signaling in opposing cells is positively modulated by Src kinase and down-modulated by PTP activities. Cell rounding and cell repulsion rely on active Eph kinases, phosphotyrosine-mediated downstream signaling (see below), and disruption of the Eph-ephrin tether between cells: Possible mechanisms include (ADAM10) metalloprotease-mediated shedding of Eph-bound ephrin and endocytosis of this complex or transendocytosis of intact EphB/ephrin B complexes into either cell. Lack of active Eph signaling resulting from inactivating mutations ( ) or elevated PTPs, lack of ephrin-cleavage or transendocytosis, and kinase-independent downstream signaling lead to cell-cell adhesion and cell spreading. Eph downstream signaling components affecting cell morphology include RhoA, Rac, focal adhesion kinase (p-fak), paxillin, Crk-associated substrate (CAS): (p-), phosphorylated; ( ), increase; ( ), transient increase; ( ), decrease. EphA EphB tions of ligand than are required when the ligand binds to its preferred Eph. Apart from the classical examples including EphA4, which is activated by type A ephrins but also has a well-established biological function as a guidance receptor for ephrin B2 (see below), and ephrin A5, which can effectively activate type A Ephs as well as EphB2 (11) there may be other biologically relevant EphA ephrin B combinations. For example, the role of EphA3 as a high-affinity receptor for type A ephrins (13), in particular for ephrin A2 (14) and ephrin A5 (15 18), has been extensively elaborated (see below). Additionally, a measurable interaction between EphA3 and ephrin B2 in vitro (12, 19), as well as circumstantial evidence from analyses of retinotectal projection maps in conditional EphA3 knock-in (KI) and ephrin A2 and ephrin A3 knock-out (KO) mice (20, 21), suggests the existence of other EphA ephrin B interactions that have relevance to axonal positioning. Page 2

4 Eph type and composition Ephrin cleavage or endocytosis Ephrin type and composition Eph abundance Many of the concepts that had been used to describe the biological functions of Ephs have undergone substantial revision. For example, it was thought that the high-affinity interactions of Ephs with ephrins would dominate their activities in vivo. Although there are examples that indeed meet this expectation, there are other prominent examples in which this is certainly not the case. Also, whereas cell-cell repulsion is considered a paradigm of Eph activity, there are many situations in which the outcome from interactions between the same interaction partners (in one context) drives cell-cell repulsion and (in another context) regulates cell-cell or cell-substrate adhesion (22). Furthermore, in light of emerging reports that demonstrate direct antiproliferative activities of several Ephs and ephrins on progenitor cells, stem cells, and tumor cells (23 26), the original notion that Eph-ephrin signaling did Ephrin abundance Ephrin signal capacity Fig. 3. The concept of Eph-ephrin guided cell positioning. During directional migration, an Eph- or ephrin-expressing cell (or axon), or both, is exposed to composite gradients of interacting ephrins, Ephs, or both. Considering their capacity for promiscuous interactions and their ability to assemble into signaling complexes according to the concentration and affinities of the available Ephs and ephrins, the parameters illustrated in the figure will modulate the cell-morphological responses that make up positional cues. Furthermore, the capacity to assemble distinct forward (Eph-driven) and reverse (ephrin-driven) signaling complexes on the same cell membrane (79) provides cells with spatially separated positional cues along their cell surface. Regulated ephrin cleavage, endocytosis, or both provide the molecular switch from Eph-ephrin mediated adhesion to repulsion. Eph signal capacity not directly affect cell proliferation and differentiation (27 29) is under revision. Finally, there are now many prominent examples that show the coexpression of several Ephs and ephrins within interacting cell populations or even on the surface of a single cell, in particular during neural map development (30, 31). The dissection of the complexity of the signaling pathways and biological responses that arise from these conditions in individual or composite signaling clusters is an area of active research. There is little doubt that, to predict the outcome of any particular Eph-ephrin interaction, one must interrogate several criteria, including the slope, shape, composition, and orientation of the gradients of Ephs and interacting ephrins within the studied tissue compartment (20, 32), as well as the nature of the bidirectional signaling pathways triggered by individual interactions (Figs. 2 and 3). It is likely that precise cell positioning relies not only on the accurately graded abundance of individual Eph-ephrin pairs but also on the sum of the interactions within particular localized areas and on their modulation through crosstalk with a range of other signaling systems, such as Wnt and epidermal growth factor receptor (EGFR) pathways (33, 34). Likewise, it may not be too surprising that dysregulated expression of Eph genes substantially contributes to cell-positioning defects that underlie some developmental malformations and various stages of oncogenic development. Eph Function: Cell-Cell Repulsion, Adhesion, and Everything Else In Between The prevailing model for the function of Eph-ephrin signaling is that of a chemotactic guidance system (35), which steers moving cells to a position that is accurately predetermined by the graded abundance of the corresponding cell-surface interaction partners. Chemoattractive and chemorepulsive guidance by Ephs and ephrins has been studied in a large range of developmental programs (36 39), most extensively for patterning mechanisms that are active during retinotopic mapping in rodents and chicken (30, 31), as well as during assembly of the developing mouse vasculature from endothelial and mesenchymal components (40). Extensive KO and transgenic animal studies provide compelling evidence that Ephs and ephrins are particularly suited for the tasks of coordinated positioning and sorting of motile cells, as well as for establishing critical cell-cell contacts that are required during organogenesis. On a molecular level, Ephs function by accurately relaying a genetically predetermined spatial arrangement of multidimensional gradients (containing an array of interaction partners) into a dynamic range of finetuned cellular responses, ranging from cellcell repulsion to adhesion and from increased motility to tight adhesion (Figs. 2 and 3). Although initial functional models were based on the premise that individual Eph or ephrin family members responded preferentially by mediating either adhesive or repulsive forces, there is now little doubt that, depending on the context, the same Eph-ephrin signaling pair can elicit either response (39, 41 43). Hallmarks of a new signaling concept: The interaction mode determines the biological outcome. It seems likely that the characteristic Eph-ephrin signaling mecha- Page 3

5 nism, which relies exclusively on cell surface bound interaction partners to generate and relay signals, provides the key to the apparent paradox by which a given Ephephrin pair mediates either adhesion or repulsion (Fig. 2). By default, activation of Eph tyrosine kinases activates pathways that modulate cytoskeletal plasticity, contraction of the cytoskeleton, loss of focal adhesions, cell rounding, and cell-cell repulsion or detachment (38, 44). However, Ephs and ephrins initially form heterotetrameric complexes (10), which then assemble into large signaling clusters (45) that involve several distinct Ephephrin interaction sites (46, 47) and tether the interacting cells. A distinctive feature of Ephephrin signaling the phenomenon of bidirectional signaling in the Eph-bearing cell (forward signaling) and in the ephrin-bearing cell (reverse signaling) is an extensively reviewed property that is essential for the understanding of the Normal epithelial monolayer ephrin B expressing biological functions of Ephs and ephrins (41, 42, 48). Reverse signaling, initially suggested by the presence of highly conserved cytoplasmic tyrosine residues in ephrin B (49), is initiated in ephrin clusters through their phosphorylation by associated Src kinases (50) to provide docking sites for adaptor molecules, in particular growth factor receptor bound protein 4 (Grb4), and for the initiation of signaling pathways that modulate the actin cytoskeleton (51). Very little is known about ephrin A reverse signaling (41), likely as a result of the difficulty in dissecting pathways of GPI-anchored proteins. Genetic studies in C. elegans, however, clearly suggest roles for these ephrins as signal transducers (52, 53). It is likely that Eph-ephrin mediated cell positioning can be viewed conclusively only by considering forward and reverse signaling as being equally important components, and there is mounting evidence for essential roles for reverse signaling in nerve guidance, synaptogenesis, and vascular patterning. For cell-cell repulsion to proceed after Eph-ephrin interactions, the resulting multivalent molecular tethers between opposing cells must be broken (Fig. 2): a key event that not only provides a switch between cell-cell repulsion and adhesion (54 57), but also determines the fate of the signaling cluster and consequently (necessarily) the type of resulting signaling cascade. It is now evident from several studies Adenoma: Sequestered aggregates EphB-expressing that, whereas clustering is clearly essential for phosphotyrosine-mediated Eph and ephrin signaling, it also triggers tyrosineindependent functions (58 60), in particular, cell adhesion and migration (61 63). Considerable experimental evidence confirms that the composition and dynamic regulation of Eph-ephrin signaling cluster assembly and disassembly and signal relay (Fig. 3) determine the nature and strength of the responses [reviewed in (39, 64)]. First, Eph function is regulated by phosphorylation of the activation loop tyrosine and two juxtamembrane tyrosines, which together modulate the conformation, accessibility, and activity of the kinase domain but also provide Src homology 2 domain (SH2 domain) docking sites for downstream molecules (65 67). Clearly, the ability to activate downstream pathways necessarily depends on Eph tyrosine kinase signaling capacity, and modulating the ratio of kinase-active to kinase-inactive receptors will switch responses from repulsion to adhesion (22). Protein tyrosine phosphatases (PTPs) will play important roles in modulating Eph function (68) (Fig. 2), although evidence for Eph-specific PTPs is currently limited. One potential regulator of Eph kinase activity is low molecular weight (LMW) PTP, which is believed to modulate EphB2-induced cell adhesion and capillary assembly (69), to mediate the dephosphorylation of EphA2 (70, 71), and to participate in signaling downstream of Invasive cancer with loss of Eph abundance EphB-deficient Fig. 4. A model of Eph-ephrin mediated cell positioning in epithelial cancers (carcinomas). (Left) Eph-ephrin interactions between Eph-expressing endothelial and ephrin-expressing stromal cells control the integrity of the normal epithelial monolayer. (Middle) During the initial phases of tumorigenesis (adenoma), repulsive signaling through stromal ephrin maintains the tumor cells as isolated islands (188). (Right) Loss of Eph expression during later stages allows for tumor spread and invasion. EphA2 (72 74). In addition, PTP receptor type O (Ptpro) dephosphorylates EphA4 and EphB2 in neuronal cells, thereby controlling the sensitivity of neuronal axons to ephrins (75). Other PTPs also negatively regulate reverse signaling of type B ephrins (76). Second, the abundance of Eph and ephrin in gradients directly influences the signaling outcome, and the underlying principles involved have been extensively explored in vitro (63, 69, 77 80) and in vivo [reviewed in (31, 32)]. There is also good evidence that several Eph and ephrin family members are found on the same cells during development, which raises important questions about the coexistence and regulation of forward and reverse signaling in the same cell (81, 82). Somewhat unexpectedly, the alternative scenario, in which different Eph family members that can potentially bind to a common ephrin coexist on the same cell, remains to be addressed. In light of the extensive cross-reactivity between different Eph-ephrin family members (11, 83, 84), one would expect that Ephs that can bind to common ephrins would assemble in the same signaling cluster, whereby differences in individual binding affinities would have a major effect on the composition, size, and stability of the signaling complex and its resulting signal. Third, it is apparent that regulated disruption of the molecular Eph-ephrin tether between cells fulfills a gatekeeper function Page 4

6 High abundance of Eph or ephrin Invasive, metastic melanoma: High abundance of Eph or ephrin Tissue melanocyte: Low or absent Eph or ephrin in the progression to either cell-cell repulsion or adhesion (Fig. 2). Considering how critical disruption of the Eph-ephrin tether is to the ensuing signaling pathways, a feedback mechanism for this process that is tightly linked to other parameters that control Eph-ephrin signaling would seem essential. Two mechanisms have been identified that achieve controlled termination of Eph-ephrin mediated cell-cell contacts (Fig. 2). In fibroblast monolayers of opposing cells that contain either EphB2 or EphB4 and either ephrin B1 or ephrin B2, Rac-mediated ruffling of the opposing cell membranes seems to trigger transendocytosis, whereby entire Eph-ephrin complexes, including adjacent plasma membrane components, are internalized into one of the opposing cells (55, 56). Apparently, the direction of this transendocytosis relies on the intracellular domains of the involved Eph and ephrin proteins, whereby truncation of the cytoplasmic tails of either EphB or ephrin B leads to preferential endocytosis of the Eph-ephrin complex into the ephrin- or Eph-containing cell, respectively (55, 56). Both blocking the phosphorylation of ephrin B1 (56) and exposing cells to the Rho-dependent kinase inhibitor Y [which blocks actin fiber assembly, ephrin A5 induced cell rounding, and membrane blebbing (44)] do not affect transendocytosis and cell retraction (55). The mechanism of transendocytosis is interesting, because it implies that the intact Eph-ephrin signaling cluster, which likely includes associated signaling components, would be transferred from one cell into another. Currently, very little is known about the pathways and molecules that regulate Eph internalization, but a report suggests the involvement of clathrin-mediated endocytosis (85), which is also involved in the Primary melanoma: Low or moderate abundance of Eph or ephrin Junctional nevus Fig. 5. Eph-ephrin mediated cell positioning during normal and oncogenic melanocyte biology. Pronounced Eph-ephrin interactions guide the migration of neural crest melanoblasts to their destination in the skin. Expression of Ephs and ephrins is absent in normal tissue melanocytes and nonmalignant nevi. The increasing abundance of EphA2, EphA3, ephrin A1, and ephrin B2 in primary melanoma may provide signals for the invasive growth of tumor cells in the radial growth phase and during progression into invasive melanomas. internalization of other RTKs. It seems plausible that, similar to other RTKs, critical Eph signaling steps, in particular those leading to changes in cell morphology (86), will persist in various endocytic compartments [reviewed in (87)]. Loss of Eph or ephrin during transendocytosis would obviously preclude such persistent signaling, which suggests that morphological changes in these cells may be triggered during the initial cell-cell contact and therefore executed independently of the Eph-ephrin signaling cluster. As for EphA-ephrin A mediated cellcell contacts, ephrin-shedding by the transmembrane metalloprotease ADAM10 (a Disintegrin and Metalloprotease 10), also known as Kuzbanian (54, 57), releases the molecular tether between the opposing cells (Fig. 2). In general, regulated RTK ligand cleavage by ADAM proteases fulfills essential functions during normal and pathological tissue and organ development (88), which is supported by the similarities between mice deficient in ADAM10, ADAM17, Notch, Eph, erbb1, or epidermal growth factor (EGF) (89, 90). Not surprisingly, the cleavage of ephrins is tightly regulated, whereby only the intact Eph-ephrin complex provides the critical high-affinity binding site for ADAM10, which then positions its protease domain into a conformation that allows efficient cleavage of only Eph-bound ephrin (57). Similar to other RTK ligands (89, 90), ADAM10-mediated shedding of ephrins is inhibited by tyrosine kinase inhibitors (54) and relies on Eph kinase activation, a feedback control that ensures disruption of the Eph-ephrin tether only under conditions of ongoing, repulsive Eph-ephrin signaling. To date, ADAMmediated shedding of type B ephrins has not been demonstrated; however, the finding that rhomboid-like protein 2, a rhomboid serine protease, efficiently cleaves ephrin B3 raises the possibility that ephrin shedding may also play a part in the endocytosis of EphB ephrin B complexes (91). Ephs communicate with other signaling systems. Considering the interest that Ephephrin biology has attracted, and the large number of molecules that are known to partake in downstream signaling cascades, the understanding of the pathways that execute the various responses attributed to Eph-ephrin signaling is surprisingly limited. To some extent, this may reflect the difficulty of dissecting pathways that rely on kinase activation and the generation of SH2 domain docking sites, as well as on the assembly of multimeric receptor clusters (even in the absence of kinase activity). Important roles for signaling components that execute Eph- and ephrin-triggered changes in cell morphology, motility, adhesion, and repulsion including Src and Abl kinases, phosphotyrosine-binding adaptors, PDZ domain containing proteins, the 85-kD subunit of phosphoinositide 3-kinase (PI3K), and modulators of Ras and Rho family small guanosine triphosphatases (GTPases) have been extensively reviewed (38 41, 92, 93). Not Page 5

7 surprisingly, there is crosstalk between Ephs and signaling mechanisms that control cell adhesion and cytoskeletal plasticity, such as integrin and PI3K pathways and Ras-ERK (extracellular signal regulated kinase) signaling. Initially, the notion that Eph activation seems to inhibit mitogen-activated protein kinase (MAPK) signaling was interpreted to mean that Eph signaling is largely independent of this classical mitogenic RTK signaling pathway (42). However, the finding that cyclic adenosine monophosphate response element binding protein (CREB) mediated activation of ephrin B2 transcription [downstream of MAP/ERK kinase (MEK) and ERK activation] leads to increased EphB activity, which in turn elevates N-methyl-D-aspartate (NMDA) receptor phosphorylation and activity, thus causing epileptic seizures, has brought crosstalk between Eph and MAPK signaling back into the limelight (94). The Ras- MAPK pathway is a central component of many RTK signaling mechanisms, usually activated when RTK autophosphorylation through the recruitment of Grb2 and SOS1 leads to Ras- and Raf-mediated phosphorylation of MEK1 and MEK2 and the activation of ERK1 and ERK2 (95). The integration of Eph and MAPK signaling pathways seems to be highly conserved, because this crosstalk is critical for the regulation of oocyte maturation in C. elegans (96, 97). The activity of the single C. elegans Eph receptor VAB-1 inhibits MAPK signaling and, in parallel with the C. elegans NMDA receptor, inhibits oocyte maturation a block that is relieved upon the recruitment of VAB-1 to the actin homolog, major sperm protein (96). Likewise, mammalian Ephs, in particular EphB2 and EphA2, inhibit MAPK signaling, which was initially described in neuronal and epithelial cells (98, 99). In the case of EphB2, recruitment of the GTPase activating protein (GAP) p120-ras (p120-rasgap) to the activated receptor (100, 101), which leads to reduced GTP-bound Ras and subsequent inhibition of Ras-MAPK signaling, is necessary for ephrin-induced neurite retraction (98). EphA2-mediated inhibition of the MAPK pathway in endothelial and epithelial cell lines even attenuates MAPK activation by growth factor receptor signaling (99), which agrees with the inefficient mitogenic signaling previously observed in cells that contain activated Eph proteins (27). Although subsequent studies revealed that ablation of both p120-rasgap binding sites and the introduction of an exogenous Grb2-docking site were needed to convert ephrin-b1 activated EphB2 from a MAPK-signaling repressor into an activator of ERK in neuronal cells (102), transient expression of wild-type EphB2 alone causes MAPK activation in human embryonic kidney 293T cells (67). Adding to the debate, other authors have suggested that a Shc-mediated interaction between EphA2 and Grb2 leads to ERK activation and cell detachment in breast and prostate cancer cell lines (103), whereas ephrin A1 dependent stimulation of endogenous EphA2 in wild-type mouse embryo fibroblasts (MEFs), but not in p120-rasgap / MEFs, robustly inhibits ERK phosphorylation and activation (102). However, in P19 mouse embryonic carcinoma cells, recruitment of Grb2 and Shc to activated EphB1, which leads to phosphorylation of Src and Shc and to ERK activation, seems necessary for the attachment and directed migration of these cells (104). Taken together, these findings confirm a definite involvement of Ras-MAPK signaling downstream of several Ephs in the execution of a range of disparate Eph functions, in particular the inhibition of MAPK activity. There is now also very good evidence that Ephs act downstream of MAPK signaling. In primary cortical neurons, ephrin B2 activated EphB (through associated Src) promotes NMDA receptor phosphorylation to potentiate Ca 2+ influx and activate signaling pathways involved in the specification and maturation of synaptic connections (105). It is now known that one of the genes whose transcription is activated after MAPK activation and phosphorylation of CREB is ephrin B2 itself, which suggests that, in this case, positive feedback leads to epileptic seizures (96). So how is it possible for Ras-MAPK signaling to execute such a diverse array of Eph receptor functions as well as to mediate similarly complex signaling outcomes for many other RTKs, such as EGFR and nerve growth factor receptor (NGFR)? The first clues to answer this question have come from the application of systems biology strategies (106) to unravel ERK responses in PC12 neuronal cells to inputs by EGFR and NGFR that promote proliferation and differentiation, respectively (107). These studies suggest that, depending on the type of signal relayed from the activated RTK, transient Ras-MAPK activation (by EGFR) triggers a negative feedback mechanism that results in proliferation, whereas sustained activation results in a positive feedback loop that leads to cell differentiation (107). It is tempting to speculate that a similar concept might explain the seemingly conflicting range of biological responses that emanate from activated Eph receptors, because it is highly plausible that their well-established ability to trigger opposing signaling pathways (such as MAPK activation or inhibition) reflects the capacity for dynamic regulation of the size and composition of their signaling clusters. Another example of crosstalk that may well affect Eph function in normal and oncogenic development (clearly in the intestinal epithelium) involves the communication between the Eph and Wnt signaling pathways (34, 108). Wnt signaling controls a complex program of precursor cell proliferation and renewal, Paneth cell differentiation and compartmentalization, and the ordered migration of epithelial cells along the colonic crypts, a stage that is controlled by the graded abundance of both EphB and ephrin (109). The abundance of EphB and ephrin B is tightly controlled through the β-catenin:tcf (T cell factor) transcription factor complex and, not surprisingly, EphB2 / EphB3 / mice exhibit pronounced intermingling of differentiated and precursor cell populations. It seems very likely that this role of Ephs and their regulation by Wnt signaling have major implications for tumor progression, which suggests the potential role of EphB receptors as tumor suppressors. Promiscuous or Convenient Relationships: Strong Binding Affinity Does Not Determine Function Retinotopic patterning: More than axon repulsion. According to the chemoaffinity hypothesis (110), the assembly of topographic neural maps relies on the action of axon guidance molecules, which are present in spatially restricted, complementary gradients on projecting axons and their targets and serve to position axonal termination zones that faithfully reflect the origin of individual axons. Ephs and ephrins appear to be the only protein families to date that fulfill these criteria, and their presence and function are absolutely essential for accurate topographic map assembly, a role that has been conserved from C. elegans (6, 7) to vertebrates (64). The original thinking was that Eph- and ephrin-mediated guidance operated in a straightforward manner. The pattern of the Page 6

8 low (anterior) to high (posterior) abundance of the retinal ganglion guidance ligands ephrin A5 (15) and ephrin A2 (14, 111) in the chick brain tectum or the mouse brain superior colliculus matched the low-to-high, nasal-to-temporal (NT) graded abundance of EphA3 (112) in the chick retina (111). In vitro studies that showed that these ephrins strongly repel axons from the temporal retina (in which EphA3 is highly abundant), but not from the nasal retina [in which EphA3 is low in abundance (15)], and that targeted deletion of ephrin A2 and ephrin A5 results in severe anterior-posterior (AP) mapping defects (21, 113) ended the 20-year-long search for the elusive affinity labels that had been predicted to guide retinotectal projection (110). In an apparent variation of this concept, functional analysis of EphB2- and EphB3-deficient mice revealed that ventral retinal axons, which have a high abundance of EphB2, EphB3, and EphB4, project to targets in the medial colliculus that contain a high abundance of ephrin B1, whereas dorsal axons that contain a low abundance of EphB project to the lateral part of the collicular gradient, in which the abundance of ephrin B1 is low (114). This suggested chemoattraction rather than repulsion as the underlying force that guides the projection of dorsoventral (DV) graded retinal axons into lateral-medial (LM) positions in the tectum or superior colliculus (115, 116). It seems, however, that these two apparently contradictory findings reflect aspects of an underlying common axon guidance mechanism. Generally, all retinal axons in rodents or chicken embryos, in contrast to those in fish and frogs, initially cross the superior colliculus or tectum in a nontopographic manner, substantially overshooting their correct AP and LM termination zones. Accurate positioning of the axon arbors is then established by promoting and inhibiting (back-)branching toward the correct and aberrant termination zones, respectively [reviewed in (64)]. For both axes of the projection map, this axon branching is guided by ephrins that act as liganddensity sensors (78, 117), which direct branch distribution and directional bias toward the correct position by eliciting repulsive or attractive responses according to the abundance of the Eph receptors that they encounter [reviewed in (30, 64, 116)]. Important insights into the ligand-density sensor concept have been gained from functional genetics approaches, which compared perturbations in the NT gradient of retinal EphA5 and EphA6, through the ectopic expression of EphA3, on a random proportion of retinal ganglion cells of either wild-type or EphA4-deficient mice (20, 32). Analysis of their retinotopic patterning defects demonstrated that the relative rather than the absolute abundance of Eph and ephrin, together with signaling activity, determines the projection position of retinal axons (31). Retinal axons find their correct positions by competing with all other axons for the available collicular space. Thus, axons with the highest or lowest abundance of EphA always project to either the anterior-most or posterior-most positions, respectively; ectopic overexpression of EphA3 does not lead to arborizations outside of the map. These studies also demonstrate that the slope of the gradient, which reflects the combined activities of Ephs at each position in the gradient, determines the fidelity, or the discrimination limit, at which two neighboring retinal axons project to distinct collicular positions. By considering that the profile of axon guidance molecules in the retina and superior colliculus is far more complex than initially anticipated, and that most Ephs (apart from EphA1, EphA2, EphB5, and EphB6) and ephrins (apart from ephrin A1, ephrin 3, and ephrin 4) are present in overlapping gradients on retinal ganglion cells and on collicular or tectal targets [reviewed in (64)], the finding that the relative abundance of Eph or ephrin determines axon projections has considerable implications. First, current studies and derived models have focused on selected Eph-ephrin interactions that are considered relevant because of high-affinity binding data derived in vitro. It is now obvious that the characteristic property of ephrins to promiscuously activate a range Ephs within, or outside of, a given subclass (84) and to act as signal transducers in their own right (41, 81, 118, 119) has to be taken into account to understand Eph- and ephrincontrolled positioning. As an example, retinal axons in ephrin A2 / ephrin A5 / mice show unexplained DV mapping defects (21) that are typical for EphB2 / EphB3 / mice, which suggests a potentially relevant interaction between ephrin A5 and EphB2 during map formation. Second, the presence of overlapping countergradients, and thus the coexpression on the same axon of ephrins and Ephs that would normally interact in trans, will likely affect the overall response of such an axon in the target zone. This is a topic of current research and dispute. The notion that retinal and tectal countergradients of ephrins and Ephs may help to establish and maintain the graded abundance of Eph and ephrin in these sites currently lacks experimental support. The phenotypes of ephrin A2 / ephrin A5 / mice revealed apparently undisturbed retinal gradients of EphA5 and EphA6, and likewise the ephrin B2 gradient seemed unaffected in EphB2 / EphB3 / mice (21, 114). However, analysis of ephrin A2 / ephrin A3 / ephrin A5 / mice revealed that these ephrins, in addition to their role in axon positioning (120), are essential for the establishment and internal organization of the thalamocortical projection map (121). Although the presence of different guidance signals and their integrated regulation on migrating cells seems ideally suited as the basis for high-fidelity vertebrate axon positioning, its faithful analysis in vitro with axon repulsion as readout (15, 122) provides a considerable experimental challenge. Thus, the apparently increased sensitivity of ephrin A2 / ephrin A5 / nasal axons to repulsive signals in stripe assays (21, 122) led to a model whereby retinal ephrin A2 and ephrin A5 act to inhibit repulsive signaling by EphA4 (which is present uniformly across the retina) by interacting in an antagonistic manner on the same axon membrane in cis (82, 123). Overall, this would result in a NT gradient of nonfunctional EphA4 receptors on retinal axons, complementing and thus sharpening the repulsive gradient of EphA5 and EphA6 (82). According to this model, targeted deletion of EphA4 should not affect the projections of nasal axons, because it is nonfunctional in the retinal axons of wildtype mice. However, this is not the case, and pronounced abnormal projections of these axons are observed in EphA4 / mice (32), which suggests a very significant contribution of EphA4 forward signaling to the gradient. Notably, potential axon guidance effects of ephrin reverse signaling (41, 81, 118, 119), and of interactions between EphA4 and ephrin B2 on the dorsal half of nasal axons (83) or between ephrin A5 and EphB2 on the lateral half of anterior targets (11), were not considered in the model. Likewise, structural and kinetic observations, suggesting that the binding domains of Ephs and ephrins preferentially interact in trans (11, 46, 47, 124), had not been contemplated in the context of this cis-interaction model. Third, the coexistence of various Ephs and ephrins on a single axon raises intrigu- Page 7

9 ing questions about how signaling on such a cell surface is regulated to achieve specific outcomes. In this context, it is important to consider that opposing axons with different Eph and ephrin family members on each of their surfaces will be in sufficient contact to allow the assembly of signaling complexes between neighboring axons in trans to occur, both in vivo (that is, in axon bundles entering the superior colliculus) and in vitro [such as in stripe assays in the monitoring of outgrowth from retinal ganglion cell explants (15, 122)]. Do these interactions actually take place, and if so, do they occur throughout the axon, or are there mechanisms in place that bias signaling activity and the resulting responses toward the axon growth and termination zones? The first answers to these questions were provided in a study that convincingly demonstrated that EphA receptors and GPI-anchored ephrin A ligands, coexpressed on the membrane of the same motor axon, separate into distinct domains. This lateral segregation of repulsive EphA and attractive ephrin A signaling clusters allows distinct signaling outcomes to occur in separate areas of the axon membrane (81). Importantly, the same study confirmed that overexpression of ephrin A in cells that contain both ephrin A and EphA modulates reverse ephrin A signaling and function in controlling growth cone spreading, but has no effect on EphA forward signaling mediated growth cone repulsion and collapse. Together, these findings offer an insight into how the effective uncoupling of Ephs and ephrins into distinct functional cell-membrane domains (i) provides a mechanism that controls concurrent, yet distinct, cell-morphological responses on the same cell surface, and (ii) (by mutual exclusion) effectively prevents Eph-ephrin interactions in cis. Functions of Ephs during vascular patterning. The first evidence to associate Eph signaling with angiogenesis, which was discovered before any other function of Ephs had been established, came from experiments that showed that recombinant ephrin A1 acts as a trigger of rat corneal angiogenesis in vivo and that demonstrated the chemotaxis of EphA2-containing bovine adrenal capillary endothelial cells (125) and capillary-like assembly (126) in vitro. Although the roles of EphA2 and ephrin A1 in adult angiogenic remodeling and some of the underlying signaling pathways have been confirmed (127, 128), it is now clear that type B Eph receptors and ephrins are the essential regulators of the assembly, maturation, and maintenance of blood vessels and the patterning of the vascular system in vertebrates [reviewed in (37, 40, 41)]. Similar to the concepts first described for Eph-guided axonal mapping, but less dependent on countergradients, initial models were based on the preferential abundance in functionally distinct regions (129, 130), in which ephrin B2 and EphB4 were thought to demarcate arterial and venous endothelium, respectively. Given the very similar defects that result from targeted deletion of either gene and lead to embryonic lethality (129, 131) which include the failure of angiogenic remodeling in the yolk sac and head, disrupted cardinal veins, and insufficient or missing myocardial trabeculation of the heart (formation of protrusions on the luminal wall of the early heart tube that maintain blood flow before the development of cardiac valves) it was tempting to speculate that bidirectional signaling between ephrin B2 (reverse signals) on the arterial endothelium that contacts the neighboring EphB4 on venous endothelial cells (forward signals) is essential for appropriate remodeling of the embryonic vasculature (132). Indeed, transgenic mice that express a mutant form of ephrin B2 (ephrin B2-ΔC), in which its entire cytoplasmic domain is replaced by the hemaggultinin (HA) tag sequence, show a vascular phenotype very similar to that of the classical ephrin B2 / mouse (133). This suggests that reverse signaling of ephrin B2 is essential for angiogenic remodeling (133). However, comparison of this transgenic mouse with a mouse that expresses ephrin B2 in which the cytoplasmic domain was replaced with the β-galactosidase (β-gal) protein domain revealed lethal vascular defects only in the ephrin B2-ΔC mouse. Expression of the ephrin B2 β-gal fusion protein allowed for normal angiogenesis and normal embryonic development to live-born pups, which suggests that ephrin B2 signaling is not essential for vascular development and angiogenic remodeling (118). It transpires that deletion of the cytoplasmic domain of ephrin B2 effectively inhibits its trafficking to the plasma membrane. This results in ephrin B2 being undetectable at the cell surface and acting as a null mutation that replicates the phenotype of ephrin B2 / mice (118). Thus, it seems likely that, during embryonic vascular remodeling, ephrin B2 functions only as a ligand to trigger EphB4 forward signaling. It is now evident that ephrin B2 is more widely abundant than was initially appreciated. Its coexistence with EphB2 on mesenchymal cells, including pericytes and vascular smooth muscle cells (130, ), and the inappropriate vascularization of somites in ephrin B2 / and in EphB2 / EphB3 / mice suggested a role for ephrin B2 in endothelial and mesenchymal cell-cell communication to regulate blood vessel growth into the intersomitic space (130, 131, 134). Indeed, the inappropriate ectopic expression of ephrin B2 in Xenopus (137) or mouse embryos (135) results in the abnormal migration of intersomitic veins into the adjacent somitic tissue and atypical recruitment of vascular smooth muscle cells to the ascending aorta (135). This is in line with EphB ephrin-b2 signals guiding endothelial and mesenchymal cell-cell interactions and blood vessel maturation during embryogenesis. It is tempting to extrapolate this finding to predict a mechanism whereby ephrin B2 expression in mesenchymal elements is crucially important in vascular patterning. However, endothelial-specific deletion of ephrin B2 in a targeting approach that leaves mesenchymal ephrin B2 intact, phenocopies the angiogenic remodeling defects in the yolk sac, head, trunk, and heart of conventional ephrin B2 / mice with 100% penetrance (138). This finding clearly suggests an additional and distinct role for mesenchymal ephrin B2, which does not compensate for the loss of endothelial remodeling by ephrin B2 (138). So what is the role of ephrin B2 on pericytes and smooth muscle cells? A complementary approach for the targeted deletion of ephrin B2 in mural cells that express platelet-derived growth factor receptor β on their surface has provided some important answers (139). Loss of ephrin B2 expression by this approach is lethal shortly after birth as a result of hemorrhaging of several organs, including the skin, intestine, kidney, and lung, as well as the vascular plexus of the central nervous system, which is suggestive of disrupted microvessel architecture. Detailed analysis revealed compromised or missing endothelial and smooth muscle cell (pericyte) interactions resulting from defects in the adhesion, spreading, and directional migration of ephrin B2 / smooth muscle cells. Overall, it appears that, during angiogenic remodeling, ephrin B2 acts in an adhesive manner to facilitate adequate and effective cell-cell contacts between pericytes and smooth muscle cells as Page 8

10 well as with the underlying endothelial cell layer: a model that is also consistent with the expression pattern of the relevant EphB receptors on these cells (139). Finally, emerging evidence suggests that an essential role in vasculogenesis and angiogenesis is not limited to ephrin B2 and EphB4. Several additional Ephs and ephrins are found in less-restricted arterial and venous expression patterns, including the presence of ephrin B1, EphB3, and EphB4 on venous endothelial cells and of EphB3 on some arteries (130). Also, ephrin B1, EphB2, and EphB3 are found in addition to ephrin B2 and EphB4 on the primitive vasculature: EphB2 is found on embryonic mesenchyme, whereas EphB3 and EphB4 are found on all major veins and on aortic arches, and ephrin B1 mrna is detected in all major blood vessel primordia. Apparently, these additional Eph signals fulfill partially overlapping functions in vascular development, and targeted deletion of EphB2 and EphB3 results in a 30% penetrance of vascular defects that are similar but not identical to those of ephrin B2 / mice. In particular, defective endothelial cell guidance leads to absent or abnormally shaped primordial vessels, poorly developed head vasculature, less extensively folded traberculae in the heart, and premature death at embryonic day 11.5 as a result of these vascular remodeling defects (130). In this context, it is noteworthy that the gene expression profile and cell behavior in the endothelial lining of the endocardium are reminiscent of those of the rest of the vasculature. In the heart, cell contact dependent and tissue-dependent morphological changes result in the typical twochambered morphology, the development of the atrioventricular, aortic, and pulmonary valves, and endothelial cell lined myocardial trabeculations. Consistent with their preferential expression in the endocardium, targeted deletion of either EphB2, EphB3, EphB4, or ephrin B2 results in arrested development and little or no myocardial trabeculation ( ), whereas other typical heart structures seem unaffected. Intriguingly, characterization of the perinatal-lethal phenotype of mice that lack EphA3, an Eph family member not previously implicated in cardiovascular development, reveals its critical role in the development of the atrioventricular valves of the heart (140). During cardiogenesis, EphA3 is present in the mesenchymal cells of the endocardial cushions, whereas in a complementary fashion ephrin A1 is present on the surrounding endothelial lining. Of all the EphA3-binding ephrins, ephrin A1 has the lowest affinity for EphA3 (12, 13, 141), which could indicate that such a low-affinity interaction is required for specific cellcell adhesion to establish the contact between these two cell layers, similar to the interaction between mesenchymal ephrin B2 and endothelial EphB4 discussed earlier. In this context, an earlier study revealing distinct localization in neonatal rat cardiomyocytes of EphA3, whose abundance is reduced after exposure to the proinflammatory cytokine interleukin-1 (IL-1) (142), is of note. This study, which analyzed the modulation in expression of cytokine-responsive genes upon injury (heart failure), revealed the IL-1 mediated increase in the abundance of ADAM10 concurrent with a decrease in the abundance of EphA3. In light of ADAM10 s role in cell-cell contact dependent cleavage of EphA3-bound ephrin (57), it is tempting to speculate that an increase in the ratio between ephrin A1 and EphA3 concurrent with an increase in ADAM10 abundance leads to repulsive signaling and increased mobility between mesenchymal and endothelial cells, which is required during regenerative tissue remodeling. Weak EphA4 interactions mediate axon guidance in the spinal cord. Based on structural features (143, 144), EphA4 is a typical class A receptor. In particular, the H-I loop of the N-terminal β-sandwich jellyroll structure, which is critically involved in ligand binding, consists of only 7 amino acid residues in EphA receptors, as compared with 17 residues in EphB receptors (143). This region appears to mediate the low-affinity ephrin interaction, which is required for heterotetramer formation (47, 124). As for other Ephs, EphA4 binds to both ephrin A and ephrin B ligands, although with much lower affinity to the latter (83, 145). In contrast to the countergradient mode of action discussed above, in several situations, EphA4 and its interacting ligands appear to function in discrete zones that exclude one cell population from another or act as repulsive barriers to pioneering axons. As another striking feature, EphA4 interacts in these developmental processes involving discrete zones or repulsive barriers with ephrin B ligands for which it has only very modest affinities. The exclusion of cell populations is perhaps best exemplified by the critical role of EphA4 in hindbrain development. Indeed, EphA4 (also known as segmental Eph-like kinase-1) was initially isolated in a search for genes involved in hindbrain segmentation (146). Apart from its expression in alternating hindbrain rhombomeres, a dynamic pattern of expression was observed in the forebrain, spinal cord, and presomitic mesoderm. In the hindbrain, the presence of EphA4 in the r3 and r5 rhombomeres is contrasted with that of ephrin B in the alternating rhombomeres. It was demonstrated that EphA4 interacts preferentially with ephrin B2 to establish rhombomere boundaries through a contact repulsion mechanism (147). Perhaps the best-characterized developmental role of EphA4 is in the formation of motor tracts in the spinal cord. The observation that EphA4 / mice have an abnormal gait led to the discovery of abnormal lateralization of developing corticospinal tract (CST) axons in these mice (148). Generally, once pioneering CST axons cross the midline, they do not recross it. The motor interneurons that regulate central pattern generation the regulators of gait are similarly patterned through the interaction of EphA4 with ephrin B3 (149). Using EphA4 mutant mice, researchers demonstrated that kinase-dependent EphA4 signaling triggered by ephrin B3, which is highly abundant in a uniform band at the midline, acts as an impenetrable barrier to prevent axons from recrossing the midline by causing growth cone collapse and repulsion (150). Axon collapse and repulsion involve the differential regulation of Rho family proteins (151) and, perhaps not surprisingly, the same CST axon guidance phenotype was recently observed in a spontaneous mouse mutant, termed miffy, in which disruption of the gene encoding the Rac-GAP α- chimerin results in defects similar to those observed in EphA4 / and ephrin B3 / mice (152). Elaboration of the underlying defect led to the identification of α- chimerin as a downstream target of ephrininduced EphA4 signaling in motor neurons ( ). Interestingly, knock-out of another downstream target of EphA4 signaling, the Rho guanine nucleotide exchange factor ephexin1, did not produce a neural phenotype, which suggests that there may be functional redundancy within the ephexin family (155). A secondary phenotype in EphA4 / mice, which manifests as a partially penetrant abnormality in the formation of the anterior commissure (148), is not evident in mice that express a kinase-deficient mu- Page 9