GPR4 Antibody

What is GPR4 and why is it significant in biomedical research?

GPR4 is a proton-sensing G protein-coupled receptor that plays crucial roles in various physiological processes, including pH sensing, immune response regulation, inflammation, and angiogenesis. Its significance in research stems from its widespread distribution across human tissues, including lung, kidney, liver, and heart tissues, and its involvement in numerous pathological conditions. GPR4 functions primarily as a sensor of extracellular acidification, activating various downstream signaling pathways that lead to cellular responses including inflammatory cytokine production and vascular remodeling. The receptor has emerged as a potential therapeutic target for conditions characterized by tissue acidosis, particularly cancer, inflammatory disorders, and cardiovascular diseases .

What experimental models are suitable for studying GPR4 function?

Several experimental models have demonstrated utility in GPR4 research. In vitro models include human cell lines like the chondrocyte SW1353 cell line for inflammation studies and vascular endothelial cells for angiogenesis research. For animal models, GPR4 knockout mice (Gpr4−/−) have proven particularly valuable for loss-of-function studies. The destabilization of the medial meniscus (DMM) surgery model in mice has been effective for investigating GPR4's role in osteoarthritis development. Researchers can also employ lentiviral overexpression systems (like Lenti-Gpr4) for gain-of-function studies in mouse joint tissues. When selecting an experimental model, researchers should consider the specific physiological context of their research question, as GPR4 functions differently across tissue types and pathological conditions .

How should GPR4 antibodies be validated before experimental use?

Proper validation of GPR4 antibodies is essential to ensure experimental reliability. Start with Western blot analysis using positive control samples known to express GPR4, such as LO2 or Raji cell lines, to confirm the antibody detects a band at the expected molecular weight (approximately 45kDa for GPR4). Cross-validate with immunofluorescence staining paired with confocal microscopy to verify proper cellular localization, particularly in the plasma membrane where GPR4 is predominantly expressed. For advanced validation, compare staining patterns between wild-type and GPR4 knockout tissues to confirm specificity. Additionally, peptide competition assays using the immunizing peptide (such as the sequence corresponding to amino acids 283-362 of human GPR4 or the peptide corresponding to residues 162-177 of rat GPR4) can verify binding specificity. Always optimize antibody concentration through titration experiments to determine the optimal dilution (typically in the range of 1:500 to 1:2000 for Western blot applications) .

What are the optimal conditions for GPR4 antibody-based Western blot analysis?

For optimal GPR4 detection via Western blot, prepare protein samples from tissues or cells under non-denaturing conditions when possible, as GPR4's seven-transmembrane structure can be sensitive to harsh detergents. Use a protein extraction buffer containing 1% NP-40 or a similar mild detergent, supplemented with protease inhibitors. Load 20-40μg of total protein per lane and separate on an 8-12% SDS-PAGE gel. When transferring to membranes, PVDF membranes generally provide better results than nitrocellulose for this transmembrane protein. Block with 5% non-fat milk in TBST for 1 hour at room temperature. Incubate with the GPR4 antibody at a dilution of 1:500 to 1:2000 (optimize for your specific antibody) overnight at 4°C. The expected molecular weight for GPR4 is approximately 41kDa calculated, though it often appears around 45kDa in gels due to post-translational modifications. Always include positive control samples known to express GPR4, such as LO2 or Raji cell lines .

How can immunofluorescence techniques be optimized for GPR4 localization studies?

For optimal immunofluorescence detection of GPR4, tissue preparation is critical. For frozen sections, fix tissues briefly (8-10 minutes) with 4% paraformaldehyde to preserve membrane proteins. For paraffin sections, use citrate buffer (pH 6.0) for antigen retrieval, applying moderate heat to avoid epitope damage. When blocking, include 0.3% Triton X-100 for membrane permeabilization. Incubate with GPR4 antibody at 1:100 to 1:500 dilution overnight at 4°C. For visualization, secondary antibodies conjugated with bright fluorophores like Alexa Fluor 488 or 594 provide optimal signal-to-noise ratio. When studying vascular expression of GPR4, co-staining with endothelial markers such as CD105 is informative, as these proteins have been found to co-localize in tumor vasculature. Confocal microscopy with z-stacking is recommended for precise subcellular localization. For quantification, measure the average fluorescence intensity across multiple fields, and compare expression between different regions of the tissue (e.g., tumor margin versus central tumor regions, as GPR4 shows differential expression in these areas) .

What controls should be included when using GPR4 antibodies in research applications?

A comprehensive control strategy is essential for GPR4 antibody experiments. Primary controls should include positive control samples with confirmed GPR4 expression (e.g., LO2 or Raji cell lines) and negative controls using GPR4 knockout tissues or cells when available. Technical controls should include isotype controls using non-specific IgG from the same host species as the GPR4 antibody, and secondary antibody-only controls to assess non-specific binding. For blocking peptide controls, pre-incubate the GPR4 antibody with excess immunizing peptide (5-10 fold molar excess) before application to verify signal specificity. When conducting comparative studies, include internal normalization controls such as housekeeping proteins (β-actin, GAPDH) for Western blot or nuclear counterstains (DAPI) for immunofluorescence. For functional studies, compare results using multiple GPR4 antibodies targeting different epitopes, or complement antibody-based techniques with gene expression analysis (qPCR) to corroborate protein-level findings .

How can GPR4 antibodies be used to investigate the relationship between tissue acidosis and angiogenesis?

To investigate GPR4's role in acidosis-induced angiogenesis, researchers can employ a multi-faceted approach combining in vitro and in vivo methodologies. Begin with endothelial cell cultures exposed to controlled pH gradients (pH 7.4 normal vs. pH 6.8-6.4 acidic) and assess GPR4 activation using phospho-specific antibodies targeting downstream signaling molecules like cAMP or protein kinase A. Co-immunoprecipitation with GPR4 antibodies can identify interaction partners that change under acidic conditions. For angiogenesis assessment, perform tube formation assays with endothelial cells under varying pH conditions, with and without GPR4 neutralizing antibodies. In mouse models, implant pH-sensing probes alongside tumor xenografts to correlate local pH with GPR4 expression and microvessel density. Immunofluorescence co-staining for GPR4 and endothelial markers like CD105 in tumor sections can reveal spatial relationships between acidic microenvironments (using pH-sensitive dyes) and neovascularization. This approach has revealed that GPR4 expression positively correlates with microvessel density in hepatocellular carcinoma and is preferentially expressed in tumor margins where angiogenesis is most active .

What approaches can be used to study GPR4's role in inflammatory signaling pathways?

To elucidate GPR4's role in inflammatory signaling, implement a comprehensive investigative approach. Begin with chromatin immunoprecipitation (ChIP) assays using antibodies against transcription factors downstream of GPR4 activation (such as CREB) to identify inflammation-related gene promoters activated during GPR4 signaling. Complement this with RNA-seq analysis comparing wild-type and GPR4-deficient cells under inflammatory stimuli to establish the GPR4-dependent transcriptome. For protein-level validation, use phospho-specific antibodies in Western blot or immunofluorescence to track activation of key inflammatory signaling nodes (p38 MAPK, NF-κB) following GPR4 stimulation. In tissue explant cultures (such as human cartilage explants for osteoarthritis studies), apply GPR4-specific antagonists like NE52-QQ57 and measure inflammatory cytokine production using multiplex ELISA. For in vivo validation, utilize GPR4 knockout mice in disease models characterized by inflammatory components, such as the destabilization of medial meniscus (DMM) model for osteoarthritis, and assess both histological outcomes and local cytokine production. This integrative approach has revealed GPR4's critical role in promoting inflammatory processes in conditions like osteoarthritis through modulation of pathways such as CXCL12/CXCR7 signaling .

How should researchers address the challenge of GPR4 antibody cross-reactivity with other pH-sensing GPCRs?

Addressing potential cross-reactivity between GPR4 antibodies and related pH-sensing GPCRs (OGR1, TDAG8, G2A) requires a systematic approach to ensure experimental specificity. First, perform detailed epitope analysis – select antibodies targeting unique regions with minimal sequence homology to related receptors, preferably in the extracellular or C-terminal domains where divergence is greatest. Validate specificity using expression systems where cells overexpress individual pH-sensing GPCRs one at a time, and perform parallel Western blots to confirm single-band detection only in GPR4-expressing cells. For definitive validation, employ knockout/knockdown controls – compare antibody signals between wild-type tissues and tissues from GPR4 knockout models or cells with CRISPR-mediated GPR4 deletion. When cross-reactivity cannot be fully eliminated, employ compensatory strategies such as using multiple antibodies targeting different GPR4 epitopes and confirming concordant results, or complementing antibody-based detection with nucleic acid-based approaches that offer greater specificity through unique sequence targeting. Additionally, consider functional validation through selective pharmacological inhibition using GPR4-specific antagonists like NE52-QQ57 to confirm that observed phenotypes are indeed GPR4-dependent .

How can GPR4 antibodies be utilized to study its role in cancer progression and tumor angiogenesis?

To investigate GPR4's role in cancer progression and tumor angiogenesis, researchers should implement a multi-modal approach integrating both human samples and experimental models. For human tumor analyses, perform immunohistochemistry with GPR4 antibodies on paired tumor and adjacent normal tissues across multiple cancer types, with particular attention to hepatocellular carcinoma, ovarian cancer, and head and neck squamous cell carcinoma where GPR4 overexpression has been documented. Quantify microvessel density using CD105 co-staining and correlate with GPR4 expression patterns, noting that GPR4 expression is often elevated in tumor margins compared to central regions. For mechanistic studies, establish knockdown/knockout cancer cell lines using CRISPR-Cas9 targeting GPR4, followed by xenograft implantation to assess tumor growth rates and vascular development in vivo. Complement this with intravital microscopy using fluorescently-labeled GPR4 antibodies to track receptor expression dynamics during tumor progression. For therapeutic targeting evaluation, test GPR4 antagonists in combination with conventional anti-angiogenic agents in preclinical models. This approach has revealed that GPR4 promotes tumor angiogenesis through multiple mechanisms, including VEGF secretion via p38 signaling and modulation of endothelial cell proliferation and migration, making it a potential prognostic factor and therapeutic target in multiple cancer types .

What are the best practices for using GPR4 antibodies in osteoarthritis research?

For optimal use of GPR4 antibodies in osteoarthritis research, implement a comprehensive methodology spanning both human and animal models. When working with human samples, collect paired cartilage specimens from OA patients and non-OA controls, stratifying by disease severity using standard radiographic grading. For immunohistochemical analysis, use GPR4 antibodies at optimized dilutions (typically 1:100-1:200) with appropriate antigen retrieval methods, and quantify expression levels across different cartilage zones. In animal models, the destabilization of medial meniscus (DMM) surgical approach provides a reliable OA model in mice, where GPR4 antibody staining should be performed at multiple time points post-surgery to track temporal expression changes. For intervention studies, administer GPR4 antagonists such as NE52-QQ57 via intra-articular injection (30μM in 10μl, twice weekly) and assess outcomes through histological analyses (OARSI scoring), micro-CT for bone changes, and immunostaining for matrix degradation markers. Ex vivo cartilage explant cultures provide an intermediate system to test pH modulation effects on GPR4 activity and cartilage homeostasis. This methodological approach has revealed that GPR4 expression increases in OA cartilage and contributes to disease progression by modulating inflammatory signaling pathways, including the CXCL12/CXCR7 axis .

How can researchers investigate the role of GPR4 in inflammatory vascular disorders?

To investigate GPR4's role in inflammatory vascular disorders, employ a translational research approach spanning molecular, cellular, and in vivo methodologies. Begin with primary human endothelial cell cultures exposed to inflammatory stimuli (TNF-α, IL-1β) under varying pH conditions to mimic disease microenvironments. Use GPR4 antibodies for immunofluorescence to track receptor localization changes during inflammatory activation, particularly at cell-cell junctions relevant to vascular permeability. Implement real-time impedance measurements to assess endothelial barrier function while manipulating GPR4 activity through genetic approaches (siRNA) or pharmacological intervention (GPR4 antagonists). For in vivo studies, utilize vascular-specific GPR4 knockout models or inducible systems to avoid developmental confounding effects, as constitutive GPR4 deficiency causes perinatal mortality and spontaneous bleeding. In these models, induce inflammatory vascular conditions such as atherosclerosis (ApoE-/- background) or vascular injury, then analyze vessels using a combination of intravital microscopy and post-mortem immunohistochemistry with GPR4 antibodies. Assessment of vascular permeability using tracers like Evans Blue dye can correlate functional outcomes with GPR4 expression patterns. This approach has revealed that GPR4 contributes to vascular inflammation by regulating the expression of adhesion molecules (VCAM1, ICAM1) and mediating stress responses in the endothelium, potentially offering a therapeutic target for inflammatory vascular conditions .

How can new technological approaches enhance GPR4 antibody-based research?

Emerging technologies are revolutionizing GPR4 antibody applications in research. Single-cell proteomics combined with GPR4 antibodies allows for unprecedented resolution in heterogeneous tissues, revealing cell-specific expression patterns not detectable with bulk analysis. This is particularly valuable for understanding GPR4's differential expression across various cell types within the tumor microenvironment. Proximity ligation assays can detect protein-protein interactions of GPR4 with downstream effectors in situ, providing spatial context to signaling events. For dynamic studies, genetically encoded pH sensors paired with fluorescently-labeled GPR4 antibodies in live-cell imaging enable real-time correlation of local pH changes with receptor activation and trafficking. CRISPR-Cas9 gene editing can generate epitope-tagged endogenous GPR4, allowing for antibody-based detection without overexpression artifacts. Mass cytometry (CyTOF) using metal-conjugated GPR4 antibodies permits simultaneous detection of receptor expression alongside dozens of other proteins in single cells. Additionally, antibody-based biosensors incorporating GPR4 detection elements can monitor receptor activation in real-time in response to physiological pH fluctuations. These technological advances provide researchers with powerful tools to dissect GPR4's complex roles in physiological and pathological processes with previously unattainable precision and contextual understanding .

What considerations are important when developing therapeutic strategies targeting GPR4?

Developing effective therapeutic strategies targeting GPR4 requires careful consideration of several key factors. First, understand the context-dependent roles of GPR4 – while GPR4 inhibition shows therapeutic potential in inflammatory conditions like osteoarthritis and certain cancers, complete blockade may compromise physiological pH sensing and vascular stability, as evidenced by bleeding tendencies in GPR4-null mice. When designing targeting approaches, consider the specific binding properties of therapeutic antibodies or small molecule antagonists like NE52-QQ57, which has demonstrated efficacy in preclinical models at concentrations of 30μM. Receptor internalization dynamics must be accounted for, as pH-dependent activation can alter GPR4 surface expression and accessibility to therapeutic agents. Develop companion diagnostic approaches using validated GPR4 antibodies to identify patient populations most likely to benefit from GPR4-targeted therapies based on receptor expression levels. For delivery strategies, consider targeted approaches for tissue-specific intervention – intra-articular injection has proven effective for osteoarthritis applications (twice weekly administration), while systemic delivery may be necessary for metastatic cancers. Finally, investigate combination approaches pairing GPR4 inhibitors with established therapies such as anti-inflammatory agents or conventional cancer treatments to potentially achieve synergistic effects through complementary mechanism targeting .

How can researchers address discrepancies in GPR4 antibody-based detection results across different experimental systems?

Addressing discrepancies in GPR4 antibody detection across experimental systems requires systematic troubleshooting and validation approaches. Begin by implementing standardized positive and negative controls across all experimental platforms – cell lines with confirmed GPR4 expression (LO2, Raji) serve as reliable positive controls, while GPR4 knockout tissues provide definitive negative controls. When conflicting results emerge, perform epitope mapping to determine if post-translational modifications or protein-protein interactions might mask antibody binding sites in certain contexts. GPR4's calculated molecular weight is 41kDa, but it often appears at 45kDa due to glycosylation and other modifications that may vary across tissue types. For cross-platform validation, complement antibody-based detection with nucleic acid analyses like RNAscope, which can verify transcript presence with cellular resolution. Consider the impact of experimental conditions on receptor conformation – acidic pH activates GPR4 and may alter epitope accessibility, potentially explaining detection variability between physiological and acidic environments. When working with formalin-fixed tissues, optimize antigen retrieval methods specifically for membrane proteins, as standard protocols may inadequately expose GPR4 epitopes. Finally, maintain detailed documentation of antibody lot numbers, as manufacturing variations can contribute to inconsistent results. By implementing these systematic approaches, researchers can resolve discrepancies and establish reliable detection protocols for GPR4 across diverse experimental systems .

Table 1: GPR4 Expression Across Tissue Types and Disease States

Abbreviations: HCC - Hepatocellular Carcinoma; OA - Osteoarthritis; IF - Immunofluorescence; WB - Western Blot; IHC - Immunohistochemistry; qPCR - Quantitative Polymerase Chain Reaction

Table 2: GPR4 Antibody Applications and Optimized Protocols

PFA - Paraformaldehyde; FACS - Fluorescence-Activated Cell Sorting