LPAR2 Antibody, Biotin conjugated

What is LPAR2 and what biological functions does it mediate?

LPAR2 (also known as EDG4 or LPA-2) is a G protein-coupled receptor for lysophosphatidic acid (LPA), a mediator of diverse cellular activities. This receptor is primarily located in the cell membrane and plays key roles in multiple signaling pathways. LPAR2 appears to couple to multiple G protein families including G(i)/G(o), G(12)/G(13), and G(q), allowing it to participate in various downstream cellular processes .

It plays a critical role in the phospholipase C-beta (PLC-beta) signaling pathway and stimulates phospholipase C activity in a manner that is independent of RALA activation . In the context of immunity, LPAR2 has been shown to promote T cell motility, contrasting with LPAR6 which primarily suppresses T cell migration .

What are the key specifications of commercially available LPAR2 polyclonal antibodies with biotin conjugation?

Commercially available LPAR2 polyclonal antibodies with biotin conjugation typically have the following specifications:

These specifications ensure consistent performance in various experimental applications and appropriate cross-reactivity with relevant species .

How does LPAR2 differ functionally from other lysophosphatidic acid receptors?

LPAR2 exhibits distinct functional characteristics compared to other LPA receptors:

• While LPAR1 is primarily associated with tumor cell migration and metastasis, LPAR2 has been specifically linked to T cell motility .

• Unlike LPAR6, which preferentially couples to the Gα12/13-RhoA pathway that drives cytoskeletal contraction and suppression of cell motility, LPAR2 couples to Gi-mediated Rac GTPase activation and promotes the random motility of T cells .

• The signaling pathway distinction is critical: LPAR2 activation generally enhances cell movement, while LPAR6 restricts it through different G-protein coupling mechanisms .

• In cancer biology, LPAR2 expression correlates with different prognoses across cancer types - associated with worse outcomes in some cancers (ACC, KIRC, MESO) but improved prognosis in others (HNSC, OV, STAD) .

This functional diversity highlights the importance of receptor-specific research approaches when investigating LPA signaling pathways.

What are the optimal application conditions for LPAR2 antibody in immunohistochemistry experiments?

For optimal immunohistochemistry results with biotin-conjugated LPAR2 antibodies:

For Paraffin-Embedded Sections (IHC-P):

• Recommended dilution: 1:200-400

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0)

• Blocking: 5-10% normal serum from the same species as the secondary antibody

• Incubation time: Overnight at 4°C for primary antibody

• Detection: Streptavidin-HRP system followed by DAB or similar chromogen

• Counterstain: Hematoxylin for nuclear visualization

For Frozen Sections (IHC-F):

• Recommended dilution: 1:100-500

• Fixation: Acetone or 4% paraformaldehyde

• Blocking: 5-10% normal serum with 0.1-0.3% Triton X-100

• Incubation time: 1-2 hours at room temperature

• Detection: Same as IHC-P

• Controls: Include negative controls (omitting primary antibody) and positive controls (tissues known to express LPAR2)

Optimization may be necessary for each specific tissue type, and validation with alternative detection methods is advised for conclusive results.

How should researchers design experiments to investigate LPAR2's role in immune cell function?

Based on recent research findings, experiments investigating LPAR2's role in immune cell function should consider:

• Cell Migration Assays:

  • Transwell migration assays comparing LPAR2-expressing vs. LPAR2-knockout or silenced immune cells

  • Live cell imaging to track T cell motility patterns following LPA stimulation

  • Chemokinesis vs. chemotaxis assessment using gradient-free and gradient conditions

• Signaling Pathway Analysis:

  • Assessment of Gi-mediated Rac GTPase activation following LPAR2 stimulation

  • Comparative analysis with other LPA receptors, particularly LPAR6 which mediates opposing effects

  • Phosphorylation studies of downstream targets using phospho-specific antibodies

• Immune Microenvironment Context:

  • Co-culture systems with tumor cells that secrete autotaxin (ATX) to produce LPA

  • Analysis of T cell infiltration patterns in the presence of ATX/LPA

  • Investigation of LPAR2 expression correlation with immune cell markers in tumor samples

• Specificity Controls:

  • Use of specific LPAR2 antagonists alongside LPAR6 antagonists (such as XAA)

  • Comparison between multiple immune cell subsets that express different levels of LPAR2

  • Genetic approaches using CRISPR/Cas9 or siRNA to confirm antibody specificity

This multi-faceted approach enables researchers to delineate the specific contributions of LPAR2 to immune function while controlling for potential confounding effects of other LPA receptors.

What methods can be used to validate LPAR2 antibody specificity for research applications?

Validating antibody specificity is crucial for reliable research outcomes. For LPAR2 antibodies, the following validation methods are recommended:

• Genetic Approaches:

  • CRISPR/Cas9 knockout of LPAR2 in relevant cell lines

  • siRNA or shRNA knockdown of LPAR2 followed by immunostaining

  • Overexpression of tagged LPAR2 (e.g., FLAG-tag) and co-localization analysis

• Peptide Competition Assays:

  • Pre-incubation of antibody with the immunizing peptide (derived from human LPAR2 regions 1-100/351)

  • Gradual reduction in signal should be observed with increasing peptide concentration

• Multiple Antibody Validation:

  • Use of alternative antibodies targeting different epitopes of LPAR2

  • Comparison of staining patterns across various detection methods

• Cross-Reactivity Assessment:

  • Testing against cell lines expressing other LPA receptors but not LPAR2

  • Examination of predicted cross-reactive species (Human, Dog, Cow, Sheep, Pig, Horse, Rabbit)

• Western Blot Analysis:

  • Verification of band size corresponding to LPAR2 (approximately 39 kDa)

  • Analysis of cells with differential LPAR2 expression

• Orthogonal Validation:

  • Correlation of protein detection with mRNA expression (RT-PCR or RNA-seq)

  • Mass spectrometry confirmation of the immunoprecipitated protein

These methods collectively provide robust validation of antibody specificity, which is essential before employing these reagents in critical research applications.

How does LPAR2 signaling impact tumor microenvironment and immune infiltration?

LPAR2 signaling appears to have complex, context-dependent effects on the tumor microenvironment and immune infiltration:

• Correlation with Immune Cell Infiltration:
  Recent studies show that LPAR2 expression significantly correlates with the presence of various immune markers in immune cell subsets of head and neck squamous cell carcinoma (HNSC) and kidney renal clear cell carcinoma (KIRC) . This suggests LPAR2 may influence the composition of tumor-infiltrating immune cells.

• Dual Role in Cancer Prognosis:
  High LPAR2 expression correlates with improved survival in certain cancers (BLCA, CESC, HNSC, TYHM, UCEC) but worse prognosis in others (ACC, KIRC, MESO) . This suggests tissue-specific functions of LPAR2 in modulating anti-tumor immunity.

• Interaction with ATX/LPA Axis:
  While LPAR2 promotes T cell motility, the ATX-LPA axis can have chemorepulsive effects on tumor-infiltrating lymphocytes. This creates a complex signaling environment where the balance between different LPA receptors determines immune cell infiltration patterns .

• Clinical Significance:
  Survival analyses reveal that LPAR2 expression significantly impacts patient outcomes:

  • Improved OS in HNSC (HR = 0.65 [0.49-0.86], P = 0.0023)

  • Improved OS in UCEC (HR = 0.59 [0.38-0.9], P = 0.014)

  • Worse OS in KIRC (HR = 1.99 [1.46-2.71], P < 0.001)

These findings highlight the potential of LPAR2 as both a prognostic biomarker and therapeutic target, particularly in cancers where it influences immune infiltration.

What is the relationship between LPAR2 and G-protein signaling in cell invasion mechanisms?

LPAR2 demonstrates specific G-protein coupling preferences that influence cell invasion mechanisms:

• G-Protein Coupling Specificity:
  LPAR2 couples to multiple G protein families, including G(i)/G(o), G(12)/G(13), and G(q), allowing it to participate in various downstream cellular processes . This multi-pathway activation enables diverse cellular responses.

• Role in Entotic Invasion:
  LPAR2 has been identified as a signal transducer specifically required for actively invading cells during entosis (homotypic cell-in-cell invasion). This process depends on G12/13 signaling and PDZ-RhoGEF activation .

• Contrasting Functions with LPAR6:
  While LPAR2 couples to Gi-mediated pathways to promote cell motility, LPAR6 preferentially couples to the Gα12/13-RhoA pathway, driving cytoskeletal contraction and suppression of cell motility . This differential G-protein coupling explains their opposing effects on cell movement.

• Downstream Effector Activation:
  LPAR2 plays a key role in phospholipase C-beta (PLC-beta) signaling and stimulates PLC activity in a manner independent of RALA activation . This pathway is critical for calcium signaling and protein kinase C activation.

• Actin Cytoskeletal Regulation:
  G12/13 signaling downstream of LPAR2 influences blebbing and actin dynamics, which are essential for the mechanical process of cell invasion .

Understanding these specific G-protein coupling preferences and downstream signaling cascades is crucial for developing targeted interventions that modulate LPAR2-mediated cellular processes in disease contexts.

How can researchers integrate LPAR2 expression data with clinical outcomes in cancer research?

Researchers seeking to integrate LPAR2 expression data with clinical outcomes should consider these methodological approaches:

This integrated approach provides robust evidence for the clinical relevance of LPAR2 in specific cancer contexts and may identify patient subgroups most likely to benefit from therapies targeting the LPAR2 signaling axis.

What are common pitfalls when using biotin-conjugated antibodies for LPAR2 detection?

Researchers working with biotin-conjugated LPAR2 antibodies should be aware of these common pitfalls:

• Endogenous Biotin Interference:

  • Tissues may contain endogenous biotin that can cause non-specific binding

  • Solution: Use biotin blocking steps (streptavidin followed by biotin) before applying the biotin-conjugated primary antibody

  • Alternative: Consider using avidin-biotin blocking kits specifically designed for this purpose

• Storage-Related Degradation:

  • Biotin conjugates may lose activity if not stored properly at -20°C as recommended

  • Repeated freeze-thaw cycles can degrade antibody performance

  • Solution: Aliquot antibodies upon receipt and maintain strict storage temperature conditions

• Cross-Reactivity Challenges:

  • While predicted to react with multiple species, actual cross-reactivity may vary

  • Solution: Validate antibody in each species before conducting full experiments

  • Alternative: Use species-specific positive controls alongside experimental samples

• Detection System Sensitivity Issues:

  • Streptavidin-based detection systems vary in sensitivity

  • Solution: Titrate antibody concentration for each application (ELISA: 1:500-1000; IHC-P: 1:200-400; IHC-F: 1:100-500)

  • Alternative: Consider amplification steps for low-expressing samples

• Background in Highly Biotin-Rich Tissues:

  • Tissues like liver, kidney, and brain contain high endogenous biotin

  • Solution: Use alternative non-biotin detection methods for these tissues

  • Alternative: Increase washing steps and blocking concentrations

• Competitive Binding with Lysophosphatidic Acid:

  • Native LPA may compete with antibody binding in certain sample preparations

  • Solution: Consider fixation methods that preserve epitope accessibility while removing endogenous ligands

Addressing these challenges proactively will improve experimental outcomes and data reliability when working with biotin-conjugated LPAR2 antibodies.

How should researchers interpret contradictory results between LPAR2 expression and functional outcomes?

When faced with contradictory results between LPAR2 expression and functional outcomes, researchers should consider:

• Context-Dependent Signaling:

  • LPAR2 couples to multiple G-protein families (G(i)/G(o), G(12)/G(13), G(q)) , enabling different functional outcomes depending on the cellular context

  • Examine which G-protein coupling predominates in your specific experimental system

  • Consider the balance between different LPA receptors, as LPAR2 and LPAR6 can have opposing effects

• Cancer-Type Specificity:

  • LPAR2 demonstrates opposite associations with survival in different cancer types:

    • Associated with worse prognosis in ACC, KIRC, MESO

    • Associated with better prognosis in HNSC, OV, STAD

  • Always interpret results within the specific tissue/cancer context

• Methodological Considerations:

  • Antibody detection may not reflect functional receptor status

  • Evaluate both protein expression (IHC, Western blot) and functional assays (signaling, migration)

  • Consider receptor internalization, which may reduce membrane expression despite active signaling

• Competing Pathways:

  • The ATX-LPA axis involves multiple receptors with potentially conflicting effects

  • Autotaxin can produce LPA that affects T cell migration through both LPAR2 and LPAR6

  • Measure activity of competing pathways when interpreting LPAR2-specific effects

• Analysis Framework:

  • Use multivariate analyses that control for confounding factors

  • Consider non-linear relationships between expression and function

  • Employ multiple statistical approaches (parametric and non-parametric)

• Validation Strategies:

  • Use genetic approaches (knockout, knockdown) to confirm antibody-based findings

  • Apply pharmacological tools (specific agonists/antagonists) to distinguish receptor-specific effects

  • Consider the temporal dynamics of receptor expression and downstream signaling

Through systematic consideration of these factors, researchers can better interpret apparently contradictory results and develop a more nuanced understanding of LPAR2 biology.

What controls should be included when evaluating LPAR2 expression in tumor samples?

Comprehensive control strategies for evaluating LPAR2 expression in tumor samples should include:

• Technical Controls:

  • Positive Controls: Known LPAR2-expressing tissues/cell lines (preferably matched to tumor type)

  • Negative Controls: Primary antibody omission on serial sections

  • Isotype Controls: Rabbit IgG at the same concentration as the primary antibody

  • Absorption Controls: Primary antibody pre-incubated with immunizing peptide

• Biological Reference Controls:

  • Adjacent Normal Tissue: For comparison with tumor expression

  • Tissue Microarrays: Multiple tumor and normal samples processed identically

  • Gradient of Expression: Include samples with known high, medium, and low LPAR2 expression

• Validation Controls:

  • Orthogonal Methods: Validate IHC findings with Western blot or RT-PCR

  • Multiple Antibodies: Use antibodies targeting different epitopes of LPAR2

  • In Situ Hybridization: Confirm protein expression with mRNA detection

• Contextual Controls:

  • Correlation Controls: Assess relationship with known regulators or targets of LPAR2

  • Functional Pathway Controls: Evaluate downstream signaling elements (e.g., phospholipase C-beta activation)

  • Other LPA Receptors: Measure LPAR1, LPAR6, etc., to understand receptor balance

• Clinical Correlation Controls:

  • Survival Data: Compare LPAR2 expression with patient outcomes

  • Treatment Response: Analyze expression relative to therapy response

  • Immune Infiltration: Correlate with immune cell markers in the same samples

• Quantification Controls:

  • Inter-observer Validation: Multiple pathologists scoring the same samples

  • Digital Pathology Standards: Calibration controls for automated scoring

  • Batch Controls: Reference samples included in each staining batch

What emerging techniques might enhance the detection and functional analysis of LPAR2 in complex tissues?

Several cutting-edge methodologies show promise for advancing LPAR2 research:

• Spatial Transcriptomics and Proteomics:

  • Techniques like 10x Genomics Visium or GeoMx DSP allow simultaneous visualization of LPAR2 expression and spatial context

  • These approaches can reveal microenvironmental relationships between LPAR2-expressing cells and immune infiltrates

  • Integration with multiplexed immunofluorescence can map receptor distribution relative to signaling nodes

• Single-Cell Multi-Omics:

  • Single-cell RNA sequencing combined with protein detection (CITE-seq) can correlate LPAR2 transcript levels with surface protein expression

  • This can identify cellular subpopulations with unique LPAR2 signaling properties

  • Trajectory analysis can reveal how LPAR2 expression changes during cellular differentiation or tumor progression

• Live Cell Biosensors:

  • FRET-based biosensors for detecting LPAR2 activation and G-protein coupling in real-time

  • Optogenetic approaches to spatiotemporally control LPAR2 signaling

  • Luminescent biosensors to track downstream second messenger production

• Advanced Microscopy Techniques:

  • Super-resolution microscopy (STORM, PALM) to visualize LPAR2 nanoclustering at the membrane

  • Lattice light-sheet microscopy for long-term 3D imaging of LPAR2-mediated cell dynamics

  • Correlative light and electron microscopy to connect receptor distribution with ultrastructural features

• Engineered Antibody Formats:

  • Nanobodies or single-domain antibodies for improved tissue penetration

  • Bispecific antibodies targeting LPAR2 and downstream effectors

  • Photoswitchable antibodies for selective visualization in specific regions

• Computational Approaches:

  • Machine learning algorithms for pattern recognition in LPAR2 distribution

  • Systems biology modeling of LPAR2 signaling networks

  • Digital pathology tools for automated quantification across large tissue cohorts

These innovative approaches will provide deeper insights into LPAR2 biology and potentially reveal new therapeutic opportunities in cancer and immune modulation.

How might targeting LPAR2 signaling impact cancer immunotherapy approaches?

The relationship between LPAR2 signaling and cancer immunotherapy presents several intriguing research directions:

• Modulation of T Cell Infiltration:

  • Since LPAR2 promotes T cell motility , targeting this receptor could potentially enhance T cell trafficking into tumors

  • Conversely, the ATX-LPA axis can have chemorepulsive effects on tumor-infiltrating lymphocytes

  • Combination approaches targeting both LPAR2 and LPAR6 might optimize T cell infiltration patterns

• Cancer Type-Specific Approaches:

  • Given the differential prognostic significance of LPAR2 across cancer types , immunotherapy strategies should be tailored accordingly:

    • In HNSC where high LPAR2 correlates with better outcomes (HR = 0.65 [0.49-0.86], P = 0.0023) , enhancing LPAR2 signaling might be beneficial

    • In KIRC where high LPAR2 correlates with worse outcomes (HR = 1.99 [1.46-2.71], P < 0.001) , inhibiting LPAR2 might improve immunotherapy response

• Combination with Immune Checkpoint Inhibitors:

  • LPAR2 modulation could potentially sensitize tumors to anti-PD-1/PD-L1 therapy

  • Research should investigate whether LPAR2 expression correlates with checkpoint inhibitor response

  • Sequential approaches might optimize immune infiltration before checkpoint blockade

• Targeting the ATX-LPA-LPAR2 Axis:

  • Autotaxin inhibitors could reduce LPA production, affecting both tumor cells and immune cells

  • Selective LPAR2 modulators might enhance beneficial immune functions while limiting pro-tumor effects

  • Antibody-drug conjugates targeting LPAR2 could deliver immunomodulatory compounds directly to the tumor microenvironment

• Biomarker Development:

  • LPAR2 expression patterns could serve as predictive biomarkers for immunotherapy response

  • The ratio of different LPA receptors might provide more comprehensive predictive power

  • Monitoring changes in LPAR2 expression during treatment could indicate developing resistance mechanisms

• Cell-Based Therapeutic Approaches:

  • Engineered T cells with modified LPAR2 signaling might demonstrate enhanced tumor infiltration

  • Dendritic cell vaccines targeting LPAR2-expressing tumors could generate more specific immune responses

  • Ex vivo modification of tumor-infiltrating lymphocytes to optimize LPAR2-mediated motility

These approaches highlight the potential of LPAR2-targeted strategies to enhance existing immunotherapies and develop novel treatment paradigms in cancer.

What are the most promising research directions for understanding LPAR2's role in disease mechanisms beyond cancer?

Beyond cancer, LPAR2 presents exciting research opportunities in several disease areas:

• Inflammatory and Autoimmune Disorders:

  • Given LPAR2's role in T cell motility , investigating its contribution to inflammatory cell recruitment in autoimmune diseases

  • Examining the balance between pro-inflammatory and regulatory T cell migration mediated by LPAR2

  • Developing targeted therapies that modulate LPAR2 signaling to restore immune homeostasis

• Cardiovascular Disease:

  • Exploring LPAR2's role in atherosclerosis development and progression

  • Investigating potential connections between LPAR2 signaling and cardiac remodeling after injury

  • Examining relationships between LPAR2 and lipoprotein metabolism, particularly given the existence of antibodies against both LPAR2 and lipoprotein a

• Fibrotic Disorders:

  • Investigating LPAR2's contribution to fibroblast activation and extracellular matrix deposition

  • Examining LPAR2-mediated signaling in organ-specific fibrosis (liver, lung, kidney)

  • Developing therapeutic strategies targeting the ATX-LPA-LPAR2 axis to mitigate fibrotic processes

• Neurological Disorders:

  • Analyzing LPAR2 expression patterns in neurodegenerative diseases

  • Exploring the receptor's role in neuroinflammation and blood-brain barrier integrity

  • Investigating potential neuroprotective or neurotoxic effects of LPAR2 signaling

• Metabolic Diseases:

  • Examining LPAR2's role in adipose tissue inflammation and insulin resistance

  • Investigating connections between LPAR2 signaling and hepatic metabolism

  • Exploring potential links to obesity-related immune dysfunction

• Reproductive Biology:

  • Analyzing LPAR2's contribution to embryo implantation and placental development

  • Investigating its role in reproductive tract immunity

  • Exploring connections to fertility disorders and pregnancy complications

• Tissue Repair and Regeneration:

  • Examining how LPAR2-mediated cell migration contributes to wound healing

  • Investigating the receptor's role in stem cell mobilization and differentiation

  • Developing LPAR2-targeted approaches to enhance tissue regeneration

• Infectious Disease Responses:

  • Analyzing how LPAR2 signaling affects immune responses to pathogens

  • Investigating whether infectious agents exploit or modulate LPAR2 signaling

  • Exploring the receptor as a potential target for immunomodulatory anti-infective strategies

These diverse research directions highlight LPAR2's significance beyond cancer and suggest potential therapeutic applications across multiple disease areas.