(15), re2. [DOI: 10.1126/stke.115re2] 1Science SignalingMartin Lackmann and Andrew W. Boyd (15 April 2008) Complexity?Eph, a Protein Family Coming of Age: More Confusion, Insight, or

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Features of a Young Receptor Tyro-sine Kinase FamilyIt was in 1987, in the lead-up to the humangenome project when “homology cloning”was the buzz term, that a previously unrec-ognized receptor tyrosine kinase (RTK) genewas discovered in a hepatoma cell line (1). Itbecame apparent very quickly that Eph wasthe founding member of the most populousRTK family. The genes that encode Ephs andtheir ephrin ligands are present throughoutthe animal kingdom and have an origin thatpossibly predates the parazoan-eumetazoanbifurcation (2). An exponentially growing in-terest in these proteins over the past twodecades (Fig. 1) leaves us today with an in-triguingly complex picture that characterizesthis protein family.

Conservation of both the structure andfunction of Eph and ephrin gene productsthroughout evolution (3) contrasts with thedramatic increase in the number of mem-bers of each family in vertebrates. Consid-ering signaling by RTKs as one of the uni-versal concepts of cell-cell communication(4), it is tempting to speculate that the ex-pansion of the Ephs to the largest of all

RTK families invertebrates re-flects their role asa critical cell-po-sitioning system,such that the in-creasing numberof discrete recep-tors was essentialfor the evolutionof the complexvertebrate bodyplan. In this con-text, it is interest-ing to considerthat the cell-posi-tioning function ofEph started with asingle, primordialCaenorhabdi t i selegans Eph receptor VAB-1 (5), whichinteracts with not one but four ephrins(EFN 1 to EFN 4) (6) to control kinase-dependent and kinase-independent tasks bypromoting cell-cell repulsion (7) or adhe-sion (8) in different cell types and duringdifferent stages of embryogenesis.

The expansion in the numbers of Ephsand ephrins in vertebrates (to 16 Ephs and9 ephrins) exceeds, particularly in the caseof the receptors, what one would expectfrom the two putative genome duplicationsthat occurred during the evolution of thevertebrate body plan. In bony fish—in par-ticular, zebrafish (Danio rerio), in which

Eph and ephrin expression and functionhave been studied in detail—only some ofthe duplicated genes that arose from a fur-ther genome duplication have persisted.One explanation for this is that in thesecases each of the duplicated genes has tak-en on a complementary role, and the sumof these preserved all of the functions ofthe ancestral gene (9). A large body of re-search in a range of vertebrates over thepast two decades suggests that the conceptof exploiting various Eph-ephrin combina-tions in different cellular contexts to modu-late motile cell behavior has been a verysuccessful mechanism that was conse-quently conserved and expanded duringvertebrate evolution.

Thus, the 16 vertebrate Ephs and 9 verte-brate ephrins, many of which regulateimportant cellular interactions, are in-volved in guiding organ development andpatterning of the vascular, skeletal, andnervous systems. The glycosylphos-

phatidylinositol (GPI)–anchored ephrinproteins expanded into a subfamily of sixtype A ephrins, which is complemented bya smaller subfamily of three type I trans-membrane proteins known as type Bephrins. Based on structural features intheir ligand-binding domains and theirephrin-binding preferences (10), Ephs areclassified into 10 EphA and 6 EphB recep-tors, which preferentially bind to the typeA and type B ephrins, respectively (Fig. 2).It is now emerging that this distinction maybe oversimplified, because several Ephs areactivated by both type A and type B ephrinligands (11, 12), albeit at higher concentra-

Eph, a Protein Family Coming of Age:More Confusion, Insight, or Complexity?

R E V I E W

1Department of Biochemistry and MolecularBiology, Monash University, Clayton, Victoria3800, Australia. 2Leukaemia Foundation Lab-oratory, Queensland Institute of MedicalResearch, Post Office Royal Brisbane Hospi-tal, 4029, Australia.

*Corresponding author. E-mail, martin.lackmann@med.monash.edu.au

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Fig. 1. The scientific interest in Eph-ephrin biology as measured bythe number of publications. The graph illustrates the cumulativenumber of publications that appear in a PubMed search with “ephrinOR Eph receptor” as a search term, starting from the first citation in1987 (1).

Martin Lackmann1* and Andrew W. Boyd2

Published 15 April 2008

Since the mid-1980s, Eph receptors have evolved from being regarded as or-phan receptors with unknown functions and ligands to becoming one of themost complex �global positioning systems� that regulates cell traffic in multi-cellular organisms. During this time, there has been an exponentially growinginterest in Ephs and ephrin ligands, coinciding with important advances in theway biological function is interrogated through mapping of genomes and ma-nipulation of genes. As a result, many of the original concepts that used to de-fine Eph signaling and function went overboard. Clearly, the need for progressin understanding Eph-ephrin biology and the underlying molecular principlesinvolved has been compelling. Many cell-positioning programs during normaland oncogenic development�in particular, the patterning of skeletal, vascular,and nervous systems�are modulated in some way by Eph-ephrin function. Un-deniably, the complexity of the underlying signaling networks is considerable,and it seems probable that systems biology approaches are required to furtherimprove our understanding of Eph function.

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tions of ligand than are required when theligand binds to its preferred Eph. Apartfrom the “classical examples”—includingEphA4, which is activated by type Aephrins but also has a well-established bio-logical function as a guidance receptor forephrin B2 (see below), and ephrin A5,which can effectively activate type A Ephs

as well as EphB2 (11)—there may be otherbiologically relevant EphA–ephrin B com-binations. For example, the role of EphA3as a high-aff inity receptor for type Aephrins (13), in particular for ephrin A2(14) and ephrin A5 (15–18), has been ex-tensively elaborated (see below). Addition-ally, a measurable interaction between

EphA3 and ephrin B2 in vitro (12, 19), aswell as circumstantial evidence from analy-ses of retinotectal projection maps in con-ditional EphA3 knock-in (KI) and ephrinA2 and ephrin A3 knock-out (KO) mice(20, 21), suggests the existence of otherEphA–ephrin B interactions that have rele-vance to axonal positioning.

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Fig. 2. Structural and functional features of Ephs and ephrins. Structural modules of EphA receptors (EphA1 to EphA10) include the N-ter-minal 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 Aephrins (ephrin A1 to ephrin A6) are tethered to the plasma membrane via GPI anchors, type B ephrins (ephrin B1 to ephrin B3) are trans-membrane (TM) proteins containing SH2-docking sites and PDZ-binding motifs. Eph/ephrin interactions within the A and B subfamilies areindicated by solid arrows, whereas those across Eph/ephrin subfamilies specified by broken arrows. Eph forward and ephrin reverse sig-naling in opposing cells is positively modulated by Src kinase and down-modulated by PTP activities. Cell rounding and cell repulsion relyon active Eph kinases, phosphotyrosine-mediated downstream signaling (see below), and disruption of the Eph-ephrin tether betweencells: Possible mechanisms include (ADAM10) metalloprotease-mediated shedding of Eph-bound ephrin and endocytosis of this complexor 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 adhe-sion 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.

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Many of the concepts that had been usedto describe the biological functions of Ephshave undergone substantial revision. For ex-ample, it was thought that the high-affinityinteractions of Ephs with ephrins woulddominate their activities in vivo. Althoughthere are examples that indeed meet this ex-pectation, there are other prominent exam-ples in which this is certainly not the case.Also, whereas cell-cell repulsion is consid-ered a paradigm of Eph activity, there aremany situations in which the outcome frominteractions between the same interactionpartners (in one context) drives cell-cell re-pulsion and (in another context) regulatescell-cell or cell-substrate adhesion (22). Fur-thermore, in light of emerging reports thatdemonstrate direct antiproliferative activitiesof several Ephs and ephrins on progenitorcells, stem cells, and tumor cells (23–26), theoriginal notion that Eph-ephrin signaling did

not directly affect cell proliferation and dif-ferentiation (27–29) is under revision. Final-ly, there are now many prominent examplesthat show the coexpression of several Ephsand ephrins within interacting cell popula-tions or even on the surface of a single cell,in particular during neural map development(30, 31). The dissection of the complexity ofthe signaling pathways and biological re-sponses that arise from these conditions inindividual or composite signaling clusters isan area of active research. There is littledoubt that, to predict the outcome of anyparticular Eph-ephrin interaction, one mustinterrogate several criteria, including theslope, shape, composition, and orientation ofthe gradients of Ephs and interacting ephrinswithin the studied tissue compartment (20,32), as well as the nature of the bidirectionalsignaling pathways triggered by individualinteractions (Figs. 2 and 3).

It is likely that precise cell positioningrelies not only on the accurately gradedabundance of individual Eph-ephrin pairsbut also on the sum of the interactions with-in particular localized areas and on theirmodulation through crosstalk with a rangeof other signaling systems, such as Wnt andepidermal growth factor receptor (EGFR)pathways (33, 34). Likewise, it may not betoo surprising that dysregulated expressionof Eph genes substantially contributes tocell-positioning defects that underlie somedevelopmental malformations and variousstages of oncogenic development.

Eph Function: Cell-Cell Repulsion,Adhesion, and Everything Else InBetweenThe prevailing model for the function ofEph-ephrin signaling is that of a chemotac-tic guidance system (35), which steersmoving cells to a position that is accuratelypredetermined by the graded abundance ofthe corresponding cell-surface interactionpartners. Chemoattractive and chemorepul-sive guidance by Ephs and ephrins hasbeen studied in a large range of develop-mental programs (36–39), most extensivelyfor patterning mechanisms that are activeduring retinotopic mapping in rodents andchicken (30, 31), as well as during assem-bly of the developing mouse vasculaturefrom endothelial and mesenchymal compo-nents (40). Extensive KO and transgenicanimal studies provide compelling evi-dence that Ephs and ephrins are particular-ly suited for the tasks of coordinated posi-tioning and sorting of motile cells, as wellas for establishing critical cell-cell contactsthat are required during organogenesis. Ona molecular level, Ephs function by accu-rately relaying a genetically predeterminedspatial arrangement of multidimensionalgradients (containing an array of interac-tion partners) into a dynamic range of fine-tuned cellular responses, ranging from cell-cell repulsion to adhesion and from in-creased motility to tight adhesion (Figs. 2and 3). Although initial functional modelswere based on the premise that individualEph or ephrin family members respondedpreferentially by mediating either adhesiveor repulsive forces, there is now little doubtthat, depending on the context, the sameEph-ephrin signaling pair can elicit eitherresponse (39, 41–43).

Hallmarks of a new signaling concept:The “interaction mode” determines the bi-ological outcome. It seems likely that thecharacteristic Eph-ephrin signaling mecha-

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Fig. 3. The concept of Eph-ephrin–guided cell positioning. During directional migration, anEph- or ephrin-expressing cell (or axon), or both, is exposed to composite gradients of in-teracting ephrins, Ephs, or both. Considering their capacity for promiscuous interactionsand their ability to assemble into signaling complexes according to the concentration andaffinities of the available Ephs and ephrins, the parameters illustrated in the figure willmodulate the cell-morphological responses that make up positional cues. Furthermore, thecapacity to assemble distinct forward (Eph-driven) and reverse (ephrin-driven) signalingcomplexes on the same cell membrane (79) provides cells with spatially separated posi-tional cues along their cell surface. Regulated ephrin cleavage, endocytosis, or both pro-vide the molecular switch from Eph-ephrin–mediated adhesion to repulsion.

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nism, which relies exclusively on cell sur-face–bound interaction partners to generateand relay signals, provides the key to theapparent paradox by which a given Eph-ephrin pair mediates either adhesion or re-pulsion (Fig. 2). By default, activation ofEph tyrosine kinases activates pathwaysthat modulate cytoskeletal plasticity, con-traction of the cytoskeleton, loss of focaladhesions, cell rounding, and cell-cell re-pulsion or detachment (38, 44). However,Ephs and ephrinsinitially form het-erotetrameric com-plexes (10), whichthen assemble intolarge signaling clus-ters (45) that involveseveral distinct Eph-ephrin interactionsites (46, 47) andtether the interactingcells. A distinctivefeature of Eph-ephrin signaling—the phenomenon ofbidirectional signal-ing in the Eph-bear-ing cell (forward sig-naling) and in theephrin-bearing cell(reverse signaling)—is an extensively re-viewed property thatis essential for theunderstanding of thebiological functions of Ephs and ephrins(41, 42, 48). Reverse signaling, initiallysuggested by the presence of highly con-served cytoplasmic tyrosine residues inephrin B (49), is initiated in ephrin clus-ters through their phosphorylation by asso-ciated Src kinases (50) to provide dockingsites for adaptor molecules, in particulargrowth factor receptor–bound protein 4(Grb4), and for the initiation of signalingpathways that modulate the actin cy-toskeleton (51). Very little is known aboutephrin A reverse signaling (41), likely as aresult of the difficulty in dissecting path-ways of GPI-anchored proteins. Geneticstudies in C. elegans, however, clearlysuggest roles for these ephrins as signalt ransducers (52 , 53). It is likely thatEph-ephrin–mediated cell positioning canbe viewed conclusively only by consider-ing forward and reverse signaling as beingequally important components, and thereis mounting evidence for essential rolesfor reverse signaling in nerve guidance,

synaptogenesis, and vascular patterning.For cell-cell repulsion to proceed after

Eph-ephrin interactions, the resulting mul-tivalent molecular tethers between oppos-ing cells must be broken (Fig. 2): a keyevent that not only provides a switch be-tween cell-cell repulsion and adhesion(54–57), but also determines the fate of thesignaling cluster and consequently (neces-sarily) the type of resulting signaling cas-cade. It is now evident from several studies

that, whereas clustering is clearly essentialfor phosphotyrosine-mediated Eph andephrin signaling, it also triggers tyrosine-independent functions (58–60), in particu-lar, cell adhesion and migration (61–63).Considerable experimental evidence con-firms that the composition and dynamicregulation of Eph-ephrin signaling clusterassembly and disassembly and signal relay(Fig. 3) determine the nature and strengthof the responses [reviewed in (39, 64)].

First, Eph function is regulated by phos-phorylation of the activation loop tyrosineand two juxtamembrane tyrosines, whichtogether modulate the conformation, acces-sibility, and activity of the kinase domainbut also provide Src homology 2 domain(SH2 domain)–docking sites for down-stream molecules (65–67). Clearly, theability to activate downstream pathwaysnecessarily depends on Eph tyrosine kinasesignaling capacity, and modulating the ra-tio of kinase-active to kinase-inactive re-ceptors will switch responses from repul-

sion to adhesion (22). Protein tyrosinephosphatases (PTPs) will play importantroles in modulating Eph function (68)(Fig. 2), although evidence for Eph-specificPTPs is currently limited. One potentialregulator of Eph kinase activity is lowmolecular weight (LMW)–PTP, which isbelieved to modulate EphB2-induced celladhesion and capillary assembly (69), tomediate the dephosphorylation of EphA2(70, 71), and to participate in signal-

ing downstream ofEphA2 (72–74). Inaddition, PTP re-ceptor type O (Pt-pro) dephosphory-lates EphA4 andEphB2 in neuronalcells, thereby con-trolling the sensitiv-ity of neuronal ax-ons to ephrins (75).Other PTPs alsonegatively regulatereverse signaling oftype B ephrins (76).

Second, the abun-dance of Eph andephrin in gradientsdirectly influencesthe signaling out-come, and the un-derlying principlesinvolved have beenextensively exploredin vitro (63, 69, 77–

80) and in vivo [reviewed in (31, 32)].There is also good evidence that severalEph and ephrin family members are foundon the same cells during development,which raises important questions about thecoexistence and regulation of forward andreverse signaling in the same cell (81, 82).Somewhat unexpectedly, the alternativescenario, in which different Eph familymembers that can potentially bind to acommon ephrin coexist on the same cell,remains to be addressed. In light of the ex-tensive cross-reactivity between differentEph-ephrin family members (11, 83, 84),one would expect that Ephs that can bindto common ephrins would assemble in thesame signaling cluster, whereby differencesin individual binding affinities would havea major effect on the composition, size,and stability of the signaling complex andits resulting signal.

Third, it is apparent that regulated dis-ruption of the molecular Eph-ephrin tetherbetween cells fulfills a gatekeeper function

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Fig. 4. A model of Eph-ephrin–mediated cell positioning in epithelial cancers (carcinomas).(Left) Eph-ephrin interactions between Eph-expressing endothelial and ephrin-expressingstromal cells control the integrity of the normal epithelial monolayer. (Middle) During theinitial phases of tumorigenesis (adenoma), repulsive signaling through stromal ephrinmaintains the tumor cells as isolated islands (188). (Right) Loss of Eph expression duringlater stages allows for tumor spread and invasion.

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in the progression to either cell-cell repul-sion or adhesion (Fig. 2). Considering howcritical disruption of the Eph-ephrin tetheris to the ensuing signaling pathways, afeedback mechanism for this process thatis tightly linked to other parameters thatcontrol Eph-ephrin signaling would seemessential. Two mechanisms have been iden-tified that achieve controlled terminationof Eph-ephrin–mediated cell-cell contacts(Fig. 2). In fibroblast monolayers of oppos-ing cells that contain either EphB2 orEphB4 and either ephrin B1 or ephrin B2,Rac-mediated ruffling of the opposing cellmembranes seems to trigger “transendocy-

tosis,” whereby entire Eph-ephrin complex-es, including adjacent plasma membranecomponents, are internalized into one ofthe opposing cells (55, 56). Apparently, thedirection of this transendocytosis relies onthe intracellular domains of the involvedEph and ephrin proteins, whereby trunca-tion of the cytoplasmic tails of either EphBor ephrin B leads to preferential endocyto-sis of the Eph-ephrin complex into theephrin- or Eph-containing cell, respectively(55, 56). Both blocking the phosphoryla-tion of ephrin B1 (56) and exposing cells to

the Rho-dependent kinase inhibitor Y-27632 [which blocks actin fiber assembly,ephrin A5–induced cell rounding, andmembrane blebbing (44)] do not affecttransendocytosis and cell retraction (55).The mechanism of transendocytosis is in-teresting, because it implies that the intactEph-ephrin signaling cluster, which likelyincludes associated signaling components,would be transferred from one cell into an-other. Currently, very little is known aboutthe pathways and molecules that regulateEph internalization, but a report suggeststhe involvement of clathrin-mediated endo-cytosis (85), which is also involved in the

internalization of other RTKs. It seemsplausible that, similar to other RTKs, criti-cal Eph signaling steps, in particular thoseleading to changes in cell morphology(86), will persist in various endocytic com-partments [reviewed in (87)]. Loss of Ephor ephrin during transendocytosis wouldobviously preclude such persistent signal-ing, which suggests that morphologicalchanges in these cells may be triggeredduring the initial cell-cell contact andtherefore executed independently of theEph-ephrin signaling cluster.

As for EphA-ephrin A–mediated cell-cell contacts, ephrin-shedding by the trans-membrane metalloprotease ADAM10 (aDisintegrin and Metalloprotease 10), alsoknown as Kuzbanian (54, 57), releases themolecular tether between the opposing cells(Fig. 2). In general, regulated RTK ligandcleavage by ADAM proteases fulfills essen-tial functions during normal and pathologi-cal tissue and organ development (88),which is supported by the similaritiesbetween mice def icient in ADAM10,ADAM17, Notch, Eph, erbB1, or epidermalgrowth factor (EGF) (89, 90). Not surpris-ingly, the cleavage of ephrins is tightly reg-ulated, whereby only the intact Eph-ephrincomplex provides the critical high-affinitybinding site for ADAM10, which then posi-tions its protease domain into a conforma-tion that allows efficient cleavage of onlyEph-bound ephrin (57). Similar to otherRTK ligands (89, 90), ADAM10-mediatedshedding of ephrins is inhibited by tyrosinekinase inhibitors (54) and relies on Eph ki-nase activation, a feedback control that en-sures disruption of the Eph-ephrin tetheronly under conditions of ongoing, repulsiveEph-ephrin signaling. To date, ADAM-mediated shedding of type B ephrins hasnot been demonstrated; however, the find-ing that rhomboid-like protein 2, a rhom-boid serine protease, efficiently cleavesephrin B3 raises the possibility that ephrinshedding may also play a part in the endo-cytosis of EphB–ephrin B complexes (91).

Ephs communicate with other signalingsystems. Considering the interest that Eph-ephrin biology has attracted, and the largenumber of molecules that are known topartake in downstream signaling cascades,the understanding of the pathways that exe-cute the various responses attributed toEph-ephrin signaling is surprisingly limit-ed. To some extent, this may reflect the dif-ficulty of dissecting pathways that rely onkinase activation and the generation ofSH2 domain–docking sites, as well as onthe assembly of multimeric receptor clus-ters (even in the absence of kinase activi-ty). Important roles for signaling compo-nents that execute Eph- and ephrin-trig-gered changes in cell morphology, motility,adhesion, and repulsion—including Srcand Abl kinases, phosphotyrosine-bindingadaptors, PDZ domain–containing pro-teins, the 85-kD subunit of phosphoinosi-tide 3-kinase (PI3K), and modulators ofRas and Rho family small guanosinetriphosphatases (GTPases)—have been ex-tensively reviewed (38–41, 92, 93). Not

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Fig. 5. Eph-ephrin–mediated cell positioning during normal and oncogenic melanocyte biol-ogy. Pronounced Eph-ephrin interactions guide the migration of neural crest melanoblasts totheir destination in the skin. Expression of Ephs and ephrins is absent in normal tissuemelanocytes and nonmalignant nevi. The increasing abundance of EphA2, EphA3, ephrinA1, and ephrin B2 in primary melanoma may provide signals for the invasive growth of tumorcells in the radial growth phase and during progression into invasive melanomas.

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surprisingly, there is crosstalk betweenEphs and signaling mechanisms that con-trol cell adhesion and cytoskeletal plastici-ty, such as integrin and PI3K pathways andRas-ERK (extracellular signal–regulatedkinase) signaling.

Initially, the notion that Eph activationseems to inhibit mitogen-activated proteinkinase (MAPK) signaling was interpretedto mean that Eph signaling is largely inde-pendent of this “classical” mitogenic RTKsignaling pathway (42). However, the find-ing that cyclic adenosine monophosphateresponse element–binding protein(CREB)–mediated activation of ephrin B2transcription [downstream of MAP/ERKkinase (MEK) and ERK activation] leadsto increased EphB activity, which in turnelevates N-methyl-D-aspartate (NMDA) re-ceptor phosphorylation and activity, thuscausing epileptic seizures, has broughtcrosstalk between Eph and MAPK signal-ing back into the limelight (94). The Ras-MAPK pathway is a central component ofmany RTK signaling mechanisms, usuallyactivated when RTK autophosphorylationthrough the recruitment of Grb2 and SOS1leads to Ras- and Raf-mediated phosphory-lation of MEK1 and MEK2 and the activa-tion of ERK1 and ERK2 (95). The integra-tion of Eph and MAPK signaling pathwaysseems to be highly conserved, because thiscrosstalk is critical for the regulation ofoocyte maturation in C. elegans (96, 97).The activity of the single C. elegans Ephreceptor VAB-1 inhibits MAPK signalingand, in parallel with the C. elegans NMDAreceptor, inhibits oocyte maturation—ablock that is relieved upon the recruitmentof VAB-1 to the actin homolog, majorsperm protein (96). Likewise, mammalianEphs, in particular EphB2 and EphA2, in-hibit MAPK signaling, which was initiallydescribed in neuronal and epithelial cells(98, 99). In the case of EphB2, recruitmentof the GTPase activating protein (GAP)p120-Ras (p120-RasGAP) to the activatedreceptor (100, 101), which leads to reducedGTP-bound Ras and subsequent inhibitionof Ras-MAPK signaling, is necessary forephrin-induced neurite retraction (98).EphA2-mediated inhibition of the MAPKpathway in endothelial and epithelial celllines even attenuates MAPK activation bygrowth factor receptor signaling (99),which agrees with the inefficient mitogenicsignaling previously observed in cells thatcontain activated Eph proteins (27). Al-though subsequent studies revealed that ab-lation of both p120-RasGAP–binding sites

and the introduction of an exogenousGrb2-docking site were needed to convertephrin-B1–activated EphB2 from aMAPK-signaling repressor into an activa-tor of ERK in neuronal cells (102), tran-sient expression of wild-type EphB2 alonecauses MAPK activation in human embry-onic kidney 293T cells (67). Adding to thedebate, other authors have suggested that aShc-mediated interaction between EphA2and Grb2 leads to ERK activation and celldetachment in breast and prostate cancercell lines (103), whereas ephrin A1–depen-dent stimulation of endogenous EphA2 inwild-type mouse embryo f ibroblasts(MEFs), but not in p120-RasGAP−/− MEFs,robustly inhibits ERK phosphorylation andactivation (102). However, in P19 mouseembryonic carcinoma cells, recruitment ofGrb2 and Shc to activated EphB1, whichleads to phosphorylation of Src and Shcand to ERK activation, seems necessary forthe attachment and directed migration ofthese cells (104). Taken together, thesefindings confirm a definite involvement ofRas-MAPK signaling downstream of sev-eral Ephs in the execution of a range of dis-parate Eph functions, in particular the inhi-bition of MAPK activity. There is now alsovery good evidence that Ephs act down-stream of MAPK signaling. In primarycortical neurons, ephrin B2–activatedEphB (through associated Src) promotesNMDA receptor phosphorylation to poten-tiate Ca2+ influx and activate signalingpathways involved in the specification andmaturation of synaptic connections (105).It is now known that one of the geneswhose transcription is activated afterMAPK activation and phosphorylation ofCREB is ephrin B2 itself, which suggeststhat, in this case, positive feedback leads toepileptic seizures (96).

So how is it possible for Ras-MAPKsignaling to execute such a diverse array ofEph receptor functions as well as to medi-ate similarly complex signaling outcomesfor many other RTKs, such as EGFR andnerve growth factor receptor (NGFR)? Thefirst clues to answer this question havecome from the application of systems biol-ogy strategies (106) to unravel ERK re-sponses in PC12 neuronal cells to inputsby EGFR and NGFR that promote prolif-eration and differentiation, respectively(107). These studies suggest that, depend-ing on the type of signal relayed from theactivated RTK, transient Ras-MAPK acti-vation (by EGFR) triggers a negative feed-back mechanism that results in prolifera-

tion, whereas sustained activation resultsin a positive feedback loop that leads tocell differentiation (107). It is tempting tospeculate that a similar concept might ex-plain the seemingly conflicting range ofbiological responses that emanate from ac-tivated Eph receptors, because it is highlyplausible that their well-established abilityto trigger opposing signaling pathways(such as MAPK activation or inhibition)reflects the capacity for dynamic regula-tion of the size and composition of theirsignaling clusters.

Another example of crosstalk that maywell affect Eph function in normal andoncogenic development (clearly in the in-testinal epithelium) involves the communi-cation between the Eph and Wnt signalingpathways (34, 108). Wnt signaling controlsa complex program of precursor cell prolif-eration and renewal, Paneth cell differenti-ation and compartmentalization, and theordered migration of epithelial cells alongthe colonic crypts, a stage that is controlledby the graded abundance of both EphB andephrin (109). The abundance of EphB andephrin B is tightly controlled through theβ-catenin:TCF (T cell factor) transcriptionfactor complex and, not surprisingly,EphB2−/−EphB3−/− mice exhibit pro-nounced intermingling of differentiatedand precursor cell populations. It seemsvery likely that this role of Ephs and theirregulation by Wnt signaling have majorimplications for tumor progression, whichsuggests the potential role of EphB recep-tors as tumor suppressors.

Promiscuous or Convenient Rela-tionships: Strong Binding AffinityDoes Not Determine FunctionRetinotopic patterning: More than axon re-pulsion. According to the chemoaffinityhypothesis (110), the assembly of topo-graphic neural maps relies on the action ofaxon guidance molecules, which are pre-sent in spatially restricted, complementarygradients on projecting axons and their tar-gets and serve to position axonal termina-tion zones that faithfully reflect the originof individual axons. Ephs and ephrins ap-pear to be the only protein families to datethat fulfill these criteria, and their presenceand function are absolutely essential for ac-curate topographic map assembly, a rolethat has been conserved from C. elegans(6, 7) to vertebrates (64).

The original thinking was that Eph- andephrin-mediated guidance operated in astraightforward manner. The pattern of the

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low (anterior) to high (posterior) abun-dance of the retinal ganglion guidance lig-ands ephrin A5 (15) and ephrin A2 (14,111) in the chick brain tectum or themouse brain superior colliculus matchedthe low-to-high, nasal-to-temporal (NT)graded abundance of EphA3 (112) in thechick retina (111). In vitro studies thatshowed that these ephrins strongly repelaxons from the temporal retina (in whichEphA3 is highly abundant), but not fromthe nasal retina [in which EphA3 is low inabundance (15)], and that targeted deletionof ephrin A2 and ephrin A5 results in se-vere anterior-posterior (AP) mapping de-fects (21, 113) ended the 20-year-longsearch for the elusive affinity labels thathad been predicted to guide retinotectalprojection (110). In an apparent variationof this concept, functional analysis ofEphB2- and EphB3-def icient mice re-vealed that ventral retinal axons, whichhave a high abundance of EphB2, EphB3,and EphB4, project to targets in the medialcolliculus that contain a high abundance ofephrin B1, whereas dorsal axons that con-tain a low abundance of EphB project tothe lateral part of the collicular gradient, inwhich the abundance of ephrin B1 is low(114). This suggested chemoattractionrather than repulsion as the underlyingforce that guides the projection of dorso-ventral (DV) graded retinal axons into lat-eral-medial (LM) positions in the tectum orsuperior colliculus (115, 116).

It seems, however, that these two appar-ently contradictory findings reflect aspectsof an underlying common axon guidancemechanism. Generally, all retinal axons inrodents or chicken embryos, in contrast tothose in fish and frogs, initially cross thesuperior colliculus or tectum in a nontopo-graphic manner, substantially overshootingtheir correct AP and LM terminationzones. Accurate positioning of the axon ar-bors is then established by promoting andinhibiting (back-)branching toward the cor-rect and aberrant termination zones, re-spectively [reviewed in (64)]. For both axesof the projection map, this axon branchingis guided by ephrins that act as “ligand-density sensors” (78, 117), which directbranch distribution and directional bias to-ward the correct position by eliciting repul-sive or attractive responses according to theabundance of the Eph receptors that theyencounter [reviewed in (30, 64, 116)].

Important insights into the ligand-densi-ty sensor concept have been gained fromfunctional genetics approaches, which

compared perturbations in the NT gradientof retinal EphA5 and EphA6, through theectopic expression of EphA3, on a randomproportion of retinal ganglion cells of ei-ther wild-type or EphA4-deficient mice(20, 32). Analysis of their retinotopic pat-terning defects demonstrated that the rela-tive rather than the absolute abundance ofEph and ephrin, together with signaling ac-tivity, determines the projection position ofretinal axons (31). Retinal axons find theircorrect positions by competing with allother axons for the available collicularspace. Thus, axons with the highest or low-est abundance of EphA always project toeither the anterior-most or posterior-mostpositions, respectively; ectopic overexpres-sion of EphA3 does not lead to arboriza-tions outside of the map. These studies alsodemonstrate that the slope of the gradient,which reflects the combined activities ofEphs at each position in the gradient, deter-mines the fidelity, or the “discriminationlimit,” at which two neighboring retinal ax-ons project to distinct collicular positions.By considering that the profile of axonguidance molecules in the retina and supe-rior colliculus is far more complex thaninitially anticipated, and that most Ephs(apart from EphA1, EphA2, EphB5, andEphB6) and ephrins (apart from ephrin A1,ephrin 3, and ephrin 4) are present in over-lapping gradients on retinal ganglion cellsand on collicular or tectal targets [reviewedin (64)], the finding that the relative abun-dance of Eph or ephrin determines axonprojections has considerable implications.

First, current studies and derived mod-els have focused on selected Eph-ephrininteractions that are considered relevantbecause of high-affinity binding data de-rived in vitro. It is now obvious that thecharacteristic property of ephrins topromiscuously activate a range Ephs with-in, or outside of, a given subclass (84) andto act as signal transducers in their ownright (41, 81, 118, 119) has to be taken in-to account to understand Eph- and ephrin-controlled positioning. As an example,retinal axons in ephrin A2−/−ephrin A5−/−

mice show unexplained DV mapping de-fects (21) that are typical for EphB2−/−

EphB3−/− mice, which suggests a potential-ly relevant interaction between ephrin A5and EphB2 during map formation.

Second, the presence of overlappingcountergradients, and thus the coexpres-sion on the same axon of ephrins and Ephsthat would normally interact in trans, willlikely affect the overall response of such an

axon in the target zone. This is a topic ofcurrent research and dispute. The notionthat retinal and tectal countergradients ofephrins and Ephs may help to establish andmaintain the graded abundance of Eph andephrin in these sites currently lacks experi-mental support. The phenotypes of ephrinA2−/−ephrin A5−/− mice revealed apparentlyundisturbed retinal gradients of EphA5 andEphA6, and likewise the ephrin B2 gradi-ent seemed unaffected in EphB2−/−EphB3−/−

mice (21, 114). However, analysis ofephrin A2−/−ephrin A3−/−ephrin A5−/− micerevealed that these ephrins, in addition totheir role in axon positioning (120), are es-sential for the establishment and internalorganization of the thalamocortical projec-tion map (121).

Although the presence of different guid-ance signals and their integrated regulationon migrating cells seems ideally suited as thebasis for high-fidelity vertebrate axon posi-tioning, its faithful analysis in vitro with axonrepulsion as readout (15, 122) provides aconsiderable experimental challenge. Thus,the apparently increased sensitivity of ephrinA2−/−ephrin A5−/− nasal axons to repulsivesignals in “stripe assays” (21, 122) led to amodel whereby retinal ephrin A2 and ephrinA5 act to inhibit repulsive signaling byEphA4 (which is present uniformly acrossthe retina) by interacting in an antagonisticmanner on the same axon membrane in cis(82, 123). Overall, this would result in a NTgradient of nonfunctional EphA4 receptorson retinal axons, complementing and thussharpening the repulsive gradient of EphA5and EphA6 (82). According to this model,targeted deletion of EphA4 should not affectthe projections of nasal axons, because it isnonfunctional in the retinal axons of wild-type mice. However, this is not the case, andpronounced abnormal projections of theseaxons are observed in EphA4−/− mice (32),which suggests a very significant contribu-tion of EphA4 forward signaling to the gradi-ent. Notably, potential axon guidance effectsof ephrin reverse signaling (41, 81, 118, 119),and of interactions between EphA4 andephrin B2 on the dorsal half of nasal axons(83) or between ephrin A5 and EphB2 on thelateral half of anterior targets (11), were notconsidered in the model. Likewise, structuraland kinetic observations, suggesting that thebinding domains of Ephs and ephrins prefer-entially interact in trans (11, 46, 47, 124), hadnot been contemplated in the context of thiscis-interaction model.

Third, the coexistence of various Ephsand ephrins on a single axon raises intrigu-

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ing questions about how signaling on sucha cell surface is regulated to achieve specif-ic outcomes. In this context, it is importantto consider that opposing axons with dif-ferent Eph and ephrin family members oneach of their surfaces will be in sufficientcontact to allow the assembly of signalingcomplexes between neighboring axons intrans to occur, both in vivo (that is, in axonbundles entering the superior colliculus)and in vitro [such as in stripe assays in themonitoring of outgrowth from retinal gan-glion cell explants (15, 122)]. Do these in-teractions actually take place, and if so, dothey occur throughout the axon, or arethere mechanisms in place that bias signal-ing activity and the resulting responses to-ward the axon growth and terminationzones? The first answers to these questionswere provided in a study that convincinglydemonstrated that EphA receptors andGPI-anchored ephrin A ligands, coex-pressed on the membrane of the same mo-tor axon, separate into distinct domains.This lateral segregation of repulsive EphAand attractive ephrin A signaling clustersallows distinct signaling outcomes to occurin separate areas of the axon membrane(81). Importantly, the same study con-firmed that overexpression of ephrin A incells that contain both ephrin A and EphAmodulates reverse ephrin A signaling andfunction in controlling growth cone spread-ing, but has no effect on EphA forward sig-naling–mediated growth cone repulsionand collapse. Together, these findings offeran insight into how the effective uncoup-ling of Ephs and ephrins into distinct func-tional cell-membrane domains (i) providesa mechanism that controls concurrent, yetdistinct, cell-morphological responses onthe same cell surface, and (ii) (by mutualexclusion) effectively prevents Eph-ephrininteractions in cis.

Functions of Ephs during vascular pat-terning. The first evidence to associate Ephsignaling with angiogenesis, which wasdiscovered before any other function ofEphs had been established, came from ex-periments that showed that recombinantephrin A1 acts as a trigger of rat cornealangiogenesis in vivo and that demonstratedthe chemotaxis of EphA2-containingbovine adrenal capillary endothelial cells(125) and capillary-like assembly (126) invitro. Although the roles of EphA2 andephrin A1 in adult angiogenic remodelingand some of the underlying signaling path-ways have been confirmed (127, 128), it isnow clear that type B Eph receptors and

ephrins are the essential regulators of theassembly, maturation, and maintenance ofblood vessels and the patterning of the vas-cular system in vertebrates [reviewed in(37, 40, 41)]. Similar to the concepts firstdescribed for Eph-guided axonal mapping,but less dependent on countergradients,initial models were based on the preferen-tial abundance in functionally distinct re-gions (129, 130), in which ephrin B2 andEphB4 were thought to demarcate arterialand venous endothelium, respectively. Giv-en the very similar defects that result fromtargeted deletion of either gene and lead toembryonic lethality (129, 131)—which in-clude the failure of angiogenic remodelingin the yolk sac and head, disrupted cardinalveins, and insufficient or missing myocar-dial trabeculation of the heart (formation ofprotrusions on the luminal wall of the earlyheart tube that maintain blood flow beforethe development of cardiac valves)—it wastempting to speculate that bidirectional sig-naling between ephrin B2 (reverse signals)on the arterial endothelium that contactsthe neighboring EphB4 on venous endothe-lial cells (forward signals) is essential forappropriate remodeling of the embryonicvasculature (132).

Indeed, transgenic mice that express amutant form of ephrin B2 (ephrin B2-ΔC),in which its entire cytoplasmic domain isreplaced by the hemaggultinin (HA)–tagsequence, show a vascular phenotype verysimilar to that of the classical ephrin B2−/−

mouse (133). This suggests that reversesignaling of ephrin B2 is essential for an-giogenic remodeling (133). However, com-parison of this transgenic mouse with amouse that expresses ephrin B2 in whichthe cytoplasmic domain was replaced withthe β-galactosidase (β-Gal) protein domainrevealed lethal vascular defects only in theephrin B2-ΔC mouse. Expression of theephrin B2–β-Gal fusion protein allowed fornormal angiogenesis and normal embryon-ic development to live-born pups, whichsuggests that ephrin B2 signaling is not es-sential for vascular development and an-giogenic remodeling (118). It transpiresthat deletion of the cytoplasmic domain ofephrin B2 effectively inhibits its traffickingto the plasma membrane. This results inephrin B2 being undetectable at the cellsurface and acting as a null mutation thatreplicates the phenotype of ephrin B2−/−

mice (118). Thus, it seems likely that, dur-ing embryonic vascular remodeling, ephrinB2 functions only as a ligand to triggerEphB4 forward signaling.

It is now evident that ephrin B2 is morewidely abundant than was initially appreci-ated. Its coexistence with EphB2 on mes-enchymal cells, including pericytes andvascular smooth muscle cells (130,134–135), and the inappropriate vascular-ization of somites in ephrin B2−/− and inEphB2−/−EphB3−/− mice suggested a rolefor ephrin B2 in endothelial and mesenchy-mal cell-cell communication to regulateblood vessel growth into the intersomiticspace (130, 131, 134). Indeed, the inappro-priate ectopic expression of ephrin B2 inXenopus (137) or mouse embryos (135) re-sults in the abnormal migration of inter-somitic veins into the adjacent somitic tis-sue and atypical recruitment of vascularsmooth muscle cells to the ascending aorta(135). This is in line with EphB–ephrin-B2signals guiding endothelial and mesenchy-mal cell-cell interactions and blood vesselmaturation during embryogenesis. It istempting to extrapolate this finding to pre-dict a mechanism whereby ephrin B2 ex-pression in mesenchymal elements is cru-cially important in vascular patterning.However, endothelial-specific deletion ofephrin B2 in a targeting approach thatleaves mesenchymal ephrin B2 intact, phe-nocopies the angiogenic remodeling de-fects in the yolk sac, head, trunk, and heartof conventional ephrin B2−/− mice with100% penetrance (138). This finding clear-ly suggests an additional and distinct rolefor mesenchymal ephrin B2, which doesnot compensate for the loss of endothelialremodeling by ephrin B2 (138).

So what is the role of ephrin B2 on peri-cytes and smooth muscle cells? A comple-mentary approach for the targeted deletionof ephrin B2 in mural cells that expressplatelet-derived growth factor receptor β ontheir surface has provided some importantanswers (139). Loss of ephrin B2 expres-sion by this approach is lethal shortly afterbirth as a result of hemorrhaging of severalorgans, including the skin, intestine, kidney,and lung, as well as the vascular plexus ofthe central nervous system, which is sug-gestive of disrupted microvessel architec-ture. Detailed analysis revealed compro-mised or missing endothelial and smoothmuscle cell (pericyte) interactions resultingfrom defects in the adhesion, spreading,and directional migration of ephrin B2−/−

smooth muscle cells. Overall, it appearsthat, during angiogenic remodeling, ephrinB2 acts in an adhesive manner to facilitateadequate and effective cell-cell contacts be-tween pericytes and smooth muscle cells as

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well as with the underlying endothelial celllayer: a model that is also consistent withthe expression pattern of the relevant EphBreceptors on these cells (139).

Finally, emerging evidence suggests thatan essential role in vasculogenesis and an-giogenesis is not limited to ephrin B2 andEphB4. Several additional Ephs andephrins are found in less-restricted arterialand venous expression patterns, includingthe presence of ephrin B1, EphB3, andEphB4 on venous endothelial cells and ofEphB3 on some arteries (130). Also, ephrinB1, EphB2, and EphB3 are found in addi-tion to ephrin B2 and EphB4 on the primi-tive vasculature: EphB2 is found on em-bryonic mesenchyme, whereas EphB3 andEphB4 are found on all major veins and onaortic arches, and ephrin B1 mRNA is de-tected in all major blood vessel primordia.Apparently, these additional Eph signalsfulfill partially overlapping functions invascular development, and targeted dele-tion of EphB2 and EphB3 results in a 30%penetrance of vascular defects that are sim-ilar but not identical to those of ephrin B2−/−

mice. In particular, defective endothelialcell guidance leads to absent or abnormallyshaped primordial vessels, poorly devel-oped head vasculature, less extensivelyfolded traberculae in the heart, and prema-ture death at embryonic day 11.5 as a resultof these vascular remodeling defects (130).

In this context, it is noteworthy that thegene expression profile and cell behaviorin the endothelial lining of the endocardiumare reminiscent of those of the rest of thevasculature. In the heart, cell contact–dependent and tissue-dependent morpho-logical changes result in the typical two-chambered morphology, the developmentof the atrioventricular, aortic, and pul-monary valves, and endothelial cell–linedmyocardial trabeculations. Consistent withtheir preferential expression in the endo-cardium, targeted deletion of either EphB2,EphB3, EphB4, or ephrin B2 results inarrested development and little or no myo-cardial trabeculation (129–131), whereasother typical heart structures seem unaf-fected. Intriguingly, characterization of theperinatal-lethal phenotype of mice that lackEphA3, an Eph family member not previ-ously implicated in cardiovascular develop-ment, reveals its critical role in the devel-opment of the atrioventricular valves of theheart (140). During cardiogenesis, EphA3is present in the mesenchymal cells of theendocardial cushions, whereas in a comple-mentary fashion ephrin A1 is present on

the surrounding endothelial lining. Of allthe EphA3-binding ephrins, ephrin A1 hasthe lowest affinity for EphA3 (12, 13, 141),which could indicate that such a low-affini-ty interaction is required for specific cell-cell adhesion to establish the contact be-tween these two cell layers, similar to theinteraction between mesenchymal ephrinB2 and endothelial EphB4 discussed earli-er. In this context, an earlier study reveal-ing distinct localization in neonatal rat car-diomyocytes of EphA3, whose abundanceis reduced after exposure to the proinflam-matory cytokine interleukin-1 (IL-1) (142),is of note. This study, which analyzed themodulation in expression of cytokine-re-sponsive genes upon injury (heart failure),revealed the IL-1–mediated increase in theabundance of ADAM10 concurrent with adecrease in the abundance of EphA3. Inlight of ADAM10’s role in cell-cell con-tact–dependent cleavage of EphA3-boundephrin (57), it is tempting to speculate thatan increase in the ratio between ephrin A1and EphA3 concurrent with an increase inADAM10 abundance leads to repulsive sig-naling and increased mobility between mes-enchymal and endothelial cells, which is re-quired during regenerative tissue remodeling.

Weak EphA4 interactions mediate axonguidance in the spinal cord. Based onstructural features (143, 144), EphA4 is atypical class A receptor. In particular, theH-I loop of the N-terminal β-sandwich jel-lyroll structure, which is critically involvedin ligand binding, consists of only 7 aminoacid residues in EphA receptors, as com-pared with 17 residues in EphB receptors(143). This region appears to mediate thelow-affinity ephrin interaction, which isrequired for heterotetramer formation(47, 124). As for other Ephs, EphA4 bindsto both ephrin A and ephrin B ligands,although with much lower affinity to thelatter (83, 145).

In contrast to the countergradient modeof action discussed above, in several situa-tions, EphA4 and its interacting ligands ap-pear to function in discrete zones that ex-clude one cell population from another oract as repulsive barriers to pioneering ax-ons. As another striking feature, EphA4 in-teracts in these developmental processesinvolving discrete zones or repulsive barri-ers with ephrin B ligands for which it hasonly very modest affinities.

The exclusion of cell populations is per-haps best exemplified by the critical role ofEphA4 in hindbrain development. Indeed,EphA4 (also known as segmental Eph-like

kinase-1) was initially isolated in a searchfor genes involved in hindbrain segmenta-tion (146). Apart from its expression in al-ternating hindbrain rhombomeres, a dy-namic pattern of expression was observedin the forebrain, spinal cord, and pre-somitic mesoderm. In the hindbrain, thepresence of EphA4 in the r3 and r5 rhom-bomeres is contrasted with that of ephrin Bin the alternating rhombomeres. It wasdemonstrated that EphA4 interacts prefer-entially with ephrin B2 to establish rhom-bomere boundaries through a contact re-pulsion mechanism (147).

Perhaps the best-characterized develop-mental role of EphA4 is in the formation ofmotor tracts in the spinal cord. The obser-vation that EphA4−/− mice have an abnor-mal gait led to the discovery of abnormallateralization of developing corticospinaltract (CST) axons in these mice (148).Generally, once pioneering CST axonscross the midline, they do not recross it.The motor interneurons that regulate cen-tral pattern generation—the regulators ofgait—are similarly patterned through theinteraction of EphA4 with ephrin B3 (149).Using EphA4 mutant mice, researchersdemonstrated that kinase-dependentEphA4 signaling triggered by ephrin B3,which is highly abundant in a uniformband at the midline, acts as an impenetra-ble barrier to prevent axons from recross-ing the midline by causing growth conecollapse and repulsion (150). Axon col-lapse and repulsion involve the differentialregulation of Rho family proteins (151)and, perhaps not surprisingly, the sameCST axon guidance phenotype was recent-ly observed in a spontaneous mouse mu-tant, termed miffy, in which disruption ofthe gene encoding the Rac-GAP α-chimerin results in defects similar to thoseobserved in EphA4−/− and ephrin B3−/−

mice (152). Elaboration of the underlyingdefect led to the identif ication of α-chimerin as a downstream target of ephrin-induced EphA4 signaling in motor neurons(152–154). Interestingly, knock-out of an-other downstream target of EphA4 signal-ing, the Rho guanine nucleotide exchangefactor ephexin1, did not produce a neuralphenotype, which suggests that there maybe functional redundancy within the ephex-in family (155).

A secondary phenotype in EphA4−/−

mice, which manifests as a partially pene-trant abnormality in the formation of theanterior commissure (148), is not evidentin mice that express a kinase-deficient mu-

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tant EphA4 (150) or in ephrin B3−/− mice(156). Based on mRNA expression data,ephrin B2 is the ligand most likely to befound on the neurons (150). Given that thekinase function of EphA4 is not needed forthis developmental process, this phenotypehas been used as a paradigm for an axonguidance function that is mediated byephrin B reverse signaling. However, a KImouse that expresses mutant, constitutivelyactive, EphA4 has no apparent abnormali-ties in any of the developmental processeselaborated in these KO studies, but insteadshows defects in thalamocortical projec-tions and subtle abnormalities in the cen-tral pattern generator mechanism (59). Thisphenotype has been interpreted as reflect-ing the need for ephrin-induced higher-order signaling clusters in the recruitmentof adequate downstream signaling compo-nents, which does not occur with constitu-tively active receptors.

It seems somewhat counterintuitive thatthe most prominent examples for EphA4function are relayed through its interactionswith ephrin B ligands, despite EphA4 hav-ing much lower affinities for these ligandsas compared with its affinities for ephrin Aproteins (83, 145). However, there are ex-amples for EphA4–ephrin-A–mediatedregulation of developmental processes,which include neural crest migration (157)and limb bud development (158). Also, theinteraction of EphA4 with ephrin A3,which has a modest affinity for EphA4, iscritical for the development of correct hip-pocampal dendritic spine morphology(159).

Ephs in CancerA function for Ephs in oncogenesis has beenimplied since their discovery, initially basedon their unscheduled expression in humantumors or cell lines and by extrapolatingfrom insights of their roles in normal devel-opment. In general, it was assumed thatoverexpression of Eph and ephrin geneswould contribute to tumor progression bypromoting tumor spread and metastasis(160–163). However, emerging functionaldata, which suggest tumor-suppressive rolesfor Ephs in some cases and antiproliferativeroles of Ephs and ephrins in other situations,imply that this early assumption may notnecessarily hold true.

Do Ephs affect proliferation? One char-acteristic that is often regarded as a sinequa non for the cancerous state is uncon-trolled cell proliferation. At the time of thediscovery of Eph (EphA1) in cancer cells,

it was therefore assumed that Eph RTKs, incommon with other prominent RTK familymembers, would have direct effects on pro-liferation and be candidate oncogenes. In-deed, early reports suggested that EphA1(Eph) behaved as a classical oncogene,which is consistent with a role in prolifera-tion (164). Similarly, other studies suggest-ed that EphA2 and ephrin A1 had an au-tocrine proliferative effect in malignantmelanoma (165). However, many otherstudies failed to demonstrate any prolifera-tive effects, and the overwhelming evi-dence suggested a prominent role for sig-naling of Ephs in cell positioning. This fo-cused most of the attention on their role inregulating cell position and motility duringtumor metastasis and neo-angiogenesis(39, 160, 162, 163, 166). Accordingly, theconspicuous expression of Ephs in stemcell or precursor cell populations in theadult brain, colon, and skin (23, 109, 167),and the proliferative effect of ephrin B2-Fcor EphB2-Fc on adult neuroblasts (23),were initially interpreted as secondary to acell-positioning effect. However, recentstudies suggesting that the absence ofephrin A2 reverse signaling in ephrin A2−/−

mice leads to an increased number of pro-liferating neural progenitor cells withoutaffecting cell migration (25) clearly pointto a direct antimitogenic role for ephrin A2signaling. Other reports have suggestedpositive effects of Eph-ephrin signaling onthe proliferation of neural precursors (168)and for intestinal stem cells (167). Howev-er, most studies have reported antiprolifera-tive effects of Eph-ephrin signaling in bothnormal cells (168) and cancer cells (99,170, 171). As with other aspects of Eph-ephrin biology, it appears that it may de-pend on the nature of the interacting pro-teins and the cellular context if Ephs andephrins affect proliferation in a positive ornegative manner.

Do Ephs promote or suppress tumors?The high abundance of Ephs and ephrinsin many human cancers—including vari-ous carcinomas, melanoma, sarcoma, kid-ney, and brain tumors—has been previous-ly reviewed (160, 162, 163). In some cas-es, there is a clear link between this in-creased abundance and tumor progression,which is perhaps best exemplified in ma-lignant melanoma (44, 172, 173), wherethe expression of at least one of three Ephgenes is increased during melanomaprogression. A mutational screen ofmelanoma, glioblastoma, and pancreaticcancer reported a potential functional mu-

tation in EphA3 in melanoma and furthermutations in glioblastoma (174). Inglioblastoma, the increased abundance ofEphA2, EphB2, and ephrin B3 is directlyand functionally linked to cellular process-es that mediate invasiveness (175–178).Reverse signaling by ephrin B1 is alsocritically important for tumor invasivenessof gastric scirrhous cancers, which is de-pendent on the activation of Rac1, as wasobserved for ephrin B3 signaling inglioblastoma (179).

However, emerging evidence suggeststhat the notion of the high expression ofEph and ephrin genes conferring a moreinvasive or metastatic phenotype on cancercells is probably an oversimplification, par-ticularly in the case of epithelial tumors(carcinomas) (34, 171, 180). A prominentexample is EphB4, which seems to playcontradictory roles in breast and colon can-cer (26). On the one hand, the increasedabundance of EphB2 and EphB4 and theirligand ephrin B2 has been implicated inbreast and colon cancer (181–184), where-as on the other hand, EphB4 has a tumor-suppressive role in vitro (171), in mousemodels (185), and in human colorectal can-cer (180). Analysis of the EphB–ephrin B2interaction in breast cancer cells and tumorxenografts revealed that activation ofephrin B2 by cytoplasmic truncated signal-ing-compromised EphB receptors may playa pro-tumorigenic role by promoting tumorangiogenesis (151). By contrast, retroviraloverexpression of EphB4 in tumorxenografts and activation of EphB4 signal-ing switch the angiogenic program fromone of sprouting to one of circumferentialblood vessel growth and reduced vesselpermeability, obvious features of a non-neoplastic vascularization program (186).

It is highly likely not only that the ex-pression per se will determine pro- or anti-neoplastic roles of Ephs, but also that mod-ulation of Eph function by somatic muta-tion may enhance their pro-oncogenicproperties. This was shown for EphA3,which, alongside K-Ras and APC, wasidentified in a large-scale screen of somaticmutations as one of the three top coloncancer genes (187). Importantly, the modu-lation of Eph gene transcription during tu-mor progression also critically affectsoncogenic outcomes. Expression of thegenes encoding EphB2, EphB3, and theirligands is transcriptionally regulated by β-catenin and TCF to direct the sorting andmigration of precursors and differentiatingcell types within the normal and malignant

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gut epithelium (109). A gradient of de-creasing concentrations of EphB2 andEphB3 from the colon crypt to the villus iscountered by an increasing concentrationgradient of ephrin B, and cell-repulsive sig-naling helps to regulate the flow of maturecells from the crypt to the villus surface.Although further data are required to fullyunderstand the complex role of Eph andephrin functions in this epithelial cancer,one model that fits the current data arguesfor a tumor-promoting role of Ephs andephrins in the genesis of premalignantcolon tumors (polyps) and in early cancers.However, as tumors progress, heterogeneityof Eph and ephrin gene expression and ac-tivating or inactivating mutations result inapparently contradictory outcomes. Insome cases, the increased abundance ofEphs or ephrins persists, while in other tu-mors a loss of the increased Eph or ephrinabundance correlates with increased inva-siveness and metastatic behavior. A recentpaper now presents data compatible withsuch a model (188). The study reveals thatthe presence of EphB prevents invasivenessby mediating tumor cell repulsion awayfrom normal cells that contain ephrin B.The neoplastic cells form tumors but donot invade. Upon loss of EphB protein, thetumor cells become capable of invadingsurrounding tissues (Fig. 4). Whether thisreflects a difference in Eph-ephrin signal-ing in individual tumors, which in somecases is cell-repulsive and thus is likely toblock invasiveness and in other cases iscell-adhesive and enhances tumor spreadinto surrounding tissue, remains unclear.However, this explanation would fit withthe heterogeneity of Eph signaling found indifferent developmental processes. HowEph gene expression is lost during tumorevolution remains unclear. One mechanismthat has been demonstrated in colorectalcancer for both EphA7 (189) and EphB2(190) is epigenetic gene silencing. An ob-servation that may serve to provide a fur-ther mechanism is inhibition of Wnt signal-ing by hypoxia (191). Here it was arguedthat hypoxia induces the activity of hypox-ia-inducible factor 1α, which competeswith TCF1 for binding to β-catenin, thuseffectively suppressing transcription ofWnt-target genes such as EphB2 andEphB3. Such a mechanism would alsomake the EphB2 locus susceptible to per-manent silencing by gene methylation.Whether this could be seen as a generalmechanism for loss of Eph expression isunlikely, because EphA2, EphA3, and

EphB4 appear to be up-regulated by hypox-ia (192, 193).

A biphasic model, in which high Ephgene expression and oncogenic capacityearly in tumor development give way to aloss of Eph gene expression to allow tumorinvasion and metastasis, implies that Ephsignaling is tumor-suppressive at this stageand fits well for epithelial cancers. Howev-er, this is not the case for mesenchymal tu-mors. Perhaps the most studied example ofa mesenchymal tumor is melanoma.Melanocytes are neural crest derivativesthat migrate throughout the embryo beforedifferentiating into typical subepithelialmelanocytes. In the skin, for example,these cells provide pigment to overlyingkeratinocytes. Eph-ephrin signaling plays awell-characterized role in the migration ofneural crest derivatives (157, 194–196) andspecifically of neural crest melanoblasts(197). The melanocyte-to-melanoma tran-sition occurs initially in the skin but, as thetumor develops, it reacquires the high ex-pression of Eph genes (44, 172, 198) andthe migratory properties of themelanoblast. In studies of murinemelanoma, high Eph abundance and sig-naling have been directly linked to tumorinvasiveness. Thus, in this situation, Eph-ephrin signaling is clearly tumor-promot-ing (Fig. 5). Very limited evidence is avail-able for sarcomas, but these reports alsosuggest a tumor-promoting role for Ephs inthese tumors (199, 200).

Thus, as in other situations, Eph-ephrinsignaling in cancer is not simple, and muchmore information is required to understandthe heterogeneity of Eph and ephrin func-tions in neoplasia. The two scenarios posedhere rest on the very different biology ofepithelial tumors (carcinomas) as com-pared with that of “mesenchymal” tumors(melanoma, sarcoma, leukemia, and lym-phoma). However, it seems certain that thistwo-pathway model of tumor developmentis an oversimplification and will require re-finement in the face of new evidence.

OutlookThe ability of Eph-ephrin signaling to elicitquite distinct and indeed opposing effectsin different cells has made for a confusingand frustrating situation, even for thosewell versed in this f ield. There is littledoubt that the past decade of research hassubstantially increased our knowledge ofEph and ephrin functions, as well as theunderlying mechanisms involved, and hashelped to clarify some of the apparent dis-

crepancies that stemmed from earlier stud-ies. Thus, it seems now to be well estab-lished that cell-cell repulsion and adhesionare two extreme cases of a common under-lying cellular response to Eph ligation andthat a fine-tuned combination of interac-tion partners, rather than a single Eph-ephrin pair, is often what seems to deter-mine the ultimate cellular response. On theother hand, the range of activities, interac-tion partners, and molecular mechanismsunderlying Eph and ephrin functions hassteadily increased, leading to repeated, al-most continuous revision of previously es-tablished models. It is tempting to specu-late that we will eventually be able to ac-quire expression data on Ephs, ephrins, andinteracting membrane and signaling pro-teins from interacting cell types and tocombine these data with functional, inter-action, and kinetic data to feed into neuralnetwork algorithms that accurately predictoutcomes. Ultimately, only comprehensivemapping of the interconnected signalingnetworks within a cell will provide realisticviews of the mechanisms that dictate cellu-lar responses, and models addressing thistask are emerging (106). Although it seemslikely that such global approaches to char-acterize signaling complexes and pathwayswill help to decipher Eph signaling path-ways, at present, we are still far short ofhaving the required information to allowprediction of the responses of a given cellto Eph-ephrin signaling.

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