IL10RA Human, Active

What is IL10RA and what experimental approaches can best characterize its functional role?

IL10RA is a subunit of the interleukin-10 receptor complex that mediates the immunoregulatory functions of IL-10. The protein forms part of a hexameric structure with IL-10 and IL-10RB to create a functional signaling complex . When studying IL10RA function, researchers should implement multiple complementary approaches:

• Signaling assays: Measure STAT3 phosphorylation following IL-10 stimulation, as this is a direct readout of IL10RA activity

• Gene expression analysis: Quantify downstream gene expression changes using RNA-seq or qPCR

• Interaction studies: Employ co-immunoprecipitation or proximity ligation assays to examine receptor complex formation

• Cellular response assays: Assess functional outcomes in relevant immune cell populations (e.g., cytokine production, proliferation)

The choice of experimental system is critical, as IL10RA expression and function varies across cell types. HEK293 cells that naturally lack IL10RA but express IL10RB provide an excellent reconstitution system for studying variant effects .

How should researchers approach studying the structural biology of IL10RA?

IL10RA structural biology research requires multimodal approaches:

• Cryo-electron microscopy: Has successfully resolved the IL-10 signaling complex at 3.5Å resolution, revealing how IL-10 and IL10RA form a composite surface to engage IL10RB

• X-ray crystallography: The IL10RA/IL-10 complex structure (PDB: 1Y6K) provides a foundation for understanding ligand interactions

• In silico modeling: Molecular dynamics simulations can predict conformational changes and stability

• Mutagenesis: Site-directed mutagenesis validates key residues involved in structural integrity and ligand binding

When analyzing structures, researchers should focus on key functional regions including the signal peptide, N-glycosylation sites, cysteine bonds that maintain structure, JAK1 binding sites, and E3 ligase binding sites .

What methodology should be used when assessing IL10RA expression patterns?

Researchers should employ multiple complementary techniques to comprehensively characterize IL10RA expression:

• Transcriptional profiling: RNA-seq and qPCR to quantify mRNA levels

• Protein detection: Western blotting for total protein, flow cytometry for surface expression

• Spatial localization: Immunohistochemistry or immunofluorescence for tissue distribution

• Single-cell resolution: scRNA-seq to capture heterogeneity across cell populations

When designing expression studies, researchers must account for contextual variables including:

• Inflammatory status (expression may change during disease states)

• Developmental stage (expression patterns can vary temporally)

• Tissue-specific regulatory mechanisms

• Post-translational modifications affecting protein stability

How can researchers accurately evaluate IL10RA-IL10 binding interactions?

To effectively characterize IL10RA-IL10 binding interactions, researchers should implement:

• Surface Plasmon Resonance (SPR): For quantitative binding kinetics and affinity measurements

• Bio-Layer Interferometry (BLI): Alternative approach for real-time binding analysis

• Isothermal Titration Calorimetry (ITC): To determine thermodynamic parameters

• Molecular docking studies: Computational prediction of binding interfaces

• Mutagenesis validation: Experimental confirmation of predicted binding residues

Research demonstrates that point mutations in IL10RA can significantly decrease binding affinity toward IL-10, disrupting signaling . When comparing wild-type and variant IL10RA binding, standardized experimental conditions are essential to obtain reliable comparative data.

What are the known pathogenic variants of IL10RA and how should they be functionally characterized?

Numerous pathogenic IL10RA variants have been identified in monogenic inflammatory bowel disease. Systematic functional characterization should include:

Functional characterization should employ multiple approaches:

• STAT3 phosphorylation assays to assess signaling capacity

• Structural modeling to predict protein stability effects

• Binding assays to measure IL-10 interaction kinetics

• Cell-based reporter systems to evaluate downstream functional impacts

Study of these variants requires careful experimental design with appropriate controls, including wild-type IL10RA and known benign variants.

How do computational methods compare in predicting IL10RA variant pathogenicity?

Multiple computational tools exist for predicting variant pathogenicity, but their performance varies specifically for IL10RA mutations:

• Use multiple prediction algorithms concurrently

• Integrate structural information when available

• Consider evolutionary conservation across species

• Validate computational predictions with functional assays

• Assess variant frequency in population databases

The integration of protein-specific features improves prediction accuracy for IL10RA variants.

What evidence exists for natural selection acting on IL10RA variants and how should researchers investigate this?

Compelling evidence suggests natural selection has shaped IL10RA variation:

• Geographic concentration of pathogenic variants in East Asia and Northeast China

• Association with Schistosoma japonicum infection distribution, suggesting parasite-driven selection

• Partially augmented IL-10 responses in peripheral blood mononuclear cells from heterozygous carriers, indicating possible heterozygote advantage

Researchers investigating natural selection on IL10RA should implement:

• Population genetic analyses: Calculate FST, Tajima's D, and other neutrality tests

• Geographic correlation studies: Map variant frequencies against historical pathogen distribution

• Functional heterozygote studies: Compare signaling capacity in cells with heterozygous vs. homozygous genotypes

• Comparative genomics: Analyze conservation and diversification across species

• Coalescent simulations: Test selection scenarios against neutral models

These approaches can distinguish between different types of selection (purifying, balancing, positive) and identify selective pressures driving IL10RA variation.

How do IL10RA mutations mechanistically lead to inflammatory disease phenotypes?

IL10RA mutations contribute to inflammatory disease through several molecular mechanisms that researchers should systematically investigate:

• Disrupted IL-10 signaling: Measure STAT3 phosphorylation across dose ranges and time courses

• Altered receptor stability: Employ thermal shift assays and pulse-chase experiments to assess protein half-life

• Reduced binding affinity: Quantify IL-10 binding kinetics using surface plasmon resonance or similar techniques

• Disrupted receptor complex formation: Examine IL10RA/IL10RB assembly using co-immunoprecipitation or FRET/BRET approaches

• Altered cell type-specific responses: Compare effects across immune cell subsets (T cells, B cells, macrophages)

Researchers should utilize both reconstitution systems (e.g., HEK293 cells) and patient-derived cells when available. Transcriptomic profiling can identify dysregulated pathways downstream of defective IL10RA signaling that contribute to inflammatory phenotypes.

What approaches should be used to study differential IL10RA signaling across immune cell populations?

IL10RA signaling exhibits substantial differences in response thresholds across immune cell populations , necessitating specialized methodological approaches:

• Single-cell analysis: Use mass cytometry or spectral flow cytometry to simultaneously assess multiple phospho-proteins in diverse cell types

• Dose-response studies: Perform titration experiments to identify cell type-specific activation thresholds

• Temporal resolution: Capture signaling kinetics through time-course experiments

• Receptor quantification: Correlate surface expression levels with signaling capacity

• Transcriptional profiling: Identify cell type-specific gene expression changes following IL-10 stimulation

Research has demonstrated that some IL-10 receptor variants display myeloid-biased activity, suppressing macrophage activation without stimulating inflammatory CD8+ T cells . Understanding these differential effects requires careful experimental design that incorporates multiple cell types under standardized conditions.

How should researchers design therapeutic strategies targeting the IL-10/IL10RA pathway?

Developing effective therapeutics targeting the IL-10/IL10RA pathway requires consideration of complex biology and previous clinical findings:

• Recombinant IL-10 therapy limitations: Clinical trials with recombinant IL-10 have shown limited efficacy in inflammatory bowel disease and other autoimmune conditions

• Cell type selectivity: Design partial agonists that target specific immune populations while sparing others

• Endogenous IL-10 source preservation: Develop approaches to protect IL-10-producing regulatory cells from deletion

• Pathway modulation: Consider targeting upstream regulators (e.g., Fas pathway) that affect IL-10-producing cell populations

• Localized delivery: Implement strategies for tissue-specific IL-10 pathway modulation

Researchers must account for IL-10's dual role in both suppressing inflammation and potentially enhancing humoral autoimmune responses (as observed in rheumatoid arthritis) . Functional readouts should include both pro- and anti-inflammatory effects across multiple cell types.

What experimental design considerations are critical when conducting genetic association studies of IL10RA variants?

When designing genetic association studies of IL10RA variants, researchers should incorporate:

• Population stratification controls: Account for geographic clustering of variants, particularly in East Asian populations

• Age-of-onset stratification: Distinguish between early-onset (<5 years) and adult-onset disease associations

• Phenotype precision: Carefully define inflammatory bowel disease subtypes and severity metrics

• Functional validation: Include experimental characterization of novel variants

• Familial studies: Investigate inheritance patterns, particularly for rare variants

• Environmental interaction analysis: Consider pathogen exposure history, particularly parasitic infections

Researchers should utilize existing databases of published mutations in IL-10, IL10RA, and IL10RB as reference resources, while ensuring standardized variant nomenclature following HGVS guidelines. The association of specific variants with disease subtypes requires large, well-phenotyped cohorts and appropriate statistical methods.

How can structure-based approaches be employed to design IL10RA-targeted therapeutics?

Structure-based drug design targeting IL10RA should leverage available structural data:

• Structure-guided mutagenesis: Use the IL-10/IL10RA/IL10RB complex structure to design variants with altered binding properties

• Partial agonist development: Create IL-10 variants with modified IL10RB binding strength to achieve cell type selectivity

• Small molecule screening: Identify compounds that stabilize or modulate receptor complex formation

• Peptide mimetics: Design peptides that mimic key interaction interfaces

• Antibody engineering: Develop antibodies that modulate rather than block receptor function

Research demonstrates that understanding the hexameric structure of the IL-10 signaling complex enables rational design of IL-10 variants with myeloid-biased activity that can uncouple major opposing functions of IL-10 . This structure-based approach provides a blueprint for developing therapeutics with improved specificity and reduced side effects.