Recombinant Human Interleukin-13 receptor subunit alpha-1 (IL13RA1), partial (Active)

What is IL13RA1 and what is its biological function?

IL13RA1 is the alpha-1 subunit of the Interleukin-13 receptor, which forms a heterodimer with IL-4Rα. This receptor complex is critical for mediating the signaling of both IL-13 and IL-4, two key cytokines in Th2-associated immune responses. IL13RA1 plays a central role in various biological processes, particularly in asthma pathogenesis and allergic responses .

Methodological approach: To study IL13RA1 function, researchers typically use genetic knockout models (Il13ra1-/-) and assess phenotypic changes in various disease models. Studies have consistently demonstrated that IL13RA1 regulates airway resistance and mucus production in response to aeroallergen challenge . Additionally, IL13RA1 has been implicated in extracellular matrix regulation and glucose metabolism in cardiac tissue, suggesting functions beyond the immune system .

How is IL13RA1 expression regulated at the cellular level?

IL13RA1 expression is regulated by a complex network of cytokines with context-dependent effects. Research has revealed opposing regulatory mechanisms:

• Inhibitory regulation: IL-13 and IL-4 significantly decrease IL13RA1 surface expression on eosinophils (9.87 ± 1.22 and 8.42 ± 1.56 respectively, P < 0.001)

• Enhancing regulation: IFN-γ, TNF-α, and most notably TGF-β increase IL13RA1 expression (27.13 ± 2.36, 31.12 ± 3.09, P < 0.001)

• Moderate enhancement: IL-5 and GM-CSF slightly upregulate receptor expression (24.45 ± 2.30 and 25.14 ± 2.38 respectively, P < 0.001)

Methodological approach: Flow cytometry analysis using specific monoclonal antibodies is the preferred method for studying IL13RA1 expression regulation. Studies have shown that these regulatory mechanisms operate independently, as TGF-β/IFN-γ-induced upregulation does not prevent subsequent IL-13-mediated downregulation . This suggests a sophisticated regulatory network that fine-tunes cellular sensitivity to IL-13 based on the local cytokine environment.

What genetic polymorphisms exist in IL13RA1 and how do they affect function?

The IL13RA1 gene is located on chromosome Xq24. Research into genetic diversity at this locus has identified several polymorphisms, including:

• -281T>G: A single nucleotide polymorphism in the IL13RA1 promoter region

• 1365A>G: A variant in the IL13RA1 proximal 3' untranslated region

Methodological approach: Identification of these polymorphisms involves screening of the promoter and coding regions for genetic variants, followed by genotyping in relevant populations. Case-control and transmission disequilibrium test analyses are typically employed to determine genetic association with disease phenotypes.

Interestingly, studies examining the association between these polymorphisms and asthma susceptibility or severity have not found significant correlations, except for a suggestive association between the IL13RA1 -281T/1365A haplotype and raised total serum immunoglobulin E levels in adult female asthmatics . This indicates that while IL13RA1 is functionally important in asthma pathogenesis, genetic variations in this receptor may not be major determinants of asthma risk.

How does IL13RA1 differentially regulate IL-4 versus IL-13 signaling in experimental models?

Understanding the differential regulation of IL-4 and IL-13 signaling through IL13RA1 requires sophisticated experimental approaches. Research using Il13ra1-/- knockout models has revealed important distinctions:

• IL-13-dependent responses are entirely dependent on IL13RA1

• IL-4-induced chemokine production and inflammatory cell recruitment can occur independently of IL13RA1

• The balance between IL-4 and IL-13 production in situ dictates the dependency of allergen-induced responses on IL13RA1

Methodological approach: Researchers typically compare responses to IL-4 and IL-13 in wild-type vs. Il13ra1-/- mice, examining downstream signaling pathways and context-dependent responses in different tissues and cell types. Studies have shown that while airway resistance and mucus production are consistently IL13RA1-dependent, eosinophil recruitment can be IL13RA1-independent depending on the mode of allergen sensitization . This reveals the complex role of IL13RA1 in regulating different aspects of type 2 immune responses.

What experimental approaches are used to study IL13RA1 function in aeroallergen-induced lung inflammation?

Studying IL13RA1 function in aeroallergen-induced lung inflammation requires a multi-faceted experimental approach:

• Genetic knockout models:

  • Il13ra1-/- mice to assess receptor contribution to allergic airway disease

• Allergen challenge models:

  • Adjuvant-based sensitization (e.g., OVA/alum) followed by airway challenge

  • Mucosal sensitization with clinically relevant aeroallergens (e.g., Aspergillus, house dust mite)

  • Comparison of different sensitization routes to understand context-dependent effects

• Functional assessments:

  • Airway mechanics: Measurement of airway resistance and compliance in response to cholinergic stimuli

  • Histological analysis: PAS staining for mucus production and goblet cell hyperplasia

  • Immunological parameters: IgE levels, cytokine production (IL-4, IL-13, IL-5)

  • Molecular analysis: Assessment of chemokine expression (CCL11, CCL24) and TGF-β levels

These approaches have revealed that IL13RA1 regulates distinct aspects of allergic airway disease, with some features (airway resistance, mucus production) being consistently IL13RA1-dependent across different allergen models, while others (eosinophil recruitment) show model-dependent IL13RA1 dependency .

How do gene expression profiles differ between wild-type and IL13RA1-deficient tissues?

Comparative gene expression profiling between wild-type and IL13RA1-deficient systems has provided crucial insights into the biological functions regulated by this receptor:

• Lung tissue profiles:

  • Airway hyperreactivity genes: Significantly reduced expression of genes associated with smooth muscle contraction in Il13ra1-/- mice

  • Mucus-related genes: Dramatically decreased expression of mucin genes and other goblet cell-associated genes

  • Chemokines: Reduced expression of CCL11 and CCL24

• Cardiac tissue profiles:

  • Extracellular matrix pathway: Significantly enriched in wild-type compared to Il13ra1-deficient hearts (normalized enrichment score=1.90; false discovery rate q=0.005)

  • Glucose metabolism pathway: Differentially regulated (normalized enrichment score=−2.36; false discovery rate q=0)

• Macrophage gene expression:

  • Alternative activation markers: Differential regulation of genes associated with alternatively activated macrophages

  • Distinct regulation by type I vs. type II IL-4 receptors

Methodological approach: RNA-seq or microarray analysis of tissues or isolated cells, followed by gene set enrichment analysis to identify affected pathways. These studies have revealed that IL13RA1 regulates distinct gene programs in different tissues, highlighting its diverse biological roles beyond classical immune functions.

What is the role of IL13RA1 in asthma pathogenesis and how does this influence therapeutic targeting?

IL13RA1 plays a critical role in asthma pathogenesis by mediating the effects of both IL-4 and IL-13. Research using Il13ra1-/- mice has demonstrated that this receptor is essential for several hallmark features of asthma:

• Airway hyperresponsiveness: Il13ra1-/- mice are protected from allergen-induced increases in airway resistance and decreases in compliance

• Mucus production: Il13ra1-/- mice show dramatic reduction in goblet cell hyperplasia and mucus production

• Airway remodeling: IL13RA1 signaling regulates TGF-β production, which is critical for fibrotic responses

Interestingly, not all aspects of asthma are IL13RA1-dependent:

• IgE production and Th2 cytokine responses remain intact in Il13ra1-/- mice

• Eosinophilia can be IL13RA1-independent depending on the method of allergen sensitization

Methodological approach: Researchers typically compare wild-type and Il13ra1-/- mice in various models of allergic airway inflammation, including those using clinically relevant allergens like Aspergillus and house dust mite. These studies suggest that therapeutic targeting of IL13RA1 may effectively address airway hyperresponsiveness and mucus production without compromising beneficial aspects of immune responses.

How does IL13RA1 function beyond the immune system, particularly in cardiac physiology?

While IL13RA1 has been extensively studied in the context of immune responses, emerging evidence indicates important roles beyond the immune system, particularly in cardiac physiology:

• Myocardial dysfunction: Il13ra1-deficient mice develop severe myocardial dysfunction and dyssynchrony compared to wild-type mice, with significantly reduced left ventricular ejection fraction (29.7±9.9% versus 45.0±8.0%; P=0.004) and increased left ventricular end-diastolic diameter (4.2±0.2 versus 3.92±0.3; P=0.03)

• Extracellular matrix regulation: Bioinformatic analysis indicates that IL13RA1 regulates extracellular matrix pathways in the heart (normalized enrichment score=1.90; false discovery rate q=0.005)

• Metabolism: IL13RA1 appears to influence glucose metabolism in cardiac tissue (normalized enrichment score=−2.36; false discovery rate q=0)

• Collagen deposition: Il13ra1 deficiency is associated with reduced collagen deposition under both normal and pressure-overload conditions

Methodological approach: These findings emerged from studies comparing wild-type and Il13ra1-deficient mice in various cardiac function assays, combined with bioinformatic analysis of gene expression data. The downregulation of Il13ra1 and Il4ra in hearts of patients with heart failure suggests potential clinical relevance .

What are the methodological challenges in studying IL13RA1 expression on primary cells?

Studying IL13RA1 expression on primary cells presents several methodological challenges:

• Low baseline expression: IL13RA1 typically shows low surface expression on many primary cells, making detection challenging

• Antibody quality and specificity: Detection of native IL13RA1 has been problematic. Recent advances include development of monoclonal antibodies generated by genetic immunization that recognize native antigens

• Dynamic regulation: IL13RA1 expression is rapidly modulated by cytokines, requiring careful timing of analyses and consistent cell isolation procedures

• Cell type heterogeneity: Different cell populations express varying levels of IL13RA1, necessitating precise cell isolation techniques and multi-parameter analysis

Methodological solutions include:

• Use of highly sensitive flow cytometry with appropriate controls

• Development of specific monoclonal antibodies through genetic immunization

• Complementary techniques (flow cytometry, qPCR, immunohistochemistry)

• Standardized protocols for cell isolation and handling

These approaches have enabled detection and quantification of the low baseline surface expression of IL13RA1 on eosinophils and its regulation by various cytokines .

How does IL13RA1 contribute to the formation of type I versus type II IL-4 receptors?

IL13RA1 plays a pivotal role in the formation of distinct cytokine receptor complexes that mediate IL-4 and IL-13 signaling:

• Type I IL-4 receptor:

  • Composition: IL-4Rα and common gamma chain (γc)

  • Does NOT contain IL13RA1

  • Primarily expressed on hematopoietic cells

  • Exclusively binds IL-4, not IL-13

• Type II IL-4/IL-13 receptor:

  • Composition: IL-4Rα and IL13RA1

  • Expressed on both hematopoietic and non-hematopoietic cells

  • Binds both IL-4 and IL-13

  • Critical for IL-13-mediated responses

Methodological approach: Researchers study these receptor complexes using co-immunoprecipitation, FRET analysis, surface plasmon resonance, and receptor cross-linking studies. These investigations have revealed that IL13RA1 is critical for all IL-13-mediated responses, while IL-4 can signal through either the type I or type II receptor, providing greater versatility in immune regulation .

What downstream signaling pathways are activated by IL13RA1-containing receptors?

IL13RA1-containing receptors activate specific downstream signaling pathways that mediate the biological effects of IL-4 and IL-13:

• Primary signaling pathway:

  • JAK/STAT6 activation: IL13RA1 associates with JAK1, while IL-4Rα associates with JAK2/3

  • STAT6 phosphorylation leads to dimerization and nuclear translocation

  • Transcriptional activation of STAT6-dependent genes (e.g., chemokines, mucins)

• Secondary signaling pathways:

  • IRS-2/PI3K/AKT pathway: Less prominent in type II receptor signaling compared to type I

  • MAPK pathway: Activated under certain conditions

• Pathway verification methods:

  • Phospho-specific Western blotting for detection of activated signaling molecules

  • Chromatin immunoprecipitation to identify STAT6 binding sites

  • RNA-seq to profile transcriptional responses

  • Pharmacological inhibitors to dissect pathway contributions

Understanding these signaling pathways has revealed how IL13RA1 mediates diverse biological effects, including the regulation of extracellular matrix genes and glucose metabolism pathways in different tissues .

How do allergen challenge models reveal distinct IL13RA1-dependent and -independent pathways?

Allergen challenge models have been instrumental in identifying IL13RA1-dependent and -independent pathways in allergic responses:

• IL13RA1-dependent pathways:

  • Airway resistance and hyperresponsiveness: Consistently dependent on IL13RA1 across different allergen models

  • Mucus production and goblet cell hyperplasia: Entirely IL13RA1-dependent

  • TGF-β production: Regulated by IL13RA1 signaling

• IL13RA1-independent pathways:

  • IgE production: No difference observed in allergen-induced total serum IgE levels between wild-type and Il13ra1-/- mice

  • Th2 cytokine production: IL-4, IL-13, and IL-5 levels remain intact in Il13ra1-/- mice

• Context-dependent pathways:

  • Eosinophil recruitment: IL13RA1-dependent following mucosal sensitization but becomes IL13RA1-independent when alum is used as an adjuvant during sensitization

  • Chemokine production: Expression of eosinophil-selective chemokines (CCL11, CCL24) is IL13RA1-dependent, yet eosinophilia can occur independently

Methodological approach: Comparative studies of wild-type and Il13ra1-/- mice using different allergen sensitization protocols (mucosal vs. adjuvant-based) have revealed these distinct pathways. This understanding highlights the complex interplay between IL-4 and IL-13 signaling in allergic inflammation and suggests that the balance between these cytokines determines the IL13RA1-dependency of specific aspects of allergic responses .

What are the optimal methods for producing and validating recombinant IL13RA1 protein?

Production of high-quality recombinant IL13RA1 requires careful consideration of expression systems and validation methods:

• Expression system selection:

  • Mammalian expression (HEK293, CHO cells): Provides proper folding and post-translational modifications

  • Insect cell expression (Sf9, High Five): Good compromise between yield and proper folding

  • Cell-free systems: Rapid production for preliminary studies

• Protein design considerations:

  • Full-length vs. partial (extracellular domain only)

  • Strategic placement of tags to minimize functional interference

  • Codon optimization for expression host

• Purification strategy:

  • Affinity chromatography (Ni-NTA for His-tagged proteins)

  • Size exclusion chromatography for final polishing

  • Tag removal using specific proteases if necessary

• Quality control methods:

  • SDS-PAGE and Western blotting to confirm size and immunoreactivity

  • Mass spectrometry to confirm identity and modifications

  • Circular dichroism to assess secondary structure

  • Surface plasmon resonance to measure binding kinetics with IL-13 and IL-4

  • Cell-based assays to confirm biological activity

This systematic approach ensures the production of functionally active recombinant IL13RA1 suitable for research applications including structural studies, binding assays, and antibody generation.

What techniques are most effective for studying IL13RA1 in different tissue contexts?

Studying IL13RA1 across different tissue contexts requires a combination of complementary techniques:

• For lung tissue and airway function:

  • Invasive pulmonary function testing to measure airway resistance and compliance

  • Histological analysis (H&E, PAS staining) to assess mucus production and goblet cell hyperplasia

  • Bronchoalveolar lavage fluid analysis for cellular infiltration and cytokine levels

  • Precision-cut lung slices for ex vivo functional studies

• For cardiac tissue:

  • Echocardiography for functional assessment (ejection fraction, ventricular dimensions)

  • Pressure-volume relationships for detailed hemodynamic analysis

  • Histological assessment of fibrosis and extracellular matrix deposition

  • Cardiomyocyte isolation for cellular studies

• For immune cell analysis:

  • Flow cytometry for IL13RA1 surface expression and phospho-STAT6 detection

  • Cell sorting for population-specific analyses

  • Ex vivo stimulation assays to assess cytokine responses

  • Adoptive transfer studies to dissect cellular contributions

• For molecular analyses across tissues:

  • RNA-seq for comprehensive gene expression profiling

  • ChIP-seq for STAT6 binding site identification

  • Proteomics for signaling pathway analysis

  • Single-cell approaches to resolve cellular heterogeneity

This multi-faceted approach enables comprehensive characterization of IL13RA1 function across diverse physiological contexts, revealing both shared and tissue-specific mechanisms.

How can researchers design experiments to distinguish IL-4 versus IL-13 specific effects through IL13RA1?

Distinguishing IL-4 versus IL-13 specific effects through IL13RA1 requires carefully designed experimental approaches:

• Genetic approaches:

  • Compare Il13ra1-/- mice with Il4ra-/- and Il4-/- or Il13-/- mice

  • Use cell-specific conditional knockout models to isolate effects in specific cell populations

  • Employ receptor chimeras to dissect domain-specific functions

• Pharmacological approaches:

  • Use cytokine-specific neutralizing antibodies

  • Apply receptor-specific blocking antibodies

  • Utilize recombinant cytokine muteins with altered receptor specificity

• Cellular and molecular approaches:

  • Compare signaling in cells expressing only type I vs. type II receptors

  • Analyze differential gene expression in response to IL-4 vs. IL-13

  • Examine STAT6 binding patterns following IL-4 vs. IL-13 stimulation

• Experimental design considerations:

  • Dose-response studies to identify threshold differences

  • Time-course experiments to detect kinetic differences

  • Context-dependent studies (different tissues, inflammatory conditions)

  • Combined in vivo and ex vivo approaches

• Key readouts for distinguishing effects:

  • Airway resistance (consistently IL13RA1-dependent)

  • Mucus production (IL13RA1-dependent)

  • Eosinophil recruitment (context-dependent IL13RA1 requirement)

  • IgE production (IL13RA1-independent)

This systematic approach allows researchers to delineate the specific contributions of IL-4 versus IL-13 signaling through IL13RA1, providing insights into how these related cytokines mediate distinct and overlapping biological effects.