Recombinant Human Interleukin-17 receptor A (IL17RA), partial (Active)

What is the structural organization of human IL-17RA and how does it influence ligand binding?

IL-17RA possesses a complex extracellular domain (ECD) architecture that critically influences its ligand binding properties. The ECD consists of multiple fibronectin-type III-like domains organized in a specific spatial arrangement. Crystal structure analysis of human IL-17RA in the unliganded state reveals a conserved dimerization interface that facilitates receptor-receptor interactions even in the absence of ligand binding. This pre-existing dimerization interface becomes particularly important when IL-17 cytokines bind to the receptor . The ECD can be subdivided into domains D1-D2 that directly interact with IL-17 cytokines, while the D3-D4 domains serve as structural elements that position the receptor properly relative to the cell membrane .

Upon ligand binding, IL-17RA undergoes significant conformational changes, including an "elbow motion" that allows optimal positioning of the D2 domain for interactions with IL-17 family members. The C-terminal tail of IL-17 cytokines adopts an extended conformation when binding to IL-17RA, forming β-strand/β-strand interactions with the D2 domain of the receptor . This flexibility in both the receptor and ligand structures ensures optimal interaction surfaces between IL-17RA and its various ligands.

How does IL-17RA interact with other IL-17 receptor family members?

IL-17RA serves as a shared co-receptor for several IL-17 family members, including IL-17A, IL-17C, IL-17E, IL-17F, and the IL-17A/F heterodimer . Signaling through IL-17RA requires the formation of heteromeric receptor complexes with specific partner receptors depending on the cytokine:

• IL-17A, IL-17F, and IL-17A/F heterodimer: IL-17RA pairs with IL-17RC

• IL-17E: IL-17RA pairs with IL-17RB

• IL-17C: IL-17RA pairs with IL-17RE

• IL-17A can also signal through IL-17RA/IL-17RD complexes

The structural arrangement of these receptor complexes determines signaling specificity. For example, IL-17A binding induces IL-17RA dimerization, which then drives the formation of a 2:2:2 hexameric signaling assembly (signalosome) with IL-17RC . This hexameric complex has a specific geometry where the juxtamembrane regions of the two IL-17RC subunits are positioned approximately 90Å apart, which is crucial for proper intracellular signal transduction .

An interesting species-specific interaction has been documented: human IL-17RA does not appear to form productive complexes with mouse IL-17RC, highlighting the importance of considering species compatibility in experimental design .

What are the key downstream effects of IL-17RA signaling in different cell types?

IL-17RA signaling produces distinct cellular responses depending on cell type and microenvironment:

In keratinocytes:

• Potentiates expression of IL-36γ and CXCL1 mRNA

• Synergizes with TNF-α to induce inflammatory mediators

• Triggers production of antimicrobial peptides

In mesenchymal stem cells (MSCs):

• Regulates immunosuppressive properties affecting Th17 cell proliferation and differentiation

• Controls expression of VCAM1, ICAM1, and PD-L1, which mediate MSC interactions with immune cells

• Influences MSC therapeutic potential in experimental autoimmune encephalomyelitis (EAE)

In multiple cell types:

• Promotes T cell activation

• Induces production of IL-6, G-CSF, and SCF

• Stimulates production of pro-inflammatory chemokines

• Enhances neutrophil generation and recruitment to inflammation sites

These diverse effects make IL-17RA a central player in both protective immunity and pathological inflammation.

What methodologies are most effective for studying IL-17RA dimerization?

The study of IL-17RA dimerization requires specialized approaches to capture both structural arrangements and functional consequences:

Structural methods:

• X-ray crystallography has successfully revealed the dimerization interface of IL-17RA, showing critical residues like alanine 104 located within the α-helical region of the BC loop at the heart of the IL-17RA-IL-17RA protein-protein interface

• Solution-based biophysical techniques including size-exclusion chromatography, analytical ultracentrifugation, and multi-angle light scattering can confirm the dimerization status of recombinant IL-17RA

• FRET/BRET (Förster/Bioluminescence Resonance Energy Transfer) assays using differentially tagged IL-17RA constructs can monitor dimerization in living cells

Functional approaches:

• Site-directed mutagenesis targeting key residues at the dimerization interface (e.g., alanine 104) can generate dimerization-defective variants for comparative studies

• Co-immunoprecipitation experiments using differently tagged versions of IL-17RA can biochemically detect receptor oligomerization

• Signaling assays comparing wild-type IL-17RA and dimerization-defective mutants can reveal the functional importance of dimerization in downstream effects

When studying IL-17RA dimerization, researchers should consider that IL-17RA exists as a multimer on the cell surface even without ligand binding, suggesting pre-formed receptor complexes may be important for signaling initiation .

How can recombinant IL-17RA be optimally expressed and purified for structural studies?

Successful expression and purification of functional recombinant IL-17RA requires careful consideration of several factors:

Expression systems:

• Mammalian expression systems (HEK293, CHO cells) are preferred for human IL-17RA as they provide appropriate post-translational modifications and protein folding machinery

• For the extracellular domain only, insect cell expression (Sf9, Hi5) can offer higher yields while maintaining proper folding

• Bacterial systems generally yield improperly folded IL-17RA due to the complexity of its structure and need for glycosylation

Construct design:

• Including fusion tags (Fc, His, Avi-tag) can facilitate purification and detection

• For structural studies, it's often advantageous to express only the extracellular domain (ECD)

• The Avi-tag approach allows precise biotinylation at a single site, ensuring uniform protein orientation when bound to streptavidin-coated surfaces without interfering with bioactivity

Purification strategy:

• Multi-step purification typically includes affinity chromatography (based on the fusion tag), followed by ion-exchange and size-exclusion chromatography

• Buffer optimization is crucial for maintaining stability of the purified protein

• For crystallography, additional steps to remove flexible regions or heterogeneity may be necessary

Quality control:

• Biophysical techniques (circular dichroism, thermal shift assays) should be employed to confirm proper folding

• Functional binding assays with IL-17 family cytokines verify biological activity

• Mass spectrometry can confirm protein identity and assess post-translational modifications

What assays are most reliable for evaluating IL-17RA-dependent cellular responses?

Several complementary approaches can robustly measure IL-17RA-dependent cellular responses:

Signaling activation:

• Phospho-specific Western blotting to detect activation of downstream signaling molecules (e.g., NF-κB pathway components)

• Reporter gene assays using promoters of IL-17RA-responsive genes driving luciferase expression

• Flow cytometry-based phospho-flow analysis for single-cell resolution of signaling events

Gene expression:

• Quantitative PCR for measuring induction of known downstream genes like IL-36γ and CXCL1, which are potentiated by IL-17RA dimerization

• RNA-seq to comprehensively profile the IL-17RA-dependent transcriptome

• ELISA or multiplex cytokine assays to quantify secreted proteins induced by IL-17RA signaling

Cell-type specific functional assays:

• In keratinocytes: antimicrobial peptide production, barrier function assays

• In MSCs: T-cell proliferation suppression assays, analysis of immunoregulatory molecule expression (VCAM1, ICAM1, PD-L1)

• In immune cells: cytokine production profiles, cell differentiation and activation markers

For rigorous analysis, using IL-17RA knockout cells reconstituted with either wild-type or dimerization-defective IL-17RA provides a clean system to assess specific receptor functions. This approach has successfully demonstrated that IL-17RA dimerization lowers the threshold for IL-17-induced expression of downstream effector molecules .

How does IL-17RA dimerization mechanistically affect downstream signaling pathways?

IL-17RA dimerization represents a critical molecular switch that precisely controls signal transduction through several mechanisms:

Spatial organization of signaling components:

• IL-17-induced IL-17RA dimerization drives formation of a 2:2:2 hexameric signalosome with IL-17RC

• This architectural arrangement positions the juxtamembrane regions of IL-17RC subunits approximately 90Å apart

• The precise geometry of the complex determines the spatial organization of intracellular signaling components

Signal amplification:

• Dimerization of IL-17RA creates an optimal platform for recruiting adapter proteins and signaling enzymes

• The formation of the complete hexameric complex with IL-17RC enhances signaling efficiency

• Studies with dimerization-defective IL-17RA mutants show reduced expression of downstream targets like IL-36γ and CXCL1, indicating that dimerization amplifies signal strength

Pathway specificity:

• Different ligand-induced conformational changes in the IL-17RA dimer may differentially affect recruitment of various adaptor proteins

• The specific architecture of the IL-17RA-containing complex may determine which downstream pathways are preferentially activated

• IL-17RA dimerization may create unique binding surfaces for specific intracellular signaling molecules

Studies using dimerization-defective IL-17RA variants have revealed that while IL-17RC appears to play a key structural role in driving the spatial organization of intracellular signaling components, IL-17RA dimerization plays a more indirect but equally important role in defining the precise geometry of the signaling complex .

What are the consequences of IL-17RA genetic variants or mutations on receptor function?

Genetic variations in IL-17RA can substantially alter receptor function through multiple mechanisms:

Structural impacts:

• Mutations in the dimerization interface (such as at alanine 104) can disrupt receptor dimerization, reducing signaling capacity

• Variants in ligand-binding domains may alter affinity for specific IL-17 family members

• Mutations in intracellular domains can affect recruitment of signaling adaptors

Physiological consequences:

• IL-17RA deficiency in MSCs significantly impairs their immunosuppressive function and therapeutic potential in EAE, demonstrating the receptor's importance in stem cell-mediated immunomodulation

• Complete loss of IL-17RA function compromises host defense against certain microbial infections

• IL-17RA mutations may contribute to autoimmune disease susceptibility or protection

Experimental applications:

• Dimerization-defective IL-17RA variants serve as valuable tools to study the specific role of receptor dimerization in signaling

• Domain-specific mutations help identify critical regions for different IL-17RA functions

• Studying naturally occurring IL-17RA variants can reveal novel aspects of receptor biology

When designing IL-17RA mutants for research, several considerations are important:

• The location of the mutation relative to functional domains

• Whether the mutation affects only one function (e.g., dimerization) or has pleiotropic effects

• The potential impact on protein stability and expression

How can IL-17RA be targeted in therapeutic development research?

Recombinant IL-17RA serves as both a research tool and template for therapeutic development:

As a research tool:

• Soluble IL-17RA-Fc fusion proteins can act as cytokine traps in experimental systems

• Structure-based design of IL-17RA variants with altered binding properties can help dissect pathway-specific effects

• Avi-tagged IL-17RA allows uniform orientation on streptavidin surfaces for binding studies and drug screening

Therapeutic development strategies:

• Decoy receptors based on IL-17RA extracellular domain can neutralize IL-17 family cytokines

• Small molecule inhibitors targeting the IL-17RA dimerization interface could prevent signalosome formation

• Monoclonal antibodies against specific epitopes of IL-17RA can block particular ligand interactions while preserving others

Experimental models for testing interventions:

• IL-17RA knockout cell lines reconstituted with various receptor constructs provide clean systems for evaluating targeted therapeutics

• EAE animal models are valuable for testing IL-17RA-targeting strategies in neuroinflammation

• Human keratinocyte models can assess effects on skin inflammation pathways

When developing IL-17RA-targeting therapeutics, it's important to consider the receptor's involvement in both pathological inflammation and beneficial host defense, necessitating careful specificity in intervention approaches.

How does IL-17RA signaling interact with other inflammatory pathways?

IL-17RA signaling exhibits complex interactions with other inflammatory pathways, creating integrated networks that shape immune responses:

Synergy with TNF-α:

• IL-17A and IL-17F synergize with TNF-α in inducing CXCL1, G-CSF, and IL-6

• This synergistic effect requires both TNF receptor I and TNF receptor II

• The molecular mechanisms involve cooperative effects on transcription factor recruitment and chromatin remodeling

Inhibitory interactions:

• IL-17/IL-17RA interactions can inhibit TNF-α-induced upregulation of fibroblast CCL5 and VCAM-1

• Different IL-17RA-dependent responses show differential sensitivity to blocking antibodies, suggesting divergent intracellular signaling pathways

Effects on MSC immunomodulation:

• The IL-17/IL-17RA axis is critical for MSC "licensing" - the process that enhances their immunosuppressive properties

• IL-17RA-dependent signals in MSCs regulate their ability to inhibit pathogenic Th17 cells and promote regulatory T cell generation

• Pre-treatment of MSCs with IL-17A enhances their therapeutic effect in EAE, demonstrating practical applications of this pathway interaction

These complex interactions highlight the context-dependent nature of IL-17RA signaling and suggest that targeted modulation could have pathway-specific effects.

What role does IL-17RA play in disease models and potential therapeutic applications?

IL-17RA has been implicated in various disease models with significant therapeutic implications:

In autoimmune diseases:

• In the EAE model of multiple sclerosis, wild-type MSCs significantly reduced disease severity, while IL-17RA-deficient MSCs worsened disease progression

• IL-17RA expression on MSCs was required for reducing Th17 cell frequency in draining lymph nodes and for generating CD4+CD25+Foxp3+ regulatory T cells

• IL-17RA-dependent signals are critical for MSC immunosuppressive functions in autoimmune contexts

In inflammatory conditions:

• IL-17RA activity contributes to neutrophil generation and recruitment to inflammation sites

• The receptor is required for host defense against microbial infections

• IL-17RA signaling influences the progression of arthritis from inflammation to destructive joint erosion

Therapeutic implications:

• Enhancement of IL-17RA signaling in MSCs could potentially improve their therapeutic efficacy in multiple sclerosis treatment

• Modulation of the IL-17/IL-17RA axis represents a promising approach for controlling inflammatory diseases

• Targeted interventions affecting specific IL-17RA-dependent pathways could provide selective immunomodulation

These findings highlight the dual role of IL-17RA in both pathological and beneficial immune responses, necessitating careful consideration in therapeutic targeting.