LPAR2 Antibody

What is LPAR2 and what are its key characteristics?

LPAR2 (Lysophosphatidic Acid Receptor 2) is a G-protein coupled receptor that functions as a receptor for lysophosphatidic acid (LPA), which mediates diverse cellular activities. In humans, the canonical protein has a reported length of 348 amino acid residues with a molecular mass of 38.7 kDa. LPAR2 is localized in the cell membrane and is most abundantly expressed in testes and peripheral blood leukocytes, with lower expression in the pancreas, spleen, thymus, and prostate. It belongs to the G-protein coupled receptor 1 protein family and is also known by several synonyms including EDG4, LPA-2, LPA2, and LPA receptor 2 . LPAR2 gene orthologs have been identified across multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken, indicating its evolutionary conservation .

What are the primary applications for LPAR2 antibodies in research?

LPAR2 antibodies are utilized in various experimental techniques crucial for investigating its expression, localization, and function:

• Western Blot (WB): The most widely used application for detecting and quantifying LPAR2 protein in cell or tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of LPAR2 in solution

• Immunofluorescence (IF): Visualizing the subcellular localization of LPAR2

• Immunohistochemistry (IHC): Examining LPAR2 expression patterns in tissue sections

These applications allow researchers to study LPAR2's expression levels, distribution patterns, and potential roles in normal physiology and disease processes . When selecting an antibody, researchers should consider which applications have been validated for their specific research needs.

How can I confirm the specificity of a commercial LPAR2 antibody?

Confirming antibody specificity is essential for reliable results. Use these validation approaches:

• Positive controls: Test the antibody on tissues known to express high levels of LPAR2 (testes, peripheral blood leukocytes)

• Negative controls: Include tissues with minimal LPAR2 expression

• Knockout validation: Test antibody on LPAR2 knockout/knockdown models like Lpar2-/- mice

• Peptide competition assays: Pre-incubation with immunizing peptide should abolish specific staining

• Multiple antibody approach: Compare staining patterns using antibodies targeting different LPAR2 epitopes

• Western blot analysis: Confirm the antibody detects a protein of the expected molecular weight (38.7 kDa)

Thorough validation ensures experimental results accurately reflect true LPAR2 biology rather than non-specific interactions or artifacts .

How can LPAR2 antibodies be used to study its role in cancer progression?

LPAR2 expression has been correlated with different prognoses across various cancer types, making it an important subject for cancer research. Researchers can employ LPAR2 antibodies in multiple sophisticated approaches:

• Tissue microarray analysis: Examine LPAR2 expression across multiple patient samples simultaneously

• Multiplex immunofluorescence: Co-localize LPAR2 with other cancer markers to study pathway interactions

• Patient-derived xenograft models: Track LPAR2 expression during tumor evolution

• Chromatin immunoprecipitation (for transcription factors regulating LPAR2): Identify regulatory mechanisms

Research findings demonstrate that LPAR2 expression correlates with divergent outcomes depending on cancer type. Studies have shown that high LPAR2 expression is associated with worse prognosis in kidney renal clear cell carcinoma (KIRC) (HR = 1.99, p < 0.001) and adrenocortical carcinoma (ACC) (HR = 2.35, p = 0.031) . Conversely, elevated LPAR2 expression correlates with improved prognosis in head and neck squamous cell carcinoma (HNSC) (HR = 0.74, p = 0.025) and other cancers like bladder cancer (BLCA) and stomach adenocarcinoma (STAD) . These contrasting associations highlight the context-dependent role of LPAR2 in cancer biology.

What role does LPAR2 play in immune cell infiltration and tumor microenvironment?

LPAR2 expression significantly correlates with various immune markers in tumor tissue, suggesting important roles in regulating the tumor immune microenvironment. Researchers can investigate this relationship using:

• Single-cell RNA sequencing combined with protein validation using LPAR2 antibodies

• Spatial transcriptomics with LPAR2 immunostaining

• Flow cytometry panels incorporating LPAR2 with immune subset markers

• Bioinformatic analysis correlating LPAR2 expression with immune cell signature genes

Studies have revealed significant associations between LPAR2 expression and various immune cell markers, particularly in HNSC and KIRC tumors . This correlation suggests that LPAR2 may influence tumor progression partly through modulating immune cell recruitment, activation, or function. Understanding these interactions could potentially inform immunotherapeutic approaches targeting LPAR2-related pathways.

How can LPAR2 antibodies help investigate its role in NSAID-induced enteropathy?

LPAR2 has been implicated in non-steroidal anti-inflammatory drug (NSAID)-induced small intestinal injury. Researchers can employ LPAR2 antibodies to:

• Track changes in LPAR2 expression during NSAID exposure

• Identify cell types expressing LPAR2 in intestinal tissues

• Investigate LPAR2 signaling pathway activation

• Compare wild-type and Lpar2-/- mouse models

Studies using Lpar2-deficient (Lpar2-/-) mice have demonstrated that NSAID-induced mucosal damage and neutrophil recruitment occurs much earlier (at 6 hours after indomethacin treatment) in these knockout mice compared to wild-type controls . Interestingly, despite increased tissue damage, knockout mice showed lower levels of inflammatory mediators (IL-1β, TNF-α, inducible COX-2) . This suggests a dual role for LPAR2 in both protecting against immediate tissue injury and regulating subsequent inflammatory responses. Administration of selective LPAR2 agonists like DBIBB reduced mucosal injury and neutrophil activation while enhancing certain pro-inflammatory cytokines, further supporting this complex dual function .

What can LPAR2 antibodies reveal about entotic cell invasion mechanisms?

LPAR2 has been identified as a signal transducer specifically required for actively invading cells during homotypic or entotic cell-in-cell invasion. Researchers can use LPAR2 antibodies to:

• Track LPAR2 localization during invasion processes

• Identify binding partners via co-immunoprecipitation

• Visualize spatial relationships between LPAR2, RhoA, and actin

• Characterize the effects of LPAR2 agonists/antagonists on invasion dynamics

Research has shown that LPAR2 signaling is integral to entotic invasion, an integrin-independent process observed in carcinoma cells under low adhesion conditions . This process depends on RhoA and actin dynamics, with LPAR2 functioning as a key upstream regulator. Immunodetection of LPAR2 alongside cytoskeletal components can provide insights into the molecular machinery driving this process, potentially revealing new therapeutic targets for cancers exhibiting this invasive behavior.

What are optimal sample preparation techniques for different LPAR2 antibody applications?

Optimization for each specific antibody and experimental system is essential. For membrane proteins like LPAR2, particular attention should be paid to extraction efficiency and epitope preservation.

How should researchers troubleshoot weak or absent LPAR2 signal despite evidence of expression?

When LPAR2 protein detection fails despite gene expression evidence:

• Try multiple antibodies targeting different epitopes

• Optimize protein extraction specifically for membrane proteins

  • Include appropriate detergents (CHAPS, NP-40, or digitonin)

  • Consider membrane fractionation to enrich LPAR2

• Test signal amplification techniques:

  • Tyramide signal amplification for IHC/IF

  • Enhanced chemiluminescence substrates for Western blot

• Verify translation using techniques like polysome profiling

• Check for post-transcriptional regulation that may prevent protein expression

Remember that mRNA expression doesn't always correlate with protein levels due to various regulatory mechanisms. Quantitative PCR verification of LPAR2 transcript levels in parallel with protein detection attempts can help clarify whether discrepancies are technical or biological.

How can researchers distinguish between LPAR2 and other LPA receptor family members?

Ensuring specificity when studying LPAR2 among other LPA receptors requires:

• Selecting antibodies targeting unique, non-conserved regions of LPAR2

• Performing sequence alignment of LPA receptors to identify LPAR2-specific epitopes

• Including parallel detection using receptor-specific probes

• Using Lpar2-/- samples as negative controls while confirming other LPA receptors remain intact

• Combining protein studies with mRNA detection using specific primers

Cross-reactivity between LPA receptor family members can confound experimental interpretation, especially given their structural similarities as G-protein coupled receptors. When possible, utilize multiple detection methods that target different structural features of LPAR2 to confirm specificity.

What quantification methods are recommended for LPAR2 expression analysis?

For accurate LPAR2 quantification:

• Western blot: Normalize to total protein loading (Ponceau S, REVERT) rather than single housekeeping proteins

• IHC quantification:

  • H-score (combines intensity and percentage of positive cells)

  • Automated image analysis systems for objective scoring

• Flow cytometry: Report median fluorescence intensity (MFI) with appropriate isotype controls

• qPCR: Use validated reference genes stable in your experimental system

• Absolute quantification: Consider digital PCR or MRM-MS for precise measurements

When comparing samples across different experimental batches, include internal calibrators or reference standards to account for inter-assay variation. This is particularly important for longitudinal studies examining LPAR2 expression changes during disease progression.

How should researchers interpret contradictory findings about LPAR2 expression across different cancer types?

The divergent prognostic implications of LPAR2 across cancer types require careful interpretation:

• Consider tissue context: LPAR2 shows contrasting associations with prognosis - worse outcomes in KIRC (HR = 1.99, p < 0.001) and ACC (HR = 2.35, p = 0.031), but better outcomes in HNSC (HR = 0.74, p = 0.025), STAD (HR = 0.70, p = 0.035) and other cancers

• Examine signaling networks: LPAR2 may interact with different downstream effectors in various tissues

• Analyze immune context: LPAR2's significant correlation with immune markers suggests its function may depend on the immune microenvironment

• Consider genetic background: Co-occurring mutations may modify LPAR2 effects

• Evaluate epigenetic regulation: Methylation patterns of LPAR2 may vary across cancers

These contradictions likely reflect the complex, context-dependent biology of LPAR2 rather than technical artifacts. Comprehensive multi-omic analysis integrating expression data with functional studies is essential for understanding these differences.

What experimental design is recommended for studying LPAR2 in inflammatory disease models?

When investigating LPAR2 in inflammatory conditions like NSAID-induced enteropathy:

• Include both acute and chronic timepoints: LPAR2's dual role in inflammation may vary temporally

• Compare wild-type and Lpar2-/- models: Essential to distinguish direct receptor effects from compensatory mechanisms

• Use selective agonists/antagonists: DBIBB (selective LPAR2 agonist) can reveal receptor-specific effects

• Measure multiple inflammatory markers: LPAR2 modulation affects different cytokines and immune mediators differently

• Perform cell-type specific analyses: Determine which cells express LPAR2 in the disease microenvironment

Studies have shown that Lpar2-deficient mice develop accelerated intestinal damage (at 6h post-indomethacin) compared to wild-type, despite having lower inflammatory mediator levels . This suggests a complex role where LPAR2 may initially protect against tissue damage while simultaneously regulating the inflammatory response. Well-designed time-course studies with appropriate controls are essential to untangle these complex dynamics.

How can researchers validate LPAR2 as a potential therapeutic target?

To establish LPAR2 as a viable therapeutic target:

• Genetic validation: Compare phenotypes in wild-type vs. Lpar2-/- models

• Pharmacological validation: Test effects of selective LPAR2 modulators like DBIBB

• Humanized models: Evaluate human LPAR2 function in appropriate in vivo systems

• Target engagement studies: Confirm binding of compounds to LPAR2 using antibody-based techniques

• Biomarker identification: Develop reliable measurements of LPAR2 pathway activation

How can LPAR2 antibodies be used in single-cell analysis techniques?

Emerging single-cell technologies offer new opportunities for LPAR2 research:

• Mass cytometry (CyTOF): Incorporate LPAR2 antibodies into metal-tagged panels for high-dimensional analysis

• Single-cell Western blotting: Examine LPAR2 expression heterogeneity in rare cell populations

• Imaging mass cytometry: Spatially resolve LPAR2 expression in tissue microenvironments

• Spatial transcriptomics with protein validation: Correlate LPAR2 transcript and protein localization

These approaches can reveal previously undetectable heterogeneity in LPAR2 expression and signaling, potentially explaining some contradictory findings across bulk tissue analyses. They are particularly valuable for understanding LPAR2's role in complex tissues with multiple cell types, such as tumors or inflammatory lesions.

What are the current challenges in developing therapeutic antibodies targeting LPAR2?

Developing therapeutic antibodies against LPAR2 faces several challenges:

• Receptor accessibility: As a seven-transmembrane G-protein coupled receptor, LPAR2 has limited extracellular domains for antibody targeting

• Functional complexity: LPAR2's dual roles in processes like inflammation require careful consideration of partial agonism/antagonism

• Tissue-specific effects: The contradictory roles of LPAR2 across cancer types suggest potential for tissue-specific adverse effects

• Selectivity: Ensuring specificity against other LPA receptors is challenging but essential

• Mechanistic understanding: More complete elucidation of downstream signaling is needed for rational therapeutic development

Despite these challenges, LPAR2's involvement in cancer, inflammation, and cell invasion makes it an attractive target. Current research focusing on selective small molecule modulators like DBIBB provides valuable insights that could inform future therapeutic antibody development.