Recombinant Rat Interleukin-2 receptor subunit alpha (Il2ra)
Description
Molecular Structure and Production
Recombinant Rat IL-2Rα is a 55 kDa type I transmembrane glycoprotein produced using mammalian or bacterial expression systems. Key structural features include:
Production Notes:
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Carrier-Free (CF): R&D Systems’ formulation excludes BSA for applications where carrier proteins interfere (e.g., receptor-ligand binding assays) .
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Lyophilization: Reconstituted at 500 µg/mL in PBS; stable at -80°C with minimal freeze-thaw cycles .
Biological Functions
IL-2Rα is indispensable for immune homeostasis, with roles in:
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High-Affinity IL-2 Receptor Formation: Combines with IL-2Rβ (CD122) and γc (CD132) to form a ternary complex (K<sub>d</sub> ~10 pM) .
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Regulatory T Cell (Treg) Development: Essential for CD4<sup>+</sup>CD25<sup>+</sup> Treg differentiation, which suppresses autoreactive T cells .
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Activation-Induced Cell Death (AICD): Promotes apoptosis of naive T cells to prevent autoimmunity .
Key Research Findings:
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Mice with Treg-specific IL-2Rα deficiency develop fatal systemic inflammation, underscoring its non-redundant role in immune tolerance .
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STAT5 activation via IL-2Rα is critical for Treg suppressor function, independent of IL-2 sequestration .
In Vitro and In Vivo Studies
Disease Biomarker
Elevated soluble IL-2Rα levels correlate with:
Technical Considerations
Product Specs
Note: While we will prioritize shipping the format currently in stock, we are open to accommodating specific format requirements. Please indicate your preference in the order notes, and we will fulfill your request to the best of our ability.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
- Adoptive transfusion of CD4+CD25+ (Interleukin-2 Receptor alpha) Regulatory T Cells into Experimental Autoimmune Neuritis model rats has demonstrated significant therapeutic effects. PMID: 29772575
- Deletion of antigen-specific T cells through apoptosis and active suppression mediated by CD4(+)CD25(+) T-regulatory cells is a key factor in the induction of antigen-specific immune tolerance. PMID: 19884905
Q&A
What is the structural relationship between rat Il2ra and the complete IL-2 receptor complex?
The IL-2 receptor comprises three distinct components: the α-chain (Il2ra/CD25), which is cytokine specific, and the β- and γc-subunits. The Il2ra component binds IL-2 with low affinity, while the β-subunit (CD122) and γc-subunit (CD132) are shared with other cytokine receptors . The receptor can form either a high-affinity trimeric IL-2R (IL2RA/CD25, IL2RB/CD122, and IL2RG/CD132) or a low-affinity dimeric IL-2R (IL2RB and IL2RG) . When IL-2 binds to this receptor complex, it triggers oligomerization and conformational changes in the IL-2R subunits, initiating downstream signaling cascades that begin with the phosphorylation of JAK1 and JAK3 .
How is Il2ra expression regulated in different rat immune cell populations?
Il2ra is predominantly expressed on activated CD4-positive helper T cells and, to a lesser extent, on activated CD8-positive T cells and natural killer (NK) cells . Regulatory T cells (Tregs) constitutively express high levels of Il2ra, which serves as a marker for their identification (CD25+Foxp3+ T cells) . Following T cell activation, Il2ra expression is upregulated, creating a positive feedback loop as IL-2 binds to its receptor and drives T cells to proliferate while inducing further IL-2 and IL-2R alpha synthesis . This dynamic regulation of Il2ra expression is critical for the development and function of different T cell subsets, particularly regulatory T cells which require IL-2 signaling for maintenance of immune tolerance .
What experimental considerations are critical when studying Il2ra's role in regulatory T cell function?
When investigating Il2ra's role in regulatory T cell function, researchers should implement a multi-faceted approach:
Isolation and Characterization:
Begin with careful isolation of CD4+CD25+ T cells using magnetic bead separation . This technique allows separation of CD25+ (primarily Foxp3+) regulatory T cells from CD25- conventional T cells. Flow cytometry confirmation of Foxp3 expression is essential, as the majority of sorted CD25+ T cells should be positive for this T regulatory cell lineage factor .
Experimental Design:
Use congenic markers (such as CD45.1 and CD45.2) to track different T cell populations in adoptive transfer experiments . This approach enables researchers to distinguish between regulatory T cells and other T cell populations, allowing precise assessment of their interactions and functional outcomes. When evaluating regulatory T cell suppression of tumor immunity, careful selection of tumor models that express self-antigens recognized by your T cell population is critical for meaningful results .
Signaling Analysis:
Assess downstream signaling events following IL-2 engagement of the receptor complex, focusing on JAK1/3 phosphorylation and subsequent STAT5 activation . The phosphorylation status of these signaling molecules provides mechanistic insight into how IL-2 signaling through Il2ra contributes to regulatory T cell development and function.
How can researchers differentiate between the roles of Il2ra in regulatory T cells versus effector T cells?
Distinguishing the functional roles of Il2ra in different T cell populations requires specialized experimental approaches:
Cell Population Separation:
Implement precise isolation techniques that can separate regulatory T cells (CD4+CD25+Foxp3+) from activated effector T cells (CD4+CD25+Foxp3-) . This distinction is crucial as both populations express Il2ra but exhibit different functional outcomes following IL-2 stimulation.
Functional Assays:
Utilize suppression assays to evaluate regulatory T cell function, assessing their ability to inhibit the proliferation and cytokine production of responder T cells. For effector functions, measure proliferation, cytokine production, and cytolytic activity in response to IL-2 stimulation.
Signaling Pathway Resolution:
Analyze multiple nodes in IL-2 signaling pathways simultaneously. While IL-2 induces similar initial signaling events (JAK1/3 activation) in both cell types, regulatory T cells preferentially activate STAT5 pathways critical for Foxp3 maintenance, whereas effector T cells may show stronger activation of PI3K-Akt and MAPK pathways .
Genetic Approaches:
Employ conditional gene targeting or knockdown approaches specific to either regulatory or effector T cell populations to determine cell type-specific consequences of Il2ra deficiency or alteration.
What are the optimal production and purification methods for recombinant rat Il2ra protein?
Expression Systems:
While E. coli is commonly used for producing cytokines like IL-2 , mammalian expression systems such as HEK293 cells may better preserve the native structure and post-translational modifications of membrane proteins like Il2ra . For research requiring high structural fidelity, recombinant rat Il2ra produced in HEK293 cells with ≥95% purity and endotoxin levels ≤0.005 EU/μg offers optimal results .
Purification Strategy:
A multi-step purification process typically involving affinity chromatography followed by size exclusion chromatography achieves the highest purity. Verification of purity should include Coomassie-stained SDS-PAGE and mass spectrometry analysis .
Formulation Considerations:
The optimal buffer formulation depends on experimental applications. For general research use, phosphate-based buffers (e.g., 5mM NaH₂PO₄) with stabilizing agents maintain protein integrity . For carrier-free applications where the presence of BSA could interfere with experiments, specialized formulations without carrier proteins are available .
Storage Protocol:
Store recombinant proteins at -20°C to -80°C in a manual defrost freezer and avoid repeated freeze-thaw cycles to maintain biological activity . For research requiring absolute consistency, single-use aliquots should be prepared during initial protein processing.
What technical challenges must be addressed when measuring Il2ra-mediated signaling in primary rat immune cells?
Temporal Considerations:
IL-2 receptor signaling events occur rapidly after ligand binding, with initial phosphorylation events happening within minutes . Implement time-course experiments with precise timing controls to capture these dynamic events. The interaction of IL-2 with its receptor leads to oligomerization and conformational changes in the IL-2R subunits, triggering a signaling cascade that begins with JAK1/3 phosphorylation and progresses through several downstream pathways including STAT5, PI3K/Akt, and MAPK .
Cell Type Heterogeneity:
Different immune cell populations exhibit varying levels of Il2ra expression and distinct signaling outcomes following IL-2 stimulation . Flow cytometry-based approaches that allow simultaneous phenotyping and signaling analysis provide cell type-specific resolution.
Technical Limitations:
Phospho-flow cytometry requires rapid sample processing to preserve phosphorylation states. Implement protocols that include immediate fixation following stimulation to "freeze" the signaling state. Additionally, low cell numbers from primary rat samples may necessitate signal amplification strategies or highly sensitive detection methods.
Physiological Relevance:
In vitro signaling studies may not fully recapitulate the in vivo microenvironment. Consider using ex vivo analysis of freshly isolated cells or adoptive transfer approaches that allow for in vivo stimulation followed by ex vivo analysis .
How is recombinant rat Il2ra being used to study regulatory T cell development and function?
Developmental Studies:
Recombinant Il2ra, combined with IL-2, provides critical signals for the maintenance and expansion of regulatory T cells in experimental systems. Researchers utilize these proteins to establish in vitro cultures that support regulatory T cell survival and function, allowing detailed study of factors that influence their development .
Functional Characterization:
The regulatory T cell suppression assay represents a fundamental tool for assessing Treg function. In this context, recombinant Il2ra can be used to block IL-2 signaling, revealing the dependency of different Treg functions on this pathway. Studies demonstrate that CD25+Foxp3+ T cells effectively suppress immunity to tumor/self-antigens, highlighting the critical role of IL-2 signaling in this process .
Signaling Analysis:
Recombinant Il2ra, when combined with downstream signaling inhibitors, helps dissect the relative contributions of different IL-2-induced pathways to regulatory T cell function. This approach has revealed that STAT5 signaling is particularly critical for maintaining Foxp3 expression and suppressive function .
Therapeutic Applications:
Research utilizing recombinant Il2ra has informed therapeutic strategies aimed at modulating regulatory T cell activity in autoimmunity, cancer, and transplantation settings. Understanding how regulatory T cells suppress immunity to tumor/self-antigens through IL-2-dependent mechanisms has direct translational implications .
What approaches are used to investigate Il2ra trafficking and localization during immune synapse formation?
Advanced Imaging Techniques:
Super-resolution microscopy and total internal reflection fluorescence (TIRF) microscopy provide high-resolution visualization of Il2ra distribution at the immune synapse. These techniques reveal dynamic receptor clustering and internalization following T cell activation.
Molecular Tagging Strategies:
Fluorescent protein tagging or specific antibody labeling of Il2ra enables tracking of receptor movement during immune synapse formation. These approaches demonstrate that Il2ra rapidly concentrates at the central supramolecular activation cluster (cSMAC) of the immunological synapse during T cell activation.
Correlative Functional Analysis:
Simultaneous measurement of Il2ra localization and activation markers (such as calcium flux or early phosphorylation events) establishes the functional consequences of receptor redistribution. This approach has revealed that the positioning of Il2ra within the immune synapse significantly influences downstream signaling outcomes.
Methodological Considerations:
When studying Il2ra dynamics, researchers must carefully select antibodies or tagging strategies that do not interfere with receptor function. Additionally, time-lapse imaging with sufficient temporal resolution is essential to capture the rapid receptor movements that occur during synapse formation.
Comparative characteristics of recombinant rat IL-2 protein formulations
IL-2 receptor signaling pathways and functional outcomes in different T cell populations
This table illustrates the differential signaling pathways and functional outcomes of IL-2 receptor engagement across T cell subpopulations, highlighting the critical role of Il2ra in determining cellular responses to IL-2 stimulation .
What are the most common issues encountered when working with recombinant rat Il2ra and their solutions?
Protein Instability:
Recombinant proteins, particularly membrane proteins like Il2ra, may exhibit instability during storage or experimental handling. To mitigate this issue, store proteins at recommended temperatures (-20°C to -80°C) and avoid repeated freeze-thaw cycles . For applications requiring repeated access, prepare single-use aliquots during initial processing.
Low Biological Activity:
Decreased activity may result from protein denaturation or aggregation. Validate protein activity before experiments using functional assays, and consider using carrier proteins like BSA to enhance stability unless contraindicated for specific applications .
Inconsistent Results:
Variability in experimental outcomes may stem from differences in protein lots or handling procedures. Implement standardized protocols with appropriate positive and negative controls, and maintain detailed records of protein lot numbers and experimental conditions.
Non-specific Binding:
In binding assays or immunoprecipitation experiments, non-specific interactions may confound results. Include appropriate blocking agents and stringent washing steps, and validate specificity using known ligands or interaction partners.
Cross-reactivity Issues:
Antibodies against Il2ra may cross-react with related proteins. Validate antibody specificity using positive controls (cells known to express Il2ra) and negative controls (cells lacking Il2ra expression).
How can researchers optimize experimental conditions for studying Il2ra-mediated regulatory T cell development?
Culture Medium Composition:
For in vitro studies of regulatory T cell development, the medium composition significantly influences outcomes. Include appropriate concentrations of recombinant IL-2 (typically 0.04-0.2 ng/mL for biological effect) , as IL-2 signaling through Il2ra is critical for regulatory T cell maintenance and function .
Cell Isolation Purity:
The purity of starting cell populations directly impacts experimental interpretation. Implement rigorous isolation techniques, such as magnetic bead separation followed by flow cytometry confirmation of Foxp3 expression, to ensure high-purity regulatory T cell populations .
Temporal Considerations:
Regulatory T cell development and function exhibit time-dependent characteristics. Design experiments with appropriate time points, recognizing that certain phenotypic and functional changes may require extended culture periods.
Microenvironmental Factors:
The cytokine milieu and cell-cell interactions significantly influence regulatory T cell development. Consider co-culture systems that recapitulate physiological interactions, particularly when studying the suppressive function of regulatory T cells .
Functional Validation:
Phenotypic markers alone (e.g., CD25/Il2ra expression) may not fully capture regulatory T cell identity or function. Include functional assays, such as suppression assays or adoptive transfer experiments, to validate the developmental and functional status of putative regulatory T cells .
