ADGRG3 Antibody, Biotin conjugated

What is ADGRG3/GPR97 and where is it expressed?

ADGRG3 (GPR97) is a member of the adhesion G protein-coupled receptor (aGPCR) family, which comprises 33 members in humans. RNA sequencing and mass-spectrometry studies have revealed abundant expression of ADGRG3/GPR97 in granulocyte precursors and terminally differentiated neutrophilic, eosinophilic, and basophilic granulocytes . The receptor is particularly prominent in polymorphonuclear cells (PMNs) and is detected in tissue-infiltrating PMNs, with expression levels increasing during systemic inflammation . Unlike some other aGPCRs that are widely distributed, ADGRG3 shows a more restricted expression pattern primarily in granulocytes, making it a potential specific marker and functional regulator for these immune cells.

What is the molecular structure of ADGRG3?

ADGRG3 is a proteolytically processed, dichotomous, N-glycosylated receptor characterized by its unique domain architecture common to adhesion GPCRs . The receptor consists of:

• An extracellular region (ECR) containing a GAIN (GPCR Autoproteolysis-Inducing) domain

• A seven-transmembrane (7TM) domain that traverses the cell membrane

• An intracellular C-terminal domain involved in signaling

The GAIN domain undergoes autoproteolysis, resulting in a non-covalently associated N-terminal fragment and a C-terminal fragment that includes the 7TM domain . This structural organization is critical for receptor function and can be specifically targeted by antibodies for research and potentially therapeutic applications.

How are biotin-conjugated ADGRG3 antibodies generated?

Biotin-conjugated ADGRG3 antibodies are typically produced through the following methodology:

• Generation of the primary antibody: The extracellular domain (ECD) of ADGRG3 is expressed as a fusion protein (e.g., with an Fc fragment) in expression systems like HEK-293T cells

• Immunization: The purified fusion protein is used to immunize mice (typically BALB/c) following a standard immunization protocol with appropriate adjuvants

• Hybridoma generation: Splenocytes from immunized mice are fused with myeloma cells to create hybridoma cell lines

• Selection: ELISA screening identifies clones producing ADGRG3-specific antibodies

• Biotinylation: Purified antibodies are conjugated with biotin using NHS-ester chemistry or similar approaches

• Validation: The biotinylated antibodies are tested for specificity and binding efficiency

The resulting biotin-conjugated antibodies can be used with streptavidin-coupled detection systems for enhanced sensitivity in various applications.

What are the basic applications for biotin-conjugated ADGRG3 antibodies?

Biotin-conjugated ADGRG3 antibodies are valuable tools for multiple research applications:

• Flow cytometry: For detecting ADGRG3 expression on granulocytes and other cell populations

• Immunohistochemistry: To visualize ADGRG3 in tissue samples, particularly in inflammatory conditions

• Western blotting: For detecting ADGRG3 protein in cell or tissue lysates, allowing assessment of processing and glycosylation states

• Immunoprecipitation: To isolate ADGRG3 and associated proteins from complex mixtures

• ELISA: For quantitative measurement of ADGRG3 in biological samples

The biotin-streptavidin interaction provides a high-affinity, non-covalent binding that enhances detection sensitivity and allows flexible experimental design with various secondary detection systems.

How can ADGRG3 antibodies be used to study receptor activation mechanisms?

ADGRG3 antibodies can serve as valuable tools for investigating activation mechanisms through several sophisticated approaches:

• Conformational state analysis: Similar to studies on related aGPCRs, antibodies targeting specific epitopes can be used in single-molecule FRET experiments to monitor conformational changes between the extracellular region and transmembrane domain during receptor activation . This approach allows visualization of discrete conformational states and their transition dynamics.

• Receptor crosslinking studies: As demonstrated in previous research, ADGRG3 antibodies (such as G97-A mAb) can be used at 10 μg/ml, followed by crosslinking with goat anti-mouse IgG F(ab')₂ (10 μg/ml) to induce receptor activation . This methodological approach enables time-course analysis of downstream signaling events.

• Activation-specific epitope mapping: By generating a panel of antibodies targeting different domains of ADGRG3, researchers can identify epitopes that are accessible only in specific conformational states, providing insights into the structural rearrangements during receptor activation.

For optimal results in activation studies, researchers should conduct time course experiments (5-15 minutes) after antibody crosslinking at 37°C, followed by rapid cooling to halt activation processes before biochemical analysis .

What signaling pathways does ADGRG3 activate and how can antibodies help investigate them?

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

Anti-ADGRG3 antibodies can be used to trigger these pathways through receptor ligation and crosslinking. To effectively study these pathways, researchers should:

• Pre-treat cells with specific signaling inhibitors prior to antibody ligation to identify pathway dependencies

• Include appropriate controls (mouse IgG1 as negative control, LPS as positive control for inflammatory pathway activation)

• Examine the temporal dynamics of signaling by collecting lysates at multiple timepoints after antibody stimulation

Biotin-conjugated antibodies offer the additional advantage of allowing simultaneous pathway activation and visualization of receptor internalization or trafficking.

How can the functional effects of ADGRG3 activation be measured in granulocytes?

ADGRG3 activation in granulocytes triggers several antimicrobial effector functions that can be experimentally measured:

• Reactive oxygen species (ROS) production:

  • Method: Luminol-enhanced chemiluminescence or fluorescent probes (DCF-DA)

  • Expected outcome: ADGRG3 antibody ligation increases ROS production compared to isotype control

• Proteolytic enzyme activity:

  • Method: Specific enzyme substrate assays for elastase, myeloperoxidase, etc.

  • Expected outcome: Enhanced enzyme release following ADGRG3 engagement

• Bacterial uptake and killing:

  • Method: Phagocytosis assays using fluorescently-labeled bacteria followed by gentamicin protection assays

  • Expected outcome: Increased bacterial uptake and killing efficiency in ADGRG3-activated neutrophils

• Neutrophil extracellular trap (NET) formation:

  • Method: Fluorescence microscopy with DNA and neutrophil granule protein staining

  • Expected outcome: Potential enhancement of NET formation after ADGRG3 engagement

For all functional assays, it is critical to include proper controls and to verify antibody specificity to ensure that observed effects are specifically due to ADGRG3 activation rather than non-specific Fc receptor engagement.

What are the technical considerations for using biotin-conjugated ADGRG3 antibodies in flow cytometry?

When using biotin-conjugated ADGRG3 antibodies for flow cytometry, researchers should consider several technical aspects:

• Titration of antibody concentration: Establish optimal antibody concentration (typically 1-10 μg/ml) through titration experiments to ensure maximum specific signal with minimal background.

• Multiplexing considerations: When combining with other antibodies, be aware that:

  • Streptavidin conjugates are available with multiple fluorophores (PE, APC, BV421, etc.)

  • Potential spectral overlap must be accounted for in compensation controls

  • Sequential staining may be necessary (biotin-antibody first, followed by fluorophore-conjugated streptavidin)

• Blocking strategy: To reduce non-specific binding:

  • Pre-block cells with 2% normal serum from the same species as secondary reagents

  • Include 1% BSA in staining buffer

  • Consider adding 10% human AB serum when using human samples to block Fc receptors

• Fixation compatibility: If intracellular staining is required, confirm that fixation and permeabilization do not affect the epitope recognition or biotin-streptavidin interaction.

A typical protocol involves incubating cells with biotin-conjugated ADGRG3 antibody (10 μg/ml) for 30 minutes at 4°C, washing, then adding streptavidin-fluorophore conjugate for 30 minutes at 4°C before final washing and analysis .

How can I determine if my ADGRG3 antibody recognizes the native conformation of the receptor?

Confirming that an ADGRG3 antibody recognizes the native receptor conformation is crucial for functional studies and requires multiple validation approaches:

• Flow cytometry on viable cells: Positive staining of unfixed, non-permeabilized cells expressing ADGRG3 naturally (e.g., granulocytes) or through transfection suggests recognition of native, surface-expressed receptor .

• Immunoprecipitation: The ability to pull down full-length, glycosylated ADGRG3 from cell lysates prepared with mild detergents indicates recognition of properly folded protein.

• Functional assays: If antibody ligation triggers signaling responses (e.g., increased ROS production, MAPK phosphorylation), this strongly suggests binding to functionally relevant epitopes in the native conformation .

• Comparative analysis with known ligands: Competition or cooperation between the antibody and any known natural ligands for binding or functional effects would further validate conformational recognition.

• Cell-based binding assays: Using cells expressing wild-type versus mutant ADGRG3 with alterations in specific domains can help map the conformational epitope recognized by the antibody.

For most rigorous validation, researchers should employ at least three of these approaches to confirm binding to the native receptor conformation.

What are the best methods for validating ADGRG3 antibody specificity?

Thorough validation of ADGRG3 antibody specificity is essential for reliable research outcomes and should include:

• Genetic approaches:

  • Testing on ADGRG3 knockout cells/tissues (negative control)

  • Using CRISPR-Cas9 edited cell lines with deleted ADGRG3

  • Comparing wild-type versus ADGRG3-overexpressing cells (positive control)

• Biochemical methods:

  • Western blotting should show bands of appropriate molecular weight (accounting for glycosylation and proteolytic processing)

  • Pre-absorption with purified antigen should abolish signal

  • Multiple antibodies targeting different epitopes should give consistent results

• Cross-reactivity assessment:

  • Testing on related aGPCRs, particularly other ADGRG family members (ADGRG1/GPR56, ADGRG5/GPR114) to ensure specificity

  • Examining species cross-reactivity if working with models other than human

• Mass spectrometry validation:

  • Immunoprecipitated material can be analyzed by mass spectrometry to confirm identity of the captured protein

Ideally, antibody validation should be performed in the same experimental context and cell types as the planned experiments, since receptor processing and expression can vary between tissues and experimental conditions.

How can I optimize immunoprecipitation protocols using biotin-conjugated ADGRG3 antibodies?

Optimizing immunoprecipitation (IP) with biotin-conjugated ADGRG3 antibodies requires careful consideration of several parameters:

• Cell lysis conditions:

  • For membrane proteins like ADGRG3, use non-denaturing detergents (e.g., LMNG/GDN mix, Triton X-100, or CHAPS)

  • Include protease inhibitors to prevent receptor degradation

  • For studying receptor complexes, consider using crosslinking agents prior to lysis

• Capture strategy optimization:

  • Direct approach: Incubate biotin-conjugated antibody with lysate, then capture with streptavidin beads

  • Indirect approach: Pre-coat streptavidin beads with biotin-antibody, then incubate with lysate

  • Compare both methods to determine which gives better yield and lower background

• Technical parameters:

  • Antibody concentration: Typically 2-10 μg per IP reaction

  • Incubation time: 2-4 hours at 4°C or overnight for weaker interactions

  • Washing stringency: Adjust salt and detergent concentration in wash buffers to balance between specificity and yield

• Controls:

  • Include isotype control antibody (biotin-conjugated)

  • Pre-clear lysates with streptavidin beads alone to reduce non-specific binding

  • Include a condition with competing non-biotinylated ADGRG3 antibody

For co-immunoprecipitation studies of receptor-interacting proteins, gentler lysis and washing conditions may be necessary to preserve protein-protein interactions.

What are the challenges in developing function-modulating ADGRG3 antibodies?

Developing antibodies that can functionally modulate ADGRG3 activity presents several technical challenges:

• Epitope selection considerations:

  • Target functional domains like the GAIN domain or the interface between GAIN and 7TM domains

  • Antibodies against different epitopes can have opposing functional effects

  • Natural activation mechanism must be considered when designing functional antibodies

• Screening for functional effects:

  • Initial screening often requires high-throughput compatible assays (e.g., cAMP or SRE-luciferase)

  • Secondary validation in more physiologically relevant assays (ROS production, bacterial killing)

  • Both agonistic and antagonistic activities should be assessed

• Mechanistic complexity:

  • ADGRG3 has been shown to potentially switch G protein coupling from Gαs to Gαi upon activation

  • Antibody-induced activation may differ from natural ligand activation

  • Consideration of receptor internalization or desensitization after antibody binding

• Antibody format optimization:

  • Testing different formats: whole IgG, F(ab')₂, Fab, single-domain antibodies

  • Determining if crosslinking is required for functional effects

  • Evaluating if biotin conjugation affects functional properties

A methodical approach involves generating a diverse panel of antibodies against different regions, comprehensive epitope mapping, and systematic functional characterization in multiple assay systems to identify antibodies with desired modulatory properties.

How can conformational changes in ADGRG3 be studied using antibody-based approaches?

Studying ADGRG3 conformational dynamics requires sophisticated techniques using antibodies as analytical tools:

• Single-molecule FRET analysis:

  • Design: Site-specific labeling of ADGRG3 with fluorophores at the GAIN domain and 7TM region

  • Application: Antibodies binding specific epitopes can be used to stabilize or induce conformational changes

  • Expected outcome: Identification of discrete FRET states representing different conformational states, similar to what has been observed with related aGPCRs

  • Analysis: Dwell-time analysis to determine transition rates between conformational states

• Conformation-specific antibody development:

  • Approach: Generate antibodies under conditions that favor specific receptor states

  • Validation: Test binding preferentially to active or inactive receptor conformations

  • Application: Use as tools to stabilize and study specific conformational states

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Methodology: Compare deuterium incorporation patterns with and without antibody binding

  • Insight: Identifies regions protected from exchange upon antibody binding, revealing conformational changes

  • Advantage: Provides peptide-level resolution of structural changes

• Cryo-EM studies with antibody fragments:

  • Approach: Similar to studies with ADGRL3, antibody fragments (particularly Fabs) can serve as fiducial markers to improve particle alignment

  • Benefit: Helps stabilize flexible regions between domains

  • Outcome: Structural models of ADGRG3 in different functional states

These complementary approaches can provide comprehensive insights into the conformational changes associated with ADGRG3 activation and regulation.

How can ADGRG3 antibodies be used to investigate receptor trafficking and internalization?

Investigating ADGRG3 trafficking dynamics using antibody-based approaches involves several specialized techniques:

• Pulse-chase antibody labeling:

  • Method: Label surface receptors with biotin-conjugated antibodies at 4°C, warm to 37°C to allow internalization, then detect remaining surface antibody with streptavidin-fluorophore

  • Quantification: Flow cytometry or plate-based fluorescence readouts

  • Controls: Include endocytosis inhibitors to confirm specificity

• Live-cell imaging of receptor trafficking:

  • Approach: Use streptavidin-fluorophore conjugates with biotin-antibodies to visualize receptor movement in real-time

  • Analysis: Track colocalization with endosomal markers (early endosomes, recycling endosomes, lysosomes)

  • Advanced application: pH-sensitive fluorophores can distinguish surface from internalized receptors

• Biochemical trafficking analysis:

  • Surface biotinylation: Compare total receptor (western blot) with surface receptor (streptavidin pulldown)

  • Protease protection assays: Surface receptors are susceptible to extracellular proteases while internalized receptors are protected

• Antibody-induced trafficking effects:

  • Comparison: Different antibody clones may induce varying degrees of internalization

  • Mechanism investigation: Determine if antibody-induced internalization is dependent on β-arrestins or clathrin

  • Physiological relevance: Compare antibody-induced trafficking with that triggered by natural ligands or cellular activation

These methods allow comprehensive characterization of ADGRG3 dynamics at the cell surface and intracellular compartments in both basal and stimulated conditions.

What are the considerations for using ADGRG3 antibodies in in vivo inflammation models?

When designing in vivo experiments with ADGRG3 antibodies to study inflammation, researchers should consider several critical factors:

• Antibody format selection:

  • Whole IgG provides longer half-life but may trigger Fc-mediated effects

  • F(ab')₂ fragments eliminate Fc effects but have shorter half-life

  • Species matching: Use antibodies with appropriate species cross-reactivity for your model

• Dosing and administration:

  • Establish pharmacokinetics of the antibody in the model organism

  • Consider local (e.g., intranasal for lung inflammation) versus systemic administration

  • Timing: Prophylactic (before inflammatory challenge) versus therapeutic (after inflammation onset)

• Validation controls:

  • Include isotype control antibodies at equivalent doses

  • Consider ADGRG3 knockout models as negative controls

  • Validate antibody target engagement in tissues of interest

• Readout selection:

  • Granulocyte recruitment and activation (flow cytometry)

  • Tissue damage and inflammatory markers (histology, cytokine measurement)

  • Functional outcomes (bacterial clearance, resolution of inflammation)

  • Consider differences between acute and chronic models

• Potential confounding factors:

  • Expression of ADGRG3 on cells other than granulocytes may contribute to phenotypes

  • Antibody immunogenicity can limit repeated dosing

  • Compensation by related receptors (other aGPCRs) in response to ADGRG3 targeting

Given ADGRG3's upregulation during systemic inflammation , these antibodies may have particular value in models of sepsis, pneumonia, or sterile inflammation where granulocyte function is central to pathophysiology.

How can I develop quantitative assays to measure ADGRG3 receptor density using biotin-conjugated antibodies?

Developing quantitative assays for ADGRG3 receptor density measurement requires careful calibration and validation:

• Flow cytometry-based quantification:

  • Method: Use biotin-conjugated ADGRG3 antibodies with streptavidin-fluorophore having known fluorophore:protein ratio

  • Calibration: Include beads with known quantities of fluorophore (QuantiBRITE or similar)

  • Analysis: Convert median fluorescence intensity to molecules of equivalent soluble fluorophore (MESF)

  • Calculation: Determine antibody binding capacity (ABC) accounting for fluorophore:streptavidin ratio

• Saturation binding analysis:

  • Approach: Incubate cells with increasing concentrations of biotin-antibody, detect with excess streptavidin-fluorophore

  • Analysis: Generate saturation curve and Scatchard plot

  • Outcomes: Determine Bmax (receptor density) and Kd (binding affinity)

  • Control: Account for non-specific binding with excess unlabeled antibody

• ELISA-based receptor quantification:

  • Method: Capture ADGRG3 from cell lysates, detect with biotin-antibody and streptavidin-HRP

  • Calibration: Include recombinant ADGRG3-ECD at known concentrations

  • Considerations: Account for receptor processing and potential epitope masking

• Absolute quantification by mass spectrometry:

  • Approach: Immunoprecipitate with ADGRG3 antibody, perform proteomic analysis with isotope-labeled standards

  • Advantage: Provides absolute quantification independent of antibody binding efficiency

  • Challenge: Requires specialized equipment and expertise

For all methods, validation across different cell types with varying ADGRG3 expression levels is essential to establish assay robustness and dynamic range.

Why might biotin-conjugated ADGRG3 antibodies show inconsistent staining in flow cytometry?

Inconsistent flow cytometry results with biotin-conjugated ADGRG3 antibodies can stem from several factors:

• Receptor biology factors:

  • Variable expression levels: ADGRG3 expression increases during inflammation, so cell activation status matters

  • Proteolytic processing: The GAIN domain undergoes autoproteolysis, potentially affecting epitope availability

  • Receptor internalization: Antibody binding may trigger endocytosis, reducing surface staining over time

• Technical factors:

  • Over-biotinylation: Excessive biotin conjugation can interfere with antibody binding to the epitope

  • Biotin-streptavidin stoichiometry: Ensure optimal ratios for detection

  • Buffer compatibility: Some buffers contain biotin or can affect biotin-streptavidin interaction

• Procedural solutions:

  • Standardize cell preparation: Use consistent activation states and processing times

  • Optimize fixation: If fixing cells, determine if fixation affects epitope recognition

  • Titrate reagents: Both primary antibody and streptavidin-fluorophore should be titrated

  • Include blocking step: Use biotin-free BSA to reduce background

• Validation approaches:

  • Compare multiple antibody clones targeting different ADGRG3 epitopes

  • Correlate protein detection with mRNA expression data

  • Include positive control cells with known ADGRG3 expression

For the most reliable results, perform staining at 4°C to minimize receptor internalization and include appropriate positive and negative control cell populations in each experiment.

How can I differentiate between specific and non-specific effects when using ADGRG3 antibodies in functional studies?

Distinguishing specific from non-specific effects in functional studies requires rigorous controls and validation:

• Essential controls:

  • Isotype-matched control antibodies (biotin-conjugated) at equivalent concentrations

  • F(ab')₂ fragments to eliminate Fc receptor-mediated effects

  • Pre-absorption with recombinant ADGRG3 antigen to block specific binding

  • ADGRG3 knockdown/knockout cells compared with wild-type cells

• Dose-dependency assessment:

  • Perform full dose-response curves (typically 0.1-50 μg/ml)

  • Compare EC50 values from multiple functional readouts (e.g., ROS production, signaling activation)

  • Specific effects should show consistent dose-dependency across assays

• Temporal dynamics evaluation:

  • Monitor responses at multiple timepoints (e.g., 5, 10, 15 minutes for signaling; longer for functional outcomes)

  • Specific receptor-mediated effects often show characteristic kinetic profiles

  • Include both early and late timepoints to distinguish direct from secondary effects

• Pharmacological validation:

  • Use specific inhibitors of downstream signaling components

  • If ADGRG3 signals through Gαi upon activation, pertussis toxin should block specific effects

  • Test multiple antibody clones targeting different epitopes

• Comparison with physiological stimuli:

  • Compare antibody-induced effects with those triggered by known physiological activators

  • Assess additive versus competitive effects between antibodies and natural ligands

These approaches collectively provide strong evidence for attributing observed effects specifically to ADGRG3 engagement rather than experimental artifacts.

What are the critical factors affecting reproducibility in ADGRG3 antibody-based experiments?

Ensuring reproducibility in ADGRG3 antibody experiments requires attention to several critical factors:

• Antibody characteristics and handling:

  • Lot-to-lot variation: Validate each new antibody lot against previous standards

  • Storage conditions: Follow manufacturer recommendations for temperature, avoid freeze-thaw cycles

  • Degradation: Confirm antibody integrity periodically by SDS-PAGE

  • Biotinylation stability: Biotin conjugates may have limited shelf-life

• Cell preparation variables:

  • ADGRG3 expression fluctuates with cell activation state and inflammation

  • Standardize cell isolation procedures (particularly for primary granulocytes)

  • Document donor variability in primary human cells

  • Control cell density and passage number in cell lines

• Experimental conditions:

  • Buffer composition: pH, calcium concentration, and serum factors affect receptor conformation

  • Temperature: Conduct binding steps at 4°C to minimize internalization, activation steps at 37°C

  • Timing: Standardize incubation times for consistent results

  • Sequential addition: Order of reagent addition can impact outcomes

• Documentation and reporting:

  • Record complete antibody information (clone, supplier, lot, concentration)

  • Document all experimental conditions in sufficient detail for reproduction

  • Include all relevant controls in each experiment

  • Report negative and contradictory results alongside positive findings

• Validation across systems:

  • Test critical findings in multiple cell types or models

  • Use complementary techniques to confirm key observations

  • Consider independent validation by different laboratory members

By systematically controlling and documenting these variables, researchers can significantly improve the reproducibility of ADGRG3 antibody-based experiments across different laboratories and experimental conditions.

How might biotin-conjugated ADGRG3 antibodies be used in single-cell analysis technologies?

Biotin-conjugated ADGRG3 antibodies offer significant potential for integration with emerging single-cell technologies:

• Single-cell proteomics approaches:

  • Mass cytometry (CyTOF): Biotin-antibodies can be detected with metal-tagged streptavidin

  • Advantages: Allows simultaneous detection of ADGRG3 alongside dozens of other markers

  • Applications: Identify novel ADGRG3+ cell subpopulations in complex tissues

• Spatial transcriptomics integration:

  • Method: Combine antibody detection with in situ transcriptomics

  • Approach: Use biotin-antibodies with enzyme-linked streptavidin for signal amplification

  • Outcome: Correlate ADGRG3 protein expression with transcriptional programs at single-cell resolution

• Single-cell functional assays:

  • Microfluidic droplet assays: Encapsulate individual cells with biotin-antibodies

  • Functional readouts: Combine with reporter systems for activation

  • Analysis: Link receptor expression to functional heterogeneity

• Proximity labeling applications:

  • Method: Conjugate ADGRG3 antibodies with promiscuous biotin ligases (BioID, TurboID)

  • Application: Map the proximal proteome of ADGRG3 in single cells

  • Insight: Identify cell-type specific interaction partners

These approaches would be particularly valuable for understanding the heterogeneity of ADGRG3 expression and function across granulocyte subpopulations and activation states in complex inflammatory environments.

What are the potential applications of ADGRG3 antibodies in studying inflammation resolution?

ADGRG3 antibodies could provide novel insights into inflammation resolution mechanisms:

• Temporal expression dynamics:

  • Question: Does ADGRG3 expression change during the transition from inflammatory to resolution phases?

  • Method: Time-course analysis using biotin-conjugated antibodies in inflammation models

  • Hypothesis: ADGRG3 may play distinct roles in acute versus resolving inflammation

• Neutrophil phenotype transition:

  • Context: Neutrophils shift from pro-inflammatory to pro-resolving phenotypes

  • Investigation: Correlation between ADGRG3 signaling and neutrophil phenotypic changes

  • Approach: Combine ADGRG3 antibody stimulation with assessment of resolution mediator production

• Clearance of apoptotic neutrophils:

  • Question: Does ADGRG3 participate in efferocytosis signaling?

  • Method: Use antibodies to block or activate ADGRG3 during neutrophil apoptosis and clearance

  • Measurement: Quantify macrophage uptake of antibody-treated versus untreated apoptotic neutrophils

• Development of pro-resolving ADGRG3 modulators:

  • Screening: Identify antibodies that promote resolution phenotypes

  • Characterization: Determine if distinct epitopes trigger pro-inflammatory versus pro-resolving effects

  • Therapeutic potential: Engineer antibodies that selectively promote resolution functions

Since ADGRG3 has been shown to regulate antimicrobial functions and is upregulated during inflammation , understanding its potential dual role in resolution could provide new therapeutic strategies for inflammatory diseases.

How might conformational-selective ADGRG3 antibodies advance our understanding of adhesion GPCR activation?

Developing conformational-selective antibodies for ADGRG3 could transform our understanding of adhesion GPCR biology:

• Mapping the conformational landscape:

  • Approach: Generate antibodies that selectively recognize distinct ADGRG3 conformational states

  • Application: Use as probes to quantify receptor state distribution under various conditions

  • Insight: Determine if ADGRG3 exhibits multiple active conformations like other GPCRs

• Structure-function correlation:

  • Strategy: Correlate conformational states (identified by specific antibodies) with distinct signaling outcomes

  • Question: Do different conformations couple preferentially to Gαs versus Gαi pathways?

  • Method: Combine conformation-selective antibodies with pathway-specific readouts

• Comparative adhesion GPCR mechanistic studies:

  • Question: Is the conformational activation mechanism conserved across adhesion GPCRs?

  • Approach: Apply similar conformational antibody strategies to multiple aGPCRs

  • Insight: Similar to findings with ADGRL3, determine if ADGRG3 shows conformational coupling between ECR and 7TM domains

• Allosteric modulation exploration:

  • Concept: Identify antibodies that bind to allosteric sites rather than orthosteric sites

  • Application: Use as tools to stabilize specific conformations without blocking the natural ligand binding site

  • Advantage: Potential for more selective functional modulation

This approach would build upon recent structural insights from related adhesion GPCRs showing conformational coupling between extracellular and transmembrane domains and could reveal unique aspects of ADGRG3 regulation.