GPR55 Antibody, HRP conjugated

What is GPR55 and why is it significant in cannabinoid research?

GPR55 is a G-protein coupled receptor that functions as a putative cannabinoid receptor with unique signaling properties. Research has established GPR55 as a receptor for L-alpha-lysophosphatidylinositol (LPI), which induces calcium release from intracellular stores via the heterotrimeric G protein GNA13 and RHOA . GPR55 has gained significant attention due to its ability to modulate cannabinoid CB2 receptor-mediated responses, particularly in neutrophils where it potentiates migratory responses while inhibiting degranulation and reactive oxygen species (ROS) production . This receptor plays multifaceted roles in inflammation, neuropathic pain, and bone physiology, making it an important target for immunological and neurological research .

What cellular distribution patterns have been observed for GPR55?

GPR55 expression has been extensively characterized in human neutrophils at both mRNA and protein levels. Studies show that GPR55 mRNA copy numbers in neutrophils are significantly higher than those of CB2 receptors . Interestingly, GPR55 demonstrates a predominantly intracellular distribution pattern in primary neutrophils and HL60 cells, which aligns with observations in other primary cell types . When studying neutrophil-like differentiated HL60 cells (dHL60), researchers have documented a 5.5-fold increase in GPR55 mRNA levels compared to undifferentiated HL60 cells, suggesting expression changes during cellular differentiation . Immunofluorescence studies with anti-GPR55 antibodies confirm this intracellular localization pattern, which is important to consider when designing experimental protocols .

How does HRP conjugation enhance GPR55 antibody applications?

HRP conjugation provides significant technical advantages for GPR55 detection methods. The enzyme conjugation eliminates the need for secondary antibody incubation steps, streamlining experimental workflows and reducing background signal issues that can arise from secondary antibody cross-reactivity . HRP-conjugated antibodies enable highly sensitive detection through various substrates that produce colorimetric, chemiluminescent, or fluorescent signals. For GPR55 antibodies targeting specific epitopes, such as amino acids 203-222, HRP conjugation preserves epitope recognition while adding enzymatic detection capabilities . This technical enhancement is particularly valuable when studying GPR55 in complex tissues or in experiments requiring multiple antibody labeling.

What experimental approaches best demonstrate GPR55-CB2 receptor heteromerization?

Investigating GPR55-CB2 receptor heteromerization requires sophisticated methodological approaches. Research demonstrates that co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays are effective for assessing direct interaction between GPR55 and CB2 receptors in heterologous expression systems . Label-free real-time methods, particularly Epic dynamic mass redistribution and CellKey impedance assays, have proven valuable for investigating cross-talk effects on downstream signaling . When studying heteromerization, it's essential to establish proper controls using cells expressing individual receptors alongside those expressing both receptors. Expression systems like HEK293 cells stably expressing CB2 receptors (HEK-CB2R), GPR55 receptors (HEK-GPR55), or both receptors (HEK-CB2R/GPR55) provide valuable tools for comparative analyses . The detection of heteromerization effects on signaling can be assessed through ERK1/2-MAPK activation and gene reporter assays examining transcription factors like nuclear factor of activated T-cells, NF-κB, and cAMP response elements .

How does epitope selection affect experimental outcomes when using GPR55 antibodies?

The epitope specificity of GPR55 antibodies critically influences experimental outcomes. Antibodies targeting specific amino acid sequences, such as AA 203-222 in human GPR55, demonstrate differential binding characteristics compared to antibodies targeting other regions . When selecting a GPR55 antibody, researchers should consider the structural and functional domains of the receptor. For instance, antibodies targeting regions within AA 150-200 may interact with different functional domains than those targeting AA 203-222 . Epitope accessibility varies depending on cellular context and experimental conditions. GPR55's predominantly intracellular localization in neutrophils and some other cell types means that membrane permeabilization efficiency directly affects antibody binding . Differences in epitope conservation across species must be carefully evaluated, as sequence variations may alter antibody binding affinity and specificity, potentially leading to inconsistent results when comparing human and mouse samples .

What are the molecular mechanisms of GPR55 and CB2 receptor signaling cross-talk?

GPR55 and CB2 receptors demonstrate complex signal integration at the molecular level. Research reveals that these receptors interfere with each other's signaling pathways primarily at the level of small GTPases, including Rac2 and Cdc42 . This interaction leads to cellular polarization and efficient migration while simultaneously inhibiting degranulation and reactive oxygen species formation in neutrophils . Heteromerization significantly impacts downstream signaling outcomes, with GPR55-mediated activation of transcription factors (including nuclear factor of activated T-cells, NF-κB, and cAMP response elements) being reduced in the presence of CB2 receptors . Conversely, ERK1/2-MAPK activation is potentiated when CB2 receptors are co-expressed with GPR55 . The signaling effects observed are governed both by the presence/absence of the partner receptor and by the activation state of that receptor, creating a dynamic regulatory system . G-protein coupling specificity also plays a role, with GPR55 coupling to different G-protein subunits in different cellular contexts .

What validation strategies confirm GPR55 antibody specificity?

Rigorous validation of GPR55 antibody specificity requires a multi-faceted approach. Western blot analysis using positive and negative control lysates is essential, as demonstrated in studies using HEK293 cells with and without GPR55 expression . The anti-GPR55 antibody should detect bands at the appropriate molecular weight (~37 kDa for GPR55) . Immunofluorescence controls should include comparative staining between GPR55-expressing and non-expressing cells; for example, HEK-GPR55 cells should show positive immunoreactivity while untransfected HEK293 cells should not . For knockout or knockdown validation, comparing antibody reactivity in wild-type versus GPR55-deficient samples provides compelling evidence of specificity. RT-PCR and quantitative PCR correlation with protein detection helps confirm that detected signals correspond to actual GPR55 expression levels, as shown in studies of neutrophils and HL60 cells . Peptide blocking experiments using the immunizing peptide (such as the 203-222 amino acid sequence) can further demonstrate binding specificity .

What protocol modifications optimize HRP-conjugated GPR55 antibody performance?

Optimizing protocols for HRP-conjugated GPR55 antibodies requires several technical considerations. Buffer composition should include 50% glycerol with 0.01M PBS (pH 7.4) to maintain antibody stability and activity . Preservatives like 0.03% Proclin 300 help prevent microbial contamination without interfering with the HRP activity . The antibody dilution must be carefully optimized for each application; while manufacturers provide general guidelines, researchers should determine the optimal working dilution experimentally for their specific system . Signal development timing is critical with HRP-conjugated antibodies, as extended substrate exposure can increase background signal; this is especially important when detecting the predominantly intracellular GPR55 . For multiplexed detection protocols, researchers should carefully select compatible substrates and detection methods that prevent signal overlap when using multiple HRP-conjugated antibodies simultaneously.

What experimental design factors ensure reliable GPR55 detection in immune cells?

When detecting GPR55 in immune cells, particularly neutrophils, several experimental design factors are crucial. Isolation procedures significantly impact expression patterns of GPR55 and related receptors. Research shows that untouched, non-column based isolation systems used at room temperature in the absence of Ca²⁺ and Mg²⁺ ions help prevent cell stimulation that might alter receptor expression . For neutrophil studies, researchers should be aware that GPR55 is predominantly found intracellularly rather than at the cell surface, requiring appropriate permeabilization strategies during immunostaining procedures . Differentiation status affects GPR55 expression levels; for instance, differentiated neutrophil-like HL60 cells (dHL60) express significantly higher GPR55 mRNA levels than undifferentiated cells . Appropriate markers, such as CD11b, should be used to confirm cellular differentiation status when studying GPR55 in cell line models . Co-expression analysis of GPR55 with CB2 receptors provides important context for interpreting functional studies, as these receptors interact to modulate each other's signaling .

How can researchers address variable GPR55 detection in different tissue samples?

Inconsistent GPR55 detection across tissue samples requires systematic troubleshooting approaches. Fixation protocol optimization is critical, as GPR55's predominantly intracellular localization may require different fixation conditions than membrane-bound proteins . Antigen retrieval methods should be evaluated and optimized for each tissue type, particularly for formalin-fixed paraffin-embedded samples where epitope masking can occur. Signal amplification techniques like tyramide signal amplification may enhance detection of low-abundance GPR55 in certain tissues, leveraging the catalytic properties of the HRP conjugate. Different tissues may express varying GPR55 isoforms or post-translational modifications that affect antibody binding; researchers should consult literature specific to their tissue of interest and consider using multiple antibodies targeting different epitopes . Background signal issues can be addressed by implementing additional blocking steps with appropriate sera or protein blockers, and by carefully optimizing incubation times and washing procedures.

What are the key considerations for analyzing GPR55-CB2 receptor interactions in primary cells?

Analyzing GPR55-CB2 receptor interactions in primary cells presents specific challenges requiring careful experimental design. Expression level verification is essential, as primary cells may have different receptor expression patterns than cell lines. In neutrophils, for example, GPR55 mRNA levels are higher than CB2 receptor levels, which may influence interaction dynamics . Functional assays measuring migration, calcium mobilization, or ERK1/2 phosphorylation provide valuable insights into receptor interaction effects. Research shows that in neutrophils, GPR55 activation augments CB2 receptor-mediated migration while inhibiting degranulation and ROS production . Agonist and antagonist selection must be carefully considered; for GPR55, both lysophosphatidylinositol (LPI) and synthetic agonists like AM251 demonstrate chemotactic properties comparable to the CB2 agonist 2-arachidonoylglycerol (2-AG) . Receptor trafficking dynamics may differ between primary cells and cell lines, affecting interpretation of co-localization studies. In neutrophils and HL60 cells, GPR55 shows predominantly intracellular localization, unlike the typical membrane distribution seen in many overexpression systems .

What controls are essential when studying GPR55 signaling in heterologous expression systems?

Rigorous controls are critical when studying GPR55 in heterologous expression systems. Single receptor expression controls using cells expressing only GPR55 or only CB2 receptors are essential for establishing baseline signaling responses for comparison with cells expressing both receptors . Expression level normalization through quantitative assessment of receptor levels helps ensure that observed effects are not simply due to differences in expression magnitude. Western blot analysis using specific antibodies should confirm that receptors are expressed at the appropriate molecular weights (approximately 37 kDa for GPR55 and 45 kDa for CB2R) . Signaling pathway controls using specific inhibitors of downstream elements (such as small GTPase inhibitors) help delineate the mechanisms of receptor cross-talk . Vehicle controls must be included in all stimulation experiments, particularly important when using lipid-based ligands like lysophosphatidylinositol (LPI) that may have solubility considerations . Time-course experiments are valuable for capturing the dynamic nature of GPR55-CB2 receptor interactions, as signaling effects may vary significantly with duration of stimulation .

How does GPR55 signaling contribute to inflammation and immune regulation?

GPR55 plays sophisticated roles in inflammation and immune regulation through multiple mechanisms. In neutrophils, GPR55 activation creates a balanced immune response by augmenting migratory capacity toward inflammatory sites while simultaneously limiting tissue-damaging effector functions like degranulation and reactive oxygen species production . The receptor shows functional interplay with cannabinoid CB2 receptors at the level of small GTPases (Rac2 and Cdc42), leading to cellular polarization that facilitates directional migration . GPR55's ability to modulate immune cell recruitment while limiting cytotoxic effects suggests potential therapeutic applications in inflammatory conditions . Recent research indicates that GPR55 signaling may influence macrophage lipid metabolism, with receptor activation inhibiting ABCA1 translocation to plasma membrane and subsequent cholesterol efflux, potentially contributing to foam cell formation in inflammatory contexts . As a putative cannabinoid receptor, GPR55 represents an important link between the endocannabinoid system and inflammatory processes, with implications for both physiological immune regulation and pathological inflammatory conditions .

What technological advances are improving GPR55 antibody applications in research?

Technological innovations continue to enhance GPR55 antibody applications in research settings. Epitope-specific antibodies targeting defined regions, such as amino acids 203-222 or 150-200, provide researchers with tools to investigate region-specific functions and interactions of GPR55 . Directly conjugated antibodies with HRP eliminate secondary antibody requirements, streamlining workflows and reducing background in multiple detection methods . Enhanced purification methods, particularly antigen affinity purification, improve specificity and reduce non-specific binding, critical for studying receptors like GPR55 that may have structural similarities to other GPCRs . Compatibility with diverse applications, including ELISA, flow cytometry, and Western blotting, allows researchers to employ consistent reagents across multiple experimental approaches . Advances in label-free, real-time detection systems like Epic dynamic mass redistribution and CellKey impedance assays provide novel ways to investigate GPR55 signaling dynamics and receptor interactions with unprecedented temporal resolution .

What is the current understanding of GPR55 involvement in pathological conditions?

Research continues to uncover GPR55's roles in various pathophysiological processes. GPR55 has been implicated in hyperalgesia associated with inflammatory and neuropathic pain, suggesting therapeutic potential for GPR55-targeting compounds in pain management . In bone physiology, GPR55 appears to regulate osteoclast number and function, pointing to possible involvement in bone disorders and potential therapeutic applications in conditions like osteoporosis . Recent studies indicate GPR55 may contribute to cardiovascular pathology through its effects on macrophage lipid metabolism, promoting foam cell formation through inhibition of cholesterol efflux mechanisms . In cancer biology, highly metastatic breast cancer cell lines like MDA-MB231 express GPR55 and demonstrate enhanced migration toward lysophosphatidylinositol, suggesting a potential role in cancer progression and metastasis . The receptor's ability to modulate inflammatory responses through effects on neutrophil function indicates possible involvement in inflammatory disorders, with GPR55-mediated limitation of tissue-damaging neutrophil effector functions suggesting protective roles in certain inflammatory contexts .