ADGRE2 Antibody

What is ADGRE2 and what tissue distribution should researchers consider when planning experiments?

ADGRE2 (EMR2) is an adhesion G protein-coupled receptor predominantly expressed in myeloid cells, including monocytes/macrophages, dendritic cells, and all types of granulocytes. It shares 97% amino acid identity with CD97 in the EGF-like domains . Notably, ADGRE2 expression is highly regulated during monocyte/macrophage differentiation, making it an important marker for myeloid cell maturation stages . When designing experiments, researchers should focus on myeloid cell populations, as ADGRE2 is rarely expressed in other cell types, though it has been detected in some breast and colorectal adenocarcinomas . Lung mast cells and the HMC1 human mast-cell line also express ADGRE2 .

How do ADGRE2 antibodies differ from those targeting its close paralog ADGRE5 (CD97)?

While ADGRE2 and ADGRE5 (CD97) share structural similarities as adhesion GPCRs, they differ significantly in tissue distribution and function. Antibodies targeting these receptors must be carefully validated for specificity. ADGRE5 is more broadly distributed, found on all hematopoietic cells and smooth muscle cells, whereas ADGRE2 expression is restricted to myeloid lineage cells . Commercial antibodies like the monoclonal 2A1 and polyclonal AF4894 have demonstrated selectivity for ADGRE2 over CD97 in immunoprecipitation and Western blotting applications . When selecting antibodies, researchers should verify specificity using appropriate controls, as cross-reactivity could lead to misinterpretation of experimental results, particularly in samples containing multiple adhesion GPCR family members.

What are the key technical considerations when using anti-ADGRE2 antibodies for flow cytometry?

When utilizing anti-ADGRE2 antibodies for flow cytometry, researchers should consider several critical factors. First, sample preparation is crucial—fresh samples are preferred, but if frozen cells must be used, validation of epitope preservation after freezing/thawing is essential. Second, appropriate blocking steps are necessary to prevent non-specific binding, especially in myeloid cells which often display high Fc receptor expression. Third, titration of antibody concentration is vital to determine optimal signal-to-noise ratio, as ADGRE2 expression levels vary between cell types . Finally, inclusion of proper controls is mandatory, including isotype controls and comparative staining with antibodies against other myeloid markers to confirm cell identification. For multicolor panels, researchers should verify that the selected fluorophore coupled to the anti-ADGRE2 antibody has sufficient brightness for the expected expression level.

How can ADGRE2 antibodies be utilized to investigate the receptor's role in acute myeloid leukemia progression?

ADGRE2 has been identified as highly expressed in acute myeloid leukemia (AML), particularly in leukemic stem cells, with its expression associated with poor patient outcomes . Researchers investigating ADGRE2's role in AML should employ a multi-faceted approach. First, characterize ADGRE2 expression levels across different AML subtypes using validated antibodies for immunophenotyping, comparing expression between leukemic stem cells and bulk leukemia populations. Second, functional studies can utilize blocking antibodies to disrupt ADGRE2 signaling, assessing effects on leukemia cell proliferation, survival, and chemoresistance .

Mechanistic studies should focus on the ADGRE2-activated PLCβ/PKC/MEK/ERK signaling pathway, which enhances AP1 expression and drives DUSP1 expression, ultimately affecting proteostasis in AML cells . Co-immunoprecipitation experiments with anti-ADGRE2 antibodies can help identify binding partners in the signaling cascade. Importantly, xenograft models have demonstrated delayed AML progression when ADGRE2 is silenced, suggesting therapeutic potential for antibody-based targeting strategies that inhibit its function .

What methods are available to study ADGRE2 G protein-coupling using antibody-mediated activation?

ADGRE2 G protein-coupling can be investigated through several antibody-mediated methods. The polyclonal sheep anti-human EMR2 antibody (pAb AF4894) from R&D Systems has been identified as an activating antibody capable of stimulating G protein signaling in vitro . Researchers can utilize this antibody in NFAT reporter assays, where cells expressing ADGRE2 are treated with the antibody and downstream signaling is measured through the activation of an NFAT-responsive element controlling a reporter gene .

Alternative approaches include measuring second messenger responses, such as IP1 accumulation assays to detect Gα16 coupling or cAMP inhibition assays to assess Gαz coupling, following antibody treatment . For comprehensive G protein profiling, researchers can employ the yeast-based assay system where ADGRE2 is expressed alongside chimeric G proteins, allowing determination of coupling preferences across multiple G protein subtypes . When conducting these experiments, appropriate controls should include pertussis toxin treatment to distinguish between PTX-sensitive and PTX-insensitive G protein pathways, and comparison with truncated activated forms of the receptor (ADGRE2-CTF) that serve as constitutively active controls .

What experimental designs can reveal the relationship between ADGRE2 and proteostasis in myeloid cells?

Investigating the newly discovered relationship between ADGRE2 and cellular proteostasis requires sophisticated experimental approaches. Researchers should first establish baseline measurements of protein folding status in cells with normal or altered ADGRE2 expression using proteostasis sensors or chaperone activity assays. Next, examine the ADGRE2-mediated signaling cascade that affects proteostasis: the PLCβ/PKC/MEK/ERK pathway leading to AP1 activation and DUSP1 expression .

A crucial experiment involves analyzing DUSP1 phosphatase activity on Ser16 in the J-domain of co-chaperone DNAJB1, which facilitates DNAJB1-HSP70 interaction—a key process in maintaining proteostasis . This can be achieved through immunoprecipitation with anti-ADGRE2 antibodies followed by phosphorylation state analysis of downstream targets. Proteomics approaches, particularly pulse-chase experiments combined with mass spectrometry, can reveal differences in protein turnover and folding efficiency in the presence or absence of functional ADGRE2 signaling. Additionally, researchers should examine cellular responses to proteotoxic stress (heat shock, oxidative stress) when ADGRE2 signaling is intact versus when it's blocked by antibodies or genetic manipulation .

What are the optimal protocols for immunohistochemical detection of ADGRE2 in tissue samples?

For immunohistochemical detection of ADGRE2, researchers should follow a validated protocol that maximizes specificity and sensitivity. Tissue samples should undergo appropriate fixation (preferably 10% neutral buffered formalin for 24-48 hours) followed by paraffin embedding. Antigen retrieval is critical—heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be optimized, as ADGRE2's extracellular domain contains multiple EGF-like regions that may be sensitive to fixation .

Blocking should address both endogenous peroxidase activity and non-specific binding. For primary antibody incubation, researchers must determine optimal dilution and incubation conditions (typically 1:100-1:500 dilution, overnight at 4°C) for their specific anti-ADGRE2 antibody . Detection systems should be selected based on anticipated expression levels—amplification systems like tyramide signal amplification may be beneficial for detecting lower expression. Critically, validation controls must include known ADGRE2-positive tissues (myeloid cells in inflammatory lesions), ADGRE2-negative tissues, absorption controls with recombinant ADGRE2 protein, and comparison with alternative antibody clones to confirm staining patterns .

How can researchers effectively use anti-ADGRE2 antibodies to study the receptor's role in vibratory urticaria?

The discovery of a missense variant in ADGRE2 (p.C492Y) associated with autosomal dominant vibratory urticaria presents unique opportunities for antibody-based investigations . Researchers studying this condition should first develop an experimental system comparing wild-type ADGRE2 and the C492Y variant. Antibodies recognizing both forms can be used to compare expression levels, cellular localization, and conformational differences through immunofluorescence microscopy and biochemical assays.

A critical experiment involves comparing the stability of the autoinhibitory interaction between the α and β subunits of wild-type versus mutant ADGRE2. This can be accomplished through co-immunoprecipitation studies using antibodies specific to either subunit, analyzing whether the C492Y substitution affects subunit association under resting conditions and after vibratory stimulation . Functional studies should examine mast cell degranulation in response to vibration, comparing cells expressing wild-type ADGRE2 versus the C492Y variant, with anti-ADGRE2 antibodies used to block receptor function or to detect conformational changes during activation . Additionally, researchers can develop antibodies specifically recognizing the C492Y variant for diagnostic purposes in families with vibratory urticaria.

What approaches can distinguish between different functional domains of ADGRE2 using domain-specific antibodies?

ADGRE2 contains multiple functional domains, including EGF-like domains in its extracellular region, a GPCR proteolysis site (GPS), and a seven-transmembrane domain . To distinguish between these domains functionally, researchers should develop or acquire domain-specific antibodies targeting distinct regions of the receptor.

For the extracellular EGF-like domains that interact with dermatan sulfate (the endogenous ligand), researchers can use antibodies specifically targeting these regions to block ligand binding and assess functional consequences . To study the autocatalytic cleavage at the GPS, antibodies recognizing epitopes on either side of the cleavage site can determine the proportion of cleaved versus intact receptor in different cellular contexts . For the seven-transmembrane domain involved in G protein coupling, conformation-specific antibodies may detect active versus inactive receptor states .

In experimental designs, combinations of domain-specific antibodies can reveal how different domains contribute to receptor function. For example, comparing the effects of antibodies blocking the extracellular domain versus those targeting the transmembrane region can distinguish between adhesion functions and signaling functions of ADGRE2. Additionally, using epitope mapping with a panel of domain-specific antibodies can identify critical regions for protein-protein interactions in immunoprecipitation studies .

How can researchers address potential cross-reactivity issues when using anti-ADGRE2 antibodies?

Cross-reactivity remains a significant challenge when working with antibodies against ADGRE2, particularly due to its high sequence similarity with other adhesion GPCRs like CD97 (ADGRE5) . To address this issue, researchers should implement a comprehensive validation strategy. First, perform pre-absorption tests where the antibody is pre-incubated with recombinant ADGRE2 protein before application to samples—this should abolish specific staining. Second, validate antibody specificity using knockout or knockdown controls, such as CRISPR-modified cell lines or siRNA-treated samples .

For Western blotting applications, researchers should verify that the observed band size matches the predicted molecular weight of ADGRE2, keeping in mind that post-translational modifications and proteolytic processing can affect migration patterns . In immunohistochemistry or flow cytometry, compare staining patterns with known expression profiles of ADGRE2 across tissues and cell types . Additionally, using multiple antibodies targeting different epitopes of ADGRE2 and confirming concordant results provides stronger evidence of specificity. When cross-reactivity is detected, epitope mapping can identify unique regions of ADGRE2 that may serve as targets for more specific antibodies.

What control samples are essential when validating experimental results using ADGRE2 antibodies?

Rigorous validation of experimental results with ADGRE2 antibodies requires carefully selected controls. Positive controls should include cells or tissues known to express high levels of ADGRE2, such as macrophages, neutrophils, or dendritic cells . Negative controls should include cells lacking ADGRE2 expression, such as lymphocytes or cell lines of non-myeloid origin .

For genetic validation, CRISPR/Cas9-mediated knockout cell lines or siRNA knockdown samples provide definitive controls for antibody specificity . When studying ADGRE2 signaling, isotype control antibodies should be used alongside activating antibodies like 2A1 or pAb AF4894 to distinguish specific signaling effects from non-specific effects . For immunoprecipitation experiments, researchers should include "no-antibody" controls and isotype-matched non-specific antibody controls .

In disease models, such as AML studies, compare ADGRE2 antibody staining patterns between healthy and diseased samples, and correlate with other established markers of disease progression . When investigating the C492Y variant associated with vibratory urticaria, wild-type ADGRE2-expressing cells serve as essential controls for assessing functional differences . Finally, peptide competition assays, where the antibody is pre-incubated with the immunizing peptide, can confirm binding specificity.

How should researchers interpret contradictory results between different antibody-based detection methods for ADGRE2?

When faced with contradictory results between different antibody-based detection methods for ADGRE2, researchers should systematically evaluate several factors. First, consider epitope accessibility—structural differences in how ADGRE2 presents in various applications (Western blot versus flow cytometry versus immunohistochemistry) can affect antibody binding . Some epitopes may be masked in native conditions but exposed in denatured states.

Second, examine whether alternative splice variants or post-translational modifications of ADGRE2 might be differentially detected by various antibodies . The autocatalytic cleavage of ADGRE2 produces extracellular and transmembrane subunits, and antibodies targeting different regions may yield apparently conflicting results depending on the processing state of the receptor .

Third, assess potential technical issues including fixation methods, permeabilization conditions, and buffer compositions that may differentially affect epitope preservation across methods . When possible, confirm results using orthogonal techniques—for example, supplement antibody-based protein detection with mRNA analysis using RT-PCR or RNA sequencing.

Finally, consider biological variables such as cell activation state, which may affect ADGRE2 expression, localization, or conformation . Reconcile contradictory findings by developing a comprehensive model that accounts for these variables, and design definitive experiments to distinguish between competing interpretations.

What strategies can be employed to develop therapeutic antibodies targeting ADGRE2 in acute myeloid leukemia?

The identification of ADGRE2 as a potential therapeutic target in AML, particularly in leukemic stem cells, opens avenues for antibody-based intervention strategies . Researchers developing therapeutic antibodies should begin with comprehensive epitope mapping to identify regions critical for ADGRE2's role in maintaining proteostasis via the MEK/AP1/DUSP1 axis . This requires generating a panel of monoclonal antibodies against different domains and screening for those that disrupt the signaling cascade.

Antibody engineering approaches should consider format optimization—full IgG antibodies, Fab fragments, or bispecific antibodies may offer different advantages for targeting ADGRE2-expressing leukemic cells. Antibody-drug conjugates (ADCs) represent a particularly promising strategy, where anti-ADGRE2 antibodies can deliver cytotoxic payloads specifically to ADGRE2-high leukemic stem cells .

Functionality screening should assess the antibodies' ability to block the DNAJB1-HSP70 interaction downstream of ADGRE2 activation, as this appears critical for AML cell survival . Combination approaches with existing therapies should be explored, particularly with MEK inhibitors, AP1 inhibitors, and DUSP1 inhibitors, which have shown efficacy in AML xenograft models . Finally, researchers must evaluate potential off-target effects on normal myeloid cells, which also express ADGRE2, to develop a therapeutic window that selectively affects leukemic cells.

How can structural biology approaches be combined with antibody studies to understand ADGRE2 activation mechanisms?

Combining structural biology with antibody studies offers powerful insights into ADGRE2 activation mechanisms. Researchers should first develop a panel of conformation-specific antibodies that preferentially recognize active versus inactive states of ADGRE2 . These can serve as tools to trap specific conformations for structural studies.

Cryo-electron microscopy (cryo-EM) represents an ideal approach for visualizing ADGRE2 in complex with antibody fragments, potentially capturing intermediate states during receptor activation . X-ray crystallography of ADGRE2 fragments bound to Fab fragments can provide high-resolution structural details of specific domains, particularly the extracellular EGF-like domains and their interaction with ligands .

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) combined with domain-specific antibodies can reveal conformational changes during receptor activation, identifying regions with altered solvent accessibility . For studying the autocatalytic cleavage at the GPS and its role in receptor activation, researchers can use antibodies that specifically recognize the cleaved or uncleaved forms to trap these states for structural analysis .

Structure-guided antibody engineering can then create improved tools for investigating ADGRE2 function, including antibodies that stabilize specific conformations or block interactions with particular signaling partners. These structural insights, combined with functional studies, will enhance understanding of how vibration, dermatan sulfate binding, or mutations like C492Y affect ADGRE2 activation .

What experimental approaches can assess the interaction between ADGRE2 and the extracellular matrix component dermatan sulfate?

As dermatan sulfate has been identified as an endogenous ligand for ADGRE2 , understanding this interaction requires specialized experimental approaches. Researchers should develop solid-phase binding assays where recombinant ADGRE2 extracellular domain or purified ADGRE2-expressing cell membranes are immobilized, and binding of labeled dermatan sulfate is measured in the presence or absence of competing antibodies.

Surface plasmon resonance (SPR) or biolayer interferometry can quantify binding kinetics and affinities between ADGRE2 and dermatan sulfate, while also assessing how different antibodies affect this interaction . Cellular assays should measure functional outcomes of dermatan sulfate-ADGRE2 binding, such as calcium flux, ERK phosphorylation, or myeloid cell adhesion, and determine how these are modulated by various anti-ADGRE2 antibodies .

In tissue contexts, particularly skin where dermatan sulfate is the predominant glycosaminoglycan , co-localization studies using fluorescently labeled anti-ADGRE2 antibodies and dermatan sulfate-binding probes can reveal physiological interaction sites. Competition experiments with soluble dermatan sulfate, specific antibodies, and dermatan sulfate-degrading enzymes can determine the specificity and functional relevance of these interactions.

For the C492Y variant associated with vibratory urticaria, comparing wild-type and mutant ADGRE2 binding to dermatan sulfate may reveal whether altered ligand interaction contributes to the disease mechanism . These approaches collectively will enhance understanding of how ADGRE2 functions as a mechanosensory receptor in the extracellular matrix context.