EPHB4 Human

What is the molecular structure of human EPHB4?

Human EPHB4 is a 987 amino acid transmembrane receptor tyrosine kinase. Its structure includes a 15 amino acid signal sequence, a 524 amino acid extracellular domain (ECD) consisting of an N-terminal globular domain, a cysteine-rich domain, and two fibronectin type III domains. This is followed by a 21 amino acid transmembrane segment and a 427 amino acid cytoplasmic domain containing a juxtamembrane motif with two tyrosine residues (major autophosphorylation sites), a kinase domain, and a conserved sterile alpha motif (SAM) . The ECD typically spans amino acids Leu16-Ala539 and is crucial for ligand binding.

How does EPHB4 signaling differ between cell types?

EPHB4 exhibits remarkable context-dependent signaling that varies by cell type:

• In endothelial cells (HUVECs): EPHB4 activation inhibits the Ras/ERK pathway through p120 RasGAP, resulting in decreased cell proliferation

• In breast cancer cells (MCF-7): EPHB4 activation stimulates the Ras/ERK pathway via PP2A, promoting cell growth and proliferation

This dichotomy represents the first documented functional coupling between an Eph receptor and PP2A leading to activation of an oncogenic pathway. The contrasting effects highlight the complex nature of EPHB4 signaling and its potential cell-specific roles in physiological and pathological contexts.

What role does EPHB4 play in prostate cancer initiation and progression?

EPHB4 has been identified as a critical factor in prostate cancer development. In a genetic mouse model with conditional PTEN deletion in prostate epithelium, EPHB4 and ephrin-B2 are significantly induced. This induction is substantially required for tumor initiation, as demonstrated through two independent approaches:

• Genetic deletion of EPHB4 in prostate epithelium significantly reduced tumor formation

• Treatment with soluble EphB4-albumin fusion protein (sEphB4-alb) similarly inhibited tumor development

Furthermore, EPHB4-ephrin-B2 signaling remains active in castration-resistant prostate cancer models, with sEphB4-alb retaining efficacy in androgen-independent settings. These findings establish that EPHB4 not only contributes to tumor initiation in PTEN-null prostate cancer but also continues to promote progression in castration-resistant disease .

How are EPHB4 mutations linked to cardiac dysfunction?

Analysis of 573 dilated cardiomyopathy (DCM) patients identified six novel EPHB4 variants, three of which are located in the extracellular domain of EPHB4, with two specifically in the ligand binding domain. Patients carrying these variants display altered expression patterns of CD36 and CAV1 in the heart, with reduced CD36 expression and co-localization with CAV1 in cardiomyocytes .

The data suggests EPHB4 may regulate CD36 caveolar trafficking to the membrane, which appears important for maintaining cardiac homeostasis. Additionally, Eph receptor genes, including EPHB4, have been found downregulated in hypertrophic human hearts, and circulating EPHB4 is associated with poor prognosis in heart failure . These findings suggest EPHB4 plays important roles in cardiac function through regulation of fatty acid transport and metabolism.

What mechanisms underlie EPHB4's role in venous valve development?

Mutations in EPHB4 cause human venous valve aplasia, as confirmed by quantitative ultrasound studies showing substantial venous valve aplasia and deep venous reflux in affected patients . Mechanistically, EPHB4 deletion disrupts the normal endothelial expression of gap junction proteins connexin37 and connexin43 (both required for normal valve development) around reorienting valve-forming cells.

This disruption results in:

• Deficient valve-forming cell elongation

• Impaired reorientation

• Disrupted polarity

• Reduced proliferation

EPHB4 is required for both initial valve-forming cell organization and subsequent growth of valve leaflets . During development, EPHB4 expression appears stronger immediately upstream of organizing valve-forming cells, while ephrin-B2 expression is higher downstream, establishing a directional signaling gradient critical for proper valve morphogenesis.

What are the most effective experimental models for studying EPHB4 function?

Several complementary experimental systems have proven valuable for EPHB4 research:

These diverse models allow researchers to study EPHB4 at multiple levels, from molecular interactions to organismal development and disease manifestations.

How can researchers effectively measure EPHB4 activity?

Multiple complementary approaches can be used to assess EPHB4 activity:

• Biochemical Assays:

  • Receptor autophosphorylation detection using phospho-specific antibodies

  • Co-immunoprecipitation to detect EPHB4 interaction with partners like RASA1

  • Analysis of downstream pathway activation (Ras-MAPK components, ERK phosphorylation)

• Functional Cellular Assays:

  • Cell proliferation (inhibited in HUVECs, stimulated in MCF7 cells)

  • Cell-cell adhesion (typically inhibited by EPHB4)

  • Chemotaxis (inhibited by EPHB4)

  • Angiogenesis (inhibited by EPHB4)

• Molecular Probes:

  • EphrinB2 (natural ligand) to activate EPHB4

  • Agonistic monoclonal antibodies specific for EPHB4

  • Soluble EphB4 decoys to block bidirectional signaling

• Gene Expression Analysis:

  • Measurement of downstream target genes following EPHB4 activation

  • Single-nucleus RNA sequencing for cell-type specific expression patterns

When designing experiments to measure EPHB4 activity, researchers should consider the context-dependent nature of EPHB4 signaling and select appropriate readouts based on the specific cell type and biological question.

What approaches are used to study EPHB4-RASA1 interactions in lymphatic valve development?

The EPHB4-RASA1 interaction is critical for lymphatic vessel (LV) valve development through regulation of the PIEZO1→Ras-MAPK signaling axis. Researchers have employed several sophisticated approaches to study this interaction:

• Genetic Models:

  • EPHB4 2YP knockin mice that express EPHB4 unable to bind RASA1 while retaining tyrosine kinase activity

  • LV-specific inducible EPHB4-deficient mice

• Functional Rescue Experiments:

  • Inhibition of the Ras-MAPK signaling pathway to reverse LV valve development defects

  • This confirms the mechanistic link between EPHB4 and this signaling cascade

• Stimulus-Response Studies:

  • PIEZO1 activation with Yoda1 agonist in wild-type versus EPHB4 2YP embryos

  • In wild-type embryos: Yoda1 increased LV valve number

  • In EPHB4 2YP embryos: Yoda1 failed to increase LV valve number

• Molecular Pathway Analysis:

  • In vitro knockdown studies showing that:

    • Loss of EPHB4 or ephrin-B2 expression

    • Loss of RASA1 expression

    • Inhibition of EPHB4-RASA1 interaction

  • All result in dysregulated oscillatory shear stress-induced Ras-MAPK activation and impaired expression of LV specification markers

These approaches have revealed that EPHB4-RASA1 complex formation is essential for inhibiting Ras activation downstream of PIEZO1, properly regulating the Ras-MAPK pathway for valve development.

How might EPHB4 be effectively targeted in cancer therapy?

Based on preclinical evidence, EPHB4 represents a promising therapeutic target, particularly for prostate cancer. Several targeting approaches show potential:

• Blocking Bidirectional Signaling:

  • Soluble EphB4-albumin fusion protein (sEphB4-alb) with improved pharmacokinetics significantly inhibited tumor formation in prostate cancer models

  • This approach remains effective in castration-resistant prostate cancer, suggesting utility in advanced disease

• Strategic Considerations:

  • EPHB4 is induced early following PI3K pathway activation in prostate cancer

  • Targeting EPHB4 could potentially block a critical step in tumor initiation

  • Even after tumor formation, EPHB4 continues to promote progression, making it relevant throughout disease development

• Methodological Approaches:

  • Direct inhibitors of EPHB4 kinase activity

  • Antibodies preventing EPHB4-ephrinB2 interaction

  • Disruption of EPHB4's interaction with downstream effectors

Researchers should note that EPHB4's context-dependent effects present challenges for therapeutic development. The conflicting effects on cancer cells (where inhibition may be beneficial) versus endothelial cells (where inhibition could potentially promote unwanted angiogenesis) require careful consideration of delivery systems, dosing, and combination strategies .

What experimental approaches are used to study the role of EPHB4 in lymphatic valve maintenance?

Lymphatic vessel (LV) valve maintenance involves ongoing EPHB4 signaling beyond initial development. Researchers use several approaches to study this process:

• Temporal Control Models:

  • Inducible EPHB4-deficient mice allow examination of valve maintenance in adults separate from developmental effects

• Pharmacological Interventions:

  • Inhibition of the Ras-MAPK signaling pathway to determine its role in valve maintenance

  • PIEZO1 activation with Yoda1 to assess the response in mature valves

• Molecular Imaging:

  • Visualization of LV valves with specialized markers

  • Quantification of valve structure and function before and after genetic or pharmacological manipulation

• Functional Assessment:

  • Analysis of lymphatic flow and valve competence

  • Correlation of structural changes with functional outcomes

These approaches have revealed that EPHB4 is not only required for valve development but also for valve maintenance in adults, suggesting that EPHB4-targeted therapies might have implications for lymphatic valve disorders even when applied after development is complete .

How does the EPHB4-PIEZO1 signaling axis function in mechanotransduction?

The EPHB4-PIEZO1 signaling axis represents a sophisticated mechanotransduction system in lymphatic endothelium:

• Signal Initiation:

  • PIEZO1 functions as an oscillatory shear stress sensor

  • Oscillatory shear stress is considered the trigger for LV valve specification in vivo

• Signal Regulation:

  • EPHB4, when bound to ephrin-B2, recruits RASA1

  • The EPHB4-RASA1 complex inhibits Ras activation downstream of PIEZO1

  • This inhibition appropriately regulates the Ras-MAPK signaling pathway for valve development

• Experimental Evidence:

  • In human dermal lymphatic endothelial cells, disruption of EPHB4, ephrin-B2, or RASA1 results in dysregulated oscillatory shear stress-induced Ras-MAPK activation

  • Similar dysregulation occurs when cells are stimulated with the Yoda1 agonist of PIEZO1

• In Vivo Confirmation:

  • Yoda1 administration increases LV valve number in wild-type embryos

  • This effect is absent in EPHB4 2YP embryos where EPHB4 cannot bind RASA1

This signaling axis demonstrates how mechanical forces are translated into biochemical signals that drive developmental processes, with EPHB4 serving as a critical regulatory node that determines the appropriate cellular response to mechanical stimulation.

What are the major technical challenges in studying EPHB4 function?

Several technical challenges complicate EPHB4 research:

• Context-Dependent Signaling:

  • EPHB4 has opposite effects in different cell types

  • This requires careful selection of experimental systems and interpretation of results

• Bidirectional Signaling Complexity:

  • EPHB4-ephrinB2 interaction leads to both forward and reverse signaling

  • Distinguishing between these pathways requires specialized reagents and approaches

• Cell-Type Specific Coupling:

  • EPHB4 couples to different downstream effectors in different cells:

    • p120 RasGAP in endothelial cells

    • PP2A in MCF7

  • This variability means that pathway analysis must be tailored to cell type

• Protein Stability Issues:

  • Recombinant EPHB4 often requires carrier proteins like BSA to enhance stability

  • Carrier-free versions are less stable but necessary for some applications

• Human Tissue Limitations:

  • Access to human tissues with EPHB4 mutations is limited

  • This constrains translation of findings from model systems to human disease

Addressing these challenges requires multidisciplinary approaches and careful experimental design that accounts for the context-dependent nature of EPHB4 biology.

How might single-cell technologies advance our understanding of EPHB4 function?

Single-cell technologies offer promising avenues for advancing EPHB4 research:

• Cell-Type Specific Expression Patterns:

  • Single-nucleus RNA sequencing has already revealed that while EPHB4 is mainly expressed by endothelial cells in the heart, it is also present in some cardiomyocytes

  • This technique can further identify additional cell populations expressing EPHB4 across tissues

• Heterogeneity Analysis:

  • Single-cell approaches can reveal heterogeneity in EPHB4 expression and signaling within apparently homogeneous cell populations

  • This may explain variable responses to EPHB4 modulation

• Developmental Trajectories:

  • Single-cell RNA sequencing during valve development could map the temporal changes in EPHB4 expression and associated gene networks

  • This would provide insights into the dynamic regulation of EPHB4 during morphogenesis

• Spatial Transcriptomics:

  • Combining single-cell approaches with spatial information could reveal how EPHB4 expression patterns create signaling gradients important for tissue patterning

  • This is particularly relevant for understanding the complementary expression of EPHB4 and ephrin-B2 during valve development

These technologies could help resolve the context-dependent nature of EPHB4 signaling and identify new therapeutic opportunities based on cell-type specific functions.

What are the unresolved questions about EPHB4's role in human disease?

Despite significant progress, several important questions about EPHB4 in human disease remain unanswered:

• Disease Spectrum:

  • Are there additional human diseases associated with EPHB4 mutations beyond venous valve aplasia and dilated cardiomyopathy?

  • Do EPHB4 polymorphisms contribute to disease susceptibility or progression?

• Therapeutic Potential:

  • Can EPHB4-targeting therapies overcome the context-dependent effects challenge?

  • How might combination therapies address the conflicting effects on different cell types?

• Signaling Modulators:

  • What factors determine whether EPHB4 couples to inhibitory (p120 RasGAP) or stimulatory (PP2A) pathways?

  • Can these factors be manipulated to direct EPHB4 signaling therapeutically?

• Post-Translational Regulation:

  • How do post-translational modifications beyond autophosphorylation regulate EPHB4 function?

  • Do these modifications vary in disease states?

• Immunological Interactions:

  • Does EPHB4 play roles in immune cell function or immunological processes?

  • How might this impact disease pathogenesis or treatment approaches?

Addressing these questions will require integrative approaches combining genetic, molecular, cellular, and physiological studies in both model systems and human subjects.