FCER1G Antibody

What is FCER1G and what is its significance in immunology?

FCER1G (Fc receptor gamma-chain, FcRγ), also known as Fc-epsilon RI-gamma or IgE Fc receptor subunit gamma, is a critical component of the high-affinity immunoglobulin E (IgE) receptor (Fc epsilon RI). This receptor exists as a tetrameric complex comprising one alpha subunit, one beta subunit, and two disulfide-linked gamma subunits . FCER1G contains an immunoreceptor tyrosine-based activation motif (ITAM) that functions to transduce activation signals from various immunoreceptors, making it a key player in immune signaling pathways . Recent research has highlighted FCER1G's role as an adapter protein that stabilizes cell membrane partners in group 3 innate lymphoid cells (ILC3s) and promotes anti-infection immunity against bacterial and fungal infections . The significance of FCER1G extends beyond mast cells and basophils, as it influences the transcriptional state and proinflammatory cytokine production of ILC3s through the CD16-FcεR1γ signaling pathway .

How does FCER1G function at the molecular level?

At the molecular level, FCER1G functions as a critical adapter protein in immune cells. It contains an immunoreceptor tyrosine-based activation motif (ITAM) that becomes phosphorylated upon receptor engagement, initiating downstream signaling cascades . The protein stabilizes various cell membrane partners through specific amino acid residues that are essential for maintaining binding partner interactions. Alanine scanning mutagenesis analysis has identified key residues in FcεR1γ that are critical for maintaining the abundance of its binding partners . FCER1G directly impacts membrane abundance of critical cell surface receptors such as NKp46 and CD16 in ILC3s and CD64 in other cell types . The molecular weight of FCER1G is approximately 10 kDa, consisting of 87 amino acids, and it functions as a single-pass type I membrane protein localized to the cell membrane .

What cell types express FCER1G and what receptors does it associate with?

FCER1G is widely expressed across various immune cell types, not just limited to mast cells and basophils where Fc epsilon RI is exclusively found . It associates with multiple immunoreceptors beyond the high-affinity IgE receptor, including various Fc gamma receptors (FcγR). Research has identified its expression in group 3 innate lymphoid cells (ILC3s), where it plays a crucial role in stabilizing NKp46 and CD16 receptors . FCER1G is also expressed in monocytes and macrophages, with experimental evidence confirming its presence in cell lines such as RAW 264.7 and THP-1 cells . Additionally, FCER1G gene expression has been detected in peripheral blood monocytes in the context of atopic dermatitis and rheumatoid arthritis studies . The wide expression pattern of FCER1G across different immune cell populations underscores its fundamental importance in various aspects of immune function beyond traditional IgE-mediated allergic responses.

What are the recommended techniques for detecting FCER1G expression in different cell types?

Several validated techniques are available for detecting FCER1G expression in different cell types. Western blot (WB) analysis is commonly employed at dilutions of 1:500-1:1000, with positive detection confirmed in RAW 264.7 cells and THP-1 cells . Immunohistochemistry (IHC) can be performed at dilutions of 1:50-1:500, with successful detection in human ovary cancer tissue. For optimal results, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may be used as an alternative . Immunofluorescence (IF) and immunocytochemistry (ICC) at dilutions of 1:50-1:500 have been validated in THP-1 cells . Commercial antibodies targeting FCER1G have been validated for reactivity with human and mouse samples, with predicted reactivity in canine, porcine, rat, and rhesus macaque samples based on sequence homology . For immunohistochemical analysis of paraffin-embedded samples, a 1:100 dilution has been shown to be effective, as demonstrated with U873 xenograft samples .

What are the key considerations when using FCER1G antibodies for Western blot analysis?

When using FCER1G antibodies for Western blot analysis, several key considerations must be addressed for optimal results. First, researchers should note that the observed molecular weight of FCER1G is approximately 10 kDa, which aligns with its theoretical molecular weight . This small size may require special attention to gel percentage and running conditions to properly resolve the protein. Second, antibody dilution optimization is critical; a range of 1:500-1:1000 is typically recommended, but sample-dependent optimization may be necessary . Third, the storage and handling of antibodies significantly impact performance; FCER1G antibodies are typically stored at -20°C in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For protein extraction, careful consideration of lysis buffers is necessary to effectively solubilize this membrane-associated protein without disrupting its structure. Additionally, appropriate positive controls such as RAW 264.7 or THP-1 cell lysates should be included to validate antibody specificity . Lastly, be aware that post-translational modifications, cleavages, and relative charges may affect the observed molecular weight of the protein in experimental conditions .

How can researchers assess FCER1G methylation status, and what is its significance?

Researchers can assess FCER1G methylation status through several epigenetic analysis techniques. Quantitative Methylation-Specific PCR (QMSP) has been successfully employed to evaluate methylation levels at specific CpG islands within the FCER1G gene, particularly at positions −256, −93, and +57 relative to the transcription start site (TSS) . For designing methylation-specific primers, tools such as MethPrimer Software can be utilized, followed by in silico validation using BiSearch to ensure specificity . The experimental workflow should include controls using fully methylated and unmethylated DNA (such as the EpiTect PCR Control DNA Set from Qiagen) to validate primer specificity under QMSP conditions .

The methylation status of FCER1G has significant implications for disease mechanisms. Studies have shown that FCER1G hypomethylation occurs in patients with rheumatoid arthritis, suggesting potential value as an epigenetic marker of the disease that appears to be independent of disease activity . The inverse relationship between methylation status and gene expression means that lower methylation is associated with higher mRNA expression, potentially leading to increased protein production . This hypomethylation may result in activation of FcRγ-dependent pathways with proinflammatory effects, contributing to disease pathogenesis . Thus, assessing FCER1G methylation may provide insights into both disease mechanisms and potential diagnostic or prognostic biomarkers.

How does FCER1G function in innate lymphoid cells and anti-infection immunity?

FCER1G plays a crucial role in innate lymphoid cells, particularly group 3 innate lymphoid cells (ILC3s), where it functions as a key adapter protein stabilizing cell membrane receptors essential for immune responses. Genetic perturbation of FcεR1γ leads to the absence of critical cell membrane receptors NKp46 and CD16 in ILC3s, demonstrating its essential role in maintaining receptor expression . To specifically study this function, researchers have developed conditional knockout models by crossing Fcer1g flox/flox mice with Rorc-cre mice to target deletion in RORγt-expressing cells, which include ILC3s .

Mechanistically, FcεR1γ influences both the transcriptional state and proinflammatory cytokine production of ILC3s through the CD16-FcεR1γ signaling pathway . This signaling is critical for mounting effective immune responses against pathogens. Functional studies have demonstrated that FcεR1γ expression in ILC3s is essential for effective protective immunity against bacterial and fungal infections . The stabilization of receptor complexes appears to be a conserved mechanism by which FcεR1γ maintains its binding partners across different cell types . The identification of specific amino acid residues essential for maintaining binding partner abundance through alanine scanning mutagenesis has provided further insight into the molecular mechanisms underlying FCER1G function in immune cells .

What is the connection between FCER1G methylation and autoimmune diseases?

Research has established a significant connection between FCER1G methylation patterns and autoimmune diseases, particularly rheumatoid arthritis (RA). Studies have identified FCER1G gene hypomethylation in patients with RA, suggesting it may serve as a novel epigenetic marker of the disease that appears to be independent of disease activity . The epigenetic modification of FCER1G has functional consequences, as methylation status inversely correlates with gene expression levels. Lower methylation typically results in higher mRNA expression and potentially increased protein production, which may activate FcRγ-dependent pathways with proinflammatory effects .

While differences in methylation status have been observed between patients with high disease activity and those in remission, these differences did not reach statistical significance in some studies . This suggests that FCER1G hypomethylation may represent a more stable epigenetic marker of the disease rather than a dynamic indicator of current disease activity. The mechanism by which FCER1G hypomethylation contributes to autoimmune pathogenesis likely involves enhanced FcRγ signaling, leading to dysregulated immune responses and inflammation. These findings highlight the potential of epigenetic profiling of FCER1G as both a diagnostic biomarker and a window into understanding the molecular pathogenesis of autoimmune conditions, potentially guiding the development of novel therapeutic approaches that target epigenetic modifications or the FCER1G signaling pathway.

How does FCER1G contribute to antibody effector functions?

FCER1G plays a critical role in antibody effector functions through its association with various Fc receptors and subsequent activation of cellular immune responses. As an adapter protein containing an immunoreceptor tyrosine-based activation motif (ITAM), FCER1G mediates signaling from Fc receptors upon antibody binding, triggering cellular responses such as antibody-dependent cell-mediated phagocytosis (ADCP) and antibody-dependent cell-mediated cytotoxicity (ADCC) .

The involvement of FCER1G in antibody effector functions can be assessed through cellular bioassays. These assays often utilize engineered Jurkat effector cells expressing specific Fc receptors (such as FcγRI/CD64, FcγRIIa/CD32a, FcγRIIb/CD32b, and FcγRIIIa/CD16a) along with an NFAT response element driving luciferase expression . When these effector cells engage with antibody-coated target cells, the resulting signaling cascade activates NFAT-dependent transcription and luciferase production, providing a quantifiable readout of receptor engagement and activation.

Research has demonstrated that modifications to the antibody Fc region can dramatically alter interactions with Fc receptors and consequently affect FCER1G-mediated signaling. For instance, engineered antibodies with modifications such as LALA (L234A/L235A) maintain some binding to FcγRI and can still elicit cellular responses via this receptor . This highlights the complex relationship between antibody structure, Fc receptor binding, and downstream FCER1G-mediated signaling. Understanding these interactions is crucial for developing therapeutic antibodies with optimized effector functions for specific clinical applications.

What mutagenesis approaches can be used to study FCER1G binding interactions?

Alanine scanning mutagenesis has proven to be an effective approach for studying FCER1G binding interactions and identifying critical residues involved in partner stabilization. This method involves systematically replacing individual amino acids in the FCER1G protein with alanine and then assessing the impact on binding partner interactions . When implementing this approach, researchers have used lentiviral expression systems to introduce wild-type FcεR1γ or various mutants into Fcer1g–/– bone marrow-derived macrophages (BMDMs), which serve as a model system more amenable to culture and transfection compared to ILC3s .

The experimental setup typically involves: (1) designing a series of point mutations based on predicted importance of specific amino acids for binding partner interactions; (2) generating lentiviral expression constructs for wild-type and mutant FCER1G; (3) infecting Fcer1g–/– cells with these constructs; and (4) assessing membrane abundance of binding partners such as CD64 through flow cytometry or other quantitative methods . This approach has successfully identified key residues in FcεR1γ that are essential for maintaining the abundance of its binding partners. The results suggest a conserved mechanism by which FcεR1γ maintains its binding partners across different cell types . Alternative approaches could include yeast two-hybrid systems, co-immunoprecipitation followed by mass spectrometry, or proximity labeling techniques to identify novel binding partners and interaction domains.

How can researchers engineer antibodies with modified Fc regions to study FCER1G-mediated signaling?

Researchers can engineer antibodies with modified Fc regions through several sophisticated approaches to study FCER1G-mediated signaling. The process begins with designing synthetic genes encoding wild-type or variant human IgG1 heavy chain constant regions, which can be synthesized commercially and cloned into mammalian expression vectors . Site-directed mutagenesis can be employed to introduce specific amino acid substitutions in the Fc region, such as the LALA (L234A/L235A) mutations that reduce binding to certain Fc receptors . For more complex modifications, synthetic genes containing variant Fc regions can be designed with appropriate restriction sites (such as KasI and SacII) to facilitate efficient cloning into existing antibody expression vectors .

To evaluate the impact of these Fc modifications on FCER1G-mediated signaling, researchers can utilize bioassay systems that measure antibody-dependent cell-mediated functions such as phagocytosis (ADCP) or cytotoxicity (ADCC). These assays typically employ engineered effector cells (such as modified Jurkat cells) expressing specific Fc receptors coupled to a reporter system, such as NFAT-driven luciferase expression . Target cells expressing the cognate antigen (e.g., CD20+ Raji cells) are incubated with the engineered antibodies and effector cells, and the resulting activation is quantified through luciferase activity measurement . This experimental setup allows for systematic comparison of how different Fc modifications affect receptor engagement and subsequent FCER1G-dependent signaling. Additionally, researchers should consider potential immunogenicity of engineered Fc regions, which can be assessed through in vitro T cell proliferation assays testing peptides derived from the modified regions .

What are the latest techniques for studying FCER1G in tissue-specific and conditional knockout models?

Advanced techniques for studying FCER1G in tissue-specific and conditional knockout models have significantly enhanced our understanding of its function in specific cell populations. The Cre-loxP system has been successfully implemented to generate conditional FCER1G knockout models, as demonstrated by the development of Fcer1g-conditional knockout (Rorc-cre + Fcer1g f/f, CKO) mice through crossing Fcer1g flox/flox mice with Rorc-cre mice . This approach specifically targets deletion in RORγt-expressing cells, which includes ILC3s, allowing researchers to examine cell-intrinsic functions of FcεR1γ .

When implementing such models, critical validation steps include confirming the specific deletion of FcεR1γ expression in target cell populations. Flow cytometry can be used to verify the absence of FcεR1γ in RORγt-expressing cells while ensuring other cell populations maintain normal expression . Importantly, researchers must account for potential off-target effects; for instance, while the Rorc-cre system also targets CD3+ T cells (including RORγt+ Th17 cells, Treg cells, and γδT cells), these cells typically do not co-express FcεR1γ with RORγt, minimizing confounding effects in this particular model .

For functional studies, advanced techniques include: (1) ex vivo analysis of cell populations isolated from tissue-specific knockout mice to assess receptor expression, signaling pathway activation, and cytokine production; (2) in vivo challenge models with bacterial or fungal pathogens to evaluate protective immunity; (3) transcriptomic analysis to determine how FCER1G deletion affects gene expression profiles in specific cell types; and (4) high-dimensional techniques such as mass cytometry or single-cell RNA sequencing to comprehensively characterize phenotypic and functional alterations resulting from FCER1G deletion in specific cell populations .

What are common challenges in FCER1G antibody validation and how can they be addressed?

Researchers face several common challenges when validating FCER1G antibodies for experimental applications. First, the relatively small size of FCER1G (10 kDa) can make detection challenging, particularly in Western blot applications where small proteins may transfer inefficiently or run with the dye front . To address this, researchers should optimize gel percentage (12-15% or gradient gels) and transfer conditions (using PVDF membranes with 0.2 μm pore size and methanol-containing transfer buffers).

Second, cross-reactivity with similar proteins can compromise specificity. Comprehensive validation should include positive controls (such as RAW 264.7 or THP-1 cell lysates) and negative controls (such as FCER1G knockout cell lines when available) . Orthogonal validation approaches, combining multiple detection methods (WB, IHC, IF) can provide more robust confirmation of antibody specificity .

Third, variability between antibody lots can impact experimental reproducibility. Researchers should perform lot-to-lot validation when acquiring new antibody stocks and maintain detailed records of antibody performance. For immunohistochemical applications, optimization of antigen retrieval methods is critical; while TE buffer at pH 9.0 is recommended for FCER1G, citrate buffer at pH 6.0 may serve as an alternative .

Lastly, appropriate storage and handling are essential for maintaining antibody performance. FCER1G antibodies should typically be stored at -20°C, and aliquoting is recommended to avoid repeated freeze-thaw cycles that can degrade antibody quality . By addressing these challenges systematically, researchers can ensure more reliable and reproducible results when studying FCER1G expression and function.

How can researchers troubleshoot inconsistent results when detecting FCER1G in different experimental systems?

When encountering inconsistent results in FCER1G detection across different experimental systems, researchers should implement a systematic troubleshooting approach. First, evaluate sample preparation methods, as FCER1G is a membrane-associated protein that requires effective extraction techniques. Different lysis buffers (such as RIPA or NP-40 based buffers with protease inhibitors) may yield varying results depending on the cell type and application . For membrane proteins like FCER1G, inclusion of mild detergents and avoiding harsh sonication can help preserve protein integrity.

Second, optimize antibody conditions specifically for each experimental system. The recommended dilutions (1:500-1:1000 for WB, 1:50-1:500 for IHC/IF) should be considered starting points rather than absolute values . Titration experiments should be performed for each new cell type or tissue, as optimal concentrations may vary significantly depending on FCER1G expression levels and sample complexity.

Third, consider potential post-translational modifications or protein interactions that might mask epitopes in certain contexts. FCER1G contains an ITAM motif that becomes phosphorylated upon activation, potentially affecting antibody binding . Additionally, its association with various receptor complexes might influence epitope accessibility in different cell types.

Fourth, validate results using multiple antibodies targeting different epitopes of FCER1G when possible. Polyclonal antibodies recognizing the C-terminus region may provide different results than those targeting other domains . Finally, incorporate positive controls with known FCER1G expression (such as THP-1 cells) alongside experimental samples to ensure the detection system is functioning properly . By methodically addressing these factors, researchers can identify sources of inconsistency and establish reliable protocols for FCER1G detection across different experimental systems.

What is the optimal approach for quantifying FCER1G expression levels across different immune cell populations?

The optimal approach for quantifying FCER1G expression across different immune cell populations requires a multi-faceted strategy combining several complementary techniques. Flow cytometry represents a powerful method for analyzing FCER1G expression at the single-cell level while simultaneously identifying specific immune cell subsets using lineage markers. This approach allows for direct comparison of expression levels across populations within the same sample. When implementing flow cytometry, careful optimization of fixation and permeabilization protocols is essential since FCER1G is a membrane-associated protein with intracellular signaling components .

For more comprehensive analysis, quantitative PCR (qPCR) can be employed to measure FCER1G transcript levels, providing insights into gene expression regulation. When using this approach, researchers should select appropriate reference genes for normalization and consider the relationship between mRNA and protein levels, which may not always correlate directly due to post-transcriptional regulation . Western blot analysis of sorted cell populations can provide direct quantification of protein levels, though this requires larger cell numbers and may be challenging for rare populations .

For spatial context, immunohistochemistry or immunofluorescence on tissue sections allows visualization of FCER1G expression in the native microenvironment, which can reveal important information about its distribution and potential functional significance . For the most rigorous quantification, mass cytometry (CyTOF) offers advantages by allowing simultaneous detection of numerous markers with minimal spectral overlap, enabling comprehensive immune phenotyping alongside FCER1G quantification.

To account for epigenetic regulation, assessment of FCER1G methylation status using techniques like quantitative methylation-specific PCR can provide additional insights, particularly given the known inverse relationship between methylation and expression levels . By integrating data from these complementary approaches, researchers can develop a comprehensive understanding of FCER1G expression patterns across diverse immune cell populations.

What are promising areas for future research on FCER1G in immune regulation and disease?

Several promising areas for future research on FCER1G in immune regulation and disease are emerging based on recent discoveries. First, the newly established role of FCER1G in group 3 innate lymphoid cells (ILC3s) suggests potential involvement in maintaining intestinal homeostasis and mucosal immunity . Future studies should explore how FCER1G-dependent signaling in ILC3s influences interactions with the gut microbiome and contributes to inflammatory bowel diseases or protective immunity against enteric pathogens.

Second, the discovery of FCER1G hypomethylation in rheumatoid arthritis opens avenues for investigating epigenetic regulation of FCER1G in other autoimmune and inflammatory conditions . Researchers should examine whether similar epigenetic alterations occur in conditions such as systemic lupus erythematosus, psoriasis, or multiple sclerosis, potentially identifying common mechanisms across autoimmune spectrum disorders.

Third, the role of FCER1G in antibody effector functions suggests potential for therapeutic targeting in conditions where antibody responses contribute to pathology . Development of small molecules or biologics that modulate FCER1G-dependent signaling could provide novel approaches for treating antibody-mediated diseases while preserving beneficial immune functions.

Fourth, comprehensive investigation of FCER1G interactions with different Fc receptors across tissue-resident immune cells may reveal tissue-specific functions and regulation . Single-cell multi-omics approaches combining transcriptomic, epigenomic, and proteomic analyses could uncover previously unrecognized heterogeneity in FCER1G expression and function across immune cell populations in health and disease.

Finally, exploring the evolution of FCER1G across species may provide insights into conserved and divergent aspects of antibody-mediated immunity, potentially revealing fundamental principles of immune receptor signaling and offering new perspectives on therapeutic targeting strategies.

How might single-cell technologies advance our understanding of FCER1G function in diverse immune populations?

Single-cell technologies offer unprecedented opportunities to advance our understanding of FCER1G function across diverse immune populations. Single-cell RNA sequencing (scRNA-seq) can uncover the heterogeneity of FCER1G expression patterns within conventionally defined immune cell types, potentially revealing previously unrecognized subpopulations with distinct functional properties. When integrated with T-cell or B-cell receptor sequencing, this approach can link FCER1G expression to specific adaptive immune responses and clonal expansions.

CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) allows simultaneous measurement of surface protein expression and transcriptomes at single-cell resolution, enabling researchers to correlate FCER1G transcript levels with the expression of its binding partners and other functionally relevant proteins . This can provide insights into the co-regulation of receptor complexes and help identify cell states where FCER1G plays particularly critical roles.

Single-cell ATAC-seq (Assay for Transposase-Accessible Chromatin) can reveal the epigenetic landscape governing FCER1G expression across immune cell types, complementing methylation studies and providing a more comprehensive understanding of its transcriptional regulation . When combined with scRNA-seq in multi-modal analyses, this can establish direct links between chromatin accessibility, transcription factor binding, and gene expression.

Spatial transcriptomics techniques add crucial contextual information by preserving tissue architecture, allowing researchers to map FCER1G expression patterns within complex immunological niches such as lymphoid organs, mucosal surfaces, or inflammatory lesions . This spatial context is particularly important for understanding how FCER1G-expressing cells interact with other immune and non-immune cells in health and disease.

Finally, emerging technologies for single-cell proteomics and phospho-proteomics can directly assess FCER1G protein levels and activation status, providing functional information that transcriptomic approaches alone cannot capture. Together, these single-cell approaches promise to transform our understanding of FCER1G from a static component of receptor complexes to a dynamically regulated mediator of context-specific immune functions.

What therapeutic opportunities might emerge from better understanding FCER1G biology?

Advanced understanding of FCER1G biology is unveiling several promising therapeutic opportunities across multiple disease areas. In autoimmune disorders, the discovery of FCER1G hypomethylation in conditions like rheumatoid arthritis suggests potential for epigenetic therapies targeting DNA methyltransferases or using small molecules that can promote re-methylation of specific loci . Additionally, the development of antibodies or small molecules that modulate FCER1G-dependent signaling could provide targeted approaches to dampen pathological immune activation while preserving beneficial immunity.

For infectious diseases, the crucial role of FCER1G in ILC3-mediated protection against bacterial and fungal infections highlights opportunities for immune-enhancing therapies . Strategies that augment FCER1G expression or signaling in specific immune populations could boost anti-infection immunity in immunocompromised patients. Conversely, in allergic conditions where excessive IgE-mediated signaling drives pathology, targeted inhibition of FCER1G interactions with specific receptor partners might offer precision approaches to mitigate allergic inflammation.

In the expanding field of cancer immunotherapy, FCER1G plays a critical role in antibody-dependent cellular functions that are essential for therapeutic antibody efficacy . Engineering antibodies with modified Fc regions to optimize FCER1G-mediated signaling could enhance the potency of antibody therapies while minimizing off-target effects. Combined with the growing arsenal of immune checkpoint inhibitors, such optimized antibodies could provide synergistic approaches to overcoming treatment resistance.

The development of novel biomarkers represents another therapeutic opportunity. FCER1G methylation status or expression patterns across immune cell populations could serve as predictive or prognostic biomarkers in autoimmune diseases, infections, or cancer . These biomarkers might guide treatment selection, monitor disease activity, or identify patients most likely to benefit from specific immunomodulatory approaches. As multi-omics technologies continue advancing, integrated FCER1G-based signatures may emerge as valuable tools for precision medicine applications across immunological disorders.