FCGR2A Human

What is FCGR2A and what is its primary function in human immunity?

FCGR2A, also known as CD32A, is a protein-coding gene located on chromosome 1 in humans. It encodes a low-affinity receptor for the Fc region of immunoglobulin G (IgG) . This receptor is expressed on various immune cells, including macrophages and neutrophils, and plays a crucial role in immune function .

The primary function of FCGR2A involves binding to the Fc region of IgG, which triggers cellular responses against pathogens and soluble antigens. This interaction promotes important immune processes including phagocytosis of opsonized antigens . FCGR2A is specifically involved in clearing spent immune complexes from circulation and facilitating phagocytosis by immune cells .

Unlike the inhibitory FCGR2B receptor, FCGR2A contains amino acid sequences (167R and 167H) that are characteristic of an immunoreceptor tyrosine-based activation motif, indicating its role in activating immune responses .

What are the common alternative names and identifiers for FCGR2A?

Researchers should be aware of the various nomenclature used for FCGR2A in scientific literature to ensure comprehensive literature searches and proper identification in genomic and proteomic databases:

• CD32

• CD32A

• CDw32

• FCG2

• FCGR2

• FCGR2A1

• FcGR

• FcgammaRIIa

• IGFR2

This variety of identifiers reflects the evolution of naming conventions and the receptor's characterization across different research contexts and experimental systems.

What polymorphisms in FCGR2A have clinical significance?

FCGR2A exhibits several polymorphisms with significant clinical implications. The most well-studied polymorphism is the FCGR2A-131H/R variant, which affects the receptor's binding affinity to different IgG subclasses.

The 131H/H genotype has been associated with significantly elevated IFN-γ release compared to the 131R/R genotype when stimulated with certain antibodies, such as Campath and IgG1 Fc hexamer in whole blood assays . This suggests functional differences in immune response depending on genotype.

Additionally, FCGR2A polymorphisms have been associated with:

• Increased risks of nephritis and lupus

• Shorter allograft survival in transplant recipients

• Differential survival outcomes in cancer patients, particularly in head and neck squamous cell carcinoma

These polymorphisms provide important genetic markers for predicting disease susceptibility and treatment responses, making them valuable targets for personalized medicine approaches.

How does FCGR2A expression correlate with cancer prognosis and immune infiltration?

FCGR2A has emerged as a potential prognostic marker in multiple cancer types, with particularly strong evidence in head and neck squamous cell carcinoma (HNSC) and clear cell renal cell carcinoma (ccRCC).

In HNSC, FCGR2A expression is significantly higher in carcinoma tissues compared to adjacent normal tissues. Notably, FCGR2A expression varies by tumor grade, with significant differences observed between grades 1, 2, and 3. Interestingly, grade 4 tissues show the lowest FCGR2A expression .

Research has demonstrated a correlation between FCGR2A gene expression and distal HNSC metastasis, as well as patient survival and prognosis. Validation using the GSE65858 dataset confirmed that FCGR2A expression significantly correlates with:

• Total survival (p = 0.0107)

• Progression-free survival (p = 0.0362)

In ccRCC, FCGR2A expression correlates with several clinical parameters:

FCGR2A expression is higher in TNM III tumors compared to TNM II tumors, and higher in Fuhrman stage III and IV tumors compared to Fuhrman stage I tumors .

These findings suggest FCGR2A may serve as a valuable prognostic biomarker, with potential applications in risk stratification and treatment decision-making for cancer patients.

What experimental systems are available for studying FCGR2A function?

Several experimental systems have been developed to investigate FCGR2A function, ranging from protein-binding assays to genetic manipulation techniques:

AlphaLISA Binding Assay

The AlphaLISA detection system provides a sensitive method for studying FCGR2A and IgG Fc fragment binding. This assay uses:

• IgG Fc AlphaLISA acceptor beads to capture human FCGR2A

• Streptavidin-coated donor beads to capture biotinylated human FCGR2A

When donor and acceptor beads come into proximity through IgG Fc fragment binding to FCGR2A, excitation of donor beads triggers a cascade of energy transfer reactions in the acceptor beads, resulting in light emission at 615 nm .

This system can be used to test competitive binding of different IgG subclasses to FCGR2A, with typical IC50 values of 4.6, 6.4, 15.8, and 1.7 μg/mL for IgG1, IgG2, IgG3, and IgG4 respectively .

CRISPR/Cas9 Genetic Modification

CRISPR/Cas9 technology has been adapted for FCGR2A research through:

• CRISPR guide RNA designs: Specialized gRNA sequences designed by Feng Zhang's laboratory at the Broad Institute specifically target FCGR2A with minimal off-target effects. These designs follow criteria detailed in Sanjana et al. (2014) .

• Lentiviral CRISPR/Cas9 systems: Ready-to-use lentiviral particles containing CRISPR/Cas9 gene driven by an EF1A promoter, along with 5 sgRNAs targeting human FCGR2A. These replication-incompetent, HIV-based VSV-G pseudo-typed lentiviral particles can transduce almost all types of mammalian cells, including primary and non-dividing cells .

In Vitro Immune Cell Response Assays

Several assay formats have been developed to evaluate FCGR2A-dependent responses:

• PBMC-based assays: Freeze-thawed/pre-cultured peripheral blood mononuclear cells can be stimulated with various monoclonal antibodies to evaluate T-cell proliferation and cytokine release.

• Whole blood assays: These have shown greater sensitivity for detecting FCGR2A genotype-dependent responses, with significant associations observed between FCGR2A-131H/H genotype and elevated IFN-γ release when stimulated with specific antibodies .

The choice of assay format significantly impacts the ability to detect FCGR2A genotype-dependent effects, with whole blood assays demonstrating superior sensitivity compared to pre-cultured PBMC assays .

How do FCGR2A polymorphisms affect therapeutic antibody efficacy?

FCGR2A polymorphisms can substantially impact therapeutic antibody efficacy, making genotyping potentially important for personalizing immunotherapy approaches. The differential binding affinity of FCGR2A variants to IgG subclasses affects antibody-dependent cellular cytotoxicity (ADCC) and other Fc-mediated effector functions.

These findings highlight the importance of selecting appropriate assay formats when evaluating the impact of FCGR2A genotypes on therapeutic antibody responses. The lack of correlation in PBMC-based assays despite reproducible and stable donor-specific responses over time suggests that other factors beyond FCGR2A polymorphisms may influence therapeutic antibody efficacy in this experimental context .

The discrepancy between assay formats underscores the complexity of translating in vitro findings to clinical outcomes and emphasizes the need for multiple complementary approaches when assessing the impact of FCGR2A genotypes on therapeutic antibody efficacy.

What methodologies are used for FCGR2A genotyping in research and clinical settings?

Accurate FCGR2A genotyping is essential for research investigating receptor function and clinical studies examining associations with disease risk or treatment response. Several methodologies have been developed:

Multiplex Ligation-Dependent Probe Amplification (MLPA)

MLPA has been used successfully for FcγR genotyping, including FCGR2A polymorphisms, in studies involving healthy donors. This technique allows for simultaneous detection of multiple polymorphisms .

DNA Sequencing

Next-generation sequencing techniques can be employed to identify FCGR2A variants with high accuracy. This approach is particularly valuable for discovering novel polymorphisms beyond well-characterized variants.

PCR-Based Methods

Various PCR-based approaches, including allele-specific PCR and restriction fragment length polymorphism (RFLP) analysis, have been developed for targeted genotyping of common FCGR2A polymorphisms.

The selection of an appropriate genotyping method depends on the specific research question, required throughput, available resources, and need for comprehensive versus targeted polymorphism analysis.

How can FCGR2A be targeted for therapeutic development?

FCGR2A represents a potential therapeutic target, particularly in immune-related diseases and cancer. Several approaches for targeting FCGR2A in therapeutic development include:

Antibody Engineering

Therapeutic antibodies can be engineered to optimize their interaction with FCGR2A, enhancing effector functions like ADCC and phagocytosis. This approach can improve the efficacy of antibody therapies against cancer and infectious diseases.

The AlphaLISA Human FCGR2A/CD32a(167R) Binding Kit facilitates the design and development of antibody therapeutics by using competitive binding assays. With a sensitivity of IC50 7.19 μg/mL (average, with IgG whole antibody), this tool enables researchers to screen and optimize antibody candidates for FCGR2A binding .

Gene Editing Approaches

CRISPR/Cas9 technology offers the potential to modify FCGR2A expression or function in cellular therapies. Available tools include:

• FCGR2A CRISPR guide RNA with sequences designed to uniquely target the FCGR2A gene

• FCGR2A CRISPR/Cas9 Lentivirus (Integrating) systems that contain a CRISPR/Cas9 gene driven by an EF1A promoter, along with 5 sgRNAs targeting human FCGR2A

These genetic modification approaches provide platforms for developing cellular therapies with enhanced or altered FCGR2A function.

Small Molecule Modulators

Research into small molecules that can modulate FCGR2A function represents another therapeutic avenue. These could potentially enhance or inhibit FCGR2A signaling in specific disease contexts.

The development of FCGR2A-targeted therapeutics must consider polymorphic variations in the receptor, as these can significantly affect binding affinity and functional outcomes. Personalized approaches based on FCGR2A genotyping may be necessary to maximize therapeutic efficacy and minimize adverse effects.

What are best practices for experimental design when studying FCGR2A variants?

When designing experiments to study FCGR2A variants, researchers should consider several important factors to ensure valid and reproducible results:

Genotyping Considerations

• Perform comprehensive genotyping of all relevant FCGR2A polymorphisms, particularly the functionally significant 131H/R variant

• Consider genotyping additional FcγR family members (FCGR2B, FCGR2C, FCGR3A) as they may have compensatory or interactive effects

• Validate genotyping results using more than one method when possible

Assay Selection

• Choose assay formats carefully, as they significantly impact detection of FCGR2A genotype-dependent effects

• Whole blood assays have demonstrated superior sensitivity for detecting FCGR2A genotype-associated responses compared to isolated PBMC assays

• For PBMC-based assays, consider the impact of freeze-thawing and pre-culture conditions on cellular responses

• When evaluating antibody binding, the AlphaLISA system offers sensitive detection of FCGR2A-IgG interactions

Control Selection

• Include both positive and negative controls appropriate for the specific assay

• When using CRISPR/Cas9 approaches, include controls for off-target effects

• For competitive binding assays, include multiple IgG subclasses as reference standards

Statistical Considerations

• Plan for sufficient sample sizes to detect genotype-dependent effects

• Consider using multivariate analysis to account for other genetic and demographic factors

• For survival analyses, both univariate and multivariate Cox regression should be employed, as demonstrated in ccRCC studies

How should data from FCGR2A expression studies be interpreted in clinical contexts?

Interpreting FCGR2A expression data in clinical contexts requires careful consideration of several factors:

Tissue-Specific Expression Patterns

FCGR2A expression varies across different tissues and cell types. The Human Protein Atlas database provides valuable reference data on FCGR2A expression across multiple cancers and normal tissues .

Correlation with Clinical Parameters

When analyzing FCGR2A expression in relation to clinical outcomes, researchers should:

• Assess correlations with established clinical parameters (tumor size, stage, grade)

• Use appropriate statistical methods such as Pearson chi-squared test and Spearman-Rho test to explore associations between FCGR2A expression and clinical parameters

• Calculate hazard ratios using univariate and multivariate Cox regression models

• Construct receiver operating characteristic (ROC) curves to evaluate FCGR2A as a potential diagnostic marker

Integration with Other Biomarkers

FCGR2A expression should be considered in the context of other biomarkers and immune cell infiltration patterns. Protein-protein interaction networks can help identify related molecules that may contribute to observed clinical effects. Key proteins that interact with FCGR2A include CD3G, PLCG2, LAT, LYN, SYK, FCGR3A, PIK3R1, HCK, ITGAM, and ITGB2 .

Consideration of Tumor Heterogeneity

Expression patterns may vary within tumors and across patients. In HNSC, for example, FCGR2A expression varies significantly by tumor grade, with lower expression observed in grade 4 tissues despite generally higher expression in carcinoma compared to normal tissues .

What are emerging areas of investigation for FCGR2A in human disease?

Several promising research directions are emerging in the field of FCGR2A biology and its role in human disease:

FCGR2A in Immune Checkpoint Inhibitor Response

Investigating how FCGR2A polymorphisms and expression levels affect responses to immune checkpoint inhibitors represents an important frontier in cancer immunotherapy research. The role of FCGR2A in modulating antibody-dependent effects of these therapies could provide insights for patient selection and combination therapy approaches.

FCGR2A and COVID-19 Pathogenesis

The potential role of FCGR2A in COVID-19 susceptibility and severity merits investigation, given its involvement in immune complex clearance and inflammatory responses. Polymorphisms affecting binding affinity could influence disease outcomes and vaccine responses.

FCGR2A in Neurodegenerative Diseases

Emerging evidence suggests a role for immune receptors, including Fc receptors, in neurodegenerative conditions. Investigating FCGR2A expression and function in microglia and other CNS immune cells could reveal new mechanisms in diseases like Alzheimer's and Parkinson's.

FCGR2A in Cellular Therapy Development

Exploring how FCGR2A modification using CRISPR/Cas9 technology could enhance the function of adoptive cellular therapies represents an exciting avenue for improving cancer immunotherapies.

What technological advances are improving FCGR2A research capabilities?

Recent technological advances are significantly enhancing capabilities for studying FCGR2A:

Advanced CRISPR/Cas9 Systems

Increasingly sophisticated CRISPR/Cas9 systems, including those with multiple guide RNAs targeting FCGR2A , enable more precise genetic manipulation for functional studies and therapeutic development.

Single-Cell Analysis

Single-cell RNA sequencing and proteomics approaches allow for detailed characterization of FCGR2A expression and function at the individual cell level, revealing heterogeneity within immune cell populations and providing insights into cell-specific roles.

Improved Structural Biology Techniques

Advanced structural biology methods, including cryo-electron microscopy, are enabling more detailed understanding of FCGR2A-IgG interactions and how polymorphisms affect binding and signaling.

In Vitro Model Systems

Development of more physiologically relevant in vitro systems, such as organoids and microphysiological systems, provide improved platforms for studying FCGR2A function in tissue-specific contexts beyond traditional cell culture models.

These technological advances are expanding the scope and precision of FCGR2A research, promising new insights into its biological functions and therapeutic applications.