ACR4 Antibody

What is ACR4 and what cellular functions does it regulate?

ACR4 refers to two distinct proteins in scientific literature: Arabidopsis CRINKLY4 in plant biology and Atypical Chemokine Receptor 4 in mammalian systems. In plants, Arabidopsis CRINKLY4 (ACR4) functions as a receptor-like kinase (RLK) involved in global plant development, with particular importance in formative cell division processes . In mammalian systems, Atypical Chemokine Receptor 4 (ACKR4) regulates dendritic cell migration by controlling chemokine ligands, notably binding to CCR7 ligands such as CCL19 and CCL21, and is involved in tumor development in mouse models . Unlike typical chemokine receptors, ACKR4 does not induce classical G protein-coupled receptor signaling but instead facilitates chemokine degradation through β-arrestin-mediated endocytosis and lysosomal processing .

What experimental techniques have been successful in studying ACR4 protein interactions?

Multiple complementary approaches have proven effective for studying ACR4 protein interactions. For plant ACR4, researchers have successfully employed in silico analysis, tandem affinity purification (TAP), yeast two-hybrid (Y2H) assays, and phage display approaches to define ACR4-interacting proteins . For instance, TAP approaches with Arabidopsis cell suspension cultures expressing tagged ACR4 intracellular kinase domains identified potential interacting proteins including HTPA REDUCTASE 1/DAPB1, HTPA REDUCTASE 2/DAPB2, and PROTEIN PHOSPHATASE 2A-3 (PP2A-3) . In vitro validation of interactions can be performed using gel-filtration analyses and pull-down assays, while in planta confirmation can utilize coimmunoprecipitation in transient expression systems such as Nicotiana benthamiana .

How can researchers validate the specificity of newly developed ACR4 antibodies?

Validating antibody specificity requires a multi-faceted approach. For anti-mouse ACKR4 antibodies, researchers have implemented a systematic validation strategy including:

• Initial screening via enzyme-linked immunosorbent assay (ELISA) using the target peptide

• Secondary screening by flow cytometry comparing reactivity between receptor-expressing cells (e.g., CHO/mACKR4) and control cells (e.g., CHO-K1)

• Specificity confirmation through peptide blocking experiments in western blotting

• Quantitative assessment of binding affinity using flow cytometry to determine dissociation constant (KD) values

For example, western blotting validation of anti-mACKR4 antibodies (A4Mab-1 and A4Mab-2) demonstrated specific detection of mACKR4 as a ~50-kDa band in LN229/mACKR4 cell lysates but not in control LN229 cells, with this detection being successfully blocked in the presence of mACKR4 peptide .

What methodological approaches are available for developing specific antibodies against membrane proteins like ACR4?

Developing antibodies against membrane proteins presents unique challenges due to their complex structure and hydrophobic domains. Based on successful development of anti-mACKR4 antibodies, two primary approaches emerge:

• N-terminal peptide immunization strategy: This approach involves:

  • Selecting immunogenic peptide sequences from the extracellular N-terminal domain

  • Conjugating the peptide to a carrier protein (e.g., KLH)

  • Immunizing animals with the peptide-carrier conjugate using an appropriate adjuvant

  • Screening hybridomas through a two-step process: ELISA against naked peptide followed by flow cytometry to identify clones recognizing the native conformation

• Cell-Based Immunization and Screening (CBIS) method: This alternative approach has been successfully employed for developing antibodies against multiple mouse chemokine receptors and involves immunizing with receptor-expressing cells .

For mACKR4, the N-terminal peptide immunization approach successfully yielded three monoclonal antibodies (A4Mab-1, A4Mab-2, and A4Mab-3) with different binding characteristics and applications .

How do the binding properties of different ACR4 antibody clones compare, and how might this affect experimental design?

Understanding the binding characteristics of different antibody clones is critical for experimental design. The following table summarizes key properties of three anti-mACKR4 antibody clones:

These differences in binding properties should guide experimental design:

• For flow cytometry applications requiring highest sensitivity, A4Mab-3 may be preferred due to its superior affinity

• For western blotting applications, A4Mab-2 demonstrates superior reactivity

• For multi-method approaches requiring both flow cytometry and western blotting, A4Mab-1 or A4Mab-2 would be more appropriate

What are the potential confounding factors when investigating ACR4 protein-protein interactions in complex systems?

Several confounding factors must be considered when investigating ACR4 protein-protein interactions:

• Technical limitations with membrane proteins: The transmembrane nature of ACR4 presents challenges for in vitro studies. This has led researchers to focus primarily on intracellular domains for in vitro and in vivo studies .

• Limited overlap between detection methods: Different protein interaction detection approaches (e.g., TAP, Y2H, phage display) may yield distinct sets of potential interacting partners with minimal overlap. For example, studies with plant ACR4 found limited to no overlap between interaction partners identified by different methods, suggesting either method-specific detection bias or high false-positive rates .

• Expression domain considerations: Potential interacting proteins may show distinct expression patterns that only partially overlap with the ACR4 expression domain, requiring careful consideration of spatiotemporal dynamics .

• Post-translational modifications: Phosphorylation can significantly impact protein interactions. For ACR4, the interaction with some partners (like PP2A-3) may be phosphorylation-dependent, requiring careful experimental design to account for the phosphorylation state .

What protocol modifications are recommended for using ACR4 antibodies in flow cytometry applications?

When using ACR4 antibodies for flow cytometry, consider the following protocol optimizations:

• Cell preparation:

  • For adherent cells (e.g., CHO-K1, LN229), harvest cells in exponential growth phase using non-enzymatic cell dissociation solution to preserve surface epitopes

  • Wash cells twice with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)

  • Adjust cell concentration to 1 × 10⁶ cells/mL

• Antibody incubation:

  • Incubate 100 μL of cell suspension with optimized antibody concentration (typically 1 μg/mL for A4Mab-1, A4Mab-2, or A4Mab-3)

  • Maintain incubation for 30 minutes on ice

  • Wash cells twice with 1% BSA in PBS

• Secondary antibody:

  • Use appropriate fluorochrome-conjugated secondary antibody specific to the isotype (for A4Mab-1, A4Mab-2, and A4Mab-3, use anti-rat IgG)

  • Incubate for 30 minutes on ice, protected from light

  • Wash twice and resuspend in PBS containing 0.5 μg/mL propidium iodide for dead cell exclusion

For optimal results, always include appropriate isotype controls and positive/negative cell lines to establish gating strategies.

How can researchers accurately determine binding affinities for ACR4 antibodies?

Accurate determination of binding affinities is essential for characterizing antibody performance. For anti-mACKR4 antibodies, researchers employed the following methodology to determine dissociation constant (KD) values:

• Sample preparation:

  • Harvest cells expressing the target receptor (e.g., CHO/mACKR4) in exponential growth phase

  • Prepare serial dilutions of purified antibody ranging from 100 nM to 0.1 nM

• Flow cytometry analysis:

  • Incubate cells with different antibody concentrations under equilibrium conditions

  • Detect bound antibody using fluorochrome-conjugated secondary antibody

  • Measure mean fluorescence intensity (MFI) for each antibody concentration

• Data analysis:

  • Plot MFI versus antibody concentration

  • Fit data to a one-site binding model using appropriate statistical software

  • Calculate KD as the antibody concentration yielding half-maximal binding

Using this approach, researchers determined KD values of 6.0 × 10⁻⁹ M, 1.3 × 10⁻⁸ M, and 1.7 × 10⁻⁹ M for A4Mab-1, A4Mab-2, and A4Mab-3, respectively, providing quantitative measures of their binding affinities .

What controls are essential when using ACR4 antibodies in western blotting experiments?

When performing western blotting with ACR4 antibodies, incorporate the following essential controls:

• Positive and negative cell lysates:

  • Include lysates from cells confirmed to express the target protein (e.g., LN229/mACKR4) and control cells lacking expression (e.g., LN229)

  • This validates antibody specificity by demonstrating detection only in positive samples

• Loading controls:

  • Include antibodies against housekeeping proteins (e.g., β-actin) to normalize for protein loading differences

  • This ensures that differences in target protein band intensity reflect actual biological differences rather than loading variations

• Peptide blocking controls:

  • Pre-incubate antibody with excess cognate peptide before western blotting

  • Specific binding should be competitively inhibited by the peptide, resulting in reduced or absent band intensity

  • This confirms that detection is due to specific epitope recognition rather than non-specific binding

• Molecular weight markers:

  • Include appropriate molecular weight markers to confirm that detected bands appear at the expected size

  • For mACKR4, specific bands should appear at approximately 50 kDa

Implementation of these controls is exemplified in the development of anti-mACKR4 antibodies, where A4Mab-1 and A4Mab-2 demonstrated specific detection of mACKR4 as a ~50-kDa band, with this detection being blocked in the presence of mACKR4 peptide .

How might ACR4 antibodies contribute to understanding tumor development mechanisms?

ACR4 antibodies offer significant potential for understanding tumor development mechanisms, particularly through investigation of ACKR4's role in immune cell migration and tumor immunology. ACKR4 regulates dendritic cell migration by controlling chemokine ligands and has been implicated in tumor development in mouse models . Specific anti-mACKR4 antibodies enable:

• Identification and characterization of ACKR4-expressing cells in the tumor microenvironment using flow cytometry, potentially revealing novel cellular populations involved in tumor progression or suppression

• Quantification of ACKR4 expression levels in different tumor types and stages, possibly identifying correlations with disease progression or treatment response

• Investigation of ACKR4's role in chemokine-mediated immune cell trafficking within tumors, potentially revealing mechanisms by which tumors evade immune surveillance

• Development of therapeutic strategies targeting ACKR4 to modulate immune responses in cancer, as preliminary findings suggest that targeting ACKR4 might improve immunotherapy efficacy

The availability of well-characterized anti-mACKR4 monoclonal antibodies suitable for flow cytometry and western blotting provides researchers with essential tools to pursue these investigations in preclinical tumor models .

What parallels exist between plant ACR4 and mammalian ACKR4 research that might inform cross-disciplinary approaches?

While plant ACR4 (Arabidopsis CRINKLY4) and mammalian ACKR4 (Atypical Chemokine Receptor 4) are distinct proteins with different functions, several methodological parallels in their study may inform cross-disciplinary approaches:

• Protein interaction network analysis:

  • Both fields employ similar techniques (Y2H, co-immunoprecipitation) to identify interaction partners

  • Computational approaches integrating multiple interaction detection methods could benefit both fields

• Domain-focused investigations:

  • Both fields face challenges studying full transmembrane proteins and often focus on specific domains

  • Plant ACR4 research focusing on intracellular kinase domains may provide methodological insights for studying ACKR4 signaling mechanisms

• Phosphorylation dynamics:

  • Phosphorylation-dependent interactions appear important in both systems

  • Techniques developed to study phosphorylation-dependent interactions of plant ACR4 could inform similar studies of ACKR4

• Antibody development strategies:

  • Peptide-based immunization approaches successful for developing anti-mACKR4 antibodies might be adapted for generating antibodies against plant ACR4

Despite functional differences, shared methodological challenges and solutions between these research areas could accelerate progress in both fields.

How do natural antibodies against receptor proteins compare to laboratory-generated monoclonal antibodies in research applications?

Natural antibodies and laboratory-generated monoclonal antibodies each offer distinct advantages and limitations for research applications:

Natural antibodies (such as those observed in Alzheimer's disease patients against proteins like ankyrin G):

• Develop through natural immune responses to endogenous or disease-associated antigens

• May reflect physiologically relevant epitopes and binding characteristics

• Often polyclonal in nature, recognizing multiple epitopes on the target protein

• Can provide insights into disease mechanisms and potential therapeutic approaches

• May correlate with disease outcomes (e.g., AD patients with anti-ankyrin G antibodies showed stabilized or improved cognitive scores)

Laboratory-generated monoclonal antibodies (such as A4Mab-1, A4Mab-2, and A4Mab-3 against mACKR4):

• Provide consistent, reproducible reagents with defined specificity

• Can be precisely characterized for binding affinity (KD values)

• Allow targeted epitope selection through immunization strategies

• Enable controlled production and quality assessment

• Support standardized research applications (e.g., flow cytometry, western blotting)

For receptor proteins like ACR4/ACKR4, laboratory-generated monoclonal antibodies typically offer greater utility for standardized research applications, while natural antibodies may provide valuable insights into disease-relevant immune responses and potential therapeutic approaches. The complementary use of both antibody types could enhance understanding of receptor biology in both normal and pathological contexts.