ADGRG3 Antibody

What is ADGRG3 and what are its structural characteristics?

ADGRG3 (Adhesion G protein-coupled receptor G3), also known as GPR97, belongs to the adhesion G protein-coupled receptors (aGPCRs) family. This receptor consists of a seven-transmembrane domain and a GPCR-Autoproteolysis INducing (GAIN) domain . The GAIN domain facilitates auto-cleavage of the receptor, producing two subunits: the α-subunit (N-terminal fragment, NTF) and the β-subunit (C-terminal fragment, CTF) that remain non-covalently associated at the cell surface . This bipartite structure is critical for receptor function, with the extracellular domain facilitating interactions with ligands and the transmembrane domain mediating signal transduction.

ADGRG3 contains specific extracellular epitopes that are targeted by antibodies, including the amino acid residues 62-75 of human ADGRG3 (sequence CNVENLQRYWLNYE) located at the extracellular N-terminus . The receptor undergoes post-translational modifications, including N-glycosylation, which affects its processing and functional properties .

Where is ADGRG3 primarily expressed in the body?

ADGRG3 exhibits a specific expression pattern primarily within the immune system. RNA sequencing and mass spectrometry analyses have revealed abundant transcription and translation of ADGRG3 in:

• Granulocyte precursor cells

• Terminally differentiated neutrophilic, eosinophilic, and basophilic granulocytes

Additionally, immunohistochemistry and microarray analysis have confirmed ADGRG3 expression in tissue-infiltrating granulocytes . ADGRG3 expression is upregulated during systemic inflammation conditions such as pneumonia or endotoxemia . Within the aGPCRs family, ADGRG3 forms part of a subfamily G cluster alongside ADGRG1 (GPR56) and ADGRG5 (GPR114), all of which are expressed in immune cells .

In contrast to ADGRG3, other adhesion G protein-coupled receptors like ADGRV1 and ADGRF5 show different tissue distribution patterns, with ADGRF5 being prominently expressed in pulmonary alveolar type II cells where it regulates surfactant levels .

What validation techniques are recommended for ADGRG3 antibodies?

Rigorous validation of ADGRG3 antibodies is essential for experimental reliability. The following methodological approaches are recommended:

• Western Blot Validation:

  • Use mouse and rat brain membranes as positive controls

  • Include human THP-1 monocytic leukemia cell line lysates as additional controls

  • Always perform parallel experiments with blocking peptides (e.g., GPR97/ADGRG3 extracellular blocking peptide) to confirm specificity

• Immunohistochemistry Validation:

  • Test on perfusion-fixed frozen rat spinal cord sections

  • Include parallel staining with blocking peptide to demonstrate specificity

  • Use DAPI counterstaining to visualize cell nuclei

• Flow Cytometry Validation:

  • Evaluate cell surface detection using indirect flow cytometry on live intact human THP-1 monocytic leukemia cells

  • Include appropriate negative controls (cells alone, cells + secondary antibody)

• Cross-Reactivity Testing:

  • Test antibody against related adhesion GPCRs to ensure specificity for ADGRG3

Antibody suppliers typically validate their products using standardized processes to ensure quality, with validation data available for IHC, ICC-IF, and WB applications .

What types of ADGRG3 antibodies are available for research?

Researchers have access to several types of ADGRG3 antibodies, each with specific applications:

• Monoclonal Antibodies:

  • Highly specific for defined epitopes

  • Example: Anti-ADGRG3 monoclonal antibodies targeting human ADGRG3

  • Concentration typically 1.0 mg/ml

  • Provide consistent results across experiments due to homogeneity

• Domain-Specific Antibodies:

  • Target particular regions of the receptor

  • Example: Anti-GPR97 (ADGRG3)(extracellular) antibody that recognizes an extracellular epitope (amino acid residues 62-75 of human ADGRG3)

  • Useful for studying receptor topology and accessibility

• Species-Specific Antibodies:

  • Designed for human, mouse, or rat ADGRG3 variants

  • Important for cross-species studies and validation

When selecting an antibody, researchers should consider the target epitope (extracellular vs. intracellular domains), clonality (monoclonal vs. polyclonal), and validated applications to ensure experimental success.

How does ADGRG3 activation affect granulocyte function?

ADGRG3 plays a critical role in regulating granulocyte antimicrobial functions. Antibody ligation of ADGRG3 triggers several functional responses in granulocytes:

• Enhanced Reactive Oxygen Species (ROS) Production:

  • Antibody-mediated activation of ADGRG3 significantly increases ROS production in granulocytes

  • This occurs through activation of NF-κB and ERK signaling pathways

• Increased Proteolytic Enzyme Activity:

  • ADGRG3 ligation enhances the activity of proteolytic enzymes

  • This contributes to the antimicrobial functions of granulocytes

• Inflammatory Response Regulation:

  • ADGRG3 expression is induced during systemic inflammation

  • The receptor likely participates in the regulation of inflammatory responses during conditions such as pneumonia or endotoxemia

These findings indicate that ADGRG3 functions as a regulator of antimicrobial activity in human granulocytes. The specific expression of ADGRG3 in these immune cells suggests a specialized role in host defense mechanisms .

What signaling pathways are activated by ADGRG3 and how can they be studied using antibodies?

ADGRG3 engages multiple signaling pathways that can be studied using antibody-based approaches:

• G Protein-Coupled Signaling:

  • Basal state: ADGRG3 couples to Gαs, leading to cAMP elevation

  • Activated state: Upon activation, ADGRG3 can switch to Gαi coupling, resulting in decreased cAMP levels

  • This signaling switch can be detected using cAMP assays in the presence of ADGRG3-specific antibodies

• G βγ-Mediated Signaling:

  • Activation of ADGRG3 increases downstream effectors of Gβγ

  • Leads to activation of SRE and NF-κB transcriptional pathways

  • Reporter assays for these transcription factors can be used to monitor this signaling branch

• MAPK Pathway Activation:

  • ADGRG3 ligation activates the ERK signaling pathway

  • This can be detected by measuring ERK phosphorylation using phospho-specific antibodies in Western blot or flow cytometry experiments

• Glucocorticoid-Mediated Activation:

  • Recent research reveals that glucocorticoid stress hormones activate ADGRG3

  • Structural studies have shown that glucocorticoids like beclomethasone and cortisol bind to a pocket within the transmembrane domain of ADGRG3

To study these pathways, researchers can use ADGRG3 antibodies in combination with pathway-specific inhibitors, signaling protein knockdowns, or reporter assays to dissect the specific contributions of each pathway to ADGRG3-mediated cellular responses.

How can ADGRG3 antibodies be used to investigate receptor processing and autoproteolyisis?

ADGRG3 undergoes autoproteolysis via its GAIN domain, producing two subunits that remain non-covalently associated. Antibodies provide valuable tools for studying this process:

• Detection of Receptor Processing States:

  • Western blot analysis using domain-specific antibodies can distinguish between full-length ADGRG3 and its cleaved fragments

  • Extracellular domain-specific antibodies can detect the N-terminal fragment (NTF)

  • C-terminal domain-specific antibodies can identify the C-terminal fragment (CTF)

• Analysis of N-Glycosylation:

  • ADGRG3 undergoes N-glycosylation, which can be studied using glycosidase treatments followed by Western blot analysis

  • Shifts in molecular weight detected by antibodies can reveal the extent and sites of glycosylation

• Monitoring Receptor Surface Expression:

  • Flow cytometry with extracellular domain-specific antibodies can quantify surface expression levels of ADGRG3

  • This approach can determine whether receptor processing affects trafficking to the cell surface

• Immunoprecipitation Studies:

  • Antibodies can be used to immunoprecipitate ADGRG3 and its interacting partners

  • Sequential immunoprecipitation with domain-specific antibodies can reveal associations between cleaved receptor fragments and signaling molecules

These approaches have revealed that ADGRG3 exists as a proteolytically processed, N-glycosylated bipartite receptor, with important implications for its function and regulation .

What controls should be included when using ADGRG3 antibodies in experimental procedures?

To ensure experimental rigor when working with ADGRG3 antibodies, the following controls are essential:

• Blocking Peptide Controls:

  • Always include parallel experiments with specific blocking peptides, such as GPR97 (ADGRG3)(extracellular) Blocking Peptide

  • In Western blot, immunohistochemistry, and flow cytometry, blocking peptide should suppress antibody binding

• Positive Control Samples:

  • Mouse and rat brain membranes

  • Human THP-1 monocytic leukemia cell line lysates

  • Tissue-infiltrating granulocytes in inflammatory conditions

• Negative Control Samples:

  • Cell lines or tissues known not to express ADGRG3

  • Samples from ADGRG3 knockout models, if available

• Technical Controls for Flow Cytometry:

  • Unstained cells

  • Cells with secondary antibody only

  • Isotype control antibodies to assess non-specific binding

• Signal Specificity Controls:

  • For signaling studies, include specific inhibitors of pathways being studied (e.g., ERK inhibitors, NF-κB inhibitors)

  • Generate and test receptor mutants with altered signaling capabilities

These controls help validate antibody specificity and ensure that observed effects are truly attributable to ADGRG3-specific interactions rather than non-specific binding or experimental artifacts.

What is the structural basis for ligand interactions with ADGRG3?

Recent structural studies have provided significant insights into ADGRG3 ligand interactions:

• Glucocorticoid Binding:

  • Cryo-electron microscopy structures of GPR97-Go complexes have revealed that glucocorticoid stress hormones activate ADGRG3

  • Beclomethasone (an anti-inflammatory drug) and cortisol (a steroid hormone) bind to a specific pocket within the transmembrane domain of ADGRG3

  • This structural information provides a molecular basis for understanding how these ligands modulate receptor function

• Stachel Peptide Activation:

  • Like other adhesion GPCRs, ADGRG3 may be activated by its tethered agonist peptide (Stachel)

  • Antibodies targeting specific receptor domains can be used to study how the Stachel peptide interacts with the receptor core to initiate signaling

  • Similar mechanisms have been demonstrated for related receptors like ADGRL1 and ADGRF5

• GAIN Domain Structure:

  • The GAIN domain mediates autoproteolysis of ADGRG3

  • Structural studies of related adhesion GPCRs suggest that the GAIN domain plays a critical role in receptor activation and ligand recognition

  • Antibodies recognizing specific epitopes within this region can help elucidate its structural dynamics

Understanding these structural features is essential for developing targeted approaches to modulate ADGRG3 function for potential therapeutic applications.

What are the optimal conditions for using ADGRG3 antibodies in Western blot applications?

For successful Western blot detection of ADGRG3, researchers should consider the following methodological details:

• Sample Preparation:

  • For membrane proteins like ADGRG3, use membrane fractions rather than whole cell lysates to enrich the target

  • Mouse and rat brain membranes provide reliable positive controls

  • Human THP-1 monocytic leukemia cell lysates are also suitable positive controls

  • Use non-reducing conditions if targeting disulfide-containing epitopes

• Gel Electrophoresis Parameters:

  • Use gradient gels (4-12% or 4-20%) to effectively resolve the full-length ADGRG3 and its processed fragments

  • Include molecular weight markers covering the 50-250 kDa range to identify the receptor and its glycosylated forms

• Transfer Conditions:

  • For large membrane proteins like ADGRG3, use low methanol transfer buffers

  • Consider longer transfer times or semi-dry transfer systems optimized for large proteins

• Antibody Dilutions and Incubation:

  • Start with manufacturer's recommended dilutions (typically 1:500 to 1:2000)

  • Perform parallel experiments with blocking peptides at appropriate concentrations to confirm specificity

  • Optimize primary antibody incubation time and temperature (typically overnight at 4°C)

• Detection Systems:

  • Enhanced chemiluminescence (ECL) systems with extended sensitivity are recommended

  • Consider fluorescent secondary antibodies for quantitative analysis

These optimized conditions will help ensure specific detection of ADGRG3 and its processed forms in Western blot applications.

How can ADGRG3 antibodies be effectively used in immunohistochemistry and immunocytochemistry?

For optimal results in immunohistochemistry (IHC) and immunocytochemistry (ICC) applications with ADGRG3 antibodies:

• Tissue/Cell Preparation:

  • For tissues: Perfusion-fixed frozen sections yield better results than paraffin-embedded tissues for ADGRG3 detection

  • For cells: Fix with 4% paraformaldehyde and perform mild permeabilization if intracellular epitopes are targeted

• Antigen Retrieval:

  • For extracellular epitopes of ADGRG3, mild antigen retrieval methods are preferable

  • Citrate buffer (pH 6.0) heat-induced epitope retrieval may improve antibody access to target epitopes

• Blocking Conditions:

  • Use 5-10% normal serum from the species in which the secondary antibody was raised

  • Add 0.1-0.3% Triton X-100 for intracellular epitopes

  • Consider additional blocking with 1% BSA to reduce non-specific binding

• Antibody Incubation Parameters:

  • Primary antibody: Incubate at manufacturer's recommended dilution (typically 1:100 to 1:500)

  • Optimize incubation time and temperature (overnight at 4°C often yields best results)

  • Always run parallel sections/cells with blocking peptide to confirm specificity

• Detection and Visualization:

  • For fluorescence: Use appropriate fluorophore-conjugated secondary antibodies

  • Include DAPI nuclear counterstain for cellular context

  • For chromogenic detection: HRP-conjugated secondary antibodies with DAB substrate work well

  • Use confocal microscopy for co-localization studies with other cellular markers

These approaches have been successfully employed to detect ADGRG3 in rat spinal cord sections and various immune cell preparations .

What are the recommended protocols for flow cytometry detection of ADGRG3?

For effective flow cytometry detection of ADGRG3 on cell surfaces:

• Cell Preparation:

  • Use freshly isolated primary cells or cultured cell lines known to express ADGRG3 (e.g., THP-1 monocytic leukemia cells)

  • For primary granulocytes, use density gradient separation followed by immediate antibody staining to preserve cell viability and surface receptors

• Staining Protocol:

  • Keep cells in ice-cold buffer containing sodium azide to prevent receptor internalization

  • For extracellular epitopes: Use non-permeabilizing conditions

  • Titrate antibody concentration to optimize signal-to-noise ratio

  • Typical dilutions range from 1:50 to 1:200 depending on antibody affinity and target expression level

• Essential Controls:

  • Unstained cells to establish autofluorescence

  • Secondary antibody only to determine background binding

  • Isotype control antibodies matched to the primary antibody

  • Blocking peptide competition to confirm specificity

• Multi-parameter Analysis:

  • Include viability dye to exclude dead cells

  • Consider co-staining with lineage markers to identify specific cell populations

  • For granulocytes, include markers such as CD16 (neutrophils), Siglec-8 (eosinophils), or FcεRIα (basophils)

• Analysis Considerations:

  • Analyze median fluorescence intensity rather than percent positive cells for quantitative comparisons

  • Use histogram overlays to visualize shifts in ADGRG3 expression

  • For activated cells, monitor potential changes in surface expression levels

This methodology has been successfully applied to detect ADGRG3 on the surface of live intact human THP-1 cells and could be adapted for primary granulocytes and other ADGRG3-expressing cell types.

How can ADGRG3 antibodies be used to study receptor function during inflammation?

ADGRG3 expression is induced during systemic inflammation, suggesting important functional roles that can be studied using antibodies:

• Expression Profiling in Inflammatory Conditions:

  • Immunohistochemistry with ADGRG3 antibodies can track receptor expression in tissue-infiltrating granulocytes during inflammation

  • Quantitative flow cytometry can measure changes in receptor levels on circulating granulocytes during infection or inflammatory diseases

  • This approach has confirmed ADGRG3 upregulation during pneumonia and endotoxemia

• Functional Blockade Studies:

  • Neutralizing antibodies against ADGRG3 can be used to block receptor function

  • This approach can help determine the contribution of ADGRG3 to inflammatory responses

  • Measure subsequent effects on ROS production, proteolytic enzyme activity, and signaling pathway activation

• Receptor Internalization During Inflammation:

  • Fluorescently-labeled antibodies can track ADGRG3 internalization in response to inflammatory stimuli

  • Time-course studies can reveal the kinetics of receptor trafficking during the inflammatory response

• Correlation with Disease Severity:

  • Quantitative measurement of ADGRG3 expression using antibody-based techniques can be correlated with clinical parameters of inflammatory diseases

  • This may identify ADGRG3 as a potential biomarker or therapeutic target

These approaches can provide valuable insights into how ADGRG3 contributes to inflammatory responses and potentially identify new targets for anti-inflammatory therapies.

What is the relationship between ADGRG3 and glucocorticoid signaling?

Recent structural studies have revealed an unexpected relationship between ADGRG3 and glucocorticoid signaling that can be further explored using antibodies:

• Structural Basis of Interaction:

  • Cryo-electron microscopy structures have shown that glucocorticoids (beclomethasone and cortisol) bind to a specific pocket within the transmembrane domain of ADGRG3

  • Antibodies recognizing specific conformational epitopes could potentially distinguish between glucocorticoid-bound and unbound receptor states

• Functional Consequences:

  • Antibody-based approaches can be used to determine how glucocorticoid binding affects:

    • ADGRG3 surface expression

    • Receptor internalization and trafficking

    • Downstream signaling pathway activation

    • Interactions with other cellular proteins

• Physiological Context:

  • During stress responses, elevated glucocorticoid levels may modulate immune cell function through ADGRG3

  • Antibodies can help track ADGRG3 expression and activation in response to stress hormones in various physiological and pathological contexts

• Therapeutic Implications:

  • The interaction between glucocorticoids and ADGRG3 suggests potential mechanisms for the anti-inflammatory effects of glucocorticoids

  • Targeted antibodies could be developed to modulate this interaction for therapeutic purposes

This newly discovered relationship between glucocorticoids and ADGRG3 opens up an exciting area of research with implications for understanding stress responses and developing new anti-inflammatory strategies.

How do ADGRG3 antibodies compare with genetic approaches for studying receptor function?

Both antibody-based and genetic approaches offer complementary advantages for studying ADGRG3 function:

The most comprehensive understanding of ADGRG3 function will come from integrating both antibody-based and genetic approaches in research programs.

What are common challenges when working with ADGRG3 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with ADGRG3 antibodies:

• Low Signal Intensity:

  • Problem: Weak detection of ADGRG3 despite known expression

  • Solutions:

    • Optimize antibody concentration through titration experiments

    • Try alternative sample preparation methods to better preserve epitopes

    • Consider signal amplification systems (tyramide signal amplification for IHC/ICC)

    • For Western blot, use more sensitive detection reagents or longer exposure times

• Non-specific Binding:

  • Problem: Multiple bands or diffuse staining patterns

  • Solutions:

    • Always perform blocking peptide controls to confirm specificity

    • Increase blocking reagent concentration or time

    • Use more stringent washing conditions

    • For Western blot, optimize antibody dilution and incubation conditions

• Epitope Accessibility Issues:

  • Problem: Poor detection despite confirmed expression

  • Solutions:

    • For membrane proteins like ADGRG3, ensure proper membrane preparation or permeabilization

    • Try different fixation methods that better preserve the epitope

    • Consider alternative antigen retrieval methods

    • For flow cytometry, ensure cells remain viable and membrane integrity is maintained

• Variability Between Experiments:

  • Problem: Inconsistent results across experiments

  • Solutions:

    • Standardize all protocols thoroughly

    • Prepare larger batches of antibody dilutions and aliquot for consistent use

    • Include positive control samples in each experiment

    • Consider lot-to-lot variability of antibodies and maintain records

• Receptor Heterogeneity:

  • Problem: Complex banding patterns due to glycosylation and proteolytic processing

  • Solutions:

    • Use domain-specific antibodies to distinguish different receptor fragments

    • Include enzymatic treatments (glycosidases) to resolve glycosylation variants

    • Compare results with recombinant standards if available

Systematically addressing these challenges will improve the reliability and reproducibility of experiments using ADGRG3 antibodies.

How can researchers verify the specificity of ADGRG3 antibodies?

Ensuring antibody specificity is critical for reliable results. Researchers should implement multiple validation strategies:

• Blocking Peptide Competition:

  • Pre-incubate antibody with excess immunogenic peptide before application

  • Specific binding should be significantly reduced or eliminated

  • This approach has been demonstrated effective for ADGRG3 antibodies in Western blot, immunohistochemistry, and flow cytometry applications

• Genetic Validation:

  • Test antibody on samples from ADGRG3 knockout models (if available)

  • Signal should be absent in knockout samples

  • Alternatively, use siRNA or shRNA knockdown systems to reduce ADGRG3 expression

• Recombinant Expression Systems:

  • Compare antibody reactivity in cells transfected with ADGRG3 versus control

  • Test against related adhesion GPCRs to confirm specificity

  • Use epitope-tagged versions to validate antibody binding sites

• Multiple Antibody Comparison:

  • Test different antibodies targeting distinct epitopes of ADGRG3

  • Concordant results from different antibodies increase confidence in specificity

  • Disparate results warrant further investigation

• Cross-Reactivity Assessment:

  • Test antibody on tissues/cells known to express or lack ADGRG3

  • Evaluate reactivity across species (if the antibody is designed to be cross-reactive)

  • Check for unexpected patterns that might indicate off-target binding

Implementing these validation strategies will ensure that experimental findings truly reflect ADGRG3 biology rather than artifacts of non-specific antibody interactions.

How might ADGRG3 antibodies contribute to therapeutic development?

ADGRG3 antibodies have potential applications in therapeutic development based on the receptor's roles in immune function:

• Targeted Immunomodulation:

  • ADGRG3-targeting antibodies could modulate granulocyte function in inflammatory diseases

  • Inhibitory antibodies might reduce excessive neutrophil activation in conditions like sepsis or autoimmune disorders

  • Activating antibodies could potentially enhance antimicrobial responses in immunocompromised states

• Drug Discovery Tools:

  • Conformation-specific antibodies could be developed to screen for compounds that modulate ADGRG3 activity

  • These could identify small molecules that mimic or block glucocorticoid binding to ADGRG3

  • High-throughput screening assays using these antibodies could facilitate drug development

• Biomarker Development:

  • Antibodies could be used to develop diagnostic assays measuring ADGRG3 expression or activation state

  • This might identify patients likely to respond to specific anti-inflammatory therapies

  • Changes in ADGRG3 expression during inflammation suggest potential as a disease activity marker

• Targeted Drug Delivery:

  • ADGRG3 antibodies could be conjugated to therapeutic payloads for selective delivery to granulocytes

  • This approach might improve the therapeutic index of anti-inflammatory agents

While these applications are speculative, they represent promising directions for translating basic research on ADGRG3 into clinical applications using antibody-based technologies.

What are emerging techniques that could enhance ADGRG3 antibody applications?

Several cutting-edge techniques could expand the utility of ADGRG3 antibodies in research:

• Single-Cell Antibody-Based Technologies:

  • Mass cytometry (CyTOF) using metal-conjugated anti-ADGRG3 antibodies can provide high-dimensional analysis of receptor expression in heterogeneous cell populations

  • Imaging mass cytometry could map ADGRG3 distribution in tissue contexts with subcellular resolution

• Proximity Labeling Methods:

  • Antibody-enzyme conjugates (e.g., APEX2, BioID) could identify proximal proteins to ADGRG3 in living cells

  • This would help map the ADGRG3 interactome and identify new signaling partners

• Live-Cell Imaging Approaches:

  • Non-perturbing nanobodies or Fab fragments against ADGRG3 conjugated to fluorescent proteins could track receptor dynamics in real-time

  • This could reveal receptor clustering, internalization, and recycling in response to stimuli

• Cryo-Electron Tomography:

  • Gold-labeled antibodies against ADGRG3 could locate the receptor within cellular ultrastructure

  • This would provide insights into receptor organization in native membrane environments

• Antibody-Guided Structural Studies:

  • Conformationally selective antibodies could stabilize ADGRG3 in specific states for structural determination

  • This approach has been successful for other GPCRs and could complement existing structural information on glucocorticoid binding to ADGRG3