GPR132 Antibody

Basic Research Questions

• What is GPR132 and how is it identified in laboratory settings?

GPR132 is a G protein-coupled receptor with seven transmembrane domains, classified as a member of the proton-sensing GPCR subfamily. This 42.5 kDa protein (approximately 380 amino acids) is also known as G2A or probable G-protein coupled receptor 132 .

When identifying GPR132 in laboratory settings, researchers typically use:

• Western blot analysis (predicted band size: 42 kDa)

• Immunohistochemistry on tissues expressing GPR132

• Flow cytometry for cell surface detection

GPR132 antibodies recognize specific epitopes, often from the extracellular domains or C-terminal region, with several validated antibodies showing reactivity to human, mouse, and monkey samples .

• What are the primary applications for GPR132 antibodies in research?

GPR132 antibodies serve multiple research applications:

These applications help researchers investigate GPR132's role in:

• Immune cell function (particularly in macrophages and NK cells)

• Tumor microenvironment studies

• Cell cycle regulation

• Acid-sensing mechanisms

• Where is GPR132 primarily expressed and how does this inform experimental design?

GPR132 shows tissue-specific expression patterns that should guide experimental design:

• High expression: Hematopoietic tissues rich in lymphocytes (spleen, thymus), macrophages

• Moderate expression: Found in various immune cells including dendritic cells, neutrophils, mast cells, T and B lymphocytes

• Low expression: Heart and lung tissues

• Pathological contexts: Upregulated in atherosclerotic plaques (particularly around lipid cores and shoulder regions)

When designing experiments:

• Use appropriate positive controls (e.g., THP-1 monocytic leukemia cells, Raji B lymphocyte cells, or HL-60 promyelocytic leukemia cells)

• Consider activation state (GPR132 expression is reduced in activated NK cells)

• Account for potential upregulation in tumor-bearing models

• What validation methods ensure GPR132 antibody specificity?

To ensure antibody specificity for GPR132:

• Western blot analysis: Verify the band appears at the expected molecular weight (~42 kDa)

• Knockout controls: Compare staining between wild-type and GPR132 knockout samples

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to block specific binding

• Multiple antibody validation: Use antibodies targeting different epitopes of GPR132

• Cross-reactivity testing: Test reactivity in different species based on sequence homology

• Positive control tissues/cells: Use cells known to express GPR132 (e.g., THP-1, Raji, HL-60 cell lines)

Commercial antibodies are typically validated against known positive and negative samples to ensure specificity and high affinity .

Advanced Research Questions

• How can researchers effectively study the role of GPR132 in NK cell function?

Recent research has uncovered GPR132's importance in NK cell regulation through the Gαs/CSK/ZAP70/NF-κB signaling axis . To study this:

• NK cell isolation and modification:

  • Isolate NK cells from spleens of wild-type and GPR132⁻/⁻ mice

  • Use shRNA approaches to knock down GPR132 in NK92 cells (human NK cell line)

  • Compare with GPR132 agonist treatment (e.g., ONC212)

• Functional assays:

  • Cytotoxicity assays: Co-culture modified NK cells with target cells (e.g., K562, Yac-1) at different effector-to-target ratios

  • Flow cytometry: Measure expression of activation markers (CD69, CD107a), cytokines (IFN-γ), and cytotoxic molecules (GzmB)

  • Proliferation: Assess Ki67 expression

  • Apoptosis resistance: Culture without IL-2 and measure survival and Bcl-2 expression

• Mechanistic studies:

  • Analyze phosphorylation of ZAP70 and nuclear translocation of NF-κB

  • Use specific inhibitors of Gαs or CSK to confirm pathway involvement

  • Investigate receptor internalization upon activation

• In vivo models:

  • Tumor challenge models in GPR132⁻/⁻ vs. wild-type mice

  • Adoptive transfer of GPR132-modified NK cells to assess tumor control

• What approaches are most effective for investigating GPR132's role in macrophage polarization and the tumor microenvironment?

GPR132 significantly impacts macrophage polarization and tumor progression through lactate sensing:

• Macrophage polarization assessment:

  • Isolate macrophages from wild-type and GPR132⁻/⁻ mice or use siRNA/CRISPR in human macrophages

  • Treat with tumor-conditioned media or purified lactate

  • Analyze M1/M2 marker expression by flow cytometry, qPCR, and ELISA

  • Use lactate receptor antagonists to confirm specificity

• Lactate-GPR132 interaction studies:

  • Use pH-responsive fluorescence sensors (e.g., Probe 5c) to detect acidification

  • Measure calcium flux or cAMP production upon lactate exposure

  • Perform binding assays with labeled lactate

  • Compare responses in GPR132-expressing vs. control cells

• Tumor-macrophage co-culture systems:

  • Use transwell systems to assess paracrine effects

  • Evaluate cancer cell adhesion, migration, and invasion in presence of differently polarized macrophages

  • Block GPR132 using antibodies or small molecules to assess functional impact

• In vivo metastasis models:

  • Orthotopic breast cancer models in GPR132⁻/⁻ vs. wild-type mice

  • Track spontaneous metastasis (e.g., EO771.LMB cells show aggressive lung metastasis)

  • Analyze tumor-associated macrophage phenotypes by multi-parameter flow cytometry

  • Longitudinal imaging with pH-responsive probes

• How should experiments be designed to investigate GPR132's role in AML differentiation?

GPR132 activation induces differentiation in Acute Myeloid Leukemia, suggesting therapeutic potential:

• Genetic approaches:

  • Overexpress GPR132 in AML cell lines using lentiviral vectors

  • Create inducible expression systems to control timing of GPR132 activation

  • Use CRISPR-Cas9 to modify specific domains of GPR132 to determine structure-function relationships

• Pharmacological approaches:

  • Identify and validate GPR132 agonists (e.g., 8-gingerol/8GL)

  • Determine dose-response relationships and specificity

  • Assess synergy with established differentiation agents or mTOR inhibitors

• Differentiation assessment:

  • Morphological analysis (Wright-Giemsa staining)

  • Flow cytometry for myeloid differentiation markers (CD11b, CD14, CD15)

  • Colony formation assays to measure self-renewal capacity

  • Cell cycle analysis to detect G2/M checkpoint effects

• Signaling pathway analysis:

  • Focus on GPR132-Gαs-PKA pathway

  • Monitor mTOR activation (phosphorylation of downstream targets)

  • Use pathway inhibitors to confirm mechanism

• In vivo models:

  • AML xenograft models treated with GPR132 agonists

  • Assess tumor growth, survival, and differentiation markers

  • Test primary AML patient samples to confirm clinical relevance

• What are the most reliable methods for measuring GPR132 activation in pH-sensing experiments?

As a proton-sensing GPCR, GPR132 responds to extracellular pH changes. To measure this:

• Receptor activation assays:

  • BRET/FRET-based assays to measure conformational changes

  • Measurement of second messengers (cAMP, calcium, inositol phosphates)

  • β-arrestin recruitment assays

  • Receptor internalization studies using fluorescently-tagged GPR132

• pH manipulation approaches:

  • Controlled buffer systems with defined pH (typically ranging from pH 6.4-7.4)

  • Lactate treatment to mimic acidic tumor microenvironment

  • Use of proton pumps or ionophores for dynamic pH changes

  • Co-culture with glycolytic cells that naturally acidify media

• Functional readouts:

  • Transcriptional changes (qPCR for known pH-responsive genes)

  • Pathway-specific reporter assays

  • Phenotypic changes (migration, phagocytosis, cytokine production)

  • pH-sensitive fluorescent probes to correlate cellular responses with pH changes

• Controls and validation:

  • Use GPR132⁻/⁻ cells as negative controls

  • Include other pH-sensing GPCRs (GPR4, GPR65, GPR68) for comparison

  • Employ pH buffers resistant to metabolic changes

  • Use selective antagonists to confirm specificity

• What are the technical considerations for using GPR132 antibodies in live cell applications?

Working with GPR132 antibodies in live cell applications requires special consideration:

• Antibody selection criteria:

  • Choose antibodies targeting extracellular domains of GPR132

  • Verify lack of sodium azide or other toxic preservatives

  • Confirm antibody maintains binding affinity at physiological pH and temperature

  • Select appropriate fluorophore conjugates with minimal spectral overlap

• Flow cytometry optimization:

  • Titrate antibody concentration (typically starting at ~5 μg per test)

  • Use non-enzymatic cell dissociation methods to preserve surface epitopes

  • Include viability dyes to exclude dead cells

  • Set up compensation controls if using multiple fluorophores

• Live cell imaging considerations:

  • Use cell-friendly imaging buffers

  • Minimize phototoxicity with reduced laser power

  • Consider photobleaching when planning time-lapse experiments

  • Use rapid acquisition techniques for real-time trafficking studies

• Controls and validation:

  • Include isotype controls at matching concentrations

  • Block with unconjugated antibody before adding conjugated versions

  • Validate staining pattern with fixed cell immunofluorescence

  • Compare staining between known GPR132-positive and negative cell types

• How can researchers effectively use GPR132 antibodies in investigating signaling pathway cross-talk?

GPR132 interacts with multiple signaling pathways, requiring sophisticated experimental approaches:

• Co-immunoprecipitation strategies:

  • Use GPR132 antibodies to pull down receptor complexes

  • Identify interaction partners by mass spectrometry

  • Verify interactions with reverse co-IP

  • Include appropriate controls (IgG, lysate inputs)

• Signaling pathway analysis:

  • Combine GPR132 staining with phospho-specific antibodies (pZAP70, pNF-κB)

  • Use phospho-flow cytometry for single-cell resolution

  • Employ time-course experiments to capture signaling dynamics

  • Create pathway activity maps using multi-parameter readouts

• Proximity ligation assays:

  • Detect GPR132 interactions with downstream effectors at <40 nm resolution

  • Visualize signaling complexes in situ

  • Quantify interaction frequency under different conditions

• Genetic manipulation considerations:

  • Create domain-specific mutations to disrupt particular signaling branches

  • Use inducible systems to control timing of GPR132 expression

  • Combine with pathway-specific inhibitors to untangle complex networks

  • Consider compensatory mechanisms in chronic manipulation models

• Computational approaches:

  • Use systems biology tools to model GPR132 signaling networks

  • Integrate transcriptomic and proteomic data

  • Predict and validate feedback mechanisms

  • Identify potential therapeutic vulnerabilities