Recombinant Human Interleukin-23 receptor (IL23R), partial (Active)

What is the structural composition of the IL-23 receptor complex?

The IL-23 receptor complex consists of two main subunits: the IL-23R chain and the IL-12Rβ1 co-receptor chain. The IL-23R subunit is specific to the IL-23 signaling pathway, while the IL-12Rβ1 subunit is shared with the IL-12 receptor complex. This heterodimeric structure is essential for proper ligand binding and downstream signaling. The receptor complex forms through association of these subunits at their N-termini, even in the absence of ligand, though the binding of IL-23 (which itself consists of p19 and p40 subunits) stabilizes and modifies this interaction . Studies have demonstrated that the IL-23R subunit can be expressed alone on cell surfaces, but optimal signaling requires co-expression with IL-12Rβ1 .

How does IL-23R signaling contribute to autoimmune and inflammatory diseases?

IL-23R signaling is a critical factor in multiple autoimmune and inflammatory conditions through its activation of the Th17 pathway. When IL-23 binds to its receptor complex, it triggers a signaling cascade that leads to the differentiation and maintenance of Th17 cells, which produce inflammatory cytokines including IL-17 . This pathway has been shown to be active in the pathogenesis of several chronic inflammatory diseases including psoriasis, inflammatory bowel disease (IBD), rheumatoid arthritis, and multiple sclerosis .

In inflammatory bowel disease specifically, IL-23 stimulation of colonic leukocytes induces the production of IL-17, perpetuating inflammation . Similarly, in rheumatoid arthritis patients, IL-23 levels are significantly elevated in peripheral blood compared to normal controls . The importance of IL-23R signaling in these diseases makes it a valuable therapeutic target for conditions characterized by chronic inflammation.

What is the significance of the IL23R R381Q variant in inflammatory disease research?

The R381Q variant of IL23R has emerged as a critically important mutation in inflammatory disease research, particularly for inflammatory bowel disease (IBD). This naturally occurring variant provides protection against IBD development . Studies indicate that the R381Q mutation modifies IL-23-induced signaling outcomes in human monocyte-derived macrophages (MDMs) .

When compared to the wild-type receptor, the R381Q variant shows altered patterns of JAK2, TYK2, and STAT3 pathway activation following IL-23 stimulation . Specifically, MDMs transfected with the R381Q variant demonstrate different cytokine secretion profiles compared to those expressing wild-type IL23R . This variant provides valuable insights into the mechanisms of IL-23 signaling and may guide the development of therapeutic approaches that mimic this protective effect by selectively modulating IL-23 signaling pathways rather than completely blocking them.

How do IL-23 and IL-12 receptor systems overlap and differ?

The IL-23 and IL-12 receptor systems share significant structural elements but maintain distinct signaling outcomes. Both receptors utilize the IL-12Rβ1 subunit as a co-receptor, but they differ in their specific receptor chains (IL-23R for IL-23 and IL-12Rβ2 for IL-12) . This shared subunit explains why some antibodies developed against IL-12 can also affect IL-23 signaling, as they target the common p40 subunit found in both cytokines .

Despite these structural similarities, the receptors demonstrate different recycling and internalization behaviors. Research shows that IL-23 induces dynamic IL-23R cell surface regulation through clathrin and dynamin-mediated endocytosis and endocytic recycling-dependent pathways . In contrast, the IL-12Rβ1 co-receptor does not recycle with IL-23 treatment, though it does undergo internalization when stimulated with IL-12 . These differences in receptor trafficking and dynamics contribute to the specific cellular responses elicited by each cytokine system, despite their structural similarities.

What fluorescent probe methodologies are most effective for studying IL23R binding kinetics?

For studying IL23R binding kinetics, fluorescent cyclic peptide probes have proven particularly effective. Research indicates that TAMRA-labeled P630 (P630-TMR) can be used successfully to characterize IL-23R binding properties . When designing experiments with these probes, several methodological considerations are essential:

• Probe concentration optimization: P630-TMR demonstrates specific binding to cells expressing IL-23R, with studies showing comparable affinity (Kd = 51.1 ± 7.3 nM) for cells expressing various combinations of receptor constructs .

• Competition assay design: To measure binding affinities of unlabeled IL-23, competition assays where P630-TMR is displaced by increasing concentrations of IL-23 have been established . These assays reveal that IL-23 displaces P630-TMR with higher affinity when both subunits of the receptor (IL-23R and IL-12Rβ1) are co-expressed (Ki = 22.6 ± 5.2 pM) .

• Receptor expression verification: Studies should include controls to verify receptor expression levels, as binding measurements can be affected by expression variability. Standard curves using NanoLuc-labeled receptors can help predict expression levels in each experiment .

This methodological approach allows for precise measurement of binding kinetics and provides insights into how receptor subunit composition affects ligand binding.

How can BRET (Bioluminescence Resonance Energy Transfer) techniques be optimized for IL23R interaction studies?

BRET techniques represent a powerful approach for studying IL-23R interactions with both its ligand and co-receptor. To optimize these studies, researchers should consider the following methodological refinements:

• Tag positioning strategy: The position of the NanoLuc tag significantly impacts BRET efficiency. Studies show that placing the NanoLuc tag on the N-terminus of IL-23R yields higher BRET efficiency compared to tagging IL-12Rβ1 . The maximum binding (Bmax) BRET ratio was observed to be 4.04-fold higher when NL-IL23R was expressed alone compared to when co-expressed with IL-12Rβ1 .

• Data analysis approach: BRET ratio values should be generated by dividing the acceptor signal by the donor signal. For ligand binding studies, the following equation has proven effective:

$BRET\; ratio=\frac{B_{\max}[A]}{([A]+K_{D})}+((B[A])+C)$

Where Bmax is the maximum specific binding BRET signal, [A] is fluorescent probe concentration, B is the non-specific binding component's slope, and C is the Y intercept .

• Specific binding data generation: Subtract BRET values obtained in the presence of excess unlabeled probe from BRET ratios gained in the absence of unlabeled probe. The resulting specific binding data can be fit using:

$Y=\frac{B_{\max}[B]}{([B]+K_{D})}$

These optimizations enable more sensitive detection of IL-23R interactions and provide quantitative measurements of binding affinities and receptor complex formation dynamics.

What mutagenesis approaches are most informative for studying IL23R function?

Strategic mutagenesis of IL-23R provides valuable insights into receptor function, with several approaches proving particularly informative:

• Structure-function mutation analysis: The C115Y mutation in IL-23R has been successfully employed to investigate receptor functionality . This approach involves:

  • PCR-based site-directed mutagenesis using specific primers:

    • Forward primer: 5'-CAAGAGACACTAGATATGTGGAAAGACATTTC-3'

    • Reverse primer: 5'-AAAATGTTTCAGCAG-3'

  • Digestion of methylated template DNA with DpnI (37°C, 15 min)

  • Confirmation of mutations via Sanger sequencing

• Variant comparison studies: Comparing wild-type IL-23R with the naturally occurring R381Q variant provides insights into receptor signaling mechanisms . Transfection of these variants into MDMs (monocyte-derived macrophages) and subsequent analysis of downstream signaling pathways (JAK2, TYK2, STAT3) reveals how single amino acid changes can alter receptor function.

• Domain-specific mutations: Introducing mutations at the interface between IL-23R and IL-12Rβ1 helps elucidate the molecular basis of receptor complex formation and stability . These studies have demonstrated that the N-termini of the receptor subunits associate even in the absence of ligand.

These mutagenesis approaches, combined with functional assays, provide comprehensive insights into structure-function relationships within the IL-23R signaling complex.

How should experiments be designed to investigate IL23R recycling and surface regulation?

Investigating IL-23R recycling and surface regulation requires carefully designed experimental approaches that capture the dynamic nature of receptor trafficking. Based on current research, an optimal experimental design would incorporate:

• Time-course studies: IL-23R demonstrates dynamic surface regulation after IL-23 stimulation. Experimental designs should include multiple time points (0, 30 min, 1 hour, 4 hours, 8 hours) to capture the complete cycle of receptor internalization and recycling .

• Endocytosis pathway inhibition: To determine the mechanisms of IL-23R internalization, experiments should include:

  • Clathrin inhibitors to block clathrin-mediated endocytosis

  • Dynamin inhibitors to block vesicle formation

  • Controls for endocytic recycling-dependent pathways

  • Comparison with IL-12Rβ1 co-receptor, which demonstrates different recycling behavior

• Co-immunoprecipitation studies: To assess the dynamic assembly of signaling complexes, immunoprecipitate IL-23R after IL-23 treatment and analyze the recruitment of signaling molecules (JAK2, TYK2, STAT3) at various time points . Research has shown that the recruitment of these molecules parallels the changes in IL-23R surface expression.

These experimental approaches collectively provide a comprehensive view of receptor trafficking and signaling complex formation, revealing how IL-23R surface regulation influences downstream signaling events.

How should researchers analyze the binding affinity differences between IL23R variants?

Analysis of binding affinity differences between IL-23R variants requires rigorous quantitative approaches that account for multiple variables. Based on established methodologies, researchers should:

• Implement competitive binding models: When comparing binding properties of wild-type IL-23R with variants (such as R381Q), displacement curves should be analyzed using nonlinear regression to determine inhibition constants (Ki). Research shows that when both receptor subunits are expressed, IL-23 displaces fluorescent probes with a Ki of approximately 22.6 ± 5.2 pM .

• Account for receptor expression levels: Binding measurements should be normalized to receptor expression levels, which can be determined using standard curves of NanoLuc-labeled receptors. This normalization is critical since expression variability can confound binding affinity comparisons .

• Compare maximum binding capacity (Bmax) alongside affinity (Kd): Studies show that while P630-TMR binds receptor combinations with similar affinities, the maximum binding (Bmax) BRET ratio differs significantly depending on receptor composition . For instance, the Bmax for NL-IL23R expressed alone was observed to be 4.04-fold higher than when co-expressed with IL-12Rβ1.

What statistical considerations are most important when interpreting IL23R signaling pathway data?

When interpreting IL-23R signaling pathway data, several critical statistical considerations must be addressed:

• Time-dependent analysis: IL-23R signaling demonstrates significant temporal dynamics, with recruitment of signaling molecules (JAK2, TYK2, STAT3) paralleling changes in receptor surface expression . Statistical analysis should incorporate repeated measures approaches to account for these time-dependent effects.

• Multiple pathway component analysis: IL-23R activates several downstream pathways simultaneously (JAK/STAT, MAPK, NFκB) . Therefore, multivariate statistical approaches should be employed to capture the relationships between these pathways and identify potential compensatory mechanisms.

• Variance normalization across experimental conditions: Studies show that IL-23R signaling outcomes can vary based on receptor composition and expression levels . Statistical models should incorporate normalization procedures to account for these sources of variation.

• Appropriate model selection for binding data: For BRET-based receptor binding studies, data should be fitted using equations that account for both specific and non-specific binding components . These models provide more accurate estimations of binding parameters than simpler linear regression approaches.

Adhering to these statistical considerations ensures robust interpretation of complex signaling data and facilitates meaningful comparisons between experimental conditions and receptor variants.

How can researchers effectively compare IL23R dynamics across different cell types?

Comparing IL-23R dynamics across different cell types presents significant challenges due to variations in receptor expression, signaling pathway components, and cellular environments. To effectively conduct these comparisons, researchers should:

• Normalize receptor expression levels: Quantify IL-23R and IL-12Rβ1 expression in each cell type using flow cytometry or quantitative PCR before conducting functional studies . This normalization is essential since receptor density significantly impacts signaling outcomes.

• Establish cell type-specific baseline responses: IL-23R signaling in monocyte-derived macrophages (MDMs) differs from that in other immune cells. Research shows that MDMs exhibit specific patterns of IL-23R recycling and JAK2/TYK2/STAT3 recruitment after IL-23 stimulation . Each cell type should have established baseline responses before making comparisons.

• Use parallel methodological approaches: When comparing IL-23R dynamics:

  • Apply identical stimulation protocols (concentration, timing)

  • Employ the same detection methods across cell types

  • Process and analyze all samples simultaneously to minimize technical variability

• Evaluate autocrine/paracrine effects: Studies demonstrate that in MDMs, NOD2-induced cytokines depend on autocrine/paracrine IL-23, with this dependency changing over time (decreasing by 4-8 hours after stimulation) . These cell type-specific feedback mechanisms must be accounted for in comparative analyses.

By implementing these methodological approaches, researchers can generate meaningful comparisons of IL-23R dynamics across different cellular contexts while controlling for confounding variables.

How might the IL23R R381Q protective variant inform therapeutic development strategies?

The IL-23R R381Q variant provides a natural model for developing targeted therapeutics with potentially fewer side effects than complete IL-23 blockade. Research-based strategies derived from this variant include:

• Selective signaling modulation: The R381Q variant demonstrates altered patterns of JAK2, TYK2, and STAT3 pathway activation following IL-23 stimulation compared to wild-type IL-23R . Therapeutic approaches could target these specific signaling differences rather than blocking receptor-ligand interaction entirely.

• Structure-based drug design: Understanding how the R381Q mutation changes receptor conformation and function enables the development of small molecules that mimic this effect. These compounds would ideally maintain protective anti-inflammatory functions while reducing pathogenic inflammation.

• Cell type-specific targeting: Research indicates that the R381Q variant affects IL-23R signaling differently across cell types . Therapeutics could be designed to target IL-23R specifically in pathogenic cell populations while preserving normal function in protective immune cells.

• Combined receptor subunit targeting: Studies show that IL-23R functions in complex with IL-12Rβ1 . Therapeutics might target the interface between these subunits or their combined signaling output rather than individual components, potentially preserving beneficial aspects of IL-23 signaling.

These approaches represent a more nuanced strategy than current antibody therapeutics that completely block IL-23, potentially improving efficacy while reducing side effects related to complete pathway inhibition.

What are the most promising experimental approaches for targeting the IL23/IL23R pathway in inflammatory diseases?

Current research highlights several promising experimental approaches for targeting the IL-23/IL-23R pathway in inflammatory diseases:

• Receptor recycling modulation: Studies demonstrate that IL-23R undergoes clathrin and dynamin-mediated endocytosis and endocytic recycling . Therapeutics targeting this recycling process could modulate receptor availability without completely blocking IL-23 signaling.

• Selective JAK/STAT pathway inhibition: Research shows that JAK2, TYK2, and STAT3 are recruited to IL-23R following ligand binding . Selective inhibitors targeting these specific components of the signaling pathway might preserve beneficial functions while reducing pathogenic inflammation.

• Receptor subunit interface targeting: Fluorescent probe studies have characterized the binding interface between IL-23R and its ligand . Small molecules designed to modulate rather than block this interaction could fine-tune receptor activation.

• Receptor variant-inspired peptides: The protective mechanisms of the R381Q variant could be mimicked by peptides designed to interact with specific regions of the receptor, altering signaling outcomes without completely preventing activation .

These approaches represent advances beyond traditional antibody therapeutics, potentially offering improved selectivity and reduced side effects in treating inflammatory conditions like IBD, psoriasis, and rheumatoid arthritis.

How can fluorescent probes like P630-TMR advance both basic research and drug discovery for IL23R?

Fluorescent probes like P630-TMR offer versatile applications that bridge basic research and drug discovery efforts for IL-23R:

• High-throughput screening platforms: P630-TMR can be used in displacement assays to screen compound libraries for molecules that compete for IL-23R binding . This approach enables:

  • Rapid identification of potential therapeutic candidates

  • Quantitative affinity determinations (Ki values)

  • Comparison of binding profiles across receptor variants

• Real-time receptor dynamics visualization: Unlike static binding assays, fluorescent probes permit visualization of receptor trafficking and complex formation in living cells . This capability provides insights into:

  • Receptor internalization kinetics

  • Recycling pathways

  • Ligand-induced conformational changes

• Structure-function relationship elucidation: When combined with receptor mutagenesis, fluorescent probes help map the functional domains of IL-23R . Studies demonstrate that P630-TMR binding varies when receptors contain modifications, providing information about:

  • Critical binding residues

  • Allosteric modulation sites

  • Receptor subunit interfaces

• Pharmacokinetic/pharmacodynamic modeling: Fluorescent probes enable direct measurement of compound binding to IL-23R in complex biological systems, facilitating:

  • Target engagement assessment

  • Residence time determination

  • Tissue distribution visualization

The dual utility of these probes for both basic science and applied drug discovery accelerates the translation of fundamental IL-23R insights into clinically relevant therapeutic approaches.

What are the critical quality control steps for working with recombinant IL23R in experimental systems?

Working with recombinant IL-23R requires rigorous quality control to ensure experimental reliability. Critical quality control steps include:

• Verification of receptor expression and localization:

  • Confirm proper folding and cell surface expression using flow cytometry

  • Verify co-expression of IL-12Rβ1 when studying complete receptor complexes

  • Validate subcellular localization using microscopy to ensure proper trafficking

• Functional validation of receptor activity:

  • Assess IL-23-induced signaling by measuring phosphorylation of downstream mediators (JAK2, TYK2, STAT3)

  • Confirm receptor-mediated cytokine production

  • Verify ligand-induced receptor internalization and recycling dynamics

• Binding property characterization:

  • Determine binding affinity parameters (Kd, Bmax) using fluorescent probes like P630-TMR

  • Confirm specificity through competition assays with unlabeled ligands

  • Establish standard curves to normalize for expression level variations

• Sequence verification of receptor constructs:

  • Confirm sequences using Sanger sequencing, particularly after mutagenesis

  • Verify complete reading frames and tag orientations in fusion constructs

  • Check for unintended mutations that might affect receptor function

Implementation of these quality control steps ensures that experimental observations are attributable to the biological properties of IL-23R rather than technical artifacts or compromised reagents.

How should researchers address data variability in IL23R binding and signaling studies?

Addressing data variability in IL-23R studies requires systematic approaches to identify and control sources of variation:

• Standardization of receptor expression systems:

  • Use inducible expression systems to control expression levels

  • Establish standard curves using NanoLuc-labeled receptors to quantify expression

  • Include multiple biological replicates (n ≥ 5) to account for expression variability

• Optimization of experimental timing:

  • IL-23R demonstrates dynamic surface regulation and signaling complex assembly

  • Studies show dramatic changes in JAK2/TYK2/STAT3 recruitment at different time points

  • Standardize all timing aspects of experiments, including stimulation duration and measurement intervals

• Control for autocrine/paracrine effects:

  • Research demonstrates that autocrine/paracrine IL-23 significantly impacts experimental outcomes

  • This dependency changes over time (4-8 hours after stimulation)

  • Include neutralizing antibodies as controls to distinguish direct vs. indirect effects

• Data normalization and statistical approaches:

  • Apply appropriate mathematical models for binding data that account for specific and non-specific components

  • Use the equation: $BRET\; ratio=\frac{B_{\max}[A]}{([A]+K_{D})}+((B[A])+C)$ for binding analysis

  • Report both means and standard errors (SEM) for all quantitative measurements

These methodological refinements minimize experimental variability and enhance reproducibility, allowing more reliable comparison of results across different experimental conditions and research groups.

What are the most common pitfalls when studying IL23R in different cellular contexts?

When studying IL-23R across different cellular contexts, researchers should be aware of several common pitfalls that can compromise experimental validity:

• Assuming receptor complex composition is constant:

  • Different cell types may express varying levels of IL-23R and IL-12Rβ1

  • The IL-23R/IL-12Rβ1 ratio significantly impacts binding properties and signaling outcomes

  • Always quantify both receptor components independently in each cell type studied

• Overlooking dynamic receptor regulation:

  • IL-23R undergoes clathrin and dynamin-mediated endocytosis

  • Surface expression changes dramatically after stimulation

  • Sampling at single time points may miss critical aspects of receptor biology

• Neglecting autocrine signaling loops:

  • Research shows that NOD2-induced cytokines depend on autocrine/paracrine IL-23

  • This dependency changes over time (decreasing by 4-8 hours)

  • Control experiments with neutralizing antibodies are essential to distinguish direct vs. indirect effects

• Improper positioning of tags and labels:

  • Tag position significantly impacts receptor function and BRET efficiency

  • Studies demonstrate that N-terminal NanoLuc tags provide higher BRET efficiency than C-terminal tags

  • Validate that tagged constructs retain wild-type binding and signaling properties

Awareness of these pitfalls and implementation of appropriate controls enables more reliable investigation of IL-23R biology across diverse cellular contexts and experimental systems.