EPOR Human, Active

What is the molecular structure of human EPOR and how does it function?

Human EPOR is a type I single-transmembrane cytokine receptor belonging to the homodimerizing subclass that functions as ligand-induced or ligand-stabilized homodimers. The receptor has a calculated molecular mass of 59 kDa and consists of:

• An extracellular domain (ECD) responsible for ligand binding, containing:

  • A single cytokine homology module (CHM) formed by two fibronectin type III (FnIII) domains (D1 and D2)

  • Membrane-distal D1 domain with two conserved disulfides

  • Membrane-proximal D2 domain featuring a characteristic WSXWS motif

• A single-helical transmembrane domain (TMD) driving receptor dimerization

• A disordered intracellular domain (ICD) responsible for JAK2 binding and downstream signaling

When EPO binds to EPOR, it induces a conformational change initiating EPOR-associated JAK2 transphosphorylation and activating multiple downstream signal transduction cascades including STATs, PI3K/AKT, RAS/ERK1/2, and NF-κB pathways . These signaling cascades lead to activation of antiapoptotic factors, stimulation of cell differentiation, and modulation of cellular plasticity in a context-dependent manner .

Where is EPOR expressed beyond hematopoietic tissues?

While EPOR was initially characterized in erythroid progenitor cells, research has demonstrated its expression in multiple non-hematopoietic tissues:

• Central nervous system: neurons, oligodendroglia, astrocytes, and microglia

• Adipose tissue: both white adipose tissue (WAT) and brown adipose tissue (BAT)

• Skeletal muscle

• Cardiovascular system

The widespread expression of EPOR provides the molecular basis for EPO's pleiotropic effects beyond erythropoiesis, including neuroprotection, metabolic regulation, and tissue repair after injury .

How can researchers reliably detect EPOR expression in experimental systems?

Detecting EPOR has been historically challenging due to:

• Low cell surface expression, even in stimulated states

• Cross-reactivity issues with commercially available antibodies

• Scientific disputes regarding expression in extra-hematopoietic tissues

Methodological approaches for reliable EPOR detection:

• Validated specific antibodies: Use newly developed and thoroughly characterized antibodies that have been validated using EPOR-transduced cells as positive controls

• Double-labeling strategies: Implement techniques that can demonstrate both membrane expression and intracellular localization (e.g., in the Golgi apparatus)

• Recombinant protein controls: Utilize high-purity recombinant EPOR proteins (>98% as determined by SDS-PAGE) as positive controls in detection assays

• Multi-method approach: Combine protein detection (Western blot, immunohistochemistry) with functional assays and gene expression analysis

Implementing these approaches helps overcome the technical challenges that have complicated EPOR research and enables more reliable characterization of EPOR expression patterns.

What is the current understanding of EPOR's role in metabolic regulation?

Recent research has revealed that EPOR plays a significant role in energy metabolism, particularly through its expression in adipose tissue. Key findings include:

• Adipose tissue-specific functions:

  • Male mice lacking EpoR in adipose tissue exhibit increased fat mass and greater susceptibility to diet-induced obesity

  • EpoR expression in WAT mediates EPO's metabolic activities

• Metabolic improvements with EPO treatment:

  • Enhanced glucose tolerance and insulin sensitivity

  • Reduced expression of lipogenic-associated genes in WAT

  • Decreased fat mass

• Molecular mechanism:

  • EPO-EpoR signaling increases transcription factor RUNX1, which directly inhibits lipogenic gene expression

  • EPO treatment decreases WAT ubiquitin ligase FBXW7 expression and increases RUNX1 stability

  • These effects create an EPO-EpoR-RUNX1 regulatory axis controlling energy metabolism

• Tissue-specific responses:

  • Effects on fat mass and lipogenic gene expression require adipose tissue EpoR expression

  • Mice with EpoR restricted to erythroid tissue (∆EpoR E-mice) become obese, glucose intolerant, and insulin resistant

These findings suggest potential therapeutic applications for EPO or EPO mimetics in metabolic disorders, while also highlighting the importance of considering tissue-specific EPOR functions.

How does the structural dynamics of EPOR-EPO binding influence receptor activation?

The structural dynamics of EPOR-EPO binding and receptor activation involve a complex series of molecular events:

• Ligand binding and receptor dimerization:

  • A single EPO molecule binds to two EPOR molecules, forming a homodimeric complex

  • This binding follows a general activation mechanism involving ligand binding to a monomeric receptor followed by receptor dimerization

• Conformational changes:

  • EPO binding induces rotational movement of the receptor transmembrane (TM) α-helices

  • This movement causes proximity, dimerization, and activation of associated JAK2 subunits

• Structural modeling insights:

  • Three-dimensional models of EPOR complexes with EPO and JAK2 have been generated using AlphaFold Multimer

  • Due to the large size of these complexes (3220-4074 residues), modeling required stepwise assembly from smaller parts

  • Models were validated through comparisons with published experimental data

• Activation mechanisms:

  • Models support both active and inactive conformational states

  • These models help explain the molecular basis of oncogenic mutations that may involve non-canonical activation routes

Understanding these structural dynamics provides insights for developing targeted therapies that modulate EPOR signaling and may help explain how mutations in EPOR can lead to pathological conditions.

What approaches are available for engineering novel EPOR-binding proteins?

Engineering novel EPOR-binding proteins represents an important frontier in research with potential therapeutic applications. Approaches include:

• Key residue grafting strategy:

  • Scanning the Protein Data Bank for suitable scaffold proteins that can accept key interaction residues

  • Grafting the key interaction residues of human erythropoietin that bind to EPOR onto unrelated protein scaffolds

  • Making additional mutations to form stable complexes with EPOR

• Example of successful design:

  • Rat PLCδ1-PH (pleckstrin homology domain of phospholipase C-δ1) was successfully engineered to bind human EPOR

  • A designed triple mutation of PLCδ1-PH (ERPH1) bound EPOR with high affinity (KD of 24 nM and IC50 of 5.7 μM)

  • The wild-type PLCδ1-PH showed no detectable binding under the same assay conditions

• Validation methods:

  • In vitro binding assays to determine binding affinities

  • Cell-based assays to confirm functional significance

  • Correlation between computational binding affinities and in vitro measurements

• Activity assessment:

  • Measuring the ability of engineered proteins to inhibit EPO-dependent proliferation

  • The activity of recombinant EPOR can be measured by its ability to inhibit EPO-dependent proliferation of TF-1 human erythroleukemic cells with an ED50 of typically 15-60 ng/mL

This engineering approach demonstrates promising applications in protein engineering targeting protein-protein interfaces and could lead to novel therapeutic agents that modulate EPOR signaling.

How does EPOR expression change under pathological conditions?

EPOR expression is dynamically regulated and changes under various pathological conditions:

• Injury-induced expression:

  • In mouse models, stereotactically applied stab wounds to the motor cortex lead to distinct EPOR expression by reactive GFAP-expressing cells in the lesion vicinity

  • This suggests EPOR upregulation as part of the tissue response to injury

• Neurological disorders:

  • In human epilepsy patients, neurons and oligodendrocytes of the hippocampus strongly express EPOR

  • This may represent an adaptive response to neural tissue distress

• Potential role in carcinogenesis:

  • EPO and EPOR may be involved in carcinogenesis, angiogenesis, and invasion

  • Oncogenic mutations may involve non-canonical activation routes of the EPOR signaling pathway

• Metabolic disorders:

  • Mice with EpoR restricted to erythroid tissue (lacking EPOR in other tissues) become obese, glucose intolerant, and insulin resistant

  • This suggests that normal EPOR expression in metabolic tissues is protective against metabolic dysfunction

Understanding these pathological changes in EPOR expression provides insights into disease mechanisms and potential therapeutic opportunities for conditions ranging from neurological disorders to metabolic diseases and cancer.

What are the optimal protocols for producing and purifying recombinant human EPOR?

For researchers working with recombinant human EPOR, the following protocols and specifications are important considerations:

• Expression system selection:

  • HEK293 cells provide a mammalian expression system that ensures proper folding and post-translational modifications

  • The expressed protein typically has a molecular mass of 26.3 kDa, with apparent molecular mass of 34 kDa due to glycosylation

• Protein construction design:

  • Optimal construct includes the extracellular domain (Met 1-Pro 250) of human EPOR

  • C-terminal polyhistidine tag facilitates purification

  • Proper signal sequence ensures secretion

• Purification approach:

  • Affinity chromatography using the His-tag

  • Achieve > 98% purity as determined by reducing SDS-PAGE

• Quality control metrics:

  • Endotoxin levels should be <1.0 EU per μg as determined by the LAL method

  • Activity measurement by inhibition of EPO-dependent proliferation of TF-1 cells

  • Typical ED50: 15-60 ng/mL in the presence of 0.1 U/mL Recombinant Human EPO

• Storage and handling:

  • Lyophilized from sterile PBS, pH 7.4

  • Lyophilized proteins are stable for up to 12 months when stored at -20 to -80°C

  • Reconstituted protein solution can be stored at 4-8°C for 2-7 days

  • For longer storage, prepare aliquots to avoid freeze-thaw cycles

Following these specifications ensures the production of high-quality recombinant EPOR suitable for both structural and functional studies.

What experimental models are most appropriate for studying tissue-specific EPOR functions?

Selecting appropriate experimental models is crucial for studying tissue-specific EPOR functions:

• For neurological studies:

  • Mouse models with stereotactically applied stab wounds to study injury-induced EPOR expression

  • Human tissue samples from patients with neurological disorders (e.g., epilepsy) for clinical relevance

  • Primary cultures of neurons, astrocytes, oligodendrocytes, and microglia to study cell-specific responses

• For metabolic research:

  • Tissue-specific EPOR knockout mice (e.g., adipose-specific EPOR deletion)

  • Diet-induced obesity models to study EPO/EPOR's protective effects

  • Ex vivo adipose tissue cultures to study direct tissue effects

• For erythropoiesis studies:

  • TF-1 human erythroleukemic cells for proliferation assays

  • Mice with EPOR restricted to erythroid tissue (∆EpoR E-mice) to distinguish erythroid from non-erythroid effects

  • Bone marrow cultures for studying erythroid progenitor responses

• For structural and binding studies:

  • Recombinant protein systems with purified components

  • Cell lines expressing EPOR for functional binding assays

  • Computational modeling validated with experimental data

• For signaling pathway analysis:

  • Reporter cell lines for specific pathway activation

  • Phosphoprotein analysis in relevant tissues after EPO stimulation

  • JAK2 inhibitor studies to confirm signaling specificity

How can computational approaches complement experimental studies of EPOR?

Computational approaches provide valuable complementary tools for EPOR research:

• Structural modeling applications:

  • AlphaFold Multimer has been used to generate three-dimensional models of EPOR complexes with EPO and JAK2

  • These models support understanding of both active and inactive conformations

  • For large complexes (3220-4074 residues), stepwise assembly from smaller parts with validation against experimental data is recommended

• Membrane dynamics simulations:

  • Models equilibrated in explicit lipids of the plasma membrane provide insights into EPOR behavior in its native environment

  • These simulations help understand the rotational movement of receptor TM α-helices during activation

• Protein-protein interface design:

  • Computational scanning of the Protein Data Bank identifies suitable scaffold proteins for engineering

  • In silico prediction of binding affinities shows qualitative correlation with experimental measurements

  • This approach has successfully identified proteins that can be engineered to bind EPOR

• Oncogenic mutation analysis:

  • Computational models help elucidate the molecular basis of oncogenic mutations

  • They provide insights into non-canonical activation routes

• Integration with experimental data:

  • Models should be selected and validated through comparisons with published experimental data

  • The combination of computational prediction and experimental validation creates a powerful research approach

These computational approaches not only complement traditional experimental methods but also provide insights that might be difficult to obtain through experimental means alone, particularly for understanding dynamic molecular processes and designing novel therapeutics.

What considerations are important when developing therapeutic agents targeting EPOR?

Developing therapeutic agents targeting EPOR requires careful consideration of several factors:

• Target specificity challenges:

  • EPOR belongs to the cytokine receptor family with structural similarities to other receptors

  • Ensuring specificity for EPOR over related receptors is crucial to avoid off-target effects

  • Engineering approaches that exploit key interaction residues have shown promise in creating specific EPOR-binding proteins

• Tissue-specific targeting:

  • EPOR is expressed in multiple tissues with different functions

  • Targeting specific tissue pools of EPOR might be necessary to avoid unwanted effects

  • For example, metabolic effects without erythropoietic effects might be desirable in certain conditions

• Activation mechanism considerations:

  • Understanding the rotational movement of receptor TM α-helices that causes proximity and activation of JAK2

  • Potential to develop agents that modulate specific aspects of EPOR signaling

  • Consideration of both agonistic and antagonistic approaches depending on the therapeutic goal

• Delivery strategies:

  • Protein-based therapeutics face delivery challenges, especially for CNS targets

  • Small molecule modulators might offer advantages for certain applications

  • Tissue-specific delivery systems may improve therapeutic index

• Safety monitoring:

  • Effects on erythropoiesis need careful monitoring to avoid polycythemia

  • Potential carcinogenic effects given EPOR's potential role in carcinogenesis, angiogenesis, and invasion

  • Careful assessment of metabolic effects, especially in patients with existing metabolic disorders

• Functional validation methods:

  • Measuring ability to inhibit EPO-dependent proliferation of model cell lines like TF-1

  • Typical effective dose ranges (ED50 of 15-60 ng/mL) can serve as benchmarks

  • In vivo models should assess both intended and potential off-target effects

These considerations highlight the complexity of developing EPOR-targeted therapeutics and the importance of a comprehensive understanding of EPOR biology across different tissues and disease states.