ADGRB2 Antibody

What is ADGRB2 and why is it critical for neuroscience research?

ADGRB2 (also known as BAI2) is an orphan G protein-coupled receptor belonging to the adhesion GPCR family. It plays vital roles in cell adhesion and likely mediates cell-cell interactions . ADGRB2 is particularly important in neuroscience research because:

• It is expressed throughout the nervous system with prominent expression in synapse-dense regions of the hippocampus

• It regulates glutamatergic synapse and PSD95-associated spine development in hippocampal neurons

• It activates the NFAT-signaling pathway via the G-protein GNAZ

• Mutations in this receptor, such as the R1465W mutation, have been associated with progressive spastic paraparesis and other neurological symptoms, highlighting its clinical relevance

Understanding ADGRB2 function provides critical insights into synapse development, neurological disorders, and potential therapeutic targets within the nervous system.

What sample types can be effectively analyzed using ADGRB2 antibodies?

Based on available research data, ADGRB2 antibodies have been successfully used with:

• Paraffin-embedded formalin-fixed human brain tissue (neurons and glia)

• Primary cultured hippocampal neurons

• Transfected cell lines, including HEK293T cells expressing recombinant ADGRB2

• Brain tissue lysates for Western blotting applications

When selecting samples, researchers should consider:

• For immunohistochemistry: Formalin-fixed paraffin-embedded sections work well with specific antibodies like ab189112 at a concentration of 21 μg/ml

• For protein expression studies: Dissociated hippocampal cultures have been effectively used to study endogenous BAI2 expression and localization

• For signaling studies: Transfected cell systems provide a controlled environment to assess receptor function

How do N-terminal and C-terminal ADGRB2 antibodies differ in research applications?

The choice between N-terminal and C-terminal antibodies significantly impacts experimental results. C-terminal antibodies (like those targeting the region containing R1465) detect both cleaved and intact receptor forms, while N-terminal antibodies specifically recognize the extracellular domain, which can be shed during receptor activation .

How can antibodies be used to study ADGRB2 activation mechanisms?

Antibodies can serve as valuable tools for studying ADGRB2 activation through several approaches:

• Agonistic antibody development: Similar to findings with other aGPCRs like GPR133, specific antibodies can potentially activate ADGRB2 signaling by binding to extracellular domains. For example, research has demonstrated that polyclonal antibodies can activate EMR2 G protein signaling in vitro using an NFAT reporter assay .

• Monitoring receptor cleavage: Antibodies targeting different domains can help track the separation of N-terminal fragments (NTF) from C-terminal fragments (CTF), a key step in aGPCR activation. This approach has been successfully used with adhesion GPCRs, where antibody binding enhances NTF dissociation from CTF .

• Detecting activation-dependent interactions: Upon activation, aGPCRs like BAI2 robustly co-immunoprecipitate with β-arrestins. Antibodies can be used to isolate these complexes and assess receptor activation states .

• Signaling pathway analysis: Antibodies conjugated to beads (e.g., Dynabeads®) can enhance receptor clustering and signaling, providing a controlled method to study downstream pathways .

For optimal results, antibody-mediated activation studies should include appropriate controls, particularly testing antibody effects on cleavage-deficient receptor mutants, as antibody activation typically depends on receptor cleavage .

How do disease-associated mutations in ADGRB2 affect antibody detection and experimental design?

The R1465W mutation in ADGRB2, associated with progressive spastic paraparesis, presents specific challenges and considerations for antibody-based studies :

• Epitope accessibility: This mutation occurs in a highly conserved region of the BAI2 C-terminus, potentially altering antibody binding if the epitope encompasses or is near position 1465 .

• Protein conformation changes: The mutation increases BAI2 signaling activity and surface expression, which may affect antibody binding due to altered receptor conformation or protein-protein interactions .

• Disrupted protein interactions: The R1465W mutation disrupts BAI2's interaction with regulatory proteins like endophilin A1, which could affect antibody accessibility in co-immunoprecipitation experiments .

For researchers studying mutant forms of ADGRB2:

• Use antibodies targeting epitopes distant from mutation sites

• Include both wild-type and mutant controls to assess antibody binding differences

• Consider using multiple antibodies targeting different domains to confirm results

• Validate antibody specificity in the context of the specific mutation being studied

What methods can researchers use to study ADGRB2 G-protein coupling specificity?

Research has revealed that ADGRB2 exhibits specific G-protein coupling preferences that can be studied using antibody-based approaches:

• Yeast-based assays: While not directly demonstrated for ADGRB2, related aGPCRs like ADGRE2/EMR2 showed broad G protein-coupling in yeast-based assays where heterologous GPCRs are coupled to chimeric G proteins .

• cAMP assay systems: Studies have shown that ADGRB2 and related aGPCRs induce pertussis-toxin (PTX) insensitive inhibition of cyclic AMP (cAMP) levels in mammalian cells, suggesting coupling to Gαz .

• IP1 accumulation assays: For studying Gαq coupling, IP1 accumulation can be measured. Related aGPCRs have been shown to signal via Gα16 and Gα16/Gαz chimeras to stimulate IP1 accumulation .

• NFAT reporter assays: This system has successfully identified antibodies that activate aGPCR G protein signaling in vitro .

• Co-immunoprecipitation: The R1465W mutation in BAI2 alters G-protein coupling preferences, with wild-type BAI2 primarily coupling to Gαz and the mutant showing increased coupling to Gαi .

When designing experiments to study ADGRB2 G-protein coupling:

• Include appropriate G-protein inhibitors (e.g., pertussis toxin for Gαi)

• Use chimeric G-proteins to assess coupling specificity

• Compare truncated (activated) forms with full-length receptors

What are optimal protocols for ADGRB2 antibody validation in neuronal systems?

Comprehensive validation of ADGRB2 antibodies in neuronal systems requires multiple approaches:

• Genetic controls:

  • Compare antibody staining between wild-type and BAI2-deficient neurons

  • Use BAI2 knockout or knockdown models to confirm specificity

  • Test reactivity against related proteins (BAI1/ADGRB1 and BAI3/ADGRB3) to assess cross-reactivity

• Expression pattern verification:

  • Confirm antibody detects expected expression patterns in hippocampal cultures

  • Verify enrichment at postsynaptic sites, particularly large synapses defined by PSD95 size

  • Compare with published expression data showing upregulation during postnatal development

• Multiple detection methods:

  • Compare results across immunohistochemistry, Western blotting, and immunoprecipitation

  • For Western blotting: run non-reducing and reducing conditions to detect potential conformational epitopes

  • Use electrophoresis in 4–20% Tris-glycine gels and transfer to nitrocellulose membranes as described in published protocols

• Peptide competition assays:

  • Pre-incubate antibody with the immunizing peptide

  • Demonstrate loss of specific signal in the presence of the blocking peptide

As shown in research findings, properly validated antibodies should detect BAI2 expression throughout the nervous system with enrichment in synapse-dense regions of the hippocampus .

What protocols yield optimal results for co-immunoprecipitation studies with ADGRB2 antibodies?

Co-immunoprecipitation (co-IP) studies with ADGRB2 require careful optimization:

Recommended Protocol for ADGRB2 Co-IP Studies:

• Cell/Tissue Lysis:

  • Use mild detergents (0.5% NP-40 or 1% Triton X-100) to preserve protein-protein interactions

  • Include protease inhibitors to prevent degradation

  • For membrane proteins, consider including 150-300 mM NaCl in the lysis buffer

• Pre-clearing:

  • Pre-clear lysates with Protein A/G beads to reduce non-specific binding

  • Incubate for 1 hour at 4°C with rotation

• Antibody Binding:

  • Incubate pre-cleared lysates with ADGRB2 antibody (typically 2-5 μg per 500 μg protein)

  • For studying β-arrestin interactions, this approach has successfully demonstrated that activated forms of BAI2 robustly co-immunoprecipitate with β-arrestin2

  • Incubate overnight at 4°C with gentle rotation

• Immunoprecipitation:

  • Add Protein A/G beads and incubate for 2-4 hours at 4°C

  • Wash beads 4-5 times with cold lysis buffer containing reduced detergent

• Elution and Analysis:

  • Elute proteins by boiling in 1× Laemmli buffer

  • Analyze by Western blotting using antibodies against potential interaction partners

• Controls:

  • Include IgG control antibodies

  • Test both full-length and truncated (activated) forms of BAI2, as truncated forms show enhanced interactions with proteins like β-arrestin2

For protein detection in Western blots following co-IP, researchers have successfully used the BAI2 C-terminal antibody from Mab Technologies (Stone Mountain, GA cat. #BAI2-3) .

How should immunohistochemistry protocols be optimized for ADGRB2 detection in brain tissue?

Optimizing immunohistochemistry (IHC) for ADGRB2 detection requires careful attention to several key parameters:

• Tissue Preparation:

  • For formalin-fixed paraffin-embedded (FFPE) human brain samples: Standard processing with 10% neutral buffered formalin fixation

  • For fresh frozen sections: Rapid freezing followed by acetone or methanol fixation

  • Optimal section thickness: 5-7 μm for paraffin sections

• Antigen Retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • For paraffin sections: 20 minutes at 95-100°C

  • Allow slides to cool gradually in retrieval solution

• Blocking and Antibody Incubation:

  • Block with 5-10% normal serum (species of secondary antibody) with 0.1-0.3% Triton X-100

  • Primary antibody concentration: Commercially available antibodies like ab189112 have been successfully used at 21 μg/ml

  • Incubation: Overnight at 4°C or 1-2 hours at room temperature

• Detection Systems:

  • For chromogenic detection: HRP-polymer systems provide good signal with low background

  • For fluorescence: Tyramide signal amplification can enhance sensitivity

  • Counterstain nuclei with DAPI for fluorescence or hematoxylin for chromogenic detection

• Controls:

  • Positive control: Human brain tissue (neurons and glia)

  • Negative controls: Primary antibody omission and non-specific IgG

  • Peptide competition: Pre-absorption with immunizing peptide

• Special Considerations:

  • For co-localization studies: Use antibodies raised in different species

  • For multi-labeling: Sequential rather than simultaneous application may reduce cross-reactivity

Published research has successfully demonstrated ADGRB2 expression in human brain neurons and glia using immunohistochemical analysis with optimized protocols .

How can ADGRB2 antibodies be used to investigate synapse development and morphology?

ADGRB2 antibodies provide powerful tools for studying synapse development, as recent research has established ADGRB2 as an important regulator of glutamatergic synapse and PSD95-associated spine development :

• Co-localization Analysis with Synaptic Markers:

  • Use ADGRB2 antibodies in conjunction with pre- and post-synaptic markers

  • Pair with PSD95 antibodies to identify postsynaptic sites where ADGRB2 is highly enriched

  • Quantify co-localization with synaptic vesicle proteins (e.g., vGlut1 for glutamatergic terminals)

• Developmental Expression Studies:

  • Track ADGRB2 expression changes during critical periods of synaptogenesis

  • Compare with synapse formation timelines in culture and in vivo

  • Research has shown ADGRB2 expression is upregulated during postnatal development in the hippocampus

• Loss-of-Function Studies:

  • Use ADGRB2 antibodies to validate knockout or knockdown models

  • Quantify changes in synapse density following ADGRB2 depletion

  • Research shows BAI2 deficient neurons have decreased density of glutamatergic synapses with no change in GABAergic synapse density

• Spine Morphology Analysis:

  • Use ADGRB2 antibodies with spine markers to assess effects on spine structure

  • Quantify spine subtypes (mushroom, thin, stubby) in the presence/absence of ADGRB2

  • Studies have shown loss of BAI2 results in decreased density of mature PSD95-containing mushroom-shaped spines

• Super-resolution Microscopy Applications:

  • Utilize super-resolution techniques (STED, STORM) with ADGRB2 antibodies

  • Precisely localize ADGRB2 within the postsynaptic density

  • Measure protein distributions at nanoscale resolution

Recent findings demonstrate that ADGRB2-deficient neurons show significant alterations in spine morphology, specifically decreases in mature mushroom-shaped spines, reductions in spine volume, and reduced head diameter .

What approaches can best detect changes in ADGRB2 conformation and activation states?

Detecting changes in ADGRB2 conformation and activation states requires specialized techniques:

• Conformation-Specific Antibodies:

  • Develop or select antibodies that specifically recognize the cleaved/activated form of ADGRB2

  • Target epitopes that become exposed only after receptor activation

  • Use paired antibodies recognizing distinct domains to track conformational changes

• Activation-Dependent Protein Interactions:

  • Monitor β-arrestin recruitment as a marker of receptor activation

  • Research has shown that stalk-deficient (activated) forms of related aGPCRs robustly co-immunoprecipitate with β-arrestin2, while full-length counterparts do not

  • Use co-immunoprecipitation assays to detect these interaction changes

• FRET/BRET-Based Approaches:

  • Design biosensors with fluorescent/bioluminescent tags at different receptor domains

  • Monitor distance changes between tags during activation

  • Combine with antibody treatments to study activation mechanisms

• Trafficking and Internalization Assays:

  • Use antibodies against extracellular epitopes to track surface expression

  • Monitor antibody uptake to quantify internalization rates

  • Compare wild-type receptors with constitutively active mutants like R1465W

• G-Protein Coupling Assays:

  • Use downstream signaling reporters (cAMP, IP1, NFAT) to monitor activation

  • For related aGPCRs, antibodies have successfully activated G-protein signaling in NFAT reporter assays

  • Compare with other activation methods to validate results

Research has demonstrated that the R1465W mutation in ADGRB2 increases signaling activity, suggesting conformational changes that could be detected using these approaches .

What are common challenges when working with ADGRB2 antibodies and how can they be overcome?

Researchers frequently encounter several challenges when working with ADGRB2 antibodies:

One particularly effective approach for addressing sensitivity issues is to enhance signaling detection by using reporter assays. For example, NFAT reporter assays have successfully identified antibodies that activate related aGPCR G protein signaling in vitro .

How can researchers optimize antibody-mediated ADGRB2 activation for functional studies?

Based on research with related adhesion GPCRs, several strategies can optimize antibody-mediated ADGRB2 activation:

• Epitope Selection:

  • Target extracellular domains involved in receptor activation

  • For related aGPCRs, antibodies against specific extracellular epitopes have successfully triggered receptor signaling

  • Consider targeting the stalk region or nearby domains that influence NTF-CTF interaction

• Antibody Format Optimization:

  • Test different antibody formats (monoclonal, polyclonal, Fab fragments)

  • Consider bivalent vs. monovalent binding effects on receptor clustering

  • Research has shown that polyclonal antibodies can activate adhesion GPCR signaling in vitro

• Enhanced Clustering Approaches:

  • Conjugate antibodies to beads to increase effective concentration

  • Studies with related aGPCRs have shown coupling antibodies to Dynabeads® enhances receptor signaling

  • Compare soluble antibodies vs. immobilized formats

• Validation Controls:

  • Include cleavage-deficient receptor mutants as negative controls

  • Antibody-mediated activation typically depends on receptor cleavage

  • Compare with established activators when available

• Readout Selection:

  • Choose appropriate downstream signaling assays based on G-protein coupling

  • NFAT reporter assays have successfully identified activating antibodies for related receptors

  • Consider multiple readouts (cAMP, IP1, β-arrestin recruitment) to fully characterize activation

• Dose-Response Characterization:

  • Test a range of antibody concentrations to establish dose-dependent effects

  • Define optimal concentrations for activation studies

  • Control for any effects of storage solutions (e.g., NaN3)

Research with the related aGPCR GPR133 has demonstrated that antibody binding can enhance dissociation of the NTF from the CTF, thereby leading to increased activation .