GPR174 Antibody, HRP conjugated

What is GPR174 and what are its primary physiological functions?

GPR174 is a G-protein-coupled receptor that primarily binds lysophosphatidylserine (LysoPS) and plays diverse roles in immune response regulation. It functions predominantly as a negative regulator of regulatory T-cell (Treg) accumulation and homeostasis. Under inflammatory conditions where LysoPS production increases, GPR174 contributes to down-regulating regulatory T-cell activity, which favors effector immune responses. At the molecular level, GPR174 mediates the suppression of IL-2 production in activated T-lymphocytes, which inhibits the growth, proliferation, and differentiation of T-cells. The receptor achieves this by acting through G(12)/G(13)-containing heterotrimeric G proteins to elevate cyclic AMP levels and protein kinase A (PKA) activity, which antagonizes proximal T-cell receptor signaling pathways .

Additionally, GPR174 plays important roles in sepsis through its regulation of macrophage polarization and control of pro- and anti-inflammatory cytokine secretions. Research has also revealed that under testosterone treatment, GPR174 can function as a receptor for CCL21, triggering calcium flux through G(q)-alpha and G(12)/G(13) proteins, which leads to chemotactic effects on activated B-cells .

What is an HRP-conjugated GPR174 antibody and how does it differ from unconjugated antibodies?

An HRP-conjugated GPR174 antibody consists of an anti-GPR174 antibody chemically linked to horseradish peroxidase (HRP) enzyme. This conjugation provides a direct detection system that eliminates the need for secondary antibodies in various immunoassays. When the antibody binds to GPR174, the attached HRP enzyme can catalyze a color-producing reaction when provided with appropriate substrates (like TMB, DAB, or chemiluminescent reagents), allowing for visual or instrument-based detection of the target protein.

The primary advantage of HRP-conjugated antibodies over unconjugated alternatives is workflow efficiency; they enable single-step detection protocols that reduce background noise, decrease experimental time, and potentially enhance sensitivity. For GPR174 detection specifically, HRP-conjugated antibodies are particularly valuable in Western blot analysis, ELISA, immunohistochemistry, and flow cytometry applications where direct detection simplifies complex multi-step protocols .

What applications are GPR174 antibodies, including HRP-conjugated versions, commonly used for?

GPR174 antibodies are utilized across multiple experimental platforms in immunological and cellular research. Based on current literature and technical resources, these applications include:

• Western blot (WB): GPR174 antibodies have been validated for detecting the receptor in various tissue and cell lysates, including brain membranes, spleen lysates, and T-cell lines like MOLT-4 and Jurkat. Western blotting allows researchers to quantify expression levels and detect potential post-translational modifications of GPR174 .

• Immunocytochemistry/Immunofluorescence (ICC/IF): These techniques enable visualization of GPR174 localization within cells, confirming its expected cell membrane expression pattern and potential redistribution under different experimental conditions .

• Cell surface detection in flow cytometry: Antibodies targeting extracellular domains of GPR174 are particularly useful for detecting the receptor on live intact cells, as demonstrated with Jurkat T-cell leukemia cells. This application is critical for studying receptor expression across different immune cell populations .

• Immunohistochemistry: For examining GPR174 expression patterns in tissue sections, particularly in models like the hindlimb ischemia mouse model where GPR174's role in blood flow recovery has been investigated .

HRP-conjugated versions provide streamlined protocols for all these applications by eliminating secondary antibody steps, which is particularly valuable when working with complex tissue samples or in multiplexed detection systems.

How can GPR174 antibodies be used to investigate the receptor's role in T-cell regulation and inflammatory responses?

GPR174 antibodies serve as critical tools for dissecting the complex role of this receptor in T-cell regulation and inflammatory responses. Advanced research approaches include:

• Dual immunofluorescence staining: Combining GPR174 antibodies with markers for regulatory T-cells (such as FOXP3) enables researchers to analyze the correlation between GPR174 expression levels and Treg function. This approach has revealed that GPR174 plays a negative role in regulatory T-cell accumulation and homeostasis, particularly under inflammatory conditions where LysoPS production increases .

• Signaling pathway analysis: Using GPR174 antibodies in combination with phospho-specific antibodies against PKA substrates allows researchers to track the downstream signaling events following receptor activation. This method has demonstrated that GPR174 acts via G(12)/G(13)-containing heterotrimeric G proteins to trigger elevated cyclic AMP levels and PKA activity, which antagonizes proximal TCR signaling .

• Functional assays with receptor blocking: Pre-incubating cells with GPR174 antibodies targeting extracellular domains can block ligand binding, providing insights into receptor-specific effects on IL-2 production in T-cells. This approach has helped confirm that GPR174 mediates the suppression of IL-2 production in activated T-lymphocytes, leading to inhibition of growth, proliferation, and differentiation of T-cells .

• Immunoprecipitation with GPR174 antibodies: This technique enables the identification of protein-protein interactions, revealing that GPR174 interacts with Gαs to inhibit nuclear localization of transcription factors like EGR1 by triggering cAMP/PKA activity. These interactions have significant implications for understanding GPR174's role in regulating inflammation and endothelial cell survival after ischemic injury .

What is the significance of GPR174 in vascular recovery, and how can antibodies help investigate this function?

Recent research has uncovered a critical role for GPR174 in vascular recovery following ischemic injury. GPR174 knockout mice demonstrate enhanced blood flow recovery in hindlimb ischemia models through mechanisms involving AREG (amphiregulin) expression regulation. Antibody-based research approaches to investigate this function include:

• Immunofluorescence co-staining of tissue sections: Using GPR174 antibodies alongside endothelial cell markers (CD31) and proliferation markers (Ki67) in ischemic tissue samples allows visualization of the relationship between GPR174 expression and vascular regeneration. Studies have shown that GPR174-deficient mice exhibit increased arteriogenesis post-hindlimb ischemia, with larger lumen sizes in ischemic adductor muscles and increased numbers of endothelial cells .

• Flow cytometry-based endothelial cell quantification: GPR174 antibodies combined with endothelial markers help quantify CD45-CD31+ endothelial cells in tissue samples, confirming that GPR174 deficiency leads to increased endothelial cell numbers after ischemic injury. This approach has demonstrated elevated proliferation and reduced apoptosis of endothelial cells in GPR174 knockout mice following hindlimb ischemia .

• Hypoxia assessment in correlation with GPR174 expression: By correlating GPR174 staining patterns with hypoxic areas (detected through pimonidazole injection), researchers have determined that GPR174 knockout reduces the degree of hypoxia in ischemic muscles, consistent with increased arteriogenesis. This finding suggests that GPR174 plays a crucial inhibitory role in ischemia-induced angiogenesis and arteriogenesis .

• Ex vivo Matrigel plug assays: Using GPR174 antibodies to characterize cellular components in Matrigel plugs has helped confirm that GPR174 deletion promotes neovascularization, with GPR174 knockout mice exhibiting increases in vessel formation and hemoglobin content, indicating mature vascular formation and perfusion .

How can GPR174 antibodies be utilized in GPCR-focused drug discovery research?

GPR174 antibodies play a vital role in screening and validating potential modulators of this receptor. Advanced drug discovery applications include:

• Competitive binding assays: Using labeled GPR174 antibodies targeting the extracellular domain in flow cytometry-based competition assays with candidate compounds can help identify potential ligands or antagonists. This approach has helped characterize compounds like those in Group I (compounds 1-3) that demonstrate varying EC50 values for GPR174 activity modulation .

• Receptor internalization studies: Fluorescently labeled GPR174 antibodies enable tracking of receptor internalization upon ligand binding or compound treatment, providing insights into compound efficacy and mechanism of action. This is particularly relevant for compounds classified as antagonists or allosteric modulators of GPR174 .

• Target validation in disease models: Immunohistochemistry with GPR174 antibodies in disease tissue models helps validate the receptor as a therapeutic target for conditions where GPR174 has been implicated, including cancer, nervous system disorders, and conditions requiring neuroregeneration .

• Comparison of different screening methodologies: GPR174 antibodies support validation of hits identified through various screening platforms, such as CRA (cAMP response assay) and FLIPR (fluorescence imaging plate reader), allowing researchers to compare the effectiveness of different screening methods for GPR174 modulators, as demonstrated in comparative studies with other GPCRs .

What are the common challenges in using HRP-conjugated GPR174 antibodies for Western blot analysis, and how can they be addressed?

Western blotting with HRP-conjugated GPR174 antibodies presents several technical challenges that researchers should be prepared to address:

• High background signal: The direct detection nature of HRP-conjugated antibodies can sometimes result in higher background. This can be mitigated by:

  • Using optimized blocking solutions (5% non-fat dry milk or BSA in TBST)

  • Including additional wash steps (at least 3-5 washes of 5-10 minutes each)

  • Diluting the antibody appropriately (starting with manufacturer-recommended dilutions, typically 1:400 for GPR174 antibodies, and optimizing as needed)

• Variable band patterns across tissue types: GPR174 detection often shows different molecular weight bands in different tissue types. For example, Western blot analysis of rat brain membranes, mouse brain lysate, rat spleen lysate, and mouse spleen membranes may show different banding patterns. Verification can be performed by:

  • Including appropriate positive controls (like MOLT-4 or Jurkat T-cell leukemia cell line lysates)

  • Using blocking peptides to confirm specificity (as demonstrated with GPR174 extracellular blocking peptide)

  • Comparing results from multiple antibody clones targeting different epitopes

• Post-translational modifications affecting detection: GPR174, like many GPCRs, undergoes various post-translational modifications that can affect antibody binding. Researchers should consider:

  • Using denaturing versus non-denaturing conditions depending on the epitope location

  • Treating samples with glycosidases if glycosylation is interfering with detection

  • Including phosphatase treatment if phosphorylation status affects antibody binding

• Membrane protein extraction challenges: As a multi-pass membrane protein, GPR174 requires effective extraction methods. Optimization strategies include:

  • Using specialized membrane protein extraction buffers containing appropriate detergents

  • Avoiding excessive heating of samples to prevent aggregation

  • Including protease inhibitors to prevent degradation during extraction

How should researchers validate the specificity of GPR174 antibodies in their experimental systems?

Validating antibody specificity is crucial for producing reliable results. For GPR174 antibodies, comprehensive validation should include:

• Blocking peptide controls: Preincubation of the antibody with a specific blocking peptide (like the GPR174 extracellular blocking peptide) should eliminate or significantly reduce the signal in all applications. This has been demonstrated effectively in Western blot analysis of various tissue samples and cell lines .

• Genetic controls: Comparing wild-type samples with GPR174 knockout or knockdown samples provides the gold standard for specificity validation. This approach has been used effectively in GPR174-/Y mice models to confirm antibody specificity in both tissue sections and Western blots .

• Cross-reactivity testing: Testing the antibody against related GPCRs, particularly other lysophosphatidylserine receptors, confirms the absence of cross-reactivity. This is especially important since GPR174 (LysoPS, LYPSR3) is one of three lysophosphatidyl serine receptors belonging to the GPCR superfamily .

• Cell-type specificity confirmation: Since GPR174 is predominantly expressed in immune cells, confirming expected expression patterns across different cell types (high in T cells, B cells, and certain tissues like spleen and thymus) provides additional validation. Flow cytometry with live intact human Jurkat T-cell leukemia cells has been used to demonstrate cell surface detection of GPR174 .

• Method-specific controls: Each experimental method requires specific validation approaches:

  • For Western blot: Multiple bands should be consistently present across experiments and match predicted molecular weights (approximately 39 KDa calculated MW for GPR174)

  • For immunofluorescence: Membrane localization pattern should be consistent with GPR174 being a multi-pass membrane protein

  • For flow cytometry: Cell surface staining should be detectable only in cells expressing GPR174

What methodological considerations are important when designing experiments to study GPR174 ligand interactions using antibodies?

Studying GPR174 interactions with its ligands (primarily lysophosphatidylserine and potentially CCL21) requires careful experimental design:

• Epitope selection considerations: When selecting antibodies for ligand interaction studies, researchers should consider:

  • Using antibodies targeting non-ligand-binding regions to avoid interference with ligand binding

  • For competitive binding studies, selecting antibodies that target the extracellular domains involved in ligand recognition, such as those based on the peptide (C)DTSGNRTK(S)FVDLPTR corresponding to amino acid residues 160-175 of human GPR174 (within the 2nd extracellular loop)

  • For conformation studies, choosing antibodies that can distinguish between active and inactive receptor states

• Sample preparation for ligand binding experiments:

  • For LysoPS binding studies, researchers should control for endogenous LysoPS levels, as 18:0 LysoPS production is elevated in certain conditions like hindlimb ischemia

  • For testosterone-dependent CCL21 binding, hormone pre-treatment conditions should be carefully standardized

  • Proper membrane preparation is essential to maintain receptor conformation, especially when studying interactions that trigger G(q)-alpha and G(12)/G(13) protein-mediated calcium flux

• Downstream signaling detection approaches:

  • Select appropriate readouts based on known GPR174 signaling pathways (cAMP levels for Gs pathways, calcium flux for Gq pathways)

  • Consider using CRE (cAMP response element) activity assays, as GPR174 activation leads to increased cAMP cellular levels

  • Employ appropriate controls, as demonstrated in compound screening approaches where CRA (cAMP response assay) hits for GPR174 were validated

• Temporal considerations:

  • Since GPR174 activation leads to cellular morphological changes, cell-to-cell adhesion, and delays in cell proliferation, time-course experiments are essential

  • Consider both acute and chronic effects of ligand binding, as GPR174's roles in inflammation and T-cell regulation may have different temporal dynamics

How is GPR174 being investigated as a potential therapeutic target, and what role do antibodies play in this research?

GPR174 has emerged as a promising therapeutic target for several conditions, with antibodies playing crucial roles in target validation and mechanism elucidation:

• Ischemic vascular diseases: Recent research has identified Treg GPR174 as a potential therapeutic target for treating ischemic vascular diseases, such as peripheral arterial disease (PAD). In a hindlimb ischemia mouse model, GPR174 deficiency mitigated inflammatory response and improved endothelial cell proliferation and survival by promoting amphiregulin (AREG) secretion. Antibodies have been instrumental in confirming these effects through immunofluorescence staining of vessels, which demonstrated increased arteriogenesis post-hindlimb ischemia in GPR174-deficient mice .

• Autoimmune disorders: GPR174 has been reported as a genetic risk locus for Graves' disease, an autoimmune disorder causing hyperthyroidism. Antibody-based research is helping elucidate how GPR174 signaling contributes to autoimmune pathogenesis, potentially through its role as a negative regulator of T-cell activation. Understanding these mechanisms is critical for developing targeted therapies for autoimmune conditions .

• Small molecule inhibitor development: Researchers have identified several compounds that modulate GPR174 activity, with potential applications in cancer, nervous system diseases, and neuroregeneration. Antibodies are essential for validating these compounds' effects on receptor expression, localization, and downstream signaling. Compounds like those in Group I have shown varying EC50 values for GPR174 activity modulation and different effects on Gs signaling, suggesting diverse therapeutic potential .

• Sepsis management: GPR174 plays an important role in the initial period of sepsis through regulating macrophage polarization and pro- and anti-inflammatory cytokine secretions. Antibody-based detection of GPR174 expression and signaling pathways in sepsis models is providing insights into potential intervention strategies that could modulate the inflammatory response in this life-threatening condition .

What recent discoveries about GPR174 signaling mechanisms have been facilitated by antibody-based research?

Antibody-based research has enabled several significant discoveries about GPR174 signaling mechanisms:

How are new technological advances enhancing the applications of GPR174 antibodies in research?

Recent technological advances are expanding and refining the applications of GPR174 antibodies:

• Improved screening methodologies: Comparison of different screening platforms like CRA (cAMP response assay) and FLIPR (fluorescence imaging plate reader) has enhanced the identification of GPR174 modulators. Studies comparing these methods across different GPCRs, including GPR174, have shown varying success rates in identifying compounds that affect receptor function. These advances are accelerating the discovery of potential therapeutic agents targeting GPR174 .

• Enhanced visualization techniques: Advanced imaging techniques combined with GPR174 antibodies have enabled more detailed studies of receptor localization and trafficking. For example, cell surface detection of GPR174 in live intact human Jurkat T-cell leukemia cells has been performed using fluorescence-based flow cytometry, providing insights into receptor expression under different conditions .

• Multi-parametric flow cytometry: Integration of GPR174 antibodies into multi-parametric flow cytometry panels allows simultaneous analysis of receptor expression alongside other markers of immune cell activation and differentiation. This approach has been valuable for quantifying CD45-CD31+ endothelial cells and correlating GPR174 expression with cell phenotypes in complex tissues .

• Ex vivo models: Advanced ex vivo models like the Matrigel plug assay are being combined with GPR174 antibody detection to explore angiogenesis processes. These approaches have demonstrated that GPR174 knockout mice exhibit increases in vessel formation and hemoglobin content, indicating mature vascular formation and perfusion. Such models provide controlled environments for studying GPR174 function while maintaining physiological relevance .

What are the critical factors for optimizing immunohistochemistry protocols with GPR174 antibodies?

Successful immunohistochemistry with GPR174 antibodies requires attention to several critical factors:

• Fixation and antigen retrieval: As a multi-pass membrane protein, GPR174 epitopes can be masked during fixation. Researchers should:

  • Compare multiple fixation methods (4% paraformaldehyde, methanol, or acetone)

  • Optimize antigen retrieval conditions (heat-induced epitope retrieval in citrate buffer pH 6.0 or Tris-EDTA buffer pH 9.0)

  • For HRP-conjugated antibodies, ensure that the fixation method doesn't compromise HRP enzyme activity

• Antibody concentration and incubation conditions: The optimal antibody dilution for immunohistochemistry may differ from other applications:

  • Start with dilutions between 1:100-1:500 for most GPR174 antibodies

  • Consider longer incubation times (overnight at 4°C) for maximum sensitivity

  • For HRP-conjugated antibodies, include appropriate quenching steps to eliminate endogenous peroxidase activity

• Tissue-specific considerations: GPR174 expression varies across tissues, requiring protocol adjustments:

  • For highly vascularized tissues like those in hindlimb ischemia models, include additional blocking steps to reduce background

  • For immune tissues like spleen where GPR174 is highly expressed, titrate antibody concentrations carefully to avoid oversaturation

  • When examining endothelial cells, consider co-staining with CD31 to precisely identify vasculature structures

• Controls for validation: Include comprehensive controls to ensure specificity:

  • Positive controls like spleen tissue sections known to express GPR174

  • Negative controls using tissues from GPR174 knockout models

  • Antibody controls using blocking peptides specific to the GPR174 epitope

  • When investigating hypoxic conditions, correlate GPR174 staining with hypoxia markers like pimonidazole

How can researchers effectively use GPR174 antibodies to investigate protein-protein interactions and signaling complexes?

Investigating GPR174's protein-protein interactions and signaling complexes requires specialized approaches:

• Co-immunoprecipitation strategies: When using GPR174 antibodies for co-immunoprecipitation:

  • Select antibodies that don't interfere with protein interaction domains

  • Use gentle lysis conditions to preserve native protein complexes (non-ionic detergents like digitonin or NP-40)

  • Consider crosslinking approaches for transient interactions

  • Verify interactions bidirectionally (immunoprecipitate with GPR174 antibody and probe for partner protein, then reverse)

• Proximity ligation assays (PLA): This technique can detect interactions between GPR174 and potential partners:

  • Combine GPR174 antibodies with antibodies against suspected interaction partners (like G-proteins)

  • Optimize fixation to preserve membrane integrity where GPR174 resides

  • Include appropriate controls to validate specific interactions versus random proximity

• BRET/FRET approaches with antibody validation: While these techniques typically use tagged proteins, antibodies are essential for validation:

  • Confirm that fusion tags don't disrupt interactions identified in antibody-based methods

  • Use antibodies to verify expression levels of interaction partners

  • Validate subcellular localization of interaction complexes using immunofluorescence

• Pathway-specific investigations: Research has established that GPR174 interacts with Gαs to inhibit the nuclear localization of EGR1 by triggering cAMP/PKA activity. To investigate these pathways:

  • Use phospho-specific antibodies to track PKA substrate phosphorylation downstream of GPR174 activation

  • Combine with nuclear/cytoplasmic fractionation to monitor transcription factor localization

  • Correlate with functional readouts like AREG expression to connect signaling events to biological outcomes

What emerging areas of GPR174 research might benefit from improved antibody tools?

Several emerging research areas would benefit from advanced GPR174 antibody tools:

• Single-cell analysis of GPR174 expression heterogeneity: Current research suggests differential roles of GPR174 across immune cell subsets. Development of highly specific antibodies compatible with single-cell technologies would enable:

  • Characterization of GPR174 expression across immune cell subpopulations at single-cell resolution

  • Correlation of expression levels with functional states in heterogeneous tissues

  • Identification of unique GPR174-expressing cell populations in disease states

• Conformational state-specific antibodies: GPCRs like GPR174 can exist in different conformational states (active, inactive, ligand-bound). Antibodies capable of distinguishing between these states would allow:

  • Direct measurement of receptor activation in native tissues

  • Screening for conformation-selective modulators (biased agonists or antagonists)

  • Determination of how different ligands (LysoPS versus CCL21 with testosterone) affect receptor conformation

• Dynamic imaging of GPR174 trafficking: Antibody fragments suitable for live-cell imaging applications would enable:

  • Real-time visualization of receptor internalization and recycling

  • Analysis of how different ligands or disease states affect receptor trafficking

  • Investigation of spatial relationships between GPR174 and its signaling partners

• Therapeutic targeting opportunities: As GPR174 emerges as a potential therapeutic target for conditions ranging from ischemic vascular diseases to autoimmune disorders, antibody-based therapeutics may provide:

  • Selective modulation of specific GPR174 functions

  • Targeted delivery of drugs to GPR174-expressing cells

  • Development of antibody-drug conjugates for conditions where GPR174 expression is dysregulated

How might the development of GPR174 therapeutic antagonists benefit from antibody-based research approaches?

Antibody-based research approaches provide critical insights for the development of GPR174 therapeutic antagonists:

• Target validation and expression profiling: Before investing in antagonist development, thorough validation is essential:

  • Antibodies enable precise mapping of GPR174 expression across tissues in health and disease

  • Immunohistochemistry with GPR174 antibodies in disease models helps confirm receptor involvement

  • Flow cytometry with cell-type-specific markers identifies which populations should be targeted

• Compound screening and validation: Antibodies support multiple aspects of the drug discovery process:

  • Competition binding assays using labeled antibodies can identify compounds that bind to GPR174

  • Functional assays using GPR174 antibodies can confirm that compounds like those in Group I (compounds 1-3) modulate receptor signaling

  • Internalization assays using fluorescently labeled antibodies can characterize compound effects on receptor trafficking

• Mechanism of action studies: Understanding how antagonists affect GPR174 function is crucial:

  • Antibodies detecting downstream signaling events (like PKA activity or EGR1 nuclear localization) help characterize compound effects

  • Co-immunoprecipitation with GPR174 antibodies can reveal how antagonists alter protein-protein interactions

  • Conformational antibodies could distinguish between different antagonist binding modes

• Translational biomarkers: Antibody-based assays provide tools for clinical development:

  • Assays to monitor target engagement in clinical samples

  • Detection of pathway modulation as pharmacodynamic markers

  • Patient stratification based on GPR174 expression or activation state

Current research has already identified compounds like Compound #4 that compete with the GPR174 agonist LysoPS, providing a foundation for therapeutic antagonist development that can be further supported by comprehensive antibody-based research approaches .