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Ephrin

Ephrins are a family of membrane-anchored proteins that serve as ligands for Eph receptors, the largest subfamily of receptor tyrosine kinases (RTKs), enabling bidirectional signaling through direct cell-cell contact to regulate key developmental and physiological processes.^[1] They are classified into two subclasses: the five Ephrin-A ligands (Ephrin-A1 to A5), which are glycosylphosphatidylinositol (GPI)-anchored and primarily bind EphA receptors, and the three Ephrin-B ligands (Ephrin-B1 to B3), which are transmembrane proteins with intracellular domains that bind EphB receptors, though some cross-binding occurs between subclasses.^[2] Upon binding, Ephrins trigger forward signaling in the Eph-expressing cell via tyrosine kinase activation and reverse signaling in the Ephrin-expressing cell through phosphorylation of conserved tyrosine residues, leading to cytoskeletal rearrangements that influence cell adhesion, migration, and repulsion.^[3] In embryonic development, Ephrins are essential for establishing tissue boundaries, guiding axon pathfinding, and patterning structures across all germ layers, such as segmenting the hindbrain rhombomeres and directing topographic mapping in the retinotectal system.^[2] They also play pivotal roles in vascular morphogenesis, where Ephrin-B2 expression on arterial endothelial cells and EphB4 on venous cells promotes arteriovenous differentiation and angiogenesis.^[1] Beyond development, Ephrins contribute to adult functions including synaptic plasticity, bone remodeling, and insulin secretion, while dysregulation is implicated in pathologies such as cancer invasion and congenital disorders like primary lymphedema.^[3] Their signaling networks integrate with other pathways, such as those involving fibroblast growth factor receptors (FGFRs) and integrins, to fine-tune cellular responses in diverse contexts.^[1]

Overview

Definition and Properties

Ephrins are membrane-bound ligands for Eph receptors, the largest subfamily of receptor tyrosine kinases (RTKs) with 14 members in mammals. These proteins enable contact-dependent cell-cell communication by binding to Eph receptors on adjacent cells, thereby facilitating precise spatial and temporal regulation of cellular interactions during development and homeostasis. Unlike soluble ligands, ephrins are anchored to the plasma membrane, preventing free diffusion and ensuring that signaling occurs only at sites of direct cell-cell contact, which is crucial for processes requiring localized cues such as tissue patterning and boundary formation.^[4] A defining property of ephrins is their immobilization on the cell surface, either through glycosylphosphatidylinositol (GPI) linkage for ephrin-A subtypes or via a transmembrane domain for ephrin-B subtypes, allowing them to transduce signals in a highly restricted manner. This membrane association supports dynamic expression patterns across embryonic and adult tissues, where ephrins are prominently found in the developing nervous system, vasculature, and various organs, often exhibiting combinatorial or mutually exclusive distributions with Eph receptors to guide cellular behaviors like migration and adhesion. In mammals, eight ephrins have been identified, subdivided into A and B classes based on sequence homology and binding preferences to EphA or EphB receptors, respectively.^[5]^[4] Ephrins uniquely participate in bidirectional signaling, functioning not only as ligands that activate forward signaling in Eph-expressing cells but also as signaling receptors themselves upon clustering by Eph receptors, thereby influencing both interacting cell populations simultaneously. This dual capability arises from the ability of ephrins to recruit intracellular effectors upon engagement, leading to downstream effects on cytoskeletal dynamics and gene expression in the ligand-bearing cell. Discovered in the early 1990s as counterparts to Eph receptors, ephrins have since been recognized for their essential roles in coordinating cellular responses through this reciprocal mechanism.^[5]^[4]

Evolutionary Conservation

Ephrins represent an ancient family of signaling molecules, with ephrin-like sequences detectable as early as in cnidarians, such as Nematostella vectensis, indicating their involvement in cell-cell communication predating the evolution of centralized nervous systems.^[6] These sequences suggest that ephrins played a foundational role in the transition to multicellularity among metazoans, facilitating basic processes like cell adhesion and segregation.^[6] In non-bilaterian animals like sponges and placozoans, more rudimentary ephrin-related motifs hint at even deeper origins, potentially linked to choanoflagellate-like signaling.^[6] Across bilaterian animals, ephrins are ubiquitously present, with clear homologs identified in key invertebrate models. In Drosophila melanogaster, a single Eph receptor and a single ephrin homolog are present, contributing to developmental patterning and cell repulsion.^[6] Similarly, in Caenorhabditis elegans, the vab-2 gene encodes an ephrin ortholog essential for embryonic morphogenesis and axon guidance.^[6] These invertebrate ephrins typically number around five per genome, reflecting a simpler repertoire compared to vertebrates.^[6] The receptor-binding domains of ephrins exhibit high sequence conservation, with 30-60% identity across metazoans, underscoring their co-evolution with Eph receptors to maintain bidirectional signaling fidelity.^[7] This conservation is particularly evident in the ephrin-B subclass, which traces back to bilaterian ancestors, while ephrin-A forms emerged later in chordate lineages.^[7] In vertebrates, the Eph and ephrin gene families expanded, with mammals having 14 Eph receptors and 8 ephrins, driven by two rounds of whole-genome duplication during early vertebrate evolution, which diversified their roles in complex tissue organization.^[6]

Discovery and Classification

Historical Discovery

The discovery of ephrins began in 1990 when Holzman et al. identified B61 as a novel immediate-early response gene product in human umbilical vein endothelial cells, induced by tumor necrosis factor alpha (TNF-α) and interleukin-1; this secreted protein was later recognized as ephrin-A1. In 1994, Bartley et al. demonstrated that B61 functions as a ligand for the ECK receptor tyrosine kinase, a member of the Eph family, through affinity chromatography and autophosphorylation assays, marking the first linkage of this protein to Eph signaling.^[8] Between 1994 and 1995, further screening efforts in chick and mouse embryos uncovered additional Eph ligands. In 1994, Cheng and Flanagan cloned ELF-1 from chick brain tissue as a GPI-anchored ligand for the Mek4 and Sek Eph receptors, revealing its expression in a gradient along the tectum that complemented receptor distribution. In the same year, Davis et al. identified the first transmembrane ephrin-B ligands, such as ELK-L (later ephrin-B1), from neuroepithelioma cells, revealing a new class of Eph ligands with intracellular signaling domains.^[9] The following year, Drescher et al. identified RAGS (later ephrin-A5) from chick tectal membranes as a 25 kDa protein mediating repulsive guidance for retinal ganglion cell axons, cloned via expression screening using a growth cone collapse assay on temporal retinal axons, identifying it as a ligand for EphA receptors. These discoveries expanded the family through systematic ligand hunts targeting orphan Eph receptors in developing neural tissues. A key milestone occurred in 1997 when the Eph Nomenclature Committee established the unified term "ephrin" for all Eph ligands, derived from "Eph family receptor interacting proteins," to resolve the proliferation of disparate names like B61, ELF-1, and RAGS and clarify their distinction from traditional soluble ligands.^[10] Early functional characterization in the 1990s highlighted ephrins' role in axon repulsion using in vitro stripe assays. Cheng and Flanagan (1995) showed that ELF-1/ephrin-A1 repels temporal retinal axons on alternating lanes of chick tectal membranes, establishing topographic specificity. Similarly, Drescher et al. (1995) used stripe assays to demonstrate RAGS/ephrin-A5's dose-dependent repulsion of chick retinal axons, linking ephrins to contact-mediated guidance cues in neural mapping.

Subfamily Classification

Ephrins are classified into two main subfamilies, A and B, based on their amino acid sequence homology, membrane anchoring mechanisms, and preferential binding affinities to specific Eph receptor subclasses. This taxonomic framework was formally established in 1997 to standardize nomenclature across the family.^[10] The human genome encodes a total of eight ephrins, comprising five members of the ephrin-A subfamily (ephrin-A1 through ephrin-A5) and three members of the ephrin-B subfamily (ephrin-B1 through ephrin-B3).^[11] These subfamilies are highly conserved across vertebrate species, reflecting their fundamental roles in developmental processes.^[12] Ephrin-A proteins are anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) linkage and feature short cytoplasmic tails without dedicated signaling domains; they bind preferentially to EphA receptors and primarily elicit repulsive signaling responses.^[13]^[14] In contrast, ephrin-B proteins are transmembrane, possessing a conserved intracellular domain with PDZ-binding motifs that facilitate reverse signaling; they interact mainly with EphB receptors and support both repulsive and attractive signaling outcomes.^[13]^[14] While interactions generally follow subfamily specificity (EphA with ephrin-A and EphB with ephrin-B), binding promiscuity occurs in some cases, exemplified by ephrin-B3's ability to bind EphA4.^[15]

Molecular Structure

Core Structural Features

Ephrins, as ligands for Eph receptors, possess a highly conserved receptor-binding domain (RBD) comprising approximately 125 amino acids that forms the core of their molecular architecture. This globular domain adopts an eight-stranded β-barrel fold with Greek key topology, consisting of two antiparallel β-sheets linked by loops of varying lengths, which provides structural rigidity and enables specific interactions with Eph receptors.^[16]^[17] The β-barrel structure is stabilized by two conserved intramolecular disulfide bonds formed between four cysteine residues (Cys65–Cys104 and Cys92–Cys156 in ephrin-B2 numbering), which are absolutely conserved across all ephrins and essential for maintaining the domain's compact fold and functional integrity.^[16] Additionally, the RBD contains conserved N-glycosylation sites, such as Asn39, which are modified with N-acetylglucosamine and mannose residues; these post-translational modifications modulate ligand-receptor clustering on cell surfaces and enhance signaling efficiency by influencing oligomerization dynamics.^[16]^[18] This core domain mediates high-affinity binding to Eph receptors, with dissociation constants (K_d) typically in the low nanomolar range (approximately 10^{-9} M), as exemplified by the interaction between ephrin-B1 and EphB1 (K_d ≈ 0.9 nM).^[19] Crystal structures of ephrin ectodomains and Eph-ephrin complexes, such as the ephrin-B2 ectodomain (PDB: 1IKO) and the EphB2-ephrin-B2 complex resolved at 2.7 Å, demonstrate that the receptor-binding interface primarily involves a hydrophobic patch on the ephrin's G-H loop and β-strands, supplemented by hydrogen bonds between polar residues, facilitating the precise recognition and activation of bidirectional signaling.^[16]

Anchoring and Subfamily Differences

Ephrin-A ligands are tethered to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor attached at their C-terminus via a lipid modification. This anchoring mechanism confines ephrin-As to cholesterol-rich lipid raft microdomains, which restricts their lateral mobility within the plasma membrane and promotes cis-clustering of multiple ephrin-A molecules on the surface of the same cell.^[4]^[20] Such clustering enhances the avidity of interactions with EphA receptors on adjacent cells, facilitating localized signaling events without the need for an intracellular domain.^[4] In contrast, ephrin-B ligands employ a transmembrane anchoring strategy, consisting of a single-span α-helical transmembrane domain connected to a short intracellular cytoplasmic tail of approximately 80–90 amino acids. This tail harbors multiple conserved tyrosine residues that serve as phosphorylation sites, along with a C-terminal PDZ-binding motif that recruits adaptor proteins, such as PDZ-RGS3, to mediate intracellular signaling.00488-4.pdf)^[21] The transmembrane configuration allows ephrin-Bs to transmit signals bidirectionally, integrating extracellular cues with cytoskeletal responses inside the cell.^[22] These anchoring differences underpin distinct functional roles in cellular interactions. The GPI-linked ephrin-As primarily mediate short-range repulsion between cells, such as in axonal pathfinding, by enabling rapid, localized activation without sustained intracellular propagation.00314-2) Conversely, the transmembrane ephrin-Bs support prolonged adhesion or attraction through reverse signaling, where ligand clustering with EphB receptors on apposing cells triggers intracellular cascades that modulate cell motility and tissue organization.00267-9) Notably, the cytoplasmic tails of ephrin-Bs undergo tyrosine phosphorylation by EphB-associated kinases, creating high-affinity binding sites for SH2-domain proteins like Grb4, which in turn docks additional effectors to amplify reverse signaling.^[23]

Signaling Mechanisms

Eph Receptor Interaction

Ephrins bind to Eph receptors through high-affinity, reversible interactions mediated by the ephrin receptor-binding domain and the Eph ectodomain.^[24] These interactions form an initial 2:2 stoichiometric complex, characterized by a hydrophobic cavity on the Eph receptor that accommodates a long ephrin loop, enabling precise molecular recognition.^[24] The binding kinetics demonstrate rapid association rates on the order of 10^5 to 10^6 M^{-1} s^{-1} and dissociation rates around 10^{-2} s^{-1} for dimeric forms, resulting in nanomolar affinities that are further stabilized by cell-cell contact in physiological contexts.^[25] This reversible nature allows dynamic regulation during cellular encounters. Specificity in ephrin-Eph interactions is governed by structural determinants that favor subclass promiscuity within A or B groups while limiting cross-subclass binding. For ephrin-A ligands interacting with EphA receptors, charged residues forming an acidic patch on the ephrin contribute to selectivity by engaging basic regions on the Eph ectodomain.^[26] In contrast, ephrin-B ligands bind EphB receptors primarily through hydrophobic interfaces, including the insertion of the ephrin G-H loop into a receptor cleft, supplemented by polar interactions for high affinity.^[27] These features ensure that, for example, most EphA receptors preferentially bind ephrin-A ligands, with rare exceptions like EphA4 showing broader compatibility.^[24] Effective signaling requires multimerization of the Eph-ephrin complex beyond the initial dimer, often forming expansive clusters such as hexameric or larger assemblies to achieve full activation.^[24] Monomeric or low-avidity interactions are insufficient, but membrane-anchored or clustered ephrins (e.g., via Fc-fusion dimers) promote higher-order oligomerization, enhancing affinity by 30- to 6000-fold depending on the subclass.^[25] This clustering triggers autophosphorylation of the Eph kinase at juxtamembrane tyrosine residues, initiating forward signaling cascades.^[24]

Bidirectional Signaling Pathways

Upon binding of Eph receptors to ephrin ligands on adjacent cells, bidirectional signaling ensues, with forward signals propagating into the receptor-expressing cell and reverse signals into the ephrin-expressing cell, often resulting in coordinated cellular responses such as repulsion or adhesion.^[28] This dual activation arises from the clustering of Eph-ephrin complexes at the contact site, which triggers intracellular cascades in both directions. In forward signaling, autophosphorylation of the Eph receptor's tyrosine kinase domain recruits adaptor proteins such as Nck and RasGAP, which modulate downstream pathways leading to cytoskeletal reorganization.^[28] For instance, activation of Rho GTPases like RhoA promotes actomyosin contraction and growth cone collapse, mediating repulsive effects that inhibit cell migration or axon extension.^[20] This pathway is pivotal for contact-dependent repulsion, as demonstrated in neuronal guidance where EphA forward signaling via RhoA disrupts adhesive contacts.^[4] Reverse signaling through ephrins contrasts with forward signaling by utilizing the ligand's cytoplasmic domain (in ephrin-Bs) or associated proteins (in GPI-anchored ephrin-As). In ephrin-B ligands, phosphorylation by EphB receptors recruits PDZ- and SH2-domain-containing proteins, including PI3K, which activates pathways promoting cell attraction and adhesion.^[28] For example, ephrin-B reverse signaling via PI3K enhances synaptic maturation and cell protrusions in contexts requiring attractive cues.^[29] Ephrin-As, lacking cytoplasmic tails, transduce signals through GPI-linked adaptors such as the Src family kinase Lck, leading to phosphorylation events that support repulsion or migration.^[30] Crosstalk between forward and reverse pathways is facilitated by ephrin clustering, which induces activation of Src family kinases like Fyn, amplifying signaling in both directions and enabling fine-tuned responses.^[31] This integration allows Eph-ephrin complexes to balance repulsion and adhesion, with Src-mediated phosphorylation enhancing the recruitment of shared effectors.^[5] Mathematical models illustrate how bidirectional signaling switches between repulsion and adhesion in a threshold-dependent manner, influenced by Eph-ephrin affinity and surface density. For example, stochastic models incorporating bi-directional cues predict cell segregation when repulsive signals exceed adhesive thresholds at high ligand densities, providing a quantitative framework for tissue patterning.^[32]

Developmental Functions

Vascular Development Processes

Ephrins, particularly ephrin-B2 and ephrin-B4, play essential roles in vascular development by regulating angiogenesis, arterial-venous differentiation, and lymphangiogenesis during embryogenesis. In the initial formation of the vascular network, ephrin-B ligands interact with EphB receptors to guide endothelial cell behaviors, ensuring proper vessel assembly and remodeling. Seminal studies using knockout models demonstrated that disruption of these interactions leads to severe vascular defects, highlighting their indispensability for embryonic viability.^[33] In angiogenesis, ephrin-B2 forward signaling within endothelial cells promotes the migration of tip cells, which lead sprout extension during vessel branching. This process facilitates the directed invasion of endothelial filopodia into avascular regions, enabling capillary network expansion. Conversely, ephrin-B2 reverse signaling in pericytes enhances vessel stabilization by promoting mural cell recruitment and adhesion to nascent endothelial tubes, preventing regression and ensuring structural integrity. These bidirectional mechanisms coordinate the dynamic balance between vessel growth and maturation, as evidenced in embryonic models where ephrin-B2 deficiency impairs sprouting and pericyte coverage. Notably, ephrin-B2 and EphB4 single mutants exhibit embryonic lethality around E10.5-E11.5, characterized by defective yolk sac vasculature with a failure to remodel the primitive plexus into hierarchical arteries and veins.01234-1)^[33] Arterial-venous specification relies on ephrin-B4 expression, which restricts venous identity in endothelial progenitors and prevents ectopic arterialization. Ephrin-B4, primarily in venous endothelium and endocardium, engages bidirectional signaling with EphB4 receptors to maintain compartment boundaries and guide remodeling. This interaction repels arterial and venous cells, promoting segregation and proper fusion during vessel anastomosis. In ephrin-B4 mutants, venous domains expand inappropriately, leading to malformed intersomitic vessels and cardiac outflow tract defects.^[33]^[34] Ephrin-B2 also contributes to lymphangiogenesis by regulating lymphatic sprouting through crosstalk with VEGFR3 signaling. In lymphatic endothelial cells, ephrin-B2 facilitates VEGFR3 internalization upon VEGF-C binding, amplifying downstream pathways that drive filopodia formation and sprout initiation. This regulatory hub ensures efficient lymphatic vessel patterning from venous origins, with ephrin-B2 loss resulting in reduced lymphatic branching and edema in embryonic tissues.

Adult and Pathological Functions

Roles in Immunity and Tissue Homeostasis

Ephrins play crucial roles in regulating immune cell migration and interactions within lymphoid tissues. In the thymus, ephrin-B1 expressed on thymic epithelial cells interacts with EphB receptors on thymocytes to facilitate proper T-cell development by modulating thymocyte-epithelium attachments and three-dimensional organization.^[35] This signaling ensures efficient T-cell maturation, as disruption with ephrin-B1-Fc reduces conjugate formation between double-positive thymocytes and epithelial cells by approximately 50%, impairing TCR-dependent signaling and increasing apoptosis.^[35] Additionally, reverse signaling through ephrins on dendritic cells modulates their chemotaxis, influencing immune responses by altering migration toward chemokines like SDF-1; for instance, ephrin-B ligands inhibit G-protein-coupled chemoattraction in immune contexts, fine-tuning dendritic cell positioning during antigen presentation.^[36] Ephrins also contribute to immune synapse formation, where ephrin-A1 enhances T-cell activation by promoting colocalization with TCR signaling components, leading to increased cytokine production and proliferation, as highlighted in a comprehensive 2019 review of Eph/ephrin functions in immunity.^[36] In tissue homeostasis, ephrins maintain epithelial integrity and renewal, particularly in the intestine. Ephrin-B1, expressed in a gradient along the crypt-villus axis with higher levels at villus apices, promotes epithelial cell migration and positioning by reinforcing cell-cell adhesion through interactions with tight junction proteins like claudins-1 and -4.^[37] This PDZ-binding motif in ephrin-B1 recruits adaptor proteins such as GRIP and syntenin, enabling reverse signaling that supports crypt-villus renewal and wound healing; for example, ephrin-B1 activation enhances epithelial barrier function and directional migration in vitro, preventing disorganized proliferation.^[37]^[38] Loss of ephrin-B1 disrupts this compartmentalization, underscoring its role in sustaining intestinal homeostasis.^[39] Ephrins further regulate bone remodeling through bidirectional signaling between osteoclasts and osteoblasts. Ephrin-B2 on osteoclasts and EphB4 on osteoblasts mediate cell-cell communication that couples bone resorption and formation, maintaining skeletal homeostasis; disruption of this signaling, as in ephrin-B2 or EphB4 knockout models, leads to increased bone mass due to enhanced osteoblast activity and reduced osteoclast function.^[40] In the pancreas, EphA receptors and ephrin-A ligands on beta cells facilitate communication that regulates insulin secretion. Ephrin-A reverse signaling promotes glucose-stimulated insulin release by modulating actin cytoskeleton dynamics and cell adhesion, while EphA forward signaling inhibits secretion under basal conditions; islets from ephrin-A5-deficient mice show impaired insulin secretion in response to glucose.^[41] Ephrins further regulate adult neurogenesis by controlling progenitor dynamics in neurogenic niches. In the subventricular zone (SVZ), ephrin-B3 inhibits neural progenitor proliferation through interactions with EphB receptors, maintaining homeostasis; ephrin-B3 knockout mice exhibit a 184% increase in BrdU-positive proliferating cells in the SVZ, which is reversed by exogenous ephrin-B3 administration.^[42]^[43] Recent 2024 studies reveal a balanced EphB/ephrin interaction in the hippocampal dentate gyrus, where ephrin-B3 stimulation of EphB1 on subgranular zone progenitors regulates proliferation and migration, with disruptions altering neural stem cell self-renewal and contributing to memory-related processes.^[44] This balance ensures controlled adult neurogenesis without excessive expansion.^[44]

Involvement in Cancer and Other Diseases

Ephrins play a critical role in cancer progression through dysregulated signaling that promotes angiogenesis, invasion, and metastasis. Ephrin-B2, in particular, drives tumor angiogenesis by activating reverse signaling in endothelial cells, facilitating neovascularization in various solid tumors.^[45] For instance, in gliomas, ephrin-B2 reverse signaling via EphB4 enhances cell migration and invasion, contributing to the aggressive spread of glioblastoma multiforme.^[46] Overexpression of ephrin-B2 and related family members is commonly observed in breast and prostate cancers, where it correlates with increased tumor growth and poor prognosis.^[47]^[48] In neurological disorders, disruptions in ephrin signaling contribute to synaptic pathology and neurodevelopmental abnormalities. Ephrin-A5 signaling regulates synapse formation and stability; its dysregulation leads to reactivation of downstream effector Ephexin5, resulting in excitatory synapse loss observed in Alzheimer's disease models.^[49] This mechanism exacerbates amyloid-β-induced neurodegeneration and cognitive decline. In schizophrenia, mutations and polygenic risks affecting ephrin family genes impair topographic mapping during cortical development, leading to altered neural connectivity and spine maturation deficits.^[50]^[51] Cardiovascular pathologies also involve ephrin imbalances, particularly in vascular remodeling and plaque instability. Ephrin-B2 expression is elevated in atherosclerotic lesions, promoting endothelial inflammation and plaque formation, as evidenced by recent analyses of human samples.^[52] In congenital heart defects, haploinsufficiency or mutations in EphB receptors and ephrin-B ligands disrupt atrioventricular septation and valve development, leading to structural malformations such as ventricular septal defects.^[53]^[54] Mutations in EPHB4 cause primary lymphedema, a congenital disorder characterized by lymphatic dysfunction and swelling. Autosomal dominant EPHB4 variants impair lymphatic valve formation and vessel remodeling, leading to fluid accumulation in tissues, as observed in patients with adult-onset lymphedema.^[55] Ephrins serve as potential biomarkers for disease monitoring, with soluble forms detectable in circulation. Elevated serum levels of ephrin-A1 have been identified as a non-invasive indicator of ovarian cancer metastasis, correlating with tumor aggressiveness in recent multi-omics studies.^[56]

Therapeutic Implications

Targeting Strategies

Targeting strategies for modulating ephrin signaling primarily focus on disrupting Eph-ephrin interactions to inhibit pathological processes such as aberrant angiogenesis in cancer and vascular disorders. Small molecule inhibitors, such as the EphB4 kinase blocker NVP-BHG712, selectively target forward signaling by inhibiting the kinase activity of Eph receptors, thereby preventing downstream phosphorylation events that promote tumor cell migration and vascularization.^[57] In preclinical models of colorectal cancer, NVP-BHG712 has demonstrated efficacy in reducing VEGF-driven angiogenesis without significantly affecting VEGFR activity, highlighting its potential for cancer therapeutics.^[58] Soluble ephrin-Fc fusion proteins serve as competitive antagonists that bind to Eph receptors, disrupting ligand-receptor clustering and bidirectional signaling essential for pathological vessel formation. For instance, ephrin-A1-Fc has been shown to inhibit VEGF-induced intracellular signaling, suppressing retinal neovascularization in a dose-dependent manner in mouse models of retinopathy.^[59] These fusions mimic natural ephrin ligands but lack transmembrane domains, allowing them to act extracellularly to block signaling without triggering reverse signaling in ephrin-expressing cells. Gene therapy approaches, including CRISPR/Cas9-mediated editing, enable precise modulation of ephrin expression in vascular compartments to alter disease progression. In vascular tumor models, CRISPR knockouts of ephrin-B2 in endothelial cells have been used to demonstrate its role in regulating tumor growth and metastasis, revealing that endothelial-specific ephrin-B2 deletion normalizes vasculature and suppresses tumor progression.^[60] Similarly, monoclonal antibodies targeting the EphB4/ephrin-B2 interface inhibit both forward and reverse signaling by preventing ligand-receptor binding, as evidenced by a novel Fab fragment that blocks trans-interactions and reduces angiogenic responses in endothelial cells.^[61] Preclinical studies have reported substantial success with these strategies in reducing tumor vascularization; for example, anti-ephrin-B2 antibodies like 2B1 achieved approximately 50% inhibition of blood vessel density in xenograft mouse models of colon and lung tumors, correlating with slowed tumor growth.^[62] These interventions underscore the therapeutic potential of ephrin targeting in cancers where Eph-ephrin signaling drives vascular abnormalities, though challenges remain in achieving specificity and minimizing off-target effects on normal tissues.

Recent Research and Clinical Prospects

Recent studies from 2024 and 2025 have highlighted the Eph-ephrin system's involvement in female reproductive disorders, particularly endometriosis. A Mendelian randomization analysis identified EPHB4, the receptor for ephrin-B2, as a causal gene associated with increased endometriosis risk, with elevated EPHB4 expression correlating to higher disease susceptibility; this positions ephrin-B2/EPHB4 signaling as a potential therapeutic target for nonhormonal interventions.^[63] Complementing this, DNA-encoded chemistry screening yielded potent pan-Eph kinase inhibitors like CDD-3167, which reduced EphA2/4 phosphorylation and endometriotic cell viability in preclinical models, underscoring their promise for treating endometriosis-related lesions.^[64] In neurogenesis, Eph-ephrin signaling has emerged as a regulator in depression models. Eph receptors such as EphA4 and EphB2 modulate neural stem cell proliferation and fate in hippocampal and subventricular zones, with downstream effectors like ephexins influencing RhoA activity to link dysregulation to depression-like behaviors and cognitive deficits.^[44] Inhibition of EphA4 in demyelination models restored myelination and alleviated depression-related synaptic impairments, suggesting Eph-ephrin modulation could enhance neurogenesis-based therapies for mood disorders.^[65] Cardiovascular research has advanced EphB4-targeted strategies for coronary artery disease. A 2024 review detailed how EphB4/ephrin-B2 signaling protects against post-myocardial infarction fibrosis and rupture, with pharmacological EphB4 inhibitors reducing inflammation and neointima formation in preclinical vascular injury models.^[66] Although no Phase I/II trials are yet reported, these findings support ongoing development of EphB4 inhibitors to mitigate coronary pathologies like atherosclerosis and hypertrophy.^[53] In cancer, Ephrin-A1-related prospects include ligand-based approaches, with EFNA1-CAR-T cells demonstrating antitumor efficacy against EphA2-positive tumors in 2024 preclinical studies.^[67] For receptor-targeted therapies, the EphA2-specific antibody-drug conjugate BT5528 completed Phase I dosing in advanced solid tumors, achieving an 8.9% response rate and identifying a recommended Phase II dose of 6.5 mg/m², with 2025 updates indicating progression to expanded trials for ovarian and other EphA2-overexpressing cancers.^[68] Emerging immune applications feature ephrin-B involvement in enhancing CAR-T therapies. EphB4-targeted CAR-T cells showed feasibility for intratumoral delivery in oral squamous cell carcinoma models, improving persistence and cytotoxicity via immune modulation.^[69] Similarly, B cell co-culture boosted EphA2-CAR-T persistence and IFN-γ production against glioblastoma, highlighting ephrin/Eph augmentation of T-cell function.^[70] As of 2025, no ephrin-targeted drugs are FDA-approved, but at least five candidates—including EphA2 ADCs, EphB4 CAR-Ts, and pan-Eph inhibitors—are advancing in preclinical and early clinical pipelines for cancer, cardiovascular, and reproductive indications.

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Ligand recognition by A‐class Eph receptors: crystal structures of ...
All published structural studies of Eph receptor/ephrin complexes involve B‐class receptors. Here, we present the crystal structures of an A‐class complex ...<|control11|><|separator|>
[25]
Structural Characterization of the EphA4-Ephrin-B2 Complex ...
Here, we report the crystal structure of the EphA4 ligand-binding domain in complex with ephrin-B2, which represents the first structure of an EphA-ephrin-B ...Missing: EphA5 Kd
[26]
https://www.embopress.org/doi/10.1038/embor.2009.91
[27]
Ephrin reverse signaling controls palate fusion via a PI3 kinase ...
Activation of ephrin reverse signaling causes palate fusion ... Thus, we conclude that ephrin reverse signaling acts through the PI3K pathway to cause fusion.Eph And Ephrin Expression In... · Activation Of Ephrin Reverse... · Ephrin-Dependent Fusion...Missing: attraction | Show results with:attraction
[28]
Signaling through ephrin‐A ligand leads to activation of Src‐family ...
Jul 1, 2008 · Signaling through ephrin-A induces phosphorylation of several proteins, including the Src kinases Lck and Fyn. In addition, PI-3K is activated, ...
[29]
Eph'ective signaling: forward, reverse and crosstalk
Jul 15, 2003 · Indeed, clustering of ephrin-A molecules with EphA-Fc fusion proteins recruits the Src family kinase Fyn to lipid rafts(Davy et al., 1999). This ...
[30]
A Mathematical Model for Eph/Ephrin-Directed Segregation of ...
Dec 1, 2014 · Here we develop a stochastic, Lagrangian model that is based on Eph/ephrin biology: incorporating independent Brownian motion to describe cell movement.
[31]
Roles of ephrinB ligands and EphB receptors in cardiovascular ...
Here we report that two ephrinB ligands and three EphB receptors are expressed in and regulate the formation of the vascular network.Results · Ephrins Regulate Capillary... · Ephrin--Eph Interactions...<|control11|><|separator|>
[32]
Distinct Roles of Ephrin-B2 Forward and EphB4 Reverse Signaling ...
Ephrin-B2 forward signaling and EphB4 reverse signaling differentially affect cell adhesion and migration between arterial and venous endothelial cells.
[33]
EphrinB1‐EphB signaling regulates thymocyte‐epithelium ...
Aug 27, 2007 · Here, we describe that EphB signaling contributes to T cell development through the regulation of both attachments and three-dimensional ...
[34]
Emerging Roles for Eph Receptors and Ephrin Ligands in Immunity
This review provides an overview of the various roles of Eph receptors and ephrin ligands in immune cell development, activation, and migration.
[35]
Eph receptor and ephrin function in breast, gut, and skin epithelia
This large family of RTKs is subdivided into 2 subclasses, EphA (1–8, 10) and EphB (1–4, 6) receptors based on amino acid sequence homology and relative binding ...Missing: classification | Show results with:classification
[36]
https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2019.01473/full
[37]
https://www.tandfonline.com/doi/full/10.4161/19336918.2014.970012
[38]
EphrinB3 regulates cell proliferation and survival in adult ... - PubMed
We show that ephrinB3 contributes to the control of cell proliferation and survival in the adult subventricular zone (SVZ).Missing: progenitor | Show results with:progenitor
[39]
Master regulators of neurogenesis: the dynamic roles of Ephrin ...
Nov 6, 2024 · The first ephrin receptor (EphR), was discovered in 1987 as a new class of receptor tyrosine kinases (RTKs) [1]. Since then, 13 additional ...
[40]
EPHB2 carried on small extracellular vesicles induces tumor ...
Ephrin type-B receptor 2 (EPHB2) in extracellular vesicles drives tumor angiogenesis by activating ephrin reverse signaling.
[41]
The Phosphorylation of Ephrin-B2 Ligand Promotes Glioma Cell ...
In this study, EphB/ephrin-B signaling was identified as the driver of glioma invasion in GBM biopsy specimens. We further characterize the role of ephrin-B ...
[42]
Ephs in cancer progression: complexity and context-dependent ...
May 29, 2024 · Eph tyrosine kinases play a crucial role in a bidirectional signaling, involving forward signaling from ephrin-expressing cells to Eph receptor- ...<|control11|><|separator|>
[43]
Eph/Ephrin Profiling in Human Breast Cancer Reveals Significant ...
Independent studies reported EphB4 and EphB2 overexpression in human breast cancer [22], [25]. ... ephrin ligands expressed in prostate carcinoma cell lines.
[44]
Reducing expression of synapse-restricting protein Ephexin5 ...
Mar 27, 2017 · Accumulation of amyloid-β (Aβ) protein may cause synapse degeneration and cognitive impairment in Alzheimer's disease (AD) by reactivating expression of the ...
[45]
MIR137 polygenic risk for schizophrenia and ephrin-regulated ...
Gene mutations of the ephrin family are also implicated in neurodevelopmental disorders such as lissencephaly, polymicrogyria, or heterotopia, and ZIGA ...
[46]
The Schizophrenia Susceptibility Gene OPCML Regulates Spine ...
Oct 1, 2019 · We demonstrated a critical role of the schizophrenia-susceptible gene OPCML in spine maturation and cognitive behaviors via regulating the ephrin-EphB2-cofilin ...
[47]
Predictive value of plasma ephrinB2 levels for amputation risk ...
Jun 5, 2024 · A previous study found that ephrinB2 expression was significantly increased in human atherosclerosis ... 2024. Expression level and clinical ...
[48]
Recent advances of the Ephrin and Eph family in cardiovascular ...
Additionally, EphB4 maintains other functions of ECs, encompassing cell integrity, lipid transport, and angiogenesis., EphrinA1 mediates the migration ...Missing: modification restricts mobility
[49]
EFNB2 haploinsufficiency causes a syndromic neurodevelopmental ...
Mar 6, 2018 · 5.3 Congenital heart defects. Ephrin-B2 is a direct endocardial Notch target during ventricular trabeculation and chamber development and Notch ...Missing: imbalances | Show results with:imbalances
[50]
Integrative single-cell and exosomal multi-omics uncovers SCNN1A ...
Jul 24, 2025 · Integrative single-cell and exosomal multi-omics uncovers SCNN1A and EFNA1 as non-invasive biomarkers and drivers of ovarian cancer metastasis.
[51]
The small molecule specific EphB4 kinase inhibitor NVP-BHG712 ...
NVP-BHG712 inhibits VEGF driven vessel formation, while it has only little effects on VEGF receptor (VEGFR) activity in vitro or in cellular assays.
[52]
Optimization of the Lead Compound NVP-BHG712 as a Colorectal ...
Apr 21, 2023 · Testing in up to seven colon cancer cell lines that express EPHA2 reveals that several derivatives feature promising effects for the control of ...
[53]
EphrinA1 Inhibits Vascular Endothelial Growth Factor-Induced ...
In the dose-escalation study, intraocular injection of ephrinA1-Fc inhibited retinal neovascularization in a dose-dependent manner. Injection of 62.5 ng/eye, ...
[54]
EphB4 and ephrinB2 act in opposition in the head and neck tumor ...
Jun 20, 2022 · These data suggest that ephrinB2 plays a role in enhancing angiogenesis by disrupting vascular integrity and structural morphology and that its ...
[55]
Targeting Forward and Reverse EphB4/EFNB2 Signaling by a ...
Jan 16, 2020 · Trans-interaction between EphB4 and its membrane-bound ligand ephrin B2 (EFNB2) mediates bi-directional signaling: forward EFNB2-to-EphB4 ...
[56]
Blocking ephrinB2 with highly specific antibodies inhibits ...
May 10, 2012 · Treatment with 2B1 decreased moderately SW620 and H460 tumor growths by 35% and 25%, respectively, relative to vehicle control. Quantification ...
[57]
Identification of EPHB4 as a potential causal gene and therapeutic ...
May 31, 2025 · We found that EPHB4 is strongly associated with the risk of endometriosis, with higher levels of EPHB4 correlating with an increased risk of the condition.
[58]
Identification of potent pan-ephrin receptor kinase inhibitors ... - PNAS
May 3, 2024 · Our results also emphasize the therapeutic potential of our EPH inhibitors in cancer and endometriosis, a global health concern that causes ...
[59]
The Eph receptor A4 plays a role in demyelination and depression ...
Our experiments demonstrate that EphA4 is necessary for the demyelination and synaptic deficits seen in animal models for the study of depression, and this ...
[60]
https://www.nature.com/articles/s41467-022-31124-7
[61]
[PDF] Ephrin A1 ligand‐based CAR‐T cells for immunotherapy of EphA2 ...
Received: 2 September 2024 | Revised: 2 December ... (B) The DNA copy number of EFNA1 CAR‐T cells in the peripheral blood was detected by real‐time quantitative PCR at different time points.
[62]
Results From First-in-Human Phase I Dose-Escalation Study of a ...
Sep 4, 2024 · BT5528 is a Bicycle Toxin Conjugate, a novel class of chemically synthesized molecules, comprising a bicyclic peptide targeting EphA2 tumor antigen.
[63]
Feasibility of Intratumoral Administration With EPHB4‐CAR‐T Cells ...
In this study, we found that almost OSCC cases showed high Ephrin type‐B receptor 4 (EPHB4) expression that was mainly localized on the membrane of tumor cells.
[64]
B cells enhance EphA2 chimeric antigen receptor T cells cytotoxicity ...
Sep 7, 2024 · In this study, using ephrin type-A receptor 2 (EphA2) specific CAR T cells, we cultured B cells successfully to stimulate CAR T cells in vitro, ...