OSM Human, His

What is human Oncostatin M and what distinguishes it within the IL-6 cytokine family?

Human Oncostatin M (hOSM) is a pleiotropic cytokine of the IL-6 family that affects processes including cell differentiation, hematopoiesis, and inflammation. What makes hOSM unique within the IL-6 family is its ability to bind with high affinity to two different receptor complexes: the shared leukemia inhibitory factor receptor (LIFR)/glycoprotein 130 (gp130) complex and the specific OSM receptor (OSMR)/gp130 complex . This dual-receptor activation capability is not common among other IL-6 family members and contributes to OSM's diverse biological effects . The protein's structural characteristics include a four-helix bundle topology with specific binding sites that determine receptor interactions, with binding site III (comprising the AB loop, BC loop, and N-terminal D-helix regions) being particularly important for receptor recognition .

What is the purpose of adding a histidine tag to recombinant human OSM?

The addition of a histidine tag (His-tag) to recombinant human OSM serves several important research purposes:

• Purification efficiency: His-tagged proteins can be purified using immobilized metal affinity chromatography (IMAC), allowing for single-step purification with high yield and purity.

• Detection capabilities: His-tags enable easy detection of the recombinant protein using anti-His antibodies in various experimental applications including Western blotting, immunoprecipitation, and immunohistochemistry.

• Minimal interference: The small size of the His-tag (typically 6-10 histidine residues) generally causes minimal interference with protein folding and function when appropriately positioned.

• Orientation control: In binding studies, His-tags can provide controlled orientation of the protein when immobilized on surfaces containing nickel or cobalt ions.

When studying receptor binding properties of OSM, researchers must validate that the His-tag does not interfere with the cytokine's ability to activate downstream STAT signaling pathways, particularly through STAT3, STAT1, and STAT5 .

How do the signaling mechanisms of human OSM compare to mouse OSM?

The signaling mechanisms of human and mouse OSM exhibit significant species-specific differences despite their structural similarities:

The most striking difference is that hOSM can interact with both hOSMR/gp130 and hLIFR/gp130 with high affinity, whereas mouse OSM (mOSM) shows limited ability to interact with mLIFR/gp130 while fully activating mOSMR/gp130 . Additionally, specific amino acid differences in the AB loop of these cytokines are critical determinants of these species-specific activities .

What are the optimal expression systems for producing biologically active His-tagged human OSM?

For producing biologically active His-tagged human OSM, several expression systems can be employed, each with distinct advantages:

E. coli expression system:

• Advantages: High yield, relatively low cost, rapid production time

• Considerations: May require refolding due to inclusion body formation; lacks post-translational modifications

• Methodology: Expression typically uses pET vectors with N-terminal or C-terminal His6 tags under IPTG-inducible promoters

Mammalian expression systems (HEK293, CHO cells):

• Advantages: Proper folding and post-translational modifications; protein directly secreted into media

• Considerations: Lower yield but higher biological activity; more expensive

• Methodology: Transient or stable transfection with secretion signal sequences

Insect cell expression system (Sf9, High Five):

• Advantages: Higher yield than mammalian systems with most post-translational modifications

• Considerations: Intermediate cost; some glycosylation differences from human proteins

• Methodology: Baculovirus infection of insect cells

For human OSM specifically, mammalian expression systems are often preferred when studying receptor interactions because proper folding of the AB loop region is critical for species-specific receptor binding . Biological activity verification should include assessment of STAT3, STAT1, and STAT5 phosphorylation using Western blot analysis and reporter gene assays measuring OSM-responsive genes like TIMP1 .

How can researchers accurately measure the binding affinity of His-tagged human OSM to its receptors?

Researchers can measure the binding affinity of His-tagged human OSM to its receptors using several complementary techniques:

• Surface Plasmon Resonance (SPR):

  • Methodology: Immobilize purified OSMR or LIFR on a sensor chip and flow His-tagged OSM over the surface at different concentrations

  • Data analysis: Calculate association (ka) and dissociation (kd) rate constants to determine equilibrium dissociation constant (KD)

  • Advantage: Real-time measurement without labeling requirements

• Bio-Layer Interferometry (BLI):

  • Methodology: Similar to SPR but using optical interference patterns

  • Application: Especially useful when analyzing multiple receptor interactions simultaneously

• Isothermal Titration Calorimetry (ITC):

  • Methodology: Measures heat released or absorbed during binding

  • Advantage: Provides complete thermodynamic profile (ΔH, ΔS, ΔG)

• Fluorescence Anisotropy:

  • Methodology: Fluorescently label OSM and measure changes in rotational diffusion upon receptor binding

  • Advantage: Solution-based measurement without immobilization

• Cell-based assays:

  • Methodology: Measure downstream signaling events like STAT3 phosphorylation in cells expressing different receptor complexes

  • Analysis: Generate dose-response curves with various OSM concentrations

  • Consideration: When using His-tagged OSM, include controls with untagged OSM to ensure the tag doesn't interfere with signaling

When investigating species-specific interactions, researchers should test binding to both OSMR/gp130 and LIFR/gp130 complexes and include cross-species receptor binding experiments to fully characterize binding profiles .

What amino acid residues in human OSM determine its species-specific receptor recognition?

The species-specific receptor recognition of human OSM is predominantly determined by specific amino acid residues in the AB loop region of the protein. Based on detailed mutational studies, several key residues have been identified:

• Gly-39: This residue in human OSM (corresponding to Asn-37 in mouse OSM) is critical for human OSMR activity. Replacement of this glycine residue significantly impairs signaling through hOSMR .

• Val-42: This residue (corresponding to Thr-40 in mouse OSM) contributes to species specificity in receptor binding .

• Lys-44: This is perhaps the most critical residue determining species specificity. This positively charged lysine in human OSM (corresponding to Asp-42, a negatively charged residue in mouse OSM) prevents binding to mouse OSMR. Substitution of Lys-44 with aspartic acid (K44D) enables human OSM to activate mouse OSMR .

• F𝑋𝑋K motif in helix D: In addition to the AB loop residues, a conserved F𝑋𝑋K motif in helix D is crucial for recognition of both OSMR and LIFR receptors .

The significance of these residues has been demonstrated through chimeric cytokines and point mutations, showing that even single amino acid substitutions can dramatically alter receptor specificity. For example, introducing the human AB loop sequence into mouse OSM generates a chimera capable of activating human receptors, while the K44D mutation in human OSM enables activation of mouse OSMR .

How do structural modifications in the AB loop affect downstream signaling pathways?

Structural modifications in the AB loop of OSM have significant and differential effects on downstream signaling pathways, demonstrating the critical role of this region in determining not only receptor binding but also signal transduction specificity:

• STAT3 Signaling:

  • AB loop modifications that affect receptor binding directly impact STAT3 phosphorylation

  • The triple substitution (N37G/T40V/D42K) in mouse OSM enhances STAT3 activation through both mouse LIFR and human receptors

  • Conversely, G39N/V42T/K44D substitutions in human OSM reduce STAT3 signaling through human receptors

• STAT1 Signaling:

  • While native mouse OSM shows limited STAT1 activation via LIFR, AB loop modifications can enhance this capability

  • Humanized AB loop variants of mouse OSM show increased STAT1 phosphorylation, particularly when interacting with human receptors

• STAT5 Signaling:

  • AB loop modifications affect STAT5 activation patterns similar to their effects on STAT3 and STAT1

  • The effects on STAT5 confirm that AB loop modifications influence multiple signaling pathways, not just STAT3-dependent ones

• Temporal signaling dynamics:

  • Short-term (10 min) versus long-term (24 h) signaling patterns are differentially affected by AB loop modifications

  • Gene expression effects (e.g., TIMP1 expression via OSMR and STAT3 expression via LIFR) reflect these temporal differences

The impact of AB loop modifications on signaling extends beyond immediate phosphorylation events to functional outcomes like cell proliferation inhibition. Importantly, these effects are not limited to a single signaling pathway, indicating that the AB loop plays a multifaceted role in determining both receptor selection and the quality of downstream signaling .

How can chimeric OSM constructs be designed to overcome species barriers in preclinical models?

Designing chimeric OSM constructs to overcome species barriers in preclinical models requires strategic modifications based on the molecular determinants of species specificity. The following approaches have proven effective:

• AB Loop Replacement Strategy:

  • Replace the entire AB loop of mouse OSM with the human sequence to generate a mouse-human chimera that activates both mouse and human receptors

  • This approach generates a "universal" OSM variant that can be used across species

  • Implementation: Use site-directed mutagenesis or synthetic gene assembly to create the chimeric construct

• Targeted Point Mutations:

  • Instead of replacing the entire loop, introduce specific amino acid substitutions at key positions:

    • For mouse OSM: N37G, T40V, and D42K substitutions are sufficient to enable human receptor activation

    • For human OSM: G39N, V42T, and K44D substitutions enable mouse OSMR activation

  • This minimalist approach maintains most of the native sequence while altering receptor specificity

• Validation Protocol:

  • Test chimeric constructs in vitro using cells expressing:

    • Mouse OSMR/gp130

    • Mouse LIFR/gp130

    • Human OSMR/gp130

    • Human LIFR/gp130

  • Assess multiple signaling pathways (STAT3, STAT1, STAT5) to ensure complete functional characterization

  • Evaluate both short-term (minutes) and long-term (24h) signaling outcomes

• Functional Verification:

  • Measure downstream gene expression of OSM-responsive genes (TIMP1, STAT3)

  • Assess biological effects such as proliferation inhibition in appropriate cell lines

These chimeric approaches enable more predictive preclinical studies by allowing the same OSM variant to be used in both mouse models and human cell systems, facilitating direct translation between preclinical findings and potential human applications .

What are the challenges in developing antibodies that neutralize human OSM without cross-reactivity to other IL-6 family cytokines?

Developing antibodies that specifically neutralize human OSM without cross-reactivity to other IL-6 family cytokines presents several significant challenges:

• Structural similarity within the IL-6 family:

  • IL-6 family cytokines share a four-helix bundle structure

  • Common binding site architecture for gp130 recognition

  • Solution: Target OSM-specific epitopes, particularly in the AB loop region which shows greater sequence divergence from other family members

• Dual receptor binding capability:

  • Human OSM uniquely binds both OSMR/gp130 and LIFR/gp130 complexes

  • Challenge: Antibodies targeting only one binding interface may allow signaling through the alternative receptor

  • Strategy: Design antibodies targeting the AB loop which influences both OSMR and LIFR binding

• Epitope selection considerations:

  • Target uniquely exposed regions in human OSM

  • Potential epitopes include:

    • AB loop residues Gly-39, Val-42, and Lys-44 (species-specific determinants)

    • BC loop region (contributes to receptor binding but with different sequence from other IL-6 family members)

    • Junction regions between structural elements

• Validation approach:

  • Cross-reactivity panel: Test candidate antibodies against all IL-6 family cytokines:

    • IL-6, LIF, CNTF, CT-1, CLC, IL-11, IL-27, IL-31

  • Receptor inhibition assay: Verify blocking of both OSMR/gp130 and LIFR/gp130 signaling using phospho-STAT3 readouts

  • Species specificity characterization: Determine reactivity with mouse, rat, and non-human primate OSM

• Humanization considerations:

  • Therapeutic antibodies require humanization to reduce immunogenicity

  • Challenge: Maintaining epitope specificity during CDR grafting process

  • Solution: Conservative humanization approaches with systematic affinity maturation

The most effective approach involves structural biology-guided antibody development, targeting epitopes that include the critical species-specific residues in the AB loop while avoiding conserved regions shared with other IL-6 family cytokines .

How can researchers distinguish between OSMR-mediated and LIFR-mediated signaling in experimental systems?

Distinguishing between OSMR-mediated and LIFR-mediated signaling in experimental systems requires specialized approaches to delineate these parallel pathways:

• Genetic manipulation strategies:

  • CRISPR/Cas9 knockout: Generate OSMR-KO and LIFR-KO cell lines to isolate receptor-specific effects

  • siRNA/shRNA knockdown: For transient receptor depletion studies

  • Receptor overexpression: Express either OSMR or LIFR in receptor-negative cell lines

• Pharmacological approaches:

  • Receptor-selective ligands:

    • Use LIF (activates only LIFR/gp130) alongside OSM (activates both LIFR/gp130 and OSMR/gp130)

    • Apply engineered OSM variants with altered receptor specificity

  • Receptor-blocking antibodies: Apply specific blocking antibodies against either OSMR or LIFR

• Signal transduction differentiation:

  • Differential STAT activation:

    • OSMR/gp130 activates STAT1, STAT3, and STAT5

    • LIFR/gp130 primarily activates STAT3 with limited STAT1 activation

  • Phospho-flow cytometry: Simultaneously measure multiple phospho-proteins to identify receptor-specific signatures

  • Kinetic analysis: Monitor phosphorylation events at different time points (10 min vs. 24h)

• Transcriptomic analysis:

  • RNA-seq comparison: Compare gene expression profiles after selective receptor activation

  • Receptor-specific gene markers:

    • TIMP1 expression serves as an OSMR-specific marker

    • STAT3 expression can indicate LIFR activation

• Methodological considerations:

  • Include proper controls (untreated, LIF-treated, wild-type OSM)

  • Use dose-response curves to account for affinity differences

  • Incorporate time-course analyses (immediate vs. delayed responses)

  • Verify findings across multiple cell types

By combining these approaches, researchers can create comprehensive profiles of receptor-specific signaling events and determine the relative contributions of OSMR and LIFR to specific biological outcomes in their experimental systems .

What methodologies are most effective for studying the formation of OSM-receptor complexes in living cells?

Studying the formation of OSM-receptor complexes in living cells requires sophisticated methodologies that can capture dynamic protein-protein interactions in their native environment. The most effective approaches include:

• Advanced microscopy techniques:

  • Förster Resonance Energy Transfer (FRET):

    • Tag OSM with donor fluorophore (e.g., CFP) and receptors with acceptor fluorophore (e.g., YFP)

    • Measure energy transfer as indication of protein proximity (<10 nm)

    • Advantage: Can detect transient interactions in real-time

  • Bimolecular Fluorescence Complementation (BiFC):

    • Split fluorescent protein fragments attached to OSM and receptor components

    • Complementation produces fluorescence when proteins interact

    • Benefit: Stabilizes transient interactions for visualization

  • Single-molecule tracking:

    • Label OSM and receptors with photo-stable fluorophores

    • Track individual molecules to assess binding dynamics and complex formation

    • Provides diffusion coefficients and binding residence times

• Proximity-based labeling techniques:

  • BioID or TurboID:

    • Fuse biotin ligase to either OSM or receptor components

    • Proximity-dependent biotinylation of interacting proteins

    • Identify interactome using streptavidin pulldown and mass spectrometry

  • APEX proximity labeling:

    • Use APEX2 fusion proteins to generate short-lived radicals that label nearby proteins

    • Higher spatial resolution than BioID approaches

• Real-time binding measurements:

  • Fluorescence Recovery After Photobleaching (FRAP):

    • Photobleach fluorescently labeled receptors and measure recovery rate

    • Slowed recovery indicates complex formation with OSM

    • Provides diffusion and binding kinetics in living cells

  • Luciferase Complementation Assays:

    • Split luciferase fragments attached to OSM and receptor components

    • Luminescence signal when complex forms

    • Highly sensitive for quantitative measurements

• Multi-color single-molecule co-tracking:

  • Simultaneously track OSM, OSMR/LIFR, and gp130 with different fluorophores

  • Analyze co-localization and co-diffusion events

  • Can determine stoichiometry of receptor complexes

• Data analysis considerations:

  • Apply advanced image analysis algorithms for co-localization

  • Use hidden Markov modeling for binding state transitions

  • Implement particle tracking for diffusion analysis

  • Employ mathematical modeling to extract binding parameters

These methodologies enable researchers to determine not only whether OSM-receptor complexes form, but also their stoichiometry, stability, lateral mobility, and the sequence of assembly events in the plasma membrane of living cells .

How do differences between human and mouse OSM impact the development of therapeutic OSM antagonists?

The significant differences between human and mouse OSM create several challenges for developing therapeutic OSM antagonists, requiring careful consideration in the drug development pipeline:

• Receptor specificity challenges:

  • Human OSM binds both OSMR/gp130 and LIFR/gp130 with high affinity

  • Mouse OSM primarily signals through OSMR/gp130 with limited LIFR/gp130 activation

  • Impact: Antagonists developed against human OSM may show different efficacy profiles when tested in mouse models

• Species cross-reactivity limitations:

  • Human OSM cannot activate mouse OSMR

  • Mouse OSM cannot activate either human receptor

  • Consequence: Traditional mouse models using human OSM antagonists may not accurately predict human therapeutic outcomes

• Structural determinant considerations:

  • Key species-specific differences are localized to the AB loop, particularly residues Gly-39/Asn-37, Val-42/Thr-40, and Lys-44/Asp-42 (human/mouse)

  • Therapeutic strategy: Design antagonists targeting conserved regions between species or develop humanized mouse models

• Preclinical testing strategies:

  • Humanized mouse models:

    • Knock-in human OSMR/LIFR into mice

    • Use immunodeficient mice with human immune cell engraftment

  • Chimeric OSM approach:

    • Test antagonists against engineered mouse-human OSM chimeras

    • Use point mutants with human-like receptor specificity (N37G/T40V/D42K mouse OSM)

  • Ex vivo human tissue testing:

    • Test antagonist efficacy in human tissue explants

    • Provides better translation potential than pure animal models

• Signaling pathway considerations:

  • Differential activation of STAT pathways between species

  • Impact on functional readouts: using STAT3 phosphorylation as sole readout may miss species differences in other pathways

  • Solution: Assess multiple signaling pathways (STAT1, STAT3, STAT5) when evaluating antagonist efficacy

To overcome these challenges, the most effective approach involves using complementary strategies: (1) in vitro testing with both human and mouse cells, (2) engineered mouse OSM with humanized receptor binding properties for in vivo studies, and (3) validation in human cellular systems to confirm translational potential .

What are the considerations for using His-tagged OSM in structural biology studies aimed at drug discovery?

When using His-tagged OSM in structural biology studies for drug discovery, several critical considerations must be addressed to ensure accurate and translatable results:

• Tag position and linker design:

  • N-terminal vs. C-terminal placement:

    • Assess potential interference with receptor binding sites

    • C-terminal tags are generally preferred as the N-terminus is closer to the AB loop critical for receptor specificity

  • Linker optimization:

    • Incorporate flexible linkers (e.g., GGSGGS) between OSM and His-tag

    • Consider cleavable linkers with protease sites (TEV, PreScission) for tag removal

  • Validation requirement:

    • Compare receptor binding and signaling activity between tagged and untagged versions

    • Verify that tag doesn't alter crystal packing or solution behavior

• Structural analysis techniques:

  • X-ray crystallography considerations:

    • Screen multiple constructs with varying tag positions and linker lengths

    • Tag may provide crystal contacts that influence protein conformation

    • Validate using multiple crystal forms to identify potential artifacts

  • Cryo-EM for receptor complex studies:

    • Use His-tag for initial purification but consider removing before complex formation

    • May require larger tags (e.g., His-MBP) to increase particle size

  • NMR spectroscopy applications:

    • Combine His-tag with isotope labeling (15N, 13C, 2H)

    • Verify tag does not cause chemical shift perturbations in key binding regions

• Species considerations for drug discovery:

  • Include both human and mouse His-tagged OSM variants

  • Generate key mutants (e.g., G39N/V42T/K44D human OSM and N37G/T40V/D42K mouse OSM)

  • Compare binding sites and druggable pockets across species

• Receptor complex formation:

  • His-tag may be utilized for oriented immobilization in binding studies

  • For co-crystallization with receptors, tag removal is often preferable

  • Consider ternary complexes (OSM-OSMR-gp130 or OSM-LIFR-gp130) for complete binding interface characterization

• Quality control parameters:

  • Assess protein homogeneity by size-exclusion chromatography and DLS

  • Verify correct folding by circular dichroism

  • Confirm biological activity through cell-based assays measuring multiple STAT pathways

  • Use mass spectrometry to confirm protein integrity and post-translational modifications

By addressing these considerations, researchers can maximize the value of His-tagged OSM in structural biology studies while avoiding potential artifacts that could misdirect drug discovery efforts. The combination of proper construct design, rigorous validation, and comparative species analysis provides the strongest foundation for developing therapeutics targeting the OSM signaling pathway .

How might engineering the AB loop of human OSM create novel therapeutic cytokines with modified receptor specificities?

Engineering the AB loop of human OSM presents exciting opportunities to create novel therapeutic cytokines with customized receptor specificities and signaling properties:

• Receptor selectivity engineering:

  • OSMR-selective variants:

    • Enhance Gly-39, Val-42, and Lys-44 interactions with OSMR while disrupting LIFR binding

    • Application: Target tissues with high OSMR expression while minimizing LIFR-mediated side effects

  • LIFR-selective variants:

    • Modify AB loop to favor LIFR binding over OSMR

    • Potential: More selective activity in neural tissues where LIFR predominates

  • Cross-species active variants:

    • Incorporate N37G/T40V/D42K mutations from mouse studies to create OSM variants that activate receptors across species barriers

    • Benefit: Streamlined translation between animal studies and human applications

• Signaling bias engineering:

  • STAT pathway selectivity:

    • Design variants that preferentially activate STAT3 over STAT1/STAT5 or vice versa

    • Fine-tune AB loop structure to modify the conformation of receptor complexes

    • Applications: Anti-inflammatory (STAT3-biased) or anti-proliferative (STAT1-biased) therapeutics

  • Temporal signaling modulation:

    • Engineer variants with altered receptor binding kinetics

    • Create sustained-signaling or transient-signaling variants

• Therapeutic fusion designs:

  • Targeted delivery:

    • Fuse engineered OSM to antibody fragments targeting specific tissues

    • AB loop engineering ensures proper receptor activation despite fusion context

  • Controlled activation:

    • Incorporate protease-cleavable masks over AB loop

    • Activation occurs only in environments with specific proteases (e.g., tumor microenvironment)

• Experimental validation approaches:

  • Comprehensive receptor binding panel:

    • Test engineered variants against OSMR/gp130, LIFR/gp130, and potential off-target receptors

    • Include cross-species testing for translational applications

  • Signaling pathway profiling:

    • Assess activation of STAT1, STAT3, STAT5, MAP kinases, and PI3K pathways

    • Compare signaling duration and intensity to native OSM

  • Functional outcome assessment:

    • Measure cell type-specific responses (proliferation, differentiation, gene expression)

    • Evaluate potential immunogenicity of engineered variants

The strategic modification of the AB loop represents a promising approach for developing a new generation of cytokine therapeutics with improved specificity, reduced off-target effects, and enhanced translational potential between preclinical models and human applications .

What technological advances are needed to better understand the conformational dynamics of OSM-receptor interactions?

Understanding the conformational dynamics of OSM-receptor interactions requires significant technological advances across multiple disciplines:

• Advanced structural biology techniques:

  • Time-resolved cryo-EM:

    • Capture transient conformational states during receptor binding

    • Challenge: Requires millisecond-scale freezing methods and improved image processing algorithms

  • Single-molecule FRET with improved spatiotemporal resolution:

    • Track distance changes between labeled residues during receptor engagement

    • Need: Brighter, more photostable fluorophores and improved detection sensitivity

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

    • Map solvent accessibility changes during complex formation with improved spatial resolution

    • Advancement needed: Faster exchange protocols to capture transient states

• Computational methodology advancements:

  • Enhanced molecular dynamics simulations:

    • Simulate complete OSM-receptor complex interactions on physiologically relevant timescales

    • Requirements: Improved force fields for protein-protein interactions and enhanced sampling methods

  • Artificial intelligence approaches:

    • Deep learning models to predict conformational changes upon receptor binding

    • Integration of experimental data with simulation outputs

  • Multiscale modeling:

    • Connect atomic-level dynamics to cellular signaling networks

    • Challenge: Bridging vastly different time and length scales

• Novel biophysical tools:

  • Nanobody-based conformational sensors:

    • Develop nanobodies that recognize specific conformational states of OSM or its receptors

    • Application: Tracking receptor activation states in living cells

  • Lanthanide-based resonance energy transfer (LRET):

    • Longer distance measurements than conventional FRET

    • Advantage: Can track larger conformational changes in the complete receptor complex

  • Optical tweezers or magnetic tweezers:

    • Measure forces involved in receptor conformational changes

    • Innovation needed: Higher throughput methods compatible with membrane proteins

• Integrative structural biology approach:

  • Combine multiple experimental techniques (X-ray, NMR, cryo-EM, mass spectrometry, SAXS)

  • Develop improved computational frameworks to integrate diverse datasets

  • Create dynamic structural models that capture the entire binding and activation process

• Single-cell technologies:

  • Single-cell structural proteomics:

    • Detect receptor conformational states in individual cells

    • Correlate with downstream signaling events

  • Advanced imaging methods:

    • Super-resolution microscopy beyond current resolution limits

    • Expansion microscopy adapted for membrane protein complexes

These technological advances would enable researchers to move beyond static "snapshot" structures to dynamic models of OSM-receptor interactions, revealing how conformational changes propagate from the AB loop binding interface to initiate signaling through the transmembrane domains of receptor complexes .