B-ephrin-EphB receptor signaling modulates NMDA receptors by inducing tyrosine phosphorylation of NR2 subunits. Ephrins and EphB RTKs are localized to postsynaptic compartments in the CA1, and therefore potentially interact in a non-canonical cis- configuration. However, it is not known whether cis- configured receptor-ligand signaling is utilized by this class of RTKs, and whether this might influence excitatory synapses. We found that ablation of ephrin-B3 results in an enhancement of the NMDA receptor component of synaptic transmission relative to the AMPA receptor component in CA1 synapses. Synaptic AMPA receptor expression is reduced in ephrin-B3 knockout mice, and there is a marked enhancement of tyrosine phosphorylation of the NR2B receptor subunit. In a reduced system co-expression of ephrin-B3 attenuated EphB2-mediated NR2B tyrosine phosphorylation. Moreover, phosphorylation of EphB2 was elevated in the hippocampus of ephrin-B3 knockout mice, suggesting that regulation of EphB2 activity is lost in these mice. Direct activation of EphB RTKs resulted in phosphorylation of NR2B and a potential signaling partner, the non-receptor tyrosine kinase Pyk2. Our data suggests that ephrin-B3 limits EphB RTK-mediated phosphorylation of the NR2B subunit through an inhibitory cis- interaction which is required for the correct function of glutamatergic CA1 synapses.

Organization of signaling complexes at excitatory synapses by membrane-associated guanylate kinase (MAGUK) proteins regulates synapse development, plasticity, senescence and disease. Post-translational modification of MAGUK family proteins can drive their membrane localization, yet it is unclear how these intracellular proteins are targeted to sites of synaptic contact. Here we show using super-resolution imaging, biochemical approaches and in vivo models that the trans-synaptic organizing protein ephrin-B3 controls the synaptic localization and stability of PSD-95 and links these events to changes in neuronal activity via negative regulation of a newly identified mitogen-associated protein kinase (MAPK)-dependent phosphorylation site on ephrin-B3, Ser332. Unphosphorylated ephrin-B3 was enriched at synapses, and interacted directly with and stabilized PSD-95 at synapses. Activity-induced phosphorylation of Ser332 dispersed ephrin-B3 from synapses, prevented the interaction with PSD-95 and enhanced the turnover of PSD-95. Thus, ephrin-B3 specifies the synaptic localization of PSD-95 and likely links the synaptic stability of PSD-95 to changes in neuronal activity.

Flight in birds evolved through patterning of the wings from forelimbs and transition from alternating gait to synchronous flapping. In mammals, the spinal midline guidance molecule ephrin-B3 instructs the wiring that enables limb alternation, and its deletion leads to synchronous hopping gait. Here, we show that the ephrin-B3 protein in birds lacks several motifs present in other vertebrates, diminishing its affinity for the EphA4 receptor. The avian ephrin-B3 gene lacks an enhancer that drives midline expression and is missing in galliforms. The morphology and wiring at brachial levels of the chicken embryonic spinal cord resemble those of ephrin-B3 null mice. Dorsal midline decussation, evident in the mutant mouse, is apparent at the chick brachial level and is prevented by expression of exogenous ephrin-B3 at the roof plate. Our findings support a role for loss of ephrin-B3 function in shaping the avian brachial spinal cord circuitry and facilitating synchronous wing flapping.

Cortical networks are characterized by sparse connectivity, with synapses found at only a subset of axo-dendritic contacts. Yet within these networks, neurons can exhibit high connection probabilities, suggesting that cell-intrinsic factors, not proximity, determine connectivity. Here, we identify ephrin-B3 (eB3) as a factor that determines synapse density by mediating a cell-cell competition that requires ephrin-B-EphB signaling. In a microisland culture system designed to isolate cell-cell competition, we find that eB3 determines winning and losing neurons in a contest for synapses. In a Mosaic Analysis with Double Markers (MADM) genetic mouse model system in vivo the relative levels of eB3 control spine density in layer 5 and 6 neurons. MADM cortical neurons in vitro reveal that eB3 controls synapse density independently of action potential-driven activity. Our findings illustrate a new class of competitive mechanism mediated by trans-synaptic organizing proteins which control the number of synapses neurons receive relative to neighboring neurons.

EphA4, an Ephrins tyrosine kinase receptor, behaves as a dependence receptor (DR) by triggering cell apoptosis in the absence of its ligand Ephrin-B3. DRs act as conditional tumor suppressors, engaging cell death based on ligand availability; this mechanism is bypassed by overexpression of DRs ligands in some aggressive cancers. The pair EphA4/Ephrin-B3 favors survival of neuronal progenitors of the brain subventricular zone, an area where glioblastoma multiform (GBM) are thought to originate. Here, we report that Ephrin-B3 is highly expressed in human biopsies and that it inhibits EphA4 pro-apoptotic activity in tumor cells. Angiogenesis is directly correlated with GBM aggressiveness and we demonstrate that Ephrin-B3 also supports the survival of endothelial cells in vitro and in vivo. Lastly, silencing of Ephrin-B3 decreases tumor vascularization and growth in a xenograft mice model. Interference with EphA4/Ephrin-B3 interaction could then be envisaged as a relevant strategy to slow GBM growth by enhancing EphA4-induced cell death.

Innate emotion response to environmental stimuli is a fundamental brain function that is controlled by specific neural circuits. Dysfunction of early emotional circuits may lead to neurodevelopmental disorders such as autism and schizophrenia. However, how the functional circuits are formed to prime initial emotional behaviours remain elusive. We reveal here using gene-targeted mutations an essential role for ephrin-B3 ligand-like activity in the development of innate fear in the neonatal brain. We further demonstrate that ephrin-B3 controls axon targeting and coordinates spinogenesis and neuronal activity within the amygdala. The morphological and behavioural abnormalities in ephrin-B3 mutant mice are rescued by conditional knock-in of wild-type ephrin-B3 during the critical period when axon targeting and fear responses are initiated. Our results thus define a key axonal molecule that participates in the wiring of amygdala circuits and helps bring about fear emotion during the important adolescence period.

Guidance errors and unrefined neural map configurations appear linked to certain neurodevelopmental conditions, including autism spectrum disorders. Deficits in specific multisensory tasks that require midbrain processing are highly predictive of cognitive and behavioral phenotypes associated with such syndromes. The lateral cortex of the inferior colliculus (LCIC) is a shell region of the mesencephalon that integrates converging information from multiple levels and modalities. Mature LCIC sensory maps are discretely-organized, mimicking its compartmental micro-organization. Intermittent modular domains receive patchy somatosensory connections, while inputs of auditory origin terminate in the encompassing extramodular matrix.Eph-ephrin signaling mechanisms instruct comparable topographic arrangements in a variety of other systems. Whether Eph-ephrin interactions also govern the assembly of LCIC multimodal maps remains unaddressed. Previously, we identified EphA4 and ephrin-B2 as key mediators, with overlapping expression patterns that align with emerging LCIC modules. Here, we implicate another member of this guidance family, ephrin-B3, and quantify its transient expression with respect to neurochemically-defined LCIC compartments. Multiple-labeling studies in GAD67-GFP knock-in mice reveal extramodular ephrin-B3 expression, complementary to that of EphA4 and ephrin-B2. This distinctive pattern sharpens over the early postnatal period (birth to P8), prior to ephrin-B3 downregulation once multimodal LCIC inputs are largely segregated (P12). Channel-specific sampling of LCIC ROIs show ephrin-B3 signal periodicities that are out-of-phase with glutamic acid decarboxylase (GAD;modular marker) signal fluctuations, and match calretinin (CR) waveforms (matrix marker). Taken together, the guidance mosaic registry with emerging LCIC compartments and its interfacing afferent streams suggest a prominent role for Eph-ephrins in ordering behaviorally significant multisensory midbrain networks.

Cerebral ischemia stimulates endogenous neurogenesis. However, the functional relevance of this phenomenon remains unclear because of poor survival and low neuronal differentiation rates of newborn cells. Therefore, further studies on mechanisms regulating neurogenesis under ischemic conditions are required, among which ephrin-ligands and ephrin-receptors (Eph) are an interesting target. Although Eph/ephrin proteins like ephrin-B3 are known to negatively regulate neurogenesis under physiological conditions, their role in cerebral ischemia is largely unknown. We therefore studied neurogenesis, brain injury and functional outcome in ephrin-B3(-/-) (knockout) and ephrin-B3(+/+) (wild-type) mice submitted to cerebral ischemia. Induction of stroke resulted in enhanced cell proliferation and neuronal differentiation around the lesion site of ephrin-B3(-/-) compared to ephrin-B3(+/+) mice. However, prominent post-ischemic neurogenesis in ephrin-B3(-/-) mice was accompanied by significantly increased ischemic injury and motor coordination deficits that persisted up to 4 weeks. Ischemic injury in ephrin-B3(-/-) mice was associated with a caspase-3-dependent activation of the signal transducer and activator of transcription 1 (STAT1). Whereas inhibition of caspase-3 had no effect on brain injury in ephrin-B3(+/+) animals, infarct size in ephrin-B3(-/-) mice was strongly reduced, suggesting that aggravated brain injury in these animals might involve a caspase-3-dependent activation of STAT1. In conclusion, post-ischemic neurogenesis in ephrin-B3(-/-) mice is strongly enhanced, but fails to contribute to functional recovery because of caspase-3-mediated aggravation of ischemic injury in these animals. Our results suggest that ephrin-B3 might be an interesting target for overcoming some of the limitations of further cell-based therapies in stroke.

As the neural network becomes wired, postsynaptic signaling molecules are thought to control the growth of dendrites and synapses. However, how these molecules are coordinated to sculpt postsynaptic structures is less well understood. We find that ephrin-B3, a transmembrane ligand for Eph receptors, functions postsynaptically as a receptor to transduce reverse signals into developing dendrites of mouse hippocampal neurons. Both tyrosine phosphorylation-dependent GRB4 SH2/SH3 adaptor-mediated signals and PSD-95-discs large-zona occludens-1 (PDZ) domain-dependent signals are required for inhibition of dendrite branching, whereas only PDZ interactions are necessary for spine formation and excitatory synaptic function. PICK1 and syntenin, two PDZ domain proteins, participate with ephrin-B3 in these postsynaptic activities. PICK1 has a specific role in spine and synapse formation, and syntenin promotes both dendrite pruning and synapse formation to build postsynaptic structures that are essential for neural circuits. The study thus dissects ephrin-B reverse signaling into three distinct intracellular pathways and protein-protein interactions that mediate the maturation of postsynaptic neurons.

Ephrin‑B3 is important in the regulation of cell proliferation, differentiation and migration via cell‑cell contact, and can activate the reelin pathway during brain development. However, the effect of ephrin‑B3 on hippocampal neurogenesis and the reelin pathway in epilepsy remains to be fully elucidated. In the present study, the expression of ephrin‑B3 in pilocarpine‑induced status epilepticus (SE) rats was investigated. SYBR Green‑based reverse transcription‑quantitative polymerase chain reaction analysis, immunohistochemical labeling and western blot analysis were used to detect the gene and protein expression levels of ephrin‑B3 and reelin pathway proteins. Immunofluorescence staining of doublecortin (DCX) was utilized to analyze hippocampal neurogenesis. The data revealed that the mRNA and protein expression levels of ephrin‑B3 in the hippocampus decreased during the spontaneous seizure period. Of note, the expression of reelin and its downstream phosphorylation disabled 1 (p‑Dab1) were also notably decreased during the spontaneous seizure period, which showed similar dynamic changes as in the expression of ephrin‑B3. In addition, it was found that the number of DCX‑labeled neuronal progenitor cells was increased in the hippocampus following pilocarpine‑induced SE. To further clarify the role of ephrin‑B3 in neurogenesis and the reelin pathway in epilepsy, an exogenous ephrin‑B3 clustering stimulator, EphB3‑Fc, was infused into the bilateral hippocampus of the rats post‑SE. Following EphB3‑Fc injection, it was found that the expression levels of reelin and p‑Dab1 were significantly increased in the epileptic rats following EphB3‑Fc injection. The number of DCX‑labeled neuronal progenitor cells was reduced in the hippocampus of the epileptic rats. Furthermore, the intensity and frequency of spontaneous recurrent seizures and electroencephalographic seizures were attenuated in the epileptic rats post‑injection. These results demonstrated the critical role of ephrin‑B3 in regulation of the reelin pathway and hippocampal neurogenesis in epilepsy, providing experimental evidence that ephrin‑B3 functions as a potential protective factor in epilepsy, at least in animals.

Understanding the mechanisms of neuronal regeneration and repair in the adult central nervous system is a vital area of research. Using a rhesus lentiviral encephalitis model, we sought to determine whether recovery of neuronal metabolism after injury coincides with the induction of two important markers of synaptodendritic repair: growth-associated protein-43 (GAP-43) and ephrin B3. We examined whether the improvement of neuronal metabolism with combined anti-retroviral therapy (cART) after simian immunodeficiency virus (SIV) infection in rhesus macaques involved induction of GAP-43, also known as neuromodulin, and ephrin B3, both implicated in axonal pathfinding during neurodevelopment and regulation of synapse formation, neuronal plasticity, and repair in adult brain. We utilized magnetic resonance spectroscopy to demonstrate improved neuronal metabolism in vivo in adult SIV-infected cART animals compared to untreated and uninfected controls. We then assessed levels of GAP-43, ephrin B3, and synaptophysin, a pre-synaptic marker, in three brain regions important for cognitive function, cortex, hippocampus, and putamen, by quantitative real-time RT-PCR and immunohistochemistry. Here we demonstrate that (1) GAP-43 mRNA and protein are induced with SIV infection, (2) GAP-43 protein is higher in the hippocampus outer molecular layer in SIV-infected animals that received cART compared to those that did not, and (3) activated microglia and infiltrating SIV-infected macrophages express abundant ephrin B3, an important axonal guidance molecule. We propose a model whereby SIV infection triggers events that lead to induction of GAP-43 and ephrin B3, and that short-term cART results in increased magnitude of repair mechanisms especially in the hippocampus, a region known for high levels of adult plasticity.

Ephrin receptors (Ephs) are reported to control metastatic signaling of non-small cell lung cancer (NSCLC) and other tumors. Here we show for the first time that blocking expression of the Eph ligand Ephrin B3 inhibits NSCLC cell migration and invasion. We demonstrate that Ephrin B3 directly binds the EphAs EphA2, EphA3, EphA4, and EphA5. EphA2 Ser897 was previously shown to drive migration propensity of tumor cells and our study reveals that EphA2 stays phosphorylated on Ser897 in the Ephrin B3/EphA2 complex in NSCLC cells of different histology. Moreover, we report that within such Ephrin B3/EphA2 complex both Akt Ser 129 and p38MAPK are found indicating a potential to drive migration/proliferation. We also found the EMT marker E-cadherin expression to be maintained or increased upon Ephrin B3 blockade in NSCLC cells. Expression of Ephrin B3 was furthermore analyzed in a cohort of NSCLC stage IA-IB cases (n=200) alongside EphA2 and Ephrin A1. We found that Ephrin B3 was concomitantly expressed with EphA2 and Ephrin A1 with higher Ephrin B3 levels found in non-squamous than in squamous tumors, whereas EphA2 was higher expressed in well-differentiated than in low-differentiated tumors. In the entire NSCLC cohort, Ephrin B3 expression was not linked to patient survival, whereas a high EphA2 expression was associated with improved survival (p=0.03). In conclusion, we show that blocking Ephrin B3 expression inhibits NSCLC proliferation-, migration- and invasion capacity which calls for further studies on interference with Ephrin B3 as a possible therapeutic avenue in this tumor malignancy.

Several erythropoietin-producing hepatocellular receptor B family (EPHB) and their ligands, ephrinBs (EFNBs), are involved in blood pressure regulation in animal models. We selected 528 single nucleotide polymorphisms (SNPs) within the genes of EPHB6, EFNB2, EFNB3 and GRIP1 in the EPH/EFN signalling system to query the International Blood Pressure Consortium dataset. A SNP within the glutamate receptor interacting protein 1 (GRIP1) gene presented a p-value of 0.000389, approaching the critical p-value of 0.000302, for association with diastolic blood pressure of 60,396 individuals. According to echocardiography, we found that Efnb3 gene knockout mice showed enhanced constriction in the carotid arteries. In vitro studies revealed that in mouse vascular smooth muscle cells, siRNA knockdown of GRIP1, which is in the EFNB3 reverse signalling pathway, resulted in increased contractility of these cells. These data suggest that molecules in the EPHB/EFNB signalling pathways, specifically EFNB3 and GRIP1, are involved blood pressure regulation.

The p75 neurotrophin receptor (p75NTR) is known to transduce the signal from some myelin-associated axon growth inhibitors, including Nogo and myelin-associated glycoprotein. As ephrin-B3, a member of the ephrin family, is also expressed in myelin and inhibits axon growth, the purpose of this study was to assess the possible involvement of p75NTR in ephrin-B3 signaling. Here, we report that p75NTR is required for the inhibitory effect of ephrin-B3 on neurite growth in vitro. While ephrin-B3 inhibited neurite elongation of embryonic cortical neurons, the neurons with p75NTR knockdown or with EphA4 knockdown were less sensitive to ephrin-B3. Although no direct interaction of p75NTR with ephrin-B3 was observed, Pep5, a peptide that specifically inhibits RhoA activation mediated by p75NTR, reduced the effect of ephrin-B3. Therefore, p75NTR functions as a signal transducer for ephrin-B3. Moreover, axonal regeneration in vivo was induced by Pep5 application after optic nerve crush injury in mice. Thus, Pep5 is a promising agent that contributes to axonal regeneration in the central nervous system.

During embryonic development the preoptic area (POA) gives rise to two populations of neurons which are generated at the same time, cortical interneurons and striatal cells. POA-derived cortical interneurons take a superficial path and avoid the developing striatum (Str) when they migrate to their target region. We found that EphB1, which is expressed in the striatal anlage, prevents cortical interneurons from entering the Str via ephrin-B3 reverse signaling. In contrast, for striatal neurons which also express ephrin-B3, EphB1 acts as a stop signal. This dual role of EphB1 is due to differences in ephrin-B3 reverse signaling cascades. For striatal neurons, binding of EphB1 to ephrin-B3 reduces endogenously high levels of pSrc and pFAK, which then causes the cells to stop migration. In contrast, in cortical interneurons EphB1-ephrin-B3 reverse signaling leads to phosphorylation of Src and focal adhesion kinase (FAK) which then mediates repulsion. Consistent with these in vitro findings, in an ephrin-B3 knockout mouse line, we discovered misrouted cortical interneurons in the Str and an over-migration of striatal neurons in their target region. Thus, EphB1/ephrin-B3 reverse signaling has a different impact on two sets of neurons which are generated at the same time and place: it can act as a repulsive cue for migrating neurons or it can terminate neuronal migration, a novel role of the Eph/ephrin system.

Radiation therapy is frequently used to treat non-small cell lung cancers (NSCLCs). We have previously shown that a combination of ionizing radiation (IR) and the staurosporine analog PKC 412, but not Ro 31-8220, increases cell death in NSCLC cells. To identify genes involved in the enhancement of cell death, a total gene profiling in response to co-administration of (i) PKC 412 with IR, or (ii) Ro 31-8220 with IR was implemented. These combined treatments caused upregulation of 140 and 179 genes and downregulation of 253 and 425 genes, respectively. Certain genes were selected and verified by real-time quantitative PCR and, of these genes, robust suppression of Ephrin B3 expression was suggested as a possible cell death-inducing mechanism of combined treatment with IR and PKC 412. Indeed, silencing of Ephrin B3 using siRNA in NSCLC cells resulted in a major alteration of their morphology with an elongated phenotype, decreased proliferation and increased cell death signaling. Moreover, silencing of Ephrin B3 in combination with IR caused a decrease in IR-mediated G(2)-arrest, induced cellular senescence, inhibited MAPK ERK and p38 phosphorylation, and caused an upregulation of p27(kip1) expression. Finally, silencing of Ephrin B3 in combination with IR sensitized U-1810 cells to IR-induced apoptosis. In conclusion, we identify and describe Ephrin B3 as a putative signaling molecule involved in the response of NSCLC cells to combined treatment with PKC 412 and ionizing radiation.

A growing body of evidence has demonstrated that Eph/ephrin signalling may serve a central role in intestinal diseases. However, whether erythropoietin‑producing hepatocellular (Eph)/ephrin signalling is associated with the development of post‑infectious irritable bowel syndrome (PI‑IBS) is still unknown. In the present study, the role of Eph/Ephrin signalling in lipopolysaccharide (LPS)‑induced intestinal injury was evaluated in vivo and in vitro. LPS treatment significantly increased the levels of proinflammatory mediators [monocyte chemoattractant protein‑1, tumour necrosis factor α, interleukin (IL)‑1β, IL‑6, intercellular adhesion molecule 1 and vascular cell adhesion molecule‑1], activated the EphA2‑Ephrin A1, protein kinase B (Akt)‑nuclear factor (NF)‑κB, Src‑NF‑κB and Wnt/β‑catenin signalling pathways, and inhibited EphB1‑Ephrin B3 signalling in colon tissues, and primary cultured enteric neuronal and glial cells. Notably, EphA2 monoclonal antibody (mAb) treatment or Ephrin B3 overexpression could partially alleviate the LPS‑induced upregulation of proinflammatory mediators, and Akt‑NF‑κB, Src‑NF‑κB and Wnt/β‑catenin signalling pathways. In addition, EphA2 mAb treatment could partially inhibit LPS‑induced inactivation of EphB‑Ephrin B3 signalling, while Ephrin B3 overexpression could abrogate LPS‑induced activation of EphA2‑Ephrin A1 signalling. EphB1/Ephrin B3 signalling may antagonise the EphA2/Ephrin A1‑dependent pathway following LPS treatment. The results associated with the EphA2 signaling pathway, indicated that Eph/ephrin signalling may serve a bidirectional role in LPS‑induced intestinal injury. Eph/ephrin signalling may be a novel therapeutic target for LPS‑induced intestinal injury and potentially PI‑IBS.

In the central nervous system, myelination of axons is required to ensure fast saltatory conduction and for survival of neurons. However, not all axons are myelinated, and the molecular mechanisms involved in guiding the oligodendrocyte processes toward the axons to be myelinated are not well understood. Only a few negative or positive guidance clues that are involved in regulating axo-glia interaction prior to myelination have been identified. One example is laminin, known to be required for early axo-glia interaction, which functions through α6β1 integrin. Here, we identify the Eph-ephrin family of guidance receptors as novel regulators of the initial axo-glia interaction, preceding myelination. We demonstrate that so-called forward and reverse signaling, mediated by members of both Eph and ephrin subfamilies, has distinct and opposing effects on processes extension and myelin sheet formation. EphA forward signaling inhibits oligodendrocyte process extension and myelin sheet formation, and blocking of bidirectional signaling through this receptor enhances myelination. Similarly, EphB forward signaling also reduces myelin membrane formation, but in contrast to EphA forward signaling, this occurs in an integrin-dependent manner, which can be reversed by overexpression of a constitutive active β1-integrin. Furthermore, ephrin-B reverse signaling induced by EphA4 or EphB1 enhances myelin sheet formation. Combined, this suggests that the Eph-ephrin receptors are important mediators of bidirectional signaling between axons and oligodendrocytes. It further implies that balancing Eph-ephrin forward and reverse signaling is important in the selection process of axons to be myelinated.

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting "in trans" with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral "cis" associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.

The emergent zoonotic henipaviruses, Hendra, and Nipah are responsible for frequent and fatal disease outbreaks in domestic animals and humans. Specificity of henipavirus attachment glycoproteins (G) for highly species-conserved ephrin ligands underpins their broad host range and is associated with systemic and neurological disease pathologies. Here, we demonstrate that Cedar virus (CedV)-a related henipavirus that is ostensibly nonpathogenic-possesses an idiosyncratic entry receptor repertoire that includes the common henipaviral receptor, ephrin-B2, but, distinct from pathogenic henipaviruses, does not include ephrin-B3. Uniquely among known henipaviruses, CedV can use ephrin-B1 for cellular entry. Structural analyses of CedV-G reveal a key region of molecular specificity that directs ephrin-B1 utilization, while preserving a universal mode of ephrin-B2 recognition. The structural and functional insights presented uncover diversity within the known henipavirus receptor repertoire and suggest that only modest structural changes may be required to modulate receptor specificities within this group of lethal human pathogens.