GPR174 Antibody, Biotin conjugated

What is GPR174 and what role does it play in immune cells?

GPR174 is a G-protein-coupled receptor that recognizes lysophosphatidylserine (LysoPS) as its primary ligand. It plays critical roles in immune response regulation, particularly in T and B lymphocytes. GPR174 exerts a negative role in regulatory T-cell accumulation and homeostasis under normal physiological conditions. During inflammatory events where LysoPS production increases, GPR174 contributes to the down-regulation of regulatory T-cell activity, which favors effector immune responses. This receptor also mediates the suppression of IL-2 production in activated T-lymphocytes, leading to inhibition of growth, proliferation, and differentiation of T-cells .

How does GPR174 function at the molecular level?

At the molecular level, GPR174 operates through several distinct signaling mechanisms:

• It acts via G(12)/G(13)-containing heterotrimeric G proteins to trigger elevated cyclic AMP levels and protein kinase A (PKA) activity, which may antagonize proximal T-cell receptor signaling .

• In B cells, GPR174 signals via Gαs to modulate gene expression, including upregulation of CD86 .

• Upon testosterone treatment, GPR174 can function as a receptor for CCL21, subsequently triggering calcium flux through G(q)-alpha and G(12)/G(13) proteins, leading to chemotactic effects on activated B-cells .

• It signals via GNA13 and PKA to promote CD86 up-regulation specifically by follicular B-cells .

These multiple signaling pathways highlight GPR174's complex role in modulating immune cell behavior through diverse molecular mechanisms.

What are the known effects of GPR174 deficiency in immune cells?

GPR174 deficiency produces several notable phenotypic changes in immune cells:

• B cells lacking GPR174 show a significant defect in CD86 up-regulation in vitro, both spontaneously and following stimulation .

• GPR174-deficient B cells demonstrate enhanced survival in culture compared to wild-type cells .

• In vivo, GPR174 deficiency leads to reduced NUR77 expression in follicular B cells .

• GPR174-deficient mice have a reduced marginal zone B cell compartment .

• At the gene expression level, GPR174-deficient B cells undergo fewer transcriptional changes during culture compared to wild-type B cells, with over 1,000 genes differentially expressed between the two conditions after 4 hours of culture .

These findings suggest GPR174 plays an important regulatory role in B cell activation states and survival.

What applications are GPR174 antibodies suitable for in research settings?

Based on current research, GPR174 antibodies can be employed in several key applications:

• Western Blotting (WB): For detection and quantification of GPR174 protein in cell or tissue lysates. This application allows researchers to determine relative expression levels across different experimental conditions .

• Immunocytochemistry/Immunofluorescence (ICC/IF): For visualizing the cellular and subcellular localization of GPR174 within cells. This technique can reveal important information about receptor distribution and trafficking .

• Flow Cytometry: While not explicitly mentioned in the search results, biotin-conjugated antibodies are commonly used in flow cytometry protocols to identify cells expressing specific markers like GPR174, especially in immune cell populations.

• Immunoprecipitation: For isolation of GPR174 and associated protein complexes to study interaction partners.

Each application requires specific protocol optimizations, including antibody dilution, blocking conditions, and detection methods.

How should researchers validate the specificity of anti-GPR174 antibodies?

Validation of GPR174 antibody specificity should include multiple approaches:

• Positive and negative controls: Use cell lines or tissues known to express high levels of GPR174 (B and T lymphocytes) alongside those with minimal expression (such as CD23−CD93+ transitional B cells, which show minimal GPR174 expression) .

• Knockout validation: Compare antibody staining patterns between wild-type samples and those from GPR174-deficient models. The study cited in search result utilized mice in which the GPR174 coding exon was replaced with a tdTomato reporter, providing an excellent resource for validating antibody specificity.

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide (in this case, the human GPR174 recombinant protein fragment 291-333AA) before application to the sample. Specific binding should be blocked by this competition.

• Cross-reactivity assessment: For antibodies claimed to work across species, compare staining patterns with known expression data from resources like the Immunological Genome Project (Immgen) .

What are the optimal conditions for using biotin-conjugated GPR174 antibodies?

While the search results don't provide specific optimization protocols for biotin-conjugated GPR174 antibodies, general best practices include:

• Titration experiments: Perform dilution series (typically 1:100 to 1:2000) to determine optimal antibody concentration that maximizes specific signal while minimizing background.

• Buffer optimization: Test different blocking solutions (typically 1-5% BSA or normal serum from the same species as the secondary reagent) to reduce non-specific binding.

• Amplification systems: For detection of low-abundance targets, consider using streptavidin-based amplification systems that leverage the strong biotin-streptavidin interaction. Options include:

  • Streptavidin-HRP for enhanced chemiluminescent detection

  • Streptavidin-fluorophore conjugates for fluorescence-based applications

  • Streptavidin-gold for electron microscopy

• Storage and handling: Maintain antibody aliquots at -20°C for long-term storage and avoid repeated freeze-thaw cycles to preserve activity.

For specific applications testing GPR174 in B cells, researchers should note that uncultured cells show minimal differential expression between wild-type and GPR174-deficient samples, while cultured cells (4 hours) show significant differences . This temporal aspect should be considered when designing experiments.

How does GPR174 signaling via Gαs affect B cell gene expression programs?

GPR174 signaling through Gαs proteins profoundly impacts B cell gene expression programs. RNA sequencing analysis revealed that:

• Wild-type B cells undergo massive changes in gene expression during 4 hours of unstimulated culture, with over 1,000 genes significantly up- or down-regulated .

• GPR174-deficient B cells show dramatically fewer changes in gene expression during the same culture period, indicating that a large portion of the "spontaneous" activation program in cultured B cells is GPR174-dependent .

• Key gene expression changes mediated by GPR174 signaling include:

  • Upregulation of activation markers like CD86 (B7-2)

  • Induction of nuclear receptor subfamily 4 group A members (Nr4a1, Nr4a2, Nr4a3)

  • Increased expression of CCR7

  • Upregulation of phosphodiesterases

  • Downregulation of ITIM-containing receptors

• Temporal analysis shows that many gene expression changes occur rapidly, with significant differences detectable after just 1 hour of culture .

• The cAMP response element modulator (CREM) is more strongly expressed in wild-type cells compared to GPR174-deficient cells, consistent with cAMP signaling downstream of Gαs .

These findings demonstrate that GPR174 signaling via Gαs acts as a major driver of B cell activation states through broad transcriptional programming.

What is the relationship between LysoPS, GPR174, and B cell activation?

The relationship between lysophosphatidylserine (LysoPS), GPR174, and B cell activation represents a complex signaling axis:

• LysoPS is a bioactive lipid mediator that functions as a ligand for GPR174 . The recognition of LysoPS by GPR174 initiates signaling cascades in B cells.

• In vivo treatment of mice with LysoPS is sufficient to promote CD86 up-regulation by follicular B cells, demonstrating that ligand engagement of GPR174 can activate this pathway physiologically .

• Interestingly, addition of LysoPS to B cell cultures did not further enhance CD86 induction beyond spontaneous levels . This observation held true even when cells were cultured in the absence of fetal calf serum, suggesting:

  • Either endogenous LysoPS production is sufficient to maximally activate the pathway

  • Or constitutive/ligand-independent activity of GPR174 drives the observed effects

  • Or alternative ligands may be involved in some contexts

• The GPR174-LysoPS axis affects B cell gene expression through G protein signaling cascades, primarily via Gαs, leading to cAMP production and PKA activation .

These findings establish that while LysoPS can engage GPR174 to modulate B cell activation in vivo, the receptor may also function through more complex mechanisms in certain experimental contexts.

How can researchers effectively track GPR174 signaling in real-time experiments?

For researchers interested in monitoring GPR174 signaling dynamics in real-time, several methodological approaches can be implemented:

• FRET-based cAMP sensors: Since GPR174 signals through Gαs to increase cAMP levels, researchers can utilize genetically encoded FRET (Förster Resonance Energy Transfer) sensors that respond to changes in intracellular cAMP concentration. These sensors typically employ CFP/YFP or similar fluorophore pairs that change their FRET efficiency upon cAMP binding.

• PKA activity reporters: As GPR174 activates protein kinase A downstream of cAMP production , researchers can employ fluorescent reporters that monitor PKA substrate phosphorylation in living cells.

• Calcium flux measurements: For contexts where GPR174 triggers calcium signaling (such as in testosterone-treated conditions where GPR174 acts as a CCL21 receptor) , calcium-sensitive dyes like Fluo-4 or genetically encoded calcium indicators (GECIs) like GCaMP can be employed.

• NUR77-GFP reporter systems: The research indicates GPR174 contributes to NUR77 expression in follicular B cells . Using NUR77-GFP reporter mice or cell lines can provide a readout of this GPR174-dependent transcriptional response.

• CD86 surface expression: Given the strong dependence of CD86 upregulation on GPR174 signaling , monitoring CD86 surface levels via flow cytometry provides a reliable downstream readout of pathway activation, though not in real-time.

For all these approaches, comparing responses between wild-type and GPR174-deficient cells will help establish the specificity of the observed signals to GPR174 activation.

What controls should be included when using GPR174 antibodies in experimental settings?

A robust experimental design using GPR174 antibodies should incorporate the following controls:

• Positive Tissue/Cell Controls:

  • B and T lymphocytes, particularly follicular and marginal zone B cells, which express high levels of GPR174

  • Cell lines transfected with GPR174 expression constructs

• Negative Controls:

  • CD23−CD93+ transitional B cells, which show minimal GPR174 expression

  • GPR174-deficient cells or tissues (such as those from knockout models)

  • Isotype control antibodies matched to the primary antibody's host species and immunoglobulin subclass

• Technical Controls:

  • Secondary-only controls (omitting primary antibody) to assess non-specific binding of detection reagents

  • Antigen competition controls where primary antibody is pre-incubated with excess immunizing peptide (GPR174 peptide 291-333AA)

  • Titration series to demonstrate signal specificity and optimal antibody concentration

• Experimental Validation Controls:

  • For functional studies, forskolin and IBMX treatment can serve as positive controls for cAMP pathway activation, as they induce CD86 expression similar to GPR174 activation

  • Comparison of effects in both unstimulated and stimulated conditions (e.g., anti-IgM, anti-CD180)

Inclusion of these controls ensures experimental observations can be confidently attributed to specific GPR174 detection or modulation.

How can researchers optimize detection protocols for low-abundance GPR174 expression?

When GPR174 expression is low or difficult to detect, researchers can enhance sensitivity through several approaches:

• Signal Amplification Technologies:

  • For biotin-conjugated antibodies , employ multi-step amplification using streptavidin-biotin systems

  • Tyramide signal amplification (TSA) for immunohistochemistry and immunofluorescence

  • Proximity ligation assay (PLA) for detecting protein-protein interactions involving GPR174

• Sample Preparation Optimization:

  • Enrich for membrane fractions in Western blotting applications, as GPR174 is a membrane-localized GPCR

  • Use gentle fixation conditions for immunocytochemistry to preserve epitope accessibility

  • Consider native versus denaturing conditions based on the antibody's recognized epitope

• Culture Conditions:

  • Note that GPR174 effects become more pronounced after culture periods (1-4 hours), with substantial differences in CD86 expression between wild-type and GPR174-deficient B cells

  • This temporal window can be exploited to enhance detection of GPR174-dependent phenotypes

• Technical Considerations:

  • Increase antibody incubation time (overnight at 4°C instead of 1-2 hours)

  • Optimize blocking conditions to improve signal-to-noise ratio

  • Use highly sensitive detection systems (e.g., chemiluminescent substrates with extended signal duration for Western blots)

  • Consider super-resolution microscopy techniques for visualizing membrane receptor distribution

These optimizations can significantly improve detection sensitivity while maintaining specificity for GPR174.

What methodological approaches can be used to study GPR174-dependent changes in gene expression?

For comprehensive analysis of GPR174-dependent gene expression changes, researchers can employ the following methodological approaches:

• Transcriptomic Analysis:

  • RNA sequencing of wild-type versus GPR174-deficient cells, as demonstrated in the literature

  • Time-course experiments (0h, 1h, 4h, etc.) to capture dynamic changes in gene expression programs

  • Single-cell RNA sequencing to identify cell subpopulations with differential responsiveness to GPR174 signaling

• Targeted Gene Expression Analysis:

  • qRT-PCR for key GPR174-regulated genes such as:

    • CD86

    • Nr4a family members

    • CCR7

    • Phosphodiesterases

    • ITIM-containing receptors

• Protein-Level Validation:

  • Flow cytometry to measure surface expression of GPR174-regulated proteins (e.g., CD86, CD69, CD83)

  • Western blotting for intracellular signaling components affected by GPR174

• Mechanistic Studies:

  • Pharmacological approaches:

    • Forskolin and IBMX to manipulate cAMP levels

    • PKA inhibitors to block downstream signaling

    • LysoPS treatment to engage GPR174

  • Genetic approaches:

    • Compare effects in cells lacking GPR174 versus Gαs to establish signaling hierarchy

    • CRISPR/Cas9 targeting of GPR174 or downstream effectors

• In Vivo Models:

  • GPR174-deficient mice to study physiological relevance

  • Reporter systems like NUR77-GFP to monitor transcriptional responses

This multi-layered approach provides comprehensive insights into GPR174-dependent gene regulation, from initial receptor activation to downstream transcriptional and functional outcomes.

What are the implications of GPR174 in autoimmune disease research?

GPR174 has emerging significance in autoimmune disease research, with several important implications for investigators:

• Genetic associations have been identified between variants in the GPR174 locus and autoimmune diseases , suggesting GPR174 may be involved in pathogenesis or disease susceptibility.

• Given GPR174's role in regulating T-cell and B-cell functions, alterations in its signaling could potentially contribute to breaks in self-tolerance or inappropriate immune activation.

• The GPR174-dependent suppression of IL-2 production in activated T-lymphocytes represents a mechanism that could influence T regulatory cell development and function, which are critical for preventing autoimmunity.

• GPR174's regulatory role in macrophage polarization and cytokine secretion during sepsis suggests it may similarly influence innate immune responses in autoimmune conditions.

• The fact that GPR174 deficiency leads to enhanced B cell survival in culture raises the possibility that altered GPR174 function could affect B cell selection processes relevant to autoimmunity.

Researchers investigating autoimmune diseases should consider incorporating GPR174 analysis into their studies, particularly when examining B cell or T cell dysregulation phenotypes.

How can advanced imaging techniques be applied to study GPR174 cellular localization and trafficking?

Advanced imaging approaches offer powerful tools for investigating GPR174 biology:

• Super-resolution microscopy techniques:

  • Stimulated Emission Depletion (STED) microscopy

  • Stochastic Optical Reconstruction Microscopy (STORM)

  • Photoactivated Localization Microscopy (PALM)

  These techniques bypass the diffraction limit of conventional microscopy, allowing visualization of nanoscale receptor organization and clustering on the cell membrane.

• Live-cell imaging approaches:

  • TIRF (Total Internal Reflection Fluorescence) microscopy to visualize GPR174 dynamics specifically at the plasma membrane

  • Single-particle tracking to monitor individual receptor movement and internalization

  • FRAP (Fluorescence Recovery After Photobleaching) to study receptor mobility within membrane domains

• Functional correlation imaging:

  • Combining calcium imaging or FRET-based signaling reporters with GPR174 localization studies

  • Simultaneous imaging of GPR174 and interacting partners like G-proteins or downstream effectors

  • Correlative light and electron microscopy (CLEM) to relate GPR174 distribution to ultrastructural features

• Intravital microscopy:

  • For studying GPR174-expressing lymphocytes in their native tissue environment

  • Two-photon microscopy to track GPR174+ cells in lymphoid tissues

These advanced imaging approaches can reveal how GPR174 localization, internalization, and membrane organization contribute to its signaling functions in immune cells.

What experimental systems are optimal for studying GPR174 ligand interactions?

For researchers interested in GPR174 ligand interactions, several experimental systems offer distinct advantages:

• Cell-based functional assays:

  • B cell CD86 upregulation assay: Taking advantage of the GPR174-dependent CD86 induction , this readout can serve as a functional test for potential ligands

  • cAMP reporter cell lines: Cells expressing GPR174 alongside FRET-based or luciferase-based cAMP sensors can detect ligand-induced G-protein activation

  • Calcium mobilization assays: Useful particularly in contexts where GPR174 couples to calcium signaling

• Binding and interaction studies:

  • Surface Plasmon Resonance (SPR) using purified receptor components

  • Ligand binding assays with radiolabeled or fluorescently labeled LysoPS or candidate ligands

  • Competitive binding assays to determine relative affinity of various ligands

• In vivo systems:

  • The demonstrated ability of LysoPS administration to induce CD86 upregulation in follicular B cells in vivo provides a physiologically relevant system to test ligand activity

  • GPR174 reporter mice (such as those with tdTomato expression) can help identify tissues/contexts where the receptor is engaged

• Structural and computational approaches:

  • Molecular docking studies to predict interactions between GPR174 and potential ligands

  • Structure-activity relationship analysis of LysoPS analogs to define key binding determinants

When designing these studies, researchers should note that exogenous LysoPS did not enhance CD86 induction in vitro beyond spontaneous levels , suggesting either constitutive activity or the presence of endogenous ligands in culture conditions.