GPR174 Antibody

What is GPR174 and where is it localized in cells?

GPR174 is a G protein-coupled receptor belonging to the P2Y family, with approximately 50% sequence homology to the P2Y10 receptor. In humans, GPR174 is a 333 amino acid multi-pass membrane protein with a molecular weight of approximately 38.5 kDa . Its subcellular localization is primarily in the cell membrane, where it functions as a receptor for lysophosphatidylserine (LysoPS) and potentially other purines . The protein contains seven transmembrane domains typical of G protein-coupled receptors and is encoded by the GPR174 gene located on chromosome X, region q21, clustered with p2y10 and lpa4 genes . GPR174 is also known by the alternative names FKSG79 and GPCR17 .

What applications are GPR174 antibodies commonly used for?

GPR174 antibodies are utilized in multiple laboratory techniques for studying this receptor's expression and function:

• Western Blot (WB): The most common application for detecting GPR174 protein in tissue lysates and cell preparations

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of GPR174

• Immunocytochemistry (ICC): To visualize GPR174 in cultured cells

• Immunofluorescence (IF): For fluorescent detection of GPR174 localization

• Immunohistochemistry (IHC): To detect GPR174 in tissue sections

Researchers should select antibodies based on their specific application needs and validated reactivity with target species. For example, some commercially available antibodies show reactivity with human, mouse, rabbit, rat, bovine, and other species samples .

What cell types and tissues express GPR174?

GPR174 shows a distinct expression pattern primarily in immune tissues and cells:

• Primary Tissues: GPR174 is notably expressed in tonsil, spleen, lymph node, and bone marrow

• Immune Cell Populations: High expression is observed in:

  • T cells (particularly naïve T cells)

  • B cells (especially follicular and marginal zone B cells)

The expression pattern varies across B cell development stages. Immunological Genome Project (Immgen) RNA sequencing data shows high reporter expression in spleen follicular and marginal zone B cells, with minimal expression in CD23-CD93+ transitional B cells . Interestingly, GPR174 expression is downregulated in plasma cells and germinal center B cells . Additionally, LPS-stimulated B cells down-regulate GPR174 expression after 4 days, whereas activated CD4 T cells maintain similar expression levels to unstimulated cells .

What signaling pathways does GPR174 activate?

GPR174 activates multiple signaling pathways with cell type-specific outcomes:

• Gαs signaling pathway: Leading to increased cyclic AMP (cAMP) levels and protein kinase A (PKA) activity

• G12/G13 signaling: Activation of these G proteins can trigger transforming growth factor-α ectodomain shedding

These pathways produce distinct functional outcomes in different immune cell populations:

• In T cells: GPR174 mediates suppression of IL-2 production in activated T-lymphocytes through elevated cAMP levels and PKA activity, which antagonizes T cell receptor (TCR) signaling. This leads to inhibition of growth, proliferation, and differentiation

• In B cells: GPR174 signals via GNA13 and PKA to promote CD86 up-regulation

• During sepsis: GPR174 appears to regulate macrophage polarization and modulate pro- and anti-inflammatory cytokine secretion

The high constitutive activity of GPR174 in certain cell types may be due to endogenous lysoPS reaching levels high enough to maximally activate the receptor .

How can researchers validate GPR174 antibody specificity?

Ensuring antibody specificity is critical for reliable GPR174 research. Based on established protocols, researchers should implement multiple validation strategies:

• Pre-adsorption (blocking peptide) controls: Use GPR174 blocking peptides (like BLP-GR060) to pre-adsorb the primary antibody . This creates a negative control where the antibody's binding sites are occupied, preventing specific binding to GPR174 in the sample. A side-by-side comparison with and without blocking peptide can confirm specificity.

• Knockout/knockdown controls: Include samples from GPR174 knockout models (like the Gpr174-KO mice) or cells where GPR174 expression has been silenced as negative controls .

• Multi-technique validation: Confirm GPR174 detection using complementary techniques (Western blot, immunohistochemistry, etc.) to ensure consistent results across methods .

• Multiple epitope targeting: Compare results using antibodies targeting different regions of GPR174 (e.g., extracellular versus intracellular domains) .

• Molecular weight and expression pattern verification: Confirm that GPR174 appears at approximately 38.5 kDa in Western blots, and that expression patterns match known distribution (high in lymphoid tissues) .

Example validation data from blocking peptide experiments show complete signal elimination when Anti-GPR174 antibody is pre-incubated with blocking peptide in Western blot analysis of rat brain membranes, mouse brain lysate, rat spleen lysate, and mouse spleen membranes .

What are optimal experimental conditions for studying GPR174 function?

Experimental design for GPR174 studies should account for several critical factors:

• Dynamic expression regulation: GPR174 expression changes significantly during cell culture and activation. For instance, LPS-stimulated B cells downregulate GPR174 after 4 days, while expression remains constant in activated T cells .

• High basal activity consideration: GPR174 exhibits extremely high basal activity due to endogenous lysoPS production . The search results indicate: "The concentration of lysoPS generated by cells is likely to be higher than that required to produce the maximal effect of WT, explaining how GPR174 exhibits extremely high basal activity" . Consider using the NanoBiT Gs recruitment assay which has lower amplification than cAMP assays for measuring lysoPS potency .

• Cell culture-induced changes: Simply culturing B cells without stimulation leads to significant changes in gene expression, many promoted by GPR174 signaling via Gαs . This complicates interpretation of in vitro experiments.

• Assay selection impacts: Different assays show varying sensitivity to GPR174 activation. For example, the potencies of ligands obtained from NanoBiT assay and cAMP assay can differ by approximately 1000-fold .

• Time-dependent changes: In the sepsis CLP mouse model, peripheral Gpr174 mRNA expression significantly decreased initially and recovered after 72 hours , suggesting dynamic regulation during disease progression that must be accounted for in experimental timing.

How do GPR174 expression levels correlate with disease outcomes in sepsis?

GPR174 shows strong potential as a prognostic biomarker in sepsis based on multiple lines of evidence:

• Expression changes during sepsis: GPR174 mRNA levels are significantly decreased in septic patients compared to non-septic ICU and healthy controls . This decrease correlates with disease severity.

• Strong prognostic value: Lower GPR174 mRNA expression is associated with poor survival rates in septic patients . GPR174 mRNA levels at Day 7 had a high AUC (0.83) in predicting sepsis mortality, and was identified as an independent predictor of 90-day mortality in both logistic regression and Cox regression analyses .

• Dynamic biomarker capabilities: GPR174 mRNA showed remarkable differences between survivor and non-survivor groups (ascending in survivors and descending in non-survivors) . This pattern was unique compared to other biomarkers, including APACHE II and SOFA scores.

• Correlation with clinical parameters: GPR174 mRNA expression correlated with lymphocyte counts, C-reactive protein (CRP), and APACHE II and SOFA scores .

• Protective effects of GPR174 deficiency: In a cecal ligation and puncture (CLP) mouse model of sepsis, Gpr174-deficient mice showed dramatically decreased mortality rates (from 55% to 25%) and less severe organ damage . This protection was associated with:

  • Decreased levels of pro-inflammatory cytokines (IL-1β, TNF-α)

  • Increased levels of anti-inflammatory cytokine IL-10

  • Less severe inflammatory cell infiltration and edema in lung tissue

  • Reduced hepatic cell edema

These findings suggest GPR174 could serve as both a prognostic biomarker and potential therapeutic target in sepsis management .

What are the differential effects of GPR174 signaling in T cells versus B cells?

GPR174 exhibits distinct functional roles in different lymphocyte populations:

T cells:

• GPR174 mediates suppression of T-cell proliferation in vitro through lysoPS stimulation

• Acts via G(12)/G(13) heterotrimeric G proteins to increase cAMP levels and PKA activity

• Antagonizes proximal TCR signaling

• Suppresses IL-2 production in activated T-lymphocytes, inhibiting growth, proliferation, and differentiation

• Plays a negative role in regulatory T-cell accumulation and homeostasis

• Maintains expression levels after activation with anti-CD3 and anti-CD28

B cells:

• GPR174 and Gαs signaling promotes CD86 up-regulation in cultured B cells

• Does not affect B cell proliferation in response to lysoPS (unlike its effect on T cells)

• Expression is downregulated in LPS-stimulated B cells after 4 days

• Expression is minimal in plasma cells and germinal center B cells

• Upon testosterone treatment, can act as a receptor for CCL21, triggering calcium flux and chemotactic effects on activated B cells

These differential effects highlight GPR174's context-dependent functions in immune regulation and underscore the importance of cell type-specific analysis in GPR174 research.

What approaches are most effective for targeting GPR174 in genetic studies?

Based on published research, several strategies have proven effective for GPR174 genetic manipulation:

• Gene replacement approach: Successful studies have used mice in which "the single coding exon of Gpr174 was replaced with an in-frame tdTomato allele" . This approach both disrupts GPR174 expression and provides a fluorescent reporter to track cells that would normally express GPR174.

• X-chromosome inactivation considerations: Since GPR174 is X-linked, researchers must account for X-inactivation effects in female models. In Gpr174+/- female mice, random X inactivation yields approximately 50% reporter-positive (GPR174-deficient) cells, creating a bimodal population in follicular and marginal zone B cells .

• Knockout validation methods:

  • Quantitative PCR using specific primers: For mouse Gpr174 detection, primers used were: sense 5'-TTGGTCTGCATCAGTGTGCGAAG, antisense 5'-CAGGCAGGCAAGGCAGATGATC

  • Flow cytometry for reporter expression in knockout models with fluorescent protein replacements

• Phenotyping approaches:

  • Survival studies in disease models (e.g., CLP-induced sepsis)

  • Histopathological examination of tissues using H&E staining

  • Cytokine measurements (IL-1β, TNF-α, IL-10)

  • RNA-sequencing to identify differentially expressed genes

  • Flow cytometry for surface marker expression (e.g., CD86)

Published data indicates that Gpr174-knockout mice are viable and display significant phenotypes in sepsis models and immune cell function studies, demonstrating this approach is effective for studying GPR174 biology .

What controversies exist in current GPR174 research?

Several areas of ongoing debate and conflicting findings exist in GPR174 research:

• Ligand specificity questions: While lysoPS is confirmed as an endogenous GPR174 ligand , there appears to be context-dependent ligand binding complexity. Some research indicates GPR174 can function as a receptor for CCL21 upon testosterone treatment , suggesting possible ligand promiscuity that remains incompletely characterized.

• G protein coupling diversity: Different studies report varying G protein coupling preferences for GPR174. Some report predominant Gαs signaling leading to cAMP production , while others emphasize G12/G13 signaling pathways . The receptor may couple to multiple G protein types depending on cellular context and activation conditions.

• High constitutive activity mechanisms: GPR174 exhibits unusually high basal activity . Recent research suggests this may be due to endogenous lysoPS production reaching levels that maximally activate the receptor, but other mechanisms may contribute to this phenomenon and remain subjects of investigation.

• Contrasting cellular effects: GPR174 has opposing effects in different immune cell types. LysoPS/GPR174 signaling inhibits T cell proliferation but does not affect B cell proliferation , which could lead to seemingly contradictory findings depending on which cell populations are studied.

• Disease relevance debates: While GPR174 variants have been linked to autoimmune diseases and GPR174 shows prognostic value in sepsis , the precise mechanistic contributions to disease pathology remain under investigation. Beneficial effects of Gpr174 deficiency in sepsis models contrast with potentially detrimental effects in other contexts.

These controversies highlight areas where additional research is needed to resolve conflicting findings and develop a more comprehensive understanding of GPR174 biology.

How can transcriptomic approaches enhance GPR174 functional studies?

RNA sequencing has revealed important insights into GPR174 function and offers methodological advantages for future research:

• Gene expression program identification: RNA-seq comparison between Gpr174-knockout and wild-type mice after CLP challenge identified 360 significantly differentially expressed genes . This approach revealed that Gpr174 deficiency induces a phenotypic shift toward multiple immune response pathways in septic mice.

• Pathway analysis applications: GO analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of transcriptomic data have identified molecular and immunologic pathways impacted by GPR174 . This approach can reveal previously unknown functional connections.

• Validation methodology: Quantitative PCR validation of RNA-seq findings showed that GPR174 deficiency affected expression of immune response mediators, including:

  • Upregulation of dendritic cell-specific transmembrane protein

  • Upregulation of macrophage mannose receptor 1

  • Downregulation of interleukin-12 subunit alpha

  • Downregulation of Sialoadhesin

• Dynamic expression analysis: Transcriptomic approaches have shown that GPR174-deficient B cells are comparatively static in gene expression changes during culture, while wild-type cells undergo substantial expression changes . This provides insights into GPR174's role in maintaining cellular homeostasis.

• Multi-tissue comparative approaches: Researchers have successfully applied transcriptomic methods across multiple tissues to understand tissue-specific GPR174 functions and expression patterns .

These approaches provide powerful tools for understanding GPR174's functional roles and identifying potential therapeutic targets in diseases where GPR174 signaling is implicated.

What are the most effective antibody-based methods for studying GPR174 in tissue samples?

For optimal GPR174 detection in tissue samples, researchers should consider these methodological approaches:

• Immunohistochemistry optimization:

  • Use anti-GPR174 antibodies targeting the extracellular domain for better accessibility in fixed tissues

  • Include appropriate blocking peptide controls to confirm specificity

  • Optimize antigen retrieval methods for formalin-fixed paraffin-embedded tissues

• Immunofluorescence applications:

  • Anti-GPR174 antibodies have been successfully used for immunofluorescence in both cell lines and tissue sections

  • Co-staining with lineage markers (like CD19 for B cells, CD3 for T cells) helps identify specific GPR174-expressing cell populations

• Western blot protocols:

  • Successful detection has been achieved in:

    • Rat brain membranes

    • Mouse brain lysate

    • Rat spleen lysate

    • Mouse spleen membranes

    • Human T-cell leukemia cell lines (MOLT-4 and Jurkat)

  • Use appropriate lysis buffers containing detergents suitable for membrane protein extraction

• Flow cytometry applications:

  • Anti-GPR174 antibodies can be used for flow cytometry to quantify expression levels across immune cell populations

  • Particularly useful for monitoring dynamic changes in GPR174 expression during cell activation or disease states

• Species-specific considerations:

  • Many commercial antibodies show cross-reactivity with human, mouse, and rat GPR174

  • Validate antibody specificity for each species of interest

  • Consider species-specific differences in GPR174 expression patterns when designing experiments

Careful selection of antibodies and optimization of protocols for each specific application will maximize success in GPR174 detection across different experimental contexts.

What are the most promising clinical applications for GPR174 antibody research?

Based on current evidence, several clinical applications show strong potential:

• Sepsis biomarker development: GPR174 mRNA levels show significant prognostic value in sepsis, with changes in expression correlating with survival outcomes . Development of reliable GPR174 detection methods could yield valuable clinical biomarkers for:

  • Early sepsis diagnosis

  • Disease severity assessment

  • Mortality risk prediction

  • Treatment response monitoring

• Autoimmune disease research: GPR174 variants have been associated with autoimmune conditions including Graves' disease . GPR174 antibodies could help elucidate how these variants alter protein expression and function, providing insights into disease mechanisms.

• Inflammatory pathway targeting: GPR174's role in regulating inflammatory responses, particularly through effects on pro-inflammatory (IL-1β, TNF-α) and anti-inflammatory (IL-10) cytokines , suggests potential therapeutic applications in inflammatory disorders.

• Immune cell functional assessment: GPR174's differential effects on T cells versus B cells make it a valuable marker for assessing immune cell functionality in various disease states.

• Precision medicine applications: Understanding individual variations in GPR174 expression and function could help predict patient responses to immunomodulatory therapies and guide personalized treatment approaches.

These applications highlight the translational potential of GPR174 research beyond basic immunological studies, with particular promise in infectious and autoimmune disease contexts.

What future research directions are most likely to advance GPR174 understanding?

Several promising research directions could significantly advance GPR174 biology:

• Structure-function relationship studies: Determining the three-dimensional structure of GPR174 and how it interacts with its ligands would provide crucial insights for drug development targeting this receptor.

• Tissue-specific conditional knockout models: Developing conditional knockout systems for GPR174 in specific immune cell populations would help dissect cell-type-specific functions without confounding developmental effects.

• Signaling pathway delineation: More comprehensive characterization of GPR174 signaling networks and how they interact with other immune signaling pathways would improve understanding of its regulatory roles.

• Therapeutic modulation approaches: Development of specific GPR174 agonists and antagonists would provide valuable tools for both research and potential clinical applications in immune-related disorders.

• Single-cell analysis technologies: Applying single-cell RNA sequencing and proteomic approaches to study GPR174 expression and function at single-cell resolution would reveal heterogeneity in responses across immune cell subpopulations.

• Clinical correlation studies: Larger studies correlating GPR174 expression or genetic variants with clinical outcomes across multiple diseases would better establish its relevance as a biomarker and therapeutic target.

• Interaction with microenvironmental factors: Investigating how tissue microenvironmental factors influence GPR174 expression and function would provide context for its diverse effects in different physiological and pathological settings.

These research directions would address current knowledge gaps and advance both fundamental understanding and clinical applications of GPR174 biology.