ACKR3 Antibody

What is ACKR3 and why is it important for research?

ACKR3 (Atypical Chemokine Receptor 3) is a membrane protein that functions as an atypical chemokine receptor, controlling chemokine levels and localization via high-affinity binding. Unlike typical chemokine receptors, ACKR3 binding does not activate G-protein-mediated signal transduction but instead induces β-arrestin recruitment, leading to ligand internalization and activation of MAPK signaling pathways .

ACKR3 acts as a receptor for chemokines CXCL11 and CXCL12/SDF1 and is involved in regulating CXCR4 protein levels in migrating interneurons. In glioma cells, it transduces signals via MEK/ERK pathway, mediating resistance to apoptosis and promoting cell growth and survival . Its role in various physiological and pathological processes makes it an important target for research.

What alternative names should I be aware of when searching for ACKR3 antibodies?

When conducting literature searches or sourcing antibodies, researchers should be aware of multiple nomenclature variations for ACKR3:

This receptor underwent nomenclature changes, being previously known as CXCR7 before reclassification as an atypical chemokine receptor (ACKR) .

What species reactivity can I expect from commercially available ACKR3 antibodies?

Most commercially available ACKR3 antibodies demonstrate cross-reactivity across multiple species:

When selecting an antibody, verify the specific epitope sequence to ensure compatibility with your target species. Some antibodies, like the non-phospho-ACKR3 receptor antibody from 7TM Antibodies, target the carboxyl-terminal tail with an epitope identical across human, mouse, and rat ACKR3 .

How can I validate the specificity of an ACKR3 antibody for my experiments?

Validating ACKR3 antibody specificity is crucial given the challenges in detecting this receptor. A systematic validation approach includes:

• Overexpression systems: Compare signal between ACKR3-overexpressing cells and control cells (e.g., U87 ACKR3 vs. U87 wild-type)

• Multiple antibody comparison: Test several antibodies against the same samples to identify consistent patterns. Research has shown that while 8F11-M16 and 11G8 antibodies yield reliable results, many commercial antibodies fail to specifically detect ACKR3

• Multiple detection methods: Validate using different techniques (flow cytometry, immunofluorescence, Western blotting)

• Negative controls: Include cells known not to express ACKR3 or those expressing related receptors (e.g., CXCR4) to test cross-reactivity

The 11G8 antibody has been validated to specifically identify ACKR3 in immunostaining and immunoblotting experiments using overexpression GBM cell models and MCF-7 breast cancer cells that endogenously express ACKR3 .

ACKR3 antibodies have been validated for various applications:

The non-phospho-ACKR3 receptor antibody (7TM0080N) can also be used to isolate and enrich ACKR3 receptors from cell and tissue lysates .

What should I be aware of when interpreting Western blot results with ACKR3 antibodies?

When interpreting Western blot results for ACKR3, consider these important factors:

• Multiple bands: ACKR3/CXCR7 forms stable dimers and multimers, which will appear as additional bands at higher molecular weights. This is a characteristic feature of the receptor rather than non-specific binding

• Glycosylation patterns: ACKR3 is subject to post-translational modifications including glycosylation, which can affect apparent molecular weight

• Sample preparation: Complete solubilization of membrane proteins is critical; insufficient denaturation may result in aggregate formation

• Controls: Always include positive controls (ACKR3-overexpressing cells) and negative controls (mock-transfected cells) as demonstrated in validation studies

Research has demonstrated that the phosphorylation-independent c-terminal anti-ACKR3/CXCR7 antibody (7TM0080N) can reliably detect these multimeric forms at a dilution of 1:1000 .

How can I differentiate between ACKR3 and the related CXCR4 receptor in my samples?

Differentiating between ACKR3 and CXCR4 requires careful experimental design:

• Antibody selection: Use well-validated antibodies with demonstrated specificity. The 11G8 and 8F11-M16 antibodies have been shown not to cross-react with CXCR4

• Receptor-specific agonists: CXCL11 binds to ACKR3 but not CXCR4, while CXCL12 binds both receptors. Differential responses to these ligands can help distinguish the receptors

• Functional assays: CXCR4 couples to G proteins and directly promotes cell migration, while ACKR3 is G protein-independent. Invasion assays have shown that CXCL12 increases invasiveness of U87 CXCR4 cells but not U87 ACKR3 cells

• Conformational dynamics: Single-molecule FRET studies have revealed that apo-CXCR4 preferentially populates a high-FRET inactive state, while apo-ACKR3 shows little conformational preference and high transition probabilities among multiple conformations

These distinct molecular and functional properties can be leveraged to distinguish between the two receptors in experimental systems.

What are the recommended storage conditions for ACKR3 antibodies?

Proper storage is critical for maintaining antibody function:

For optimal preservation:

• Aliquot antibodies upon receipt to minimize freeze-thaw cycles

• For long-term storage, keep at -20°C

• Avoid repeated freeze/thaw cycles that can degrade antibody quality

• For working solutions, store at +4°C for short periods (typically 1-2 weeks)

• Follow manufacturer-specific recommendations, as formulations may vary

How can ACKR3 antibodies be used to study receptor regulation in response to ligand stimulation?

ACKR3 antibodies are valuable tools for investigating receptor regulation:

• Surface expression quantification: Flow cytometry with antibodies like 8F11-M16 can measure changes in cell surface ACKR3 levels following ligand stimulation. Studies have shown that when T018 glioma stem cells were stimulated with 10 nM CXCL12, changes in ACKR3 surface expression could be detected

• Internalization studies: Antibodies can track receptor internalization following ligand binding, a key aspect of ACKR3's chemokine scavenging function

• Phosphorylation detection: Phosphorylation-specific antibodies can be used alongside non-phospho-ACKR3 antibodies (like 7TM0080N) to study receptor phosphorylation dynamics, which are important for ACKR3 regulation of neuronal migration

• Co-localization experiments: Immunofluorescence with 11G8 antibody can reveal ACKR3 localization changes in response to stimuli

Research has demonstrated that ACKR3 regulation of neuronal migration requires receptor phosphorylation but not β-arrestin, highlighting the importance of studying these post-translational modifications .

What experimental approaches can be used to study the distinct activation mechanisms of ACKR3 compared to CXCR4?

The distinct activation mechanisms of ACKR3 and CXCR4 can be studied using:

• Single-molecule FRET: This technique has revealed fundamental differences in conformational dynamics, showing that ACKR3 populates multiple active-like conformations in response to agonists, compared to a single CXCR4 active-state

• β-arrestin recruitment assays: Since ACKR3 signaling is primarily through β-arrestin rather than G proteins, assays measuring β-arrestin recruitment are critical for studying ACKR3 activation

• Receptor mutagenesis: Studies have identified that much of the conformational heterogeneity of ACKR3 is linked to a single residue that differs between ACKR3 and CXCR4

• Ligand specificity assays: ACKR3 responds promiscuously to CXCL12, CXCL12 variants, and other peptides and proteins, while CXCR4 only responds to wild-type CXCL12 and is sensitive to mutation

• Functional outcomes: Assess differences in downstream signaling like MAPK pathway activation using phospho-specific antibodies against signaling components

These approaches have revealed that the dynamic properties of ACKR3 may underlie its inability to form productive interactions with G proteins that would drive canonical GPCR signaling .

How do I design experiments to investigate ACKR3's role in cancer using antibodies?

To investigate ACKR3's role in cancer:

• Expression profiling: Use validated antibodies like 11G8 to screen cancer cell lines and patient samples for ACKR3 expression. Studies have confirmed ACKR3 expression in MCF-7 breast cancer cells and various glioma stem cell cultures (T08, T013, T018, T033) at different levels

• Functional studies: Compare proliferation and invasion between wild-type and ACKR3-overexpressing cells. Research has shown that ACKR3 overexpression did not significantly alter proliferation in U87 or T033 glioma stem cells, nor did it modify invasive properties in Boyden chamber assays

• In vivo models: Use antibodies to confirm ACKR3 expression in xenograft models. Studies comparing T033 mRFP and T033 hACKR3 engrafted in immunodeficient mice showed that both developed large, highly invasive tumors without obvious differences in growth or invasiveness

• Microenvironment interactions: Investigate how ACKR3 expression changes in different tumor microenvironments by comparing in vitro and in vivo expression levels using RT-qPCR and immunostaining

• Therapeutic targeting: Use antibodies as potential therapeutic agents or to evaluate the efficacy of other ACKR3-targeting approaches

This multifaceted approach can provide insights into ACKR3's role in cancer progression and potential as a therapeutic target.

Why might I observe inconsistent staining patterns with ACKR3 antibodies?

Inconsistent staining patterns with ACKR3 antibodies may result from:

• Heterogeneous expression: Studies have documented heterogeneous ACKR3 expression in cell populations. In glioma stem cell cultures, only a small percentage of cells expressed ACKR3 (T08=1.22±0.76%; T013=3.91±1.78%; T018=2.90±0.66%; T033=0.78±0.18%)

• Antibody specificity issues: Many commercial antibodies fail to specifically detect ACKR3. Research comparing various antibodies found that only 11G8 and 8F11-M16 provided reliable detection

• Dynamic regulation: ACKR3 surface expression may change in response to stimuli. CXCL12 stimulation (10 nM for 24h) has been shown to alter ACKR3 expression patterns

• Cell fixation and permeabilization: Detection of membrane proteins can be sensitive to fixation methods that may mask or destroy epitopes

• Receptor internalization: As a scavenging receptor, ACKR3 undergoes constitutive and ligand-induced internalization, affecting surface availability for antibody binding

To address these issues, use positive controls with known ACKR3 expression (e.g., U87 ACKR3 cells that show 81.40±2.31% positive staining) and optimize staining protocols for each specific application .

What controls should I include when using ACKR3 antibodies?

Robust experimental design with ACKR3 antibodies requires these controls:

• Positive expression control: Include cells with confirmed ACKR3 expression (e.g., U87 ACKR3 or MCF-7 cells)

• Negative expression control: Include cells known not to express ACKR3 (e.g., U87 parental cells)

• Related receptor control: Include cells expressing related receptors like CXCR4 to control for cross-reactivity (e.g., U87 CXCR4 cells)

• Secondary antibody control: Include samples with secondary antibody only to assess background staining

• Isotype control: Include samples with non-specific antibody of the same isotype as the ACKR3 antibody

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to verify binding specificity

• Knockout/knockdown validation: If available, use ACKR3 knockout or knockdown cells as ultimate specificity controls

In published research, U87, U87 ACKR3, and U87 CXCR4 cells provided a complete control set for validating antibody specificity, with reliable antibodies showing strong signals only in U87 ACKR3 cells .

How can ACKR3 antibodies be used to study neuronal migration?

ACKR3 antibodies are valuable tools for studying neuronal migration:

• Expression mapping: Use immunohistochemistry with validated antibodies to map ACKR3 expression in developing brain regions

• Co-localization studies: Combine ACKR3 antibodies with markers for migrating neurons to assess spatial relationships

• Phosphorylation dynamics: Research has shown that ACKR3 regulation of neuronal migration requires ACKR3 phosphorylation but not β-arrestin. Using phosphorylation-specific and non-phospho antibodies can reveal these regulatory mechanisms

• Receptor-ligand interactions: Study how ACKR3 controls neuronal migration by regulating chemokine responsiveness. Research has demonstrated that ACKR3 is required for regulation of CXCR4 protein levels in migrating interneurons

• Functional blocking: Use function-blocking antibodies to interrogate ACKR3's role in migration in ex vivo or in vitro systems

Key publications in this area include work by Saaber et al. (2019) and Sánchez-Alcañiz et al. (2011), which used specific ACKR3 antibodies to demonstrate the receptor's role in neuronal migration and chemokine responsiveness regulation .

What insights can conformational studies of ACKR3 provide for drug discovery?

Conformational studies of ACKR3 offer valuable insights for drug discovery:

• Multiple conformational states: Single-molecule FRET studies have revealed that ACKR3 shows little conformational preference in its apo state and has high transition probabilities among multiple inactive, intermediate, and active conformations. This contrasts with CXCR4, which preferentially populates a high-FRET inactive state

• Activation mechanisms: ACKR3 populates multiple active-like conformations in response to agonists, compared to the single CXCR4 active-state. This suggests that ACKR3 activation may be achieved by a broader distribution of conformational states

• Critical residues: Research has identified that a single residue difference between ACKR3 and CXCR4 accounts for much of ACKR3's conformational heterogeneity

• Ligand promiscuity: ACKR3 promiscuously responds to CXCL12, CXCL12 variants, other peptides and proteins, and is relatively insensitive to mutation. This information can guide the design of selective ligands

• G-protein coupling: The dynamic properties of ACKR3 may underlie its inability to form productive interactions with G proteins. Understanding these properties could help design biased ligands

These insights can inform the development of selective ACKR3 modulators that could have therapeutic potential in conditions where ACKR3 plays a role, such as cancer or neurological disorders.