IL15RA Human

What is the molecular structure of IL15RA and how does it function in cell signaling?

Human IL15RA is a single, glycosylated polypeptide chain spanning amino acids 31-205. When produced recombinantly, it can be fused to a 242 amino acid hIgG-His Tag, resulting in a 417 amino acid protein with a molecular mass of 45.6kDa . On SDS-PAGE under reducing conditions, IL15RA displays multiple bands between 57-70kDa due to its glycosylation patterns .

Functionally, IL15RA forms a heterotrimer with IL-2 receptor beta and gamma subunits to initiate signal transduction . IL15/IL15R alpha complexes can transmit reverse signaling which promotes cellular adhesion, tyrosine phosphorylation of intracellular proteins, and cytokine secretion by IL-15/IL15R alpha expressing cells . Notably, IL15RA alone binds IL-15 with a 1000-fold higher affinity than that seen with IL-2R alpha and IL-2 . The cytoplasmic domain is dispensable for mitogenic signaling, suggesting the primary role of the alpha chain is to confer high-affinity binding rather than direct signal transduction .

How does IL15RA differ from and relate to IL2RA in structure and function?

IL15RA and IL2RA (IL-2 receptor alpha) share notable structural similarities while maintaining distinct functional properties:

What are the known isoforms of human IL15RA and what are their characteristics?

Multiple isoforms of human IL15RA have been identified through differential splicing. According to the literature, at least three differentially spliced human IL-15R alpha variants are capable of high affinity binding of IL-15 . These isoforms include:

• Interleukin 15 Receptor Alpha Isoform EM2

• Interleukin 15 Receptor Alpha Isoform IC2-IC8 (a series of 7 isoforms)

The functional differences between these isoforms remain an active area of research. The diversity in IL15RA isoforms suggests potential specialized roles in different tissue contexts or cellular responses. The cytoplasmic domains of all isoforms appear dispensable for mitogenic signaling, as their primary role is to facilitate high-affinity IL-15 binding rather than direct signal transduction .

What genomic and chromosomal characteristics define the human IL15RA gene?

The human IL15RA gene is located on chromosome 10 . Its genomic structure shows similarities to IL2RA, with comparable intron-exon organization . The gene encodes the high-affinity alpha receptor chain for IL-15, which shares structural components with the IL-2 receptor system.

Multiple reference sequences exist for the IL15RA gene:

• NC_000010.11 (Chromosome 10 Reference GRCh38.p14 Primary Assembly)

• NC_060934.1 (Chromosome 10 Alternate T2T-CHM13v2.0)

• NC_000010.10 (Chromosome 10 Reference GRCh37.p13 Primary Assembly)

Over 110 citations are associated with this gene in PubMed, reflecting its significance in immunological research . The gene's proximity to IL2RA in the genome suggests an evolutionary relationship between these cytokine receptor systems.

What methodologies are most effective for studying IL15RA-mediated signal transduction?

Multiple complementary techniques should be employed when investigating IL15RA-mediated signaling:

Protein-Protein Interaction Analysis:

• Size exclusion chromatography (SEC) for analyzing complex formation between IL15 and IL15RA

• Surface plasmon resonance (SPR) for measuring binding kinetics and affinity

• Co-immunoprecipitation for detecting protein interactions in cellular contexts

Functional Assays:

• Cell-based proliferation assays using CTLL2 mouse cytotoxic T cells (ED50 for IL15RA inhibition effect is < 2 ng/ml with Human IL-15)

• Cytokine production measurements following IL15/IL15RA stimulation

• Phosphoflow cytometry to detect activation of downstream signaling molecules

Molecular and Genetic Approaches:

• Transcriptional and chromatin profiling to identify regulated genes

• Knockout/knockdown studies of pathway components (e.g., RUNX3)

• CRISPR-Cas9 editing to modify specific domains of IL15RA

Researchers should note that different methodologies may yield contradictory results. For example, one study found that IL15Rα fusion proteins showed no IL15 complexation on SEC, but demonstrated IL15 binding ability on SPR . This highlights the importance of using multiple complementary techniques.

How can IL15/IL15RA complex formation be optimized for in vitro studies?

Optimizing IL15/IL15RA complex formation requires careful consideration of multiple experimental parameters:

Protein Design Considerations:

• Fusion protein strategy: Studies have explored fusing IL15RA to antibody fragments (e.g., F8, which targets the alternatively-spliced extra-domain A of fibronectin)

• Tag selection: Consider the impact of tags (His, Fc, etc.) on protein folding and interaction

• Expression system: SF9 Baculovirus cells have been successfully used for IL15RA production

Experimental Conditions:

• Buffer optimization: Phosphate buffered saline (pH 7.4) with 10% glycerol has been used for IL15RA protein solutions

• Storage considerations: Addition of carrier protein (0.1% HSA or BSA) for long-term storage and avoiding multiple freeze-thaw cycles

• Concentration optimization: Titrate both IL15 and IL15RA to determine optimal ratios

Validation Approaches:

• Compare multiple binding assays: SEC and SPR may yield different results

• Functional validation: Cell-based proliferation assays to confirm biological activity

• Biophysical characterization: Thermal stability analysis of complexes versus individual components

Researchers should be aware that optimization is challenging, as evidenced by findings that some IL15Rα fusions were unable to increase cellular proliferation in combination with IL15 compared to IL15 alone . This underscores the complexity of IL15/IL15RA biology and the need for thorough characterization.

What experimental approaches can resolve contradictions in IL15RA research literature?

Several contradictions exist in the IL15RA literature that require targeted experimental approaches:

Contradiction 1: Enhanced biological activity of IL15/IL15RA complexes

• Initial hypothesis: "Administering IL15 in complex with IL15Rα could increase its biological activity manifold"

• Contradicting finding: "None of the IL15Rα fusions were able to increase cellular proliferation in combination with IL15 compared to IL15 alone"

Resolution approaches:

• Compare different cell types beyond CTLL2 cells to assess context-dependent effects

• Examine dose-response relationships across wider concentration ranges

• Investigate alternative readouts beyond proliferation (cytokine production, gene expression)

• Evaluate different fusion protein designs and linker regions

• Compare different recombinant protein sources and production methods

Contradiction 2: Detection of IL15/IL15RA complexes by different methods

• Finding: IL15Rα fusion proteins showed no IL15 complexation on SEC, but F8IL15Rα displayed binding ability on SPR

Resolution approaches:

• Optimize SEC conditions (buffer composition, column selection, flow rate)

• Use orthogonal biophysical techniques (isothermal titration calorimetry, bio-layer interferometry)

• Employ native mass spectrometry to directly observe complex formation

• Analyze complex formation kinetics under various conditions

• Develop FRET-based assays to monitor interactions in real-time

These methodological approaches can help researchers address contradictions and refine our understanding of IL15RA biology.

How does IL15RA expression and function change in HIV-1 infection?

HIV-1 infection significantly impacts IL15RA-related immune pathways through multiple mechanisms:

Transcriptional Changes:

• RNA-Seq analysis reveals increased antiviral gene expression in all innate lymphoid cell (ILC) subsets from HIV-1 viremic individuals

• CD56^hi NK cells from elite controllers show increased expression of anti-inflammatory gene MYDGF

Cellular Population Shifts:

• Functionally-defective CD56^- NK cells increase in people living with HIV-1

• This increase inversely correlates with CD56^dim NK cells, ILCs, and CD4^+ T cells

Mechanistic Insights:

• CD4^+ T cells and their IL-2 production prevent CD56^dim transition to CD56^- NK cells by activating mTOR

• In people with HIV-1, plasma IL-2 correlates with CD4^+ T cell number but not with CD8^+ T cells

• TGFB1, elevated in HIV-1 infection, inhibits production of amphiregulin (AREG)

• NK cell knockout of the TGFB1-stimulated WNT antagonist RUNX3 increases AREG production

Functional Consequences:

• AREG^+ NK cell percentage correlates with numbers of ILCs and CD4^+ T cells

• AREG^+ NK cell percentage inversely correlates with inflammatory cytokine IL-6

These findings highlight the complex interplay between HIV-1 infection, cytokine networks, and IL15RA-dependent immune cell function.

What strategies exist for developing IL15RA-based therapeutic approaches?

Development of IL15RA-based therapeutics represents an active area of research with several strategic approaches:

Fusion Protein Design:

• The F8IL15Rα fusion protein targets the alternatively-spliced extra-domain A (EDA) of fibronectin, which is overexpressed in many types of cancer

• This approach aims to localize IL15 activity to the tumor microenvironment

• Both mouse and human IL15 and their corresponding Fc-fused IL15Rα subunits have been investigated

Complex Formation Optimization:

Targeted Delivery Systems:

• Antibody-cytokine fusion proteins allow specific targeting to disease sites

• IL15Rα fragments can be incorporated into various delivery platforms

• Nanoparticle formulations carrying IL15/IL15Rα complexes represent another approach

Combination Therapies:

• IL15RA-based therapeutics may synergize with checkpoint inhibitors

• Combining with agents that modulate TGFB1 signaling could be beneficial, as TGFB1 inhibits AREG production

• Strategies targeting the TCF7/WNT signaling pathway might enhance therapeutic efficacy

A key challenge in developing these therapeutics is understanding "the molecular requirements for effective IL15 signalling" . Further research into IL15Rα structure-function relationships and signaling dynamics will be critical for successful therapeutic development.

How can single-cell analysis advance understanding of IL15RA biology?

Single-cell technologies offer powerful approaches to dissect IL15RA biology with unprecedented resolution:

Single-Cell Transcriptomics:

• Can identify distinct cell populations based on IL15RA expression patterns

• Enables correlation of IL15RA with other genes in the signaling pathway

• Could reveal novel IL15RA-expressing cell subsets missed in bulk analysis

• Search result describes transcriptional profiling that separated blood ILCs into ILC2s, ILCPs, and NK cell clusters, which could be further refined with single-cell approaches

Single-Cell Proteomics:

• Mass cytometry (CyTOF) can measure IL15RA protein levels alongside dozens of other markers

• Spectral flow cytometry allows detailed characterization of IL15RA-expressing cells

• Imaging mass cytometry can map IL15RA distribution in tissue microenvironments

Functional Single-Cell Assays:

• Single-cell cytokine secretion assays can link IL15RA expression to functional outputs

• CRISPR screening at single-cell resolution can identify genes that regulate IL15RA function

• Live-cell imaging of individual cells can track IL15RA trafficking and signaling dynamics

Integrative Analysis:

• Multi-omics approaches combining transcriptomics, proteomics, and epigenomics at single-cell level

• Trajectory analysis to map developmental relationships between IL15RA-expressing cell populations

• Network analysis to identify cell-type specific IL15RA signaling pathways

These approaches could help resolve contradictions in the literature and provide a more nuanced understanding of IL15RA biology across different cell types and disease states.

What are the critical quality control parameters for IL15RA protein production and characterization?

Ensuring consistent, high-quality IL15RA protein is essential for reliable research results. Critical quality control parameters include:

Production Parameters:

• Expression system: Sf9 Baculovirus cells have been successfully used

• Purification method: Proprietary chromatographic techniques are typically employed

• Protein concentration: Standard working solution is 0.5mg/ml

• Buffer composition: Phosphate buffered saline (pH 7.4) with 10% glycerol

Analytical Characterization:

• Purity assessment: >90.0% as determined by SDS-PAGE

• Molecular weight confirmation: Expected molecular mass of 45.6kDa for the core protein

• Glycosylation analysis: Multiple bands between 57-70kDa on SDS-PAGE indicate glycosylation variants

• Protein sequence verification: Mass spectrometry to confirm amino acid sequence

• Aggregation assessment: SEC to evaluate monomer percentage

Functional Validation:

• Binding assays: SPR to confirm IL15 binding with expected affinity

• Biological activity: Inhibition of proliferation using CTLL2 mouse cytotoxic T cells (ED50 < 2 ng/ml with Human IL-15)

• Stability testing: Evaluate protein integrity after multiple freeze-thaw cycles

Storage and Handling:

• Stability requirements: Addition of carrier protein (0.1% HSA or BSA) for long-term storage

• Storage recommendations: Avoid multiple freeze-thaw cycles

• Lot-to-lot consistency: Compare critical parameters across production batches

Rigorous adherence to these quality control parameters will help ensure reproducible research results when working with IL15RA proteins.