EPHB1 Human

What is the molecular structure of EPHB1 and how does it compare to other EphB receptors?

EPHB1 belongs to the EphB subfamily comprising five members: EphB1-4 (catalytically active) and EphB6 (catalytically inactive). The first structure of the human EPHB1 tyrosine kinase domain was determined by X-ray crystallography to 2.5Å resolution, alongside new crystal forms of human EphB2 and EphB4 catalytic domains .

The high sequence and structural conservation among Eph kinase domains presents challenges for selective targeting. The EphB kinases share similar domain organization including:

• Extracellular ligand-binding domain

• Fibronectin type III domains

• Transmembrane region

• Intracellular tyrosine kinase domain

• SAM (Sterile Alpha Motif) domain

For researchers seeking to study specific EphB members, structure-driven optimization approaches are essential due to this high structural similarity across the family .

What experimental models are most appropriate for studying EPHB1 function?

Several validated experimental models can be employed depending on the research question:

Cellular models:

• Colorectal cancer cell lines for compartmentalization and phospho-proteome studies

• Primary muscle cells for pain and contractility studies

Animal models:

• Global EPHB1 knockout mice for broad functional studies

• Conditional knockout models using specific Cre drivers:

  • Vgat-Cre (GABAergic cells)

  • Emx1-Cre (cortical excitatory neurons)

• Rat models for muscle pain studies using intramuscular injections of EPHB1 activators/inhibitors

Biochemical/Structural models:

• Recombinant EPHB1 kinase domain expression in E. coli for structural studies

• Phosphorylation assays using human tissue samples

Selection criteria should include consideration of the specific physiological context being studied, as EPHB1 functions differently across tissues and developmental stages.

What are the standard methods for detecting and quantifying EPHB1 expression?

Based on published research protocols, recommended methods include:

Protein detection:

• Western blot analysis for total EPHB1 and phosphorylated EPHB1 (p-EPHB1)

• RayBio Human Phosphorylation Array Kit for quantitative phosphorylation analysis

• Immunohistochemistry for tissue localization

RNA detection:

• RT-PCR or qPCR for mRNA expression levels

• RNA-seq for comprehensive transcriptomic analysis

When assessing EPHB1 activity rather than mere expression, researchers should focus on phosphorylation status using phospho-specific antibodies, as activation state is more relevant than total protein levels for functional studies .

How do recurrent EPHB1 mutations in human cancers affect receptor function?

A comprehensive analysis of 79,151 somatic mutations across 33 different tumor types identified recurring EPHB1 mutations with functional consequences . The mutations cluster in three key domains with distinct effects:

Ligand-binding domain mutations (C61Y, R90C, R170W):

• Displayed reduced to strongly compromised cell compartmentalization

• Showed reduced ligand-induced receptor phosphorylation

Fibronectin domain mutation (R351L):

• Reduced cell compartmentalization capability

• Altered receptor activation dynamics

Kinase domain mutations:

• D762N: compromised cell compartmentalization

• R743W and G821R: enhanced compartmentalization

Methodologically, researchers found that protein 3D-structure-based mutation analysis was more effective at identifying functionally significant mutations (63% phenotype correlation) than conventional hotspot analysis (43% correlation) . This study provides a framework for prioritizing mutations for functional validation in cancer research.

What is the role of EPHB1 in peripheral pain mechanisms, particularly in myofascial trigger points (MTrPs)?

EPHB1 plays a significant role in muscle pain, particularly in myofascial trigger points (MTrPs). Key research findings include:

• P-EPHB1 expression is significantly increased in human muscles with MTrPs compared to healthy controls

• Strong correlation exists between p-EPHB1 expression and pain intensity (r = 0.723) in myofascial pain syndrome patients

• EPHB1 contributes to peripheral sensitization through:

  • Enhancing NMDA receptor function in peripheral sensory neurons

  • Possible regulation of acetylcholine release at neuromuscular junctions

  • MAPK-mediated hyperalgesia mechanisms

Experimental evidence shows that:

• Intramuscular injection of EphrinB1-Fc (EPHB1 activator) induces muscle hyperalgesia

• EphB-Fr (EPHB1 inhibitor) administration attenuates muscle hyperalgesia

This table shows significant differences between control subjects and myofascial pain syndrome patients:

Researchers studying peripheral pain mechanisms should consider EPHB1 as a potential therapeutic target, using assessment tools like the Randall-Selitto apparatus to measure mechanical hyperalgesia in animal models .

How does EPHB1 regulate axon guidance during cortical development?

EPHB1 plays a critical role in guiding cortical axon projections during brain development. Recent research revealed:

• EPHB1 functions through a cell non-autonomous mechanism, where its expression in one cell type affects the behavior of another cell type

• Conditional knockout (cKO) of EPHB1 in GABAergic cells (Vgat-Cre) reproduces the axon guidance defects seen in global EPHB1 KO mice

• EPHB1 deletion in cortical excitatory neurons (Emx1-Cre) does not produce the same defects

• In EPHB1 cKO Vgat mice, misguided axon bundles contain co-mingled striatal GABAergic and somatosensory cortical glutamatergic axons

• In wild-type mice, somatosensory axons co-fasciculate with striatal axons

For developmental neurobiology researchers, this suggests that cell-type-specific approaches are essential when studying EPHB1 function, as global knockout phenotypes may result from effects in unexpected cell populations. The use of conditional knockout strategies with different Cre drivers enables dissection of cell-autonomous versus non-autonomous functions .

What methodological approaches are optimal for studying EPHB1 kinase activity?

Researchers investigating EPHB1 kinase activity have several validated approaches:

Structural approaches:

• X-ray crystallography (to 2.5Å resolution) for detailed structural analysis of the kinase domain

• Rational protein engineering to improve crystallization properties (as demonstrated for EphB3)

Biochemical approaches:

• In vitro kinase assays using recombinant protein

• Phosphorylation site mapping using mass spectrometry

• Western blot analysis with phospho-specific antibodies

Cellular approaches:

• Compartmentalization assays to assess functional consequences of EPHB1 mutations or inhibitors

• Ligand-induced receptor phosphorylation assays

• Phospho-proteome analysis to identify downstream signaling effects

Inhibitor studies:

• Application of EphB-Fr (specific inhibitor) to assess kinase-dependent functions

• Structure-guided development of selective inhibitors based on crystal structure data

When designing kinase activity studies, researchers should consider that EPHB1 activation affects multiple downstream pathways including PI3K (particularly PIK3C2B) and MAPK signaling .

How can phospho-proteome analysis inform our understanding of EPHB1 signaling networks?

Phospho-proteome analysis provides comprehensive insights into EPHB1's role in signaling networks:

• Identifies both direct substrates and downstream effectors of EPHB1 kinase activity

• Maps signaling pathway alterations resulting from EPHB1 mutations or manipulations

• Connects receptor function to specific cellular phenotypes

In cancer research, phospho-proteome mapping of cells with EPHB1 mutations revealed:

• PI3K pathway involvement in cells with mutations causing reduced compartmentalization

• Specific phosphorylation changes in PIK3C2B associated with altered cell behavior

For researchers implementing this approach:

• Establish appropriate cellular models expressing wild-type vs. mutant EPHB1

• Confirm EPHB1 expression/activation status

• Perform global phospho-proteome analysis using mass spectrometry

• Validate key findings with targeted approaches (Western blot, kinase assays)

• Connect phosphorylation changes to functional outcomes using appropriate assays

This methodology provides mechanistic insights beyond simple correlation studies, revealing how EPHB1 integrates into broader signaling networks.

What are the challenges in developing selective EPHB1 inhibitors for therapeutic use?

Developing selective EPHB1 inhibitors presents several challenges for medicinal chemists and drug developers:

• High structural similarity among EphB family members (EphB1-4) complicates selective targeting

• Conflicting expression patterns in cancer tissues create context-dependent therapeutic requirements

• Potential off-target effects on other tyrosine kinases with similar ATP-binding pockets

Recent structural advances have provided critical tools:

• First EPHB1 tyrosine kinase domain structure determined to 2.5Å resolution

• Comparative analysis with other EphB family members

• Rational engineering approaches to improve protein stability and crystallization

For structure-based drug design efforts, researchers should leverage these structural insights while implementing rigorous selectivity profiling against related kinases. The development pipeline should include:

• Structure-guided compound design

• In vitro selectivity screening across the kinome

• Cellular target engagement validation

• Functional assays in disease-relevant models

How do EPHB1 mutations contribute to cancer progression and metastasis?

EPHB1 mutations impact cancer progression through several mechanisms:

Cell compartmentalization effects:

• Certain mutations (C61Y, R90C, R170W, R351L, D762N) reduce cell compartmentalization, potentially promoting invasion

• Other mutations (R743W, G821R) enhance compartmentalization, with unclear consequences for cancer progression

Signaling pathway alterations:

• PI3K pathway and PIK3C2B phosphorylation changes in cells with EPHB1 mutations

• Altered receptor phosphorylation correlates with compartmentalization defects

Methodological recommendations for cancer researchers:

• Screen patient samples for recurring EPHB1 mutations using targeted sequencing

• Prioritize mutations for functional validation using 3D-structure-based bioinformatic approaches

• Express mutations in appropriate cellular models

• Assess effects on:

  • Cell compartmentalization

  • Receptor phosphorylation

  • Downstream signaling pathways

  • Migration, invasion, and metastatic potential

This integrative approach combining bioinformatics, cellular models, and signaling analysis provides a robust framework for characterizing the oncogenic potential of EPHB1 mutations.

How can EPHB1-targeted approaches be applied to pain management in myofascial pain syndrome?

Based on evidence linking EPHB1 to myofascial trigger points and pain, several therapeutic approaches warrant investigation:

Potential therapeutic strategies:

• Development of selective EPHB1 inhibitors for local administration

• EphB-Fr (EPHB1 inhibitor) demonstrated efficacy in reducing muscle hyperalgesia in animal models

• Targeting downstream mediators in the EPHB1 signaling pathway (MAPK, NMDA)

Clinical assessment considerations:

• P-EPHB1 expression correlates strongly with pain intensity (r = 0.723)

• May serve as a biomarker for treatment response

Methodological approach for translational research:

• Validate EPHB1 expression in patient muscle biopsies using phosphorylation array analysis

• Correlate expression with clinical pain scores using standardized instruments

• Test EPHB1 inhibitors in preclinical models using established pain assessment methods

• Develop targeted delivery approaches to minimize systemic effects

• Design clinical trials with appropriate pain outcome measures

The strong correlation between EPHB1 activation and pain intensity suggests that EPHB1-targeted approaches could provide novel treatment options for patients with myofascial pain syndrome refractory to current therapies.

What are the best practices for engineering EPHB1 proteins for structural studies?

Based on successful approaches with EPHB family members:

Protein expression optimization:

• Expression in E. coli has been successful for kinase domains

• A single point mutation identified through rational engineering improved EphB3 soluble recombinant yield and facilitated crystallization

• This approach could be applied to EPHB1 variants that prove difficult to express

Construct design considerations:

• Careful domain boundary selection is critical

• Removal of flexible regions that might impede crystallization

• Consider surface entropy reduction mutations to promote crystal contacts

Crystallization strategies:

• Comparative crystallization analysis across EphB family members can inform conditions for EPHB1

• Wild-type forms of EphB1, EphB2, and EphB4 all crystallized readily under similar conditions

• For recalcitrant constructs, structure-guided engineering based on related family members has proven effective

Researchers should leverage the existing structural information across the EphB family to inform their approach to EPHB1 protein engineering for structural studies.

How can one distinguish the specific roles of EPHB1 from other EphB receptors in experimental systems?

Differentiating EPHB1 functions from other EphB receptors requires strategic approaches:

Genetic approaches:

• Conditional knockout models targeting specific cell types (e.g., Vgat-Cre, Emx1-Cre for EPHB1)

• CRISPR/Cas9 gene editing for precise mutations or domain deletions

• siRNA/shRNA with validated specificity for EPHB1

Biochemical approaches:

• Highly specific antibodies validated against multiple EphB receptors

• Selective inhibitors based on structural differences identified in crystallographic studies

• Recombinant proteins with mutations in key interaction domains

Experimental design considerations:

• Use multiple approaches to confirm specificity

• Include comprehensive controls for other EphB family members

• Consider redundancy and compensation among family members

The cortical axon guidance studies demonstrate the importance of cell-type-specific approaches, as EPHB1's function in GABAergic cells, rather than excitatory neurons, was found to be critical for proper axon guidance .

What are the potential roles of EPHB1 in neurodevelopmental disorders?

Given EPHB1's critical role in axon guidance during cortical development , several research directions merit exploration:

Potential neurodevelopmental implications:

• Aberrant cortical connectivity resulting from EPHB1 dysfunction

• Possible contributions to conditions characterized by altered neural circuit formation

• Role of EPHB1 in GABAergic cell function and implications for inhibitory circuit development

Recommended research approaches:

• Analysis of EPHB1 variants in neurodevelopmental disorder cohorts

• Characterization of brain connectivity in EPHB1 conditional knockout models

• Investigation of EPHB1's role in different neuronal subtypes during development

• Electrophysiological assessment of circuit function in EPHB1-deficient models

The finding that EPHB1 in GABAergic cells controls long-range cortical axon guidance through a cell non-autonomous mechanism suggests complex developmental roles that may have implications for disorders involving aberrant connectivity .

How might targeting EPHB1 address treatment resistance in cancer therapy?

Based on EPHB1's roles in cancer progression:

Potential strategies to overcome resistance:

• Combination approaches targeting EPHB1 alongside established pathways

• Identification of synthetic lethal interactions with EPHB1 mutations

• Development of mutation-specific inhibitors for precision medicine approaches

Research methodology recommendations:

• Characterize EPHB1 expression and mutation status in treatment-resistant tumors

• Correlate specific mutations with treatment response data

• Perform synthetic lethality screens in cells with EPHB1 mutations

• Develop and test combination therapies targeting EPHB1 and related pathways

The compartmentalization effects of different EPHB1 mutations suggest distinct mechanisms through which they might contribute to cancer progression and treatment response, warranting personalized therapeutic approaches based on specific mutation profiles .