Structure and thermodynamic characterization of the EphB4/Ephrin-B2 antagonist peptide complex reveals the determinants for receptor specificity

The Eph receptor tyrosine kinases and their ligands, the ephrins, regulate numerous biological processes in developing and adult tissues and have been implicated in cancer progression and in pathological forms of angiogenesis. We report the crystal structure of the EphB4 receptor in complex with a highly specific antagonistic peptide at a resolution of 1.65 angstroms. The peptide is situated in a hydrophobic cleft of EphB4 corresponding to the cleft in EphB2 occupied by the ephrin-B2 G-H loop, consistent with its antagonistic properties. Structural analysis identifies several residues within the EphB4 binding cleft that likely determine the ligand specificity of this receptor, while isothermal titration calorimetry experiments with truncated forms of the peptide define the amino acid residues of the peptide that are critical for receptor binding. These studies reveal structural features that will aid drug discovery initiatives to develop EphB4 antagonists for therapeutic applications.     

Three-dimensional structure of the EphB2 receptor in complex with an antagonistic peptide reveals a novel mode of inhibition

The Eph family of receptor tyrosine kinases has been implicated in tumorigenesis as well as pathological forms of angiogenesis. Understanding how to modulate the interaction of Eph receptors with their ephrin ligands is therefore of critical interest for the development of therapeutics to treat cancer. Previous work identified a set of 12-mer peptides that displayed moderate binding affinity but high selectivity for the EphB2 receptor. The SNEW antagonistic peptide inhibited the interaction of EphB2 with ephrinB2, with an IC50 of approximately 15 microm. To gain a better molecular understanding of how to inhibit Eph/ephrin binding, we determined the crystal structure of the EphB2 receptor in complex with the SNEW peptide to 2.3-A resolution. The peptide binds in the hydrophobic ligand-binding cleft of the EphB2 receptor, thus competing with the ephrin ligand for receptor binding. However, the binding interactions of the SNEW peptide are markedly different from those described for the TNYL-RAW peptide, which binds to the ligand-binding cleft of EphB4, indicating a novel mode of antagonism. Nevertheless, we identified a conserved structural motif present in all known receptor/ligand interfaces, which may serve as a scaffold for the development of therapeutic leads. The EphB2-SNEW complex crystallized as a homodimer, and the residues involved in the dimerization interface are similar to those implicated in mediating tetramerization of EphB2-ephrinB2 complexes. The structure of EphB2 in complex with the SNEW peptide reveals novel binding determinants that could serve as starting points in the development of compounds that modulate Eph receptor/ephrin interactions and biological activities.     

Novel strategy for selection of monoclonal antibodies against highly conserved antigens: phage library panning against ephrin-B2 displayed on yeast

Ephrin-B2 is predominately expressed in endothelium of arterial origin, involved in developmental angiogenesis and neovasculature formation through its interaction with EphB4. Despite its importance in physiology and pathological conditions, it has been challenging to produce monoclonal antibodies against ephrin-B2 due to its high conservation in sequence throughout human and rodents. Using a novel approach for antibody selection by panning a phage library of human antibody against antigens displayed in yeast, we have isolated high affinity antibodies against ephrin-B2. The function of one high affinity binder (named as 'EC8') was manifested in its ability to inhibit ephrin-B2 interaction with EphB4, to cross-react with murine ephrin-B2, and to induce internalization into ephrin-B2 expressing cells. EC8 was also compatible with immunoprecipitation and detection of ephrin-B2 expression in the tissue after standard chemical fixation procedure. Consistent with previous reports on ephrin-B2 induction in some epithelial tumors and tumor-associated vasculatures, EC8 specifically detected ephrin-B2 in tumors as well as the vasculature within and outside of the tumors. We envision that monoclonal antibody developed in this study may be used as a reagent to probe ephrin-B2 distribution in normal as well as in pathological conditions and to antagonize ephrin-B2 interaction with EphB4 for basic science and therapeutic applications.     

Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development.

Ephrin-B2 is a transmembrane ligand that is specifically expressed on arteries but not veins and that is essential for cardiovascular development. However, ephrin-B2 is also expressed in nonvascular tissues and interacts with multiple EphB class receptors expressed in both endothelial and nonendothelial cell types. Thus, the identity of the relevant receptor for ephrin-B2 and the site(s) where these molecules interact to control angiogenesis were not clear. Here we show that EphB4, a specific receptor for ephrin-B2, is exclusively expressed by vascular endothelial cells in embryos and is preferentially expressed on veins. A targeted mutation in EphB4 essentially phenocopies the mutation in ephrin-B2. These data indicate that ephrin-B2-EphB4 interactions are intrinsically required in vascular endothelial cells and are consistent with the idea that they mediate bidirectional signaling essential for angiogenesis.     

Generation of transgenic mice overexpressing EfnB2 in endothelial cells

Genetic studies have shown that ephrin-B2 and its cognate EphB4 receptor are necessary for normal embryonic angiogenesis. Moreover, there is overwhelming evidence that ephrin-B2 is involved in tumor vascularization, yet its role in adult angiogenesis has been difficult to track genetically. Here, we report the generation of transgenic mice that over-express EfnB2 specifically in endothelial cells (ECs). We show that exogenous expression of EfnB2 under the control of the Tie2 promoter/enhancer regions in ECs does not affect viability or growth of the transgenic animals. We further show that targeted expression of EfnB2 in ECs is not sufficient to rescue severe cardiovascular defects at mid-gestation stages but rescues early embryonic lethality associated with loss-of-function mutation in EfnB2. This mouse model will be useful to study the role of ephrin-B2 in physiological and pathological angiogenesis.     

The EphB4 receptor-tyrosine kinase promotes the migration of melanoma cells through Rho-mediated actin cytoskeleton reorganization

Several studies have reported the up-regulation of EphB receptor-tyrosine kinases and ephrin-B ligands in a variety of tumors, suggesting a functional relation between EphB/ephrin-B signaling and tumor progression. The ability of the EphB receptors to regulate cell migration and promote angiogenesis likely contributes to tumor progression and metastasis. Here we show that EphB receptors, and especially EphB4, regulate the migration of murine melanoma cells. Highly malignant melanoma cells express the highest levels of EphB4 receptor and migrate faster than less malignant melanoma cells. Furthermore, inhibition of EphB receptor forward signaling by overexpression of a form of EphB4 lacking the cytoplasmic portion or by treatment with competitively acting soluble EphB2-Fc results in slower melanoma cell migration. In contrast, overexpression of active EphB4 significantly enhances cell migration. The effects of EphB4 receptor on cell migration and cell morphology require its kinase activity because the inhibition of EphB4 kinase activity by overexpression of kinase dead EphB4 inhibits cell migration and affects the organization of actin cytoskeleton. Activation of EphB4 receptor with its ligand ephrin-B2-Fc enhances the migratory ability of melanoma cells and increases RhoA activity, whereas inhibiting EphB receptor forward signaling decreases RhoA activity. Moreover, expression of dominant negative RhoA blocks the effects of active EphB4 on cell migration and actin organization. These data suggest that EphB4 forward signaling contributes to the high migratory ability of invasive melanoma cells by influencing RhoA-mediated actin cytoskeleton reorganization.     

Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through alphavbeta3 and alpha5beta1 integrins

Receptors of the Eph family and their ligands (ephrins) mediate developmental vascular assembly and direct axonal guidance. Migrating cell processes identify appropriate targets within migratory fields based on topographically displayed ephrin gradients. Here, EphB1 regulated cell attachment by discriminating the density at which ephrin-B1 was displayed on a reconstituted surface. EphB1-ephrin-B1 engagement did not promote cell attachment through mechanical tethering, but did activate integrin-mediated attachment. In endothelial cells, attachment to RGD peptides or fibrinogen was mediated through alphavbeta3 integrin. EphB1 transfection conferred ephrin-B1-responsive activation of alpha5beta1 integrin-mediated cell attachment in human embryonic kidney cells. Activation-competent but signaling-defective EphB1 point mutants failed to stimulate ephrin-B1 dependent attachment. These findings lead us to propose that EphB1 functions as a 'ligand density sensor' to signal integrin-mediated cell-matrix attachment.     

The receptor tyrosine kinase EphB4 and ephrin-B ligands restrict angiogenic growth of embryonic veins in Xenopus laevis

The cues and signaling systems that guide the formation of embryonic blood vessels in tissues and organs are poorly understood. Members of the Eph family of receptor tyrosine kinases and their cell membrane-anchored ligands, the ephrins, have been assigned important roles in the control of cell migration during embryogenesis, particularly in axon guidance and neural crest migration. Here we investigated the role of EphB receptors and their ligands during embryonic blood vessel development in Xenopus laevis. In a survey of tadpole-stage Xenopus embryos for EphB receptor expression, we detected expression of EphB4 receptors in the posterior cardinal veins and their derivatives, the intersomitic veins. Vascular expression of other EphB receptors, including EphB1, EphB2 or EphB3, could however not be observed, suggesting that EphB4 is the principal EphB receptor of the early embryonic vasculature of Xenopus. Furthermore, we found that ephrin-B ligands are expressed complementary to EphB4 in the somites adjacent to the migratory pathways taken by intersomitic veins during angiogenic growth. We performed RNA injection experiments to study the function of EphB4 and its ligands in intersomitic vein development. Disruption of EphB4 signaling by dominant negative EphB4 receptors or misexpression of ephrin-B ligands in Xenopus embryos resulted in intersomitic veins growing abnormally into the adjacent somitic tissue. Our findings demonstrate that EphB4 and B-class ephrins act as regulators of angiogenesis possibly by mediating repulsive guidance cues to migrating endothelial cells.     

Expression and localisation of ephrin-B1, EphB2, and EphB4 in the mouse testis during postnatal development

The erythropoietin-producing hepatocellular receptor B (EphB) class and ephrin-B ligand have been implicated in boundary formation in various epithelia. We recently found that ephrin-B1 and EphB2/EphB4 exhibit complementary expression in the epithelia along the excurrent duct system in the adult mouse testis. Moreover, the organisation and integrity of the duct system is indispensable for the transport of spermatozoa. Here, we examined ephrin-B1, EphB2 and EphB4 expression in the mouse testis during postnatal development. RT-PCR analysis revealed that the relative expression levels of these molecules decreased with age in early postnatal development, and were similar to those of adults by four weeks of age. Furthermore, immunostaining revealed that the excurrent duct system compartments exhibiting complementary expression of ephrin-B1 and EphB2/EphB4 were formed by two weeks of age. Meanwhile, ephrin-B1 and EphB4 were effective markers for spermatogonia in the neonatal testis due to their negative expression in gonocytes. Alternatively, EphB2 was a suitable marker for assessing completion of the first wave of spermatogenesis in puberty, due to its strong expression in the elongated spermatids of seminiferous tubules. Lastly, ephrin-B1 and EphB4 proved to be markers of both foetal and adult Leydig cells during postnatal development, as they were expressed in CYP17A1-positive cells. This study is the first to investigate the expression of ephrin-B1, EphB2, and EphB4 in normal mouse testes during postnatal development. The expression patterns of ephrin-B and EphBs may represent suitable tools for examining organisation of the excurrent duct system and monitoring reproductive toxicity during postnatal development.     

EphB receptor-binding peptides identified by phage display enable design of an antagonist with ephrin-like affinity

The Eph receptor tyrosine kinases are overexpressed in many pathologic tissues and have therefore emerged as promising drug target candidates. However, there are few molecules available that can selectively bind to a single Eph receptor and not other members of this large receptor family. Here we report the identification by phage display of peptides that bind selectively to different receptors of the EphB class, including EphB1, EphB2, and EphB4. Peptides with the same EphB receptor specificity compete with each other for binding, suggesting that they have partially overlapping binding sites. In addition, several of the peptides contain amino acid motifs found in the G-H loop of the ephrin-B ligands, which is the region that mediates high-affinity interaction with the EphB receptors. Consistent with targeting the ephrin-binding site, the higher affinity peptides antagonize ephrin binding to the EphB receptors. We also designed an optimized EphB4-binding peptide with affinity comparable with that of the natural ligand, ephrin-B2. These peptides should be useful as selective inhibitors of the pathological activities of EphB receptors and as targeting agents for imaging probes and therapeutic drugs.     

Ephrin-B2 reverse signaling is required for axon pathfinding and cardiac valve formation but not early vascular development

Vascular development begins with the formation of a primary vascular plexus that is rapidly remodeled by angiogenesis into the interconnected branched patterns characteristic of mature vasculature. Several receptor tyrosine kinases and their ligands have been implicated to control early development of the vascular system. These include the vascular endothelial growth factor receptors (VEGFR-1 and VEGFR-2) that bind VEGF, the Tie-1 and Tie-2 receptors that bind the angiopoietins, and the EphB4 receptor that binds the membrane-anchored ligand ephrin-B2. Targeted mutations in the mouse germline have revealed essential functions for these molecules in vascular development. In particular, protein-null mutations that delete either EphB4 or ephrin-B2 from the mouse have been shown to result in early embryonic lethality due to failed angiogenic remodeling. The venous expression of EphB4 and arterial expression of ephrin-B2 has lead to the speculation that the interaction of these two molecules leads to bidirectional signaling into both the receptor-expressing cell and the ligand-expressing cell, and that both forward and reverse signals are required for proper development of blood vessels in the embryo. Indeed, targeted removal of the ephrin-B2 carboxy-terminal cytoplasmic tail by another group was shown to perturb vascular development and result in the same early embryonic lethality as the null mutation, leading the authors to propose that ephrin-B2 reverse signaling directs early angiogenic remodeling of the primary vascular plexus [Cell 104 (2001) 57]. However, we show here that the carboxy-terminal cytoplasmic domain of ephrin-B2, and hence reverse signaling, is not required during early vascular development, but it is necessary for neonatal survival and functions later in cardiovascular development in the maturation of cardiac valve leaflets. We further show that ephrin-B2 reverse signaling is required for the pathfinding of axons that form the posterior tract of the anterior commissure. Our results thus indicate that ephrin-B2 functions in the early embryo as a typical instructive ligand to stimulate EphB4 receptor forward signaling during angiogenic remodeling and that later in embryonic development ephrin-B2 functions as a receptor to transduce reverse signals involved in cardiac valve maturation and axon pathfinding.     

Ephrin-B2 selectively marks arterial vessels and neovascularization sites in the adult, with expression in both endothelial and smooth-muscle cells

The Eph receptor tyrosine kinases and their membrane-tethered ephrin ligands provide critical guidance cues at points of cell-to-cell contact. It has recently been reported that the ephrin-B2 ligand is a molecular marker for the arterial endothelium at the earliest stages of embryonic angiogenesis, while its receptor EphB4 reciprocally marks the venous endothelium. These findings suggested that ephrin-B2 and EphB4 are involved in establishing arterial versus venous identity and perhaps in anastamosing arterial and venous vessels at their junctions. By using a genetically engineered mouse in which the lacZ coding region substitutes and reports for the ephrin-B2 coding region, we demonstrate that ephrin-B2 expression continues to selectively mark arteries during later embryonic development as well as in the adult. However, as development proceeds, we find that ephrin-B2 expression progressively extends from the arterial endothelium to surrounding smooth muscle cells and to pericytes, suggesting that ephrin-B2 may play an important role during formation of the arterial muscle wall. Furthermore, although ephrin-B2 expression patterns vary in different vascular beds, it can extend into capillaries about midway between terminal arterioles and postcapillary venules, challenging the classical conception that capillaries have neither arterial nor venous identity. In adult settings of angiogenesis, as in tumors or in the female reproductive system, the endothelium of a subset of new vessels strongly expresses ephrin-B2, once again contrary to earlier views that such new vessels lack arterial/venous characteristics and derive from postcapillary venules. While earlier studies had focused on a role for ephrin-B2 during the earliest embryonic stages of arterial/venous determination, our current findings using ephrin-B2 as an arterial marker in the adult challenge prevailing views of the arterial/venous identity of quiescent as well as remodeling adult microvessels and also highlight a possible role for ephrin-B2 in the formation of the arterial muscle wall.     

Regulation of signaling interactions and receptor endocytosis in growing blood vessels

Blood vessels and the lymphatic vasculature are extensive tubular networks formed by endothelial cells that have several indispensable functions in the developing and adult organism. During growth and tissue regeneration but also in many pathological settings, these vascular networks expand, which is critically controlled by the receptor EphB4 and the ligand ephrin-B2. An increasing body of evidence links Eph/ephrin molecules to the function of other receptor tyrosine kinases and cell surface receptors. In the endothelium, ephrin-B2 is required for clathrin-dependent internalization and full signaling activity of VEGFR2, the main receptor for vascular endothelial growth factor. In vascular smooth muscle cells, ephrin-B2 antagonizes clathrin-dependent endocytosis of PDGFRβ and controls the balanced activation of different signal transduction processes after stimulation with platelet-derived growth factor. This review summarizes the important roles of Eph/ephrin molecules in vascular morphogenesis and explains the function of ephrin-B2 as a molecular hub for receptor endocytosis in the vasculature.     

Regulation of signaling interactions and receptor endocytosis in growing blood vessels

Blood vessels and the lymphatic vasculature are extensive tubular networks formed by endothelial cells that have several indispensable functions in the developing and adult organism. During growth and tissue regeneration but also in many pathological settings, these vascular networks expand, which is critically controlled by the receptor EphB4 and the ligand ephrin-B2. An increasing body of evidence links Eph/ephrin molecules to the function of other receptor tyrosine kinases and cell surface receptors. In the endothelium, ephrin-B2 is required for clathrin-dependent internalization and full signaling activity of VEGFR2, the main receptor for vascular endothelial growth factor. In vascular smooth muscle cells, ephrin-B2 antagonizes clathrin-dependent endocytosis of PDGFRβ and controls the balanced activation of different signal transduction processes after stimulation with platelet-derived growth factor. This review summarizes the important roles of Eph/ephrin molecules in vascular morphogenesis and explains the function of ephrin-B2 as a molecular hub for receptor endocytosis in the vasculature.     

A role of EphB4 receptor and its ligand,ephrin-B2, in erythropoiesis

Erythropoiesis is regulated not only by erythropoietin but also by microenvironments which are composed of transmembrane molecules. We have previously shown that a receptor tyrosine kinase EphB4 is predominantly expressed on human erythroid progenitors in bone marrow. EphB4 is expressed in approximately 45% of hematopoietic progenitor cells, which are CD34-positive and c-Kit-positive in human umbilical cord blood (hUCB). The transmembrane ligand for EphB4 or ephrin-B2 is expressed on bone marrow stromal cells and arterial endothelial cells. When such EphB4-positive hematopoietic progenitor cells were co-cultured with stromal cells which express ephrin-B2, they were immediately detached from stromal cells and differentiated to mature erythroid cells. At that time, expression of EphB4 immediately down-regulated. In contrast, on ephrin-B2 non-expressing stromal cells, they remained EphB4-positive cells and the generated number of mature erythroid cells was less than that on ephrin-B2 expressing stromal cells. Additionally, ephrin-B2 expression on endothelial cells up-regulated under hypoxic condition. Taken together, we propose that one of the molecular cues that regulate erythropoiesis is ephrin-B2 on stromal cells.     

Ephrin-B2 is a candidate ligand for the Eph receptor, EphB6

No ligand has hitherto been designated for the Eph receptor tyrosine kinase family member, EphB6. Here, expression of an EphB6 ligand in the pro-B leukemic cell line, Reh, is demonstrated by binding of soluble EphB6-Fc fusion protein to the Reh cells. The ligand belongs to the subgroup of membrane spanning ligands, as suggested by the fact that phosphatidylinositol-specific phospholipase C treatment did not abrogate binding of EphB6-Fc. Two transmembrane Eph receptor ligands, ephrin-B1 and ephrin-B2, were identified in Reh cells. Analysis of EphB6-Fc fusion protein binding to ephrin-B1 or ephrin-B2 transfected COS cells revealed a high-affinity saturable binding between EphB6-Fc and ephrin-B2, but not with ephrin-B1. In mice, EphB6 has previously been shown to be expressed in thymus. Here, we show expression of EphB6 in human thymus, as well as the expression of ephrin-B2 in both human and mouse thymus. We conclude that ephrin-B2 may be a physiological ligand for the EphB6 receptor.     

Association of Dishevelled with Eph tyrosine kinase receptor and ephrin mediates cell repulsion

Eph tyrosine kinase receptors and their membrane-bound ligands, ephrins, are presumed to regulate cell-cell interactions. The major consequence of bidirectional activation of Eph receptors and ephrin ligands is cell repulsion. In this study, we discovered that Xenopus Dishevelled (Xdsh) forms a complex with Eph receptors and ephrin-B ligands and mediates the cell repulsion induced by Eph and ephrin. In vitro re-aggregation assays with Xenopus animal cap explants revealed that co-expression of a dominant-negative mutant of Xdsh affected the sorting of cells expressing EphB2 and those expressing ephrin-B1. Co-expression of Xdsh induced the activation of RhoA and Rho kinase in the EphB2-overexpressed cells and in the cells expressing EphB2-stimulated ephrin-B1. Therefore, Xdsh mediates both forward and reverse signaling of EphB2 and ephrin-B1, leading to the activation of RhoA and its effector protein Rho kinase. The inhibition of RhoA activity in animal caps significantly prevents the EphB2- and ephrin-B1-mediated cell sorting. We propose that Xdsh, which is expressed in various tissues, is involved in EphB and ephrin-B signaling related to regulation of cell repulsion via modification of RhoA activity.     

Developmental expression of EphB6 in the thymus: lessons from EphB6 knockout mice

A member of the largest family of receptor protein kinases, EphB6, lacks its intrinsic kinase activity, but it is expressed in normal human tissues. To investigate the physiological function of EphB6, we generated EphB6 deficient mice. EphB6(-/-) mice developed normally, revealed no abnormality in general appearance, and were fertile. Although a developmental increase of EphB6 in the fetal thymus was confirmed, T-cell development in various lymphoid organs of EphB6(-/-) mice was comparable to those of EphB6(+/+). Even in fetal thymus organ cultures, any developmental differences of EphB6(-/-) and EphB6(+/+) thymocytes were undetectable. The different binding characteristics to ephrin-Fc proteins between EphB6(-/-) and EphB6(+/+) thymocytes demonstrated that ephrin-B2 is the unique ligand for EphB6 among eight known ephrins. While EphB6 was a dominant receptor that binds to ephrin-B2 in adult thymocytes, fetal ones also expressed another EphB that binds to ephrin-B2. Overlapping expression of the EphB subfamily in the fetal thymus might compensate for the loss of EphB6 during the thymic development.     

Complementary expression of EphB receptors and ephrin-B ligand in the pyloric and duodenal epithelium of adult mice

Eph receptors and ephrin ligands are membrane-bound cell–cell communication molecules that regulate the spatial organisation of cells in various tissues by repulsive or adhesive signals arising from contact between EphB- and ephrin-bearing cells. However, the expression and functions of Eph receptors in the gastric epithelium and Brunner’s glands are virtually unknown. We detected several EphB receptors and ephrin-B ligands in the pyloric and duodenal mucosa of the adult mouse by RT-PCR amplification. Immunostaining showed complementary expression patterns, with ephrin-B1 being preferentially expressed in the superficial part and EphB receptors in the deeper part of both epithelia. In the gastric pylorus, ephrin-B1 was expressed in pit cells and proliferating cells of the isthmus. In contrast, EphB2, EphB3, and EphB4 were expressed in pyloric glandular cells and proliferating cells of the isthmus. In the duodenum, ephrin-B1 was expressed in cells lining the ducts of Brunner’s glands as well as those covering villi and the upper portion of the crypts of Lieberkühn. In contrast, EphB2 and EphB3 were expressed in the gland segment of Brunner’s glands and the lower portion of the crypts and EphB4, in the crypts. In both mucosae, EphB2, EphB3, and EphB4 were found to be tyrosine phosphorylated, suggesting that EphB/ephrin-B signalling might occur preferentially in the isthmus, crypts, and duct-gland transition of Brunner’s glands, where the receptor and ligand expression overlaps. Based on these findings, we propose that EphB/ephrin-B signalling may regulate cell positioning within the pyloric and duodenal epithelium.     

In vivo tyrosine phosphorylation sites of activated ephrin-B1 and ephB2 from neural tissue

EphB2 is a receptor tyrosine kinase of the Eph family and ephrin-B1 is one of its transmembrane ligands. In the embryo, EphB2 and ephrin-B1 participate in neuronal axon guidance, neural crest cell migration, the formation of blood vessels, and the development of facial structures and the inner ear. Interestingly, EphB2 and ephrin-B1 can both signal through their cytoplasmic domains and become tyrosine-phosphorylated when bound to each other. Tyrosine phosphorylation regulates EphB2 signaling and likely also ephrin-B1 signaling. Embryonic retina is a tissue that highly expresses both ephrin-B1 and EphB2. Although the expression patterns of EphB2 and ephrin-B1 in the retina are different, they partially overlap, and both proteins are substantially tyrosine-phosphorylated. To understand the role of ephrin-B1 phosphorylation, we have identified three tyrosines of ephrin-B1 as in vivo phosphorylation sites in transfected 293 cells stimulated with soluble EphB2 by using mass spectrometry and site-directed mutagenesis. These tyrosines are also physiologically phosphorylated in the embryonic retina, although the extent of phosphorylation at each site may differ. Furthermore, many of the tyrosines of EphB2 previously identified as phosphorylation sites in 293 cells (Kalo, M. S., and Pasquale, E. B. (1999) Biochemistry 38, 14396-14408) are also phosphorylated in retinal tissue. Our data underline the complexity of ephrin-Eph bidirectional signaling by implicating many tyrosine phosphorylation sites of the ligand-receptor complex.