GPR37 Antibody, HRP conjugated

What is GPR37 and why is it significant in neurological research?

GPR37 (G-protein coupled receptor 37), also called ETBR-LP-1 (endothelin B receptor-like protein 1) or PAELR (Parkin-associated endothelin receptor-like receptor), is a 613 amino acid, ~55 kDa orphan 7-transmembrane receptor predominantly expressed in neuronal cells, particularly in cerebellar Purkinje cells and the hippocampus . Its significance in neurological research stems from its role as a substrate of the E3 ubiquitin ligase parkin, which becomes upregulated during endoplasmic reticulum stress. In juvenile forms of Parkinson's disease, GPR37 accumulation contributes to stress-induced neuronal cell death, making it a critical protein for understanding neurodegenerative mechanisms . Additionally, GPR37 functions as a receptor for the neuroprotective and glioprotective factor prosaposin, with ligand binding inducing endocytosis followed by an ERK phosphorylation cascade .

What are the recommended dilutions for GPR37 antibody applications in different experimental protocols?

For optimal results with GPR37 antibodies, especially HRP-conjugated versions, the following dilution ranges are recommended for various applications:

• Western Blot: 1:100-1:1000 dilution

• Immunohistochemistry (paraffin sections): 1:100-1:500 dilution

These recommendations provide starting points, but optimal dilutions should be determined by each laboratory for specific applications, as antibody performance may vary based on sample type, detection method, and experimental conditions . When establishing optimal working dilutions, a titration experiment is advisable, testing the antibody across a range of concentrations to determine the dilution that provides the best signal-to-noise ratio.

How should GPR37 antibodies be stored to maintain optimal activity?

GPR37 antibodies require proper storage to maintain their functionality and specificity. While specific storage conditions for HRP-conjugated GPR37 antibodies aren't explicitly stated in the search results, general principles for antibody storage should be followed. Typically, antibody conjugates should be stored at 2-8°C for short-term storage (1-2 weeks) and at -20°C for long-term storage, preferably in small aliquots to avoid repeated freeze-thaw cycles which can degrade both the antibody and the HRP enzyme. The antibody should be protected from light, especially important for fluorescent conjugates, and stored in glycerol or another cryoprotectant to prevent damage during freezing. Always follow manufacturer-specific instructions as storage recommendations may vary.

What controls should be included when using GPR37 antibodies in immunodetection experiments?

When designing experiments with GPR37 antibodies, particularly for immunodetection techniques, several controls should be incorporated to ensure reliable and interpretable results:

• Positive Controls: Include samples known to express GPR37, such as cerebellar Purkinje cells or hippocampal tissue samples .

• Negative Controls:

  • Tissue/cells known not to express GPR37

  • Primary antibody omission (to detect non-specific binding of secondary reagents)

  • Isotype controls (using non-specific IgG from the same species)

• Specificity Controls:

  • Blocking peptide competition assays

  • Use of GPR37 knockout tissues/cells when available

  • Western blot verification showing a band of expected size (~55 kDa)

• Technical Controls:

  • For HRP-conjugated antibodies, include peroxidase inhibition controls

  • Include loading controls for protein normalization in Western blots

These controls help distinguish true GPR37 signals from background or non-specific reactions, particularly important given the variable expression of GPR37 in different tissues and experimental conditions.

How can poor membrane expression of GPR37 be overcome in heterologous cell systems?

GPR37 characteristically exhibits poor plasma membrane expression when expressed in most cell types, which can complicate functional studies . Research has identified three independent approaches to significantly enhance GPR37 surface trafficking in heterologous cells:

• N-terminal Truncation: Removing the first 210 amino acids of GPR37 dramatically enhances its plasma membrane insertion. Complete removal of the N-terminus shows the greatest effect, but truncation of just the first 210 amino acids is nearly as effective .

• Co-expression with Other Receptors: GPR37 surface expression increases significantly when co-expressed with certain GPCRs, particularly:

  • Adenosine receptor A2AR (modest increase)

  • Dopamine receptor D2R (substantial increase)

• Co-expression with PDZ Scaffolds:

  • Syntenin-1 specifically interacts with GPR37 through its C-terminal PDZ-binding motif (G-T-H-C)

  • This interaction results in a dramatic 10-fold increase in GPR37 plasma membrane expression

These approaches can be used individually or in combination. Notably, combining N-terminal truncation with syntenin-1 co-expression works synergistically to maximize GPR37 trafficking to the plasma membrane .

What factors might affect the reactivity of GPR37 antibodies across different species?

When using GPR37 antibodies across different species, several factors must be considered that could affect reactivity and specificity:

• Sequence Homology: The extracellular portions of human and mouse GPR37 share 68% amino acid identity , which suggests potential for cross-reactivity but also the possibility of reduced affinity or specificity across species.

• Epitope Conservation: The specific epitope recognized by the antibody is critical. Some regions of GPR37 may be more conserved than others across species. Antibodies targeted against highly conserved regions will show better cross-reactivity.

• Post-translational Modifications: Species differences in glycosylation patterns or other post-translational modifications of GPR37 may affect antibody recognition.

• Verified Reactivity: Look for antibodies specifically validated for cross-reactivity. For example, some commercial antibodies have been verified to react with human, mouse, and rat GPR37 .

• Isoform Differences: Consider potential species-specific isoforms or splice variants that might affect antibody recognition.

When working with a new species, preliminary validation experiments are strongly recommended, even when using antibodies labeled as cross-reactive.

How does GPR37 interact with dopamine receptor D2R, and what are the implications for experimental design?

GPR37 forms robust complexes with dopamine receptor D2R, a finding with significant implications for both experimental design and our understanding of receptor functionality:

• Physical Interaction:

  • Co-immunoprecipitation studies demonstrate that both wild-type GPR37 and its Δ1–210 mutant robustly associate with D2R

  • This interaction occurs with both immature (unprocessed) and mature (glycosylated) forms of D2R, suggesting the interaction begins in the endoplasmic reticulum and is maintained after glycosylation

  • The Δ1–210 mutant exhibits consistently stronger interaction with D2R than wild-type GPR37, likely due to enhanced plasma membrane expression

• Functional Consequences:

  • Co-expression with Δ1–210 GPR37 alters D2R ligand-binding properties

  • Radioligand binding studies show modest but significant changes in affinity for various ligands (see Table 1 below)

Table 1: Effects of GPR37 Co-expression on D2R Ligand Binding Properties

• Experimental Design Implications:

  • When studying D2R pharmacology, consider the potential influence of endogenous GPR37 expression

  • These interactions may be relevant to the development of D2R-targeted therapeutics, particularly those treating conditions where both receptors are co-expressed

  • The magnitude of observed changes in heterologous systems likely underestimates true effects due to transfection efficiency limitations and incomplete receptor co-assembly

This interaction has particular clinical relevance given the widespread use of D2R antagonists in treating schizophrenia and the possibility of developing regionally selective compounds that preferentially target D2R/GPR37 complexes .

What are the challenges in detecting endogenous versus overexpressed GPR37 using antibody-based methods?

Detecting endogenous versus overexpressed GPR37 presents several unique challenges that researchers should consider when designing antibody-based experiments:

• Expression Level Disparities:

  • Endogenous GPR37 is often expressed at relatively low levels in most cell types

  • Overexpression systems can produce significantly higher protein levels, potentially leading to misleading localization patterns

  • Detection methods may require different optimization parameters for each scenario

• Trafficking Issues:

  • Endogenous GPR37 in neuronal cells typically exhibits better trafficking than in heterologous expression systems

  • Overexpressed GPR37 often demonstrates poor plasma membrane expression, with significant retention in the endoplasmic reticulum

  • These differences can complicate interpretation of localization studies and functional assays

• Antibody Sensitivity Considerations:

  • Higher antibody concentrations may be required to detect low-level endogenous expression

  • Signal amplification methods (e.g., tyramide signal amplification) may be necessary for endogenous detection

  • HRP-conjugated antibodies offer advantages for low-abundance targets due to enzymatic signal amplification

• Background Signal Issues:

  • Higher antibody concentrations needed for endogenous detection may increase non-specific background

  • More stringent blocking and washing conditions may be required for endogenous detection

  • Validation through multiple detection methods becomes more critical

• Sample Preparation Differences:

  • Membrane enrichment protocols may be necessary for endogenous GPR37 detection

  • Different fixation protocols might be optimal for endogenous versus overexpressed protein detection

When transitions between studying overexpressed and endogenous GPR37 are necessary, extensive reoptimization of antibody-based protocols is typically required.

How does prosaposin binding to GPR37 impact experimental design when using GPR37 antibodies?

Prosaposin has been identified as a ligand for GPR37, and its binding induces endocytosis followed by an ERK phosphorylation cascade . This interaction has several important implications for experimental design when using GPR37 antibodies:

• Receptor Internalization Effects:

  • Prosaposin binding triggers GPR37 endocytosis, potentially altering the subcellular localization pattern detected by antibodies

  • Time course experiments might show dynamic redistribution of GPR37 from membrane to intracellular compartments following ligand exposure

  • Researchers should consider the timing of fixation relative to ligand exposure when interpreting localization data

• Epitope Accessibility Concerns:

  • Ligand binding may induce conformational changes in GPR37 that could affect antibody epitope accessibility

  • Antibodies targeting different regions of GPR37 might show differential sensitivity to the receptor's activation state

  • For experiments studying receptor-ligand interactions, comparing multiple antibodies targeting different epitopes may be informative

• Signaling Pathway Activation:

  • The ERK phosphorylation cascade triggered by prosaposin binding may alter the GPR37 post-translational modification state

  • Phosphorylation or other modifications could potentially affect antibody recognition

  • Researchers may need to consider the activation state of GPR37 when interpreting antibody-based quantification results

• Experimental Timing Considerations:

  • For studies investigating GPR37 activation, careful attention to timing between ligand application and analysis is critical

  • ERK phosphorylation cascades typically peak within minutes, while receptor internalization may occur over a longer timeframe

  • Time-course experiments with antibody detection at various intervals post-stimulation may reveal dynamic changes in receptor behavior

When designing experiments to study GPR37-prosaposin interactions, researchers should consider these factors to accurately interpret antibody-based detection results and distinguish between changes in expression levels versus redistribution effects.

What are common causes of weak or non-specific signals when using GPR37 antibodies?

When working with GPR37 antibodies, particularly HRP-conjugated versions, several common issues may lead to weak or non-specific signals:

• Weak Signal Causes:

  • Insufficient GPR37 expression: As GPR37 exhibits poor plasma membrane expression in heterologous systems , detection may be challenging

  • Inadequate antibody concentration: The recommended dilution ranges (1:100-1:1000 for Western blot) may need adjustment

  • Epitope masking: Protein interactions or conformational changes may obscure the antibody recognition site

  • Excessive washing: Particularly relevant for HRP-conjugated antibodies where enzyme activity might be reduced

• Non-specific Signal Causes:

  • Insufficient blocking: Given GPR37's hydrophobic transmembrane domains, more stringent blocking may be required

  • Cross-reactivity: Antibodies may recognize structurally similar GPCRs

  • HRP-substrate incubation issues: Extended incubation with peroxidase substrates can increase background

  • Endogenous peroxidase activity: Particularly in tissue samples where incomplete quenching occurs

• Troubleshooting Approaches:

  • Enhance GPR37 expression using N-terminal truncation, co-expression with D2R, or syntenin-1

  • Optimize blocking conditions (consider protein-free blockers for HRP-conjugated antibodies)

  • Include detergents appropriate for membrane proteins in washing buffers

  • Adjust antibody concentration through careful titration experiments

  • Verify antibody specificity through knockout controls or peptide competition

• HRP-Specific Considerations:

  • Check HRP activity using a direct enzyme assay

  • Store HRP-conjugated antibodies with stabilizers to maintain enzyme activity

  • Avoid sodium azide in buffers used with HRP-conjugated antibodies

  • Consider signal enhancement methods like tyramide signal amplification for weak signals

Systematic troubleshooting addressing these potential issues can significantly improve both sensitivity and specificity when working with GPR37 antibodies.

How can researchers verify the specificity of GPR37 antibody binding in their experimental system?

Verifying GPR37 antibody specificity is crucial for experimental validity, particularly given the challenges in detecting this receptor. Researchers should consider implementing several complementary approaches:

• Molecular Weight Verification:

  • GPR37 is approximately 55 kDa in its core protein form

  • Western blotting should show bands at the expected molecular weight

  • Multiple bands may indicate post-translational modifications, processing, or degradation

• Knockout/Knockdown Controls:

  • Use GPR37 knockout tissues/cells as negative controls

  • siRNA or shRNA knockdown can provide partial reduction as validation

  • CRISPR/Cas9-mediated knockout provides definitive negative controls

• Peptide Competition Assays:

  • Pre-incubate the antibody with excess immunizing peptide

  • Specific signals should be competitively reduced/eliminated

  • Non-specific binding will remain unaffected

• Overexpression Validation:

  • Compare antibody signals in non-transfected versus GPR37-transfected cells

  • Both signal intensity and subcellular localization should differ

  • Consider using tagged versions of GPR37 to confirm co-localization with antibody signals

• Multiple Antibody Comparison:

  • Use antibodies targeting different epitopes of GPR37

  • Consistent pattern detected by multiple antibodies increases confidence

  • Disparate results may indicate isoform detection or specificity issues

• Cross-Species Reactivity Assessment:

  • Human and mouse GPR37 share 68% amino acid identity in extracellular portions

  • Expected cross-reactivity patterns should align with sequence homology

  • Unexpected reactivity patterns may indicate non-specific binding

• Mass Spectrometry Confirmation:

  • For definitive validation, immunoprecipitate with the GPR37 antibody

  • Analyze the precipitated proteins by mass spectrometry

  • Confirm the presence of GPR37-specific peptides

Implementing several of these validation approaches provides stronger evidence for antibody specificity than any single method alone.

What strategies can researchers employ when contradictory results emerge from different GPR37 antibodies?

When faced with contradictory results from different GPR37 antibodies, researchers should implement a systematic investigative approach:

• Epitope Mapping Analysis:

  • Identify the specific epitopes recognized by each antibody

  • Epitopes in different domains (N-terminus, transmembrane, C-terminus) may be differentially accessible

  • Consider that truncation studies have shown that N-terminal removal enhances GPR37 surface expression , which might affect epitope accessibility

• Conformational State Considerations:

  • GPR37 may adopt different conformations based on activation state

  • Some antibodies may preferentially recognize active, inactive, or intermediate states

  • Ligand binding (e.g., prosaposin) may alter epitope accessibility

• Post-translational Modification Impact:

  • Assess whether antibodies recognize regions subject to glycosylation, phosphorylation, or ubiquitination

  • GPR37's role as a substrate for the E3 ubiquitin ligase parkin suggests potential ubiquitination-dependent epitope masking

  • Different cell types or experimental conditions may alter modification patterns

• Isoform or Splice Variant Detection:

  • Investigate whether contradictory results stem from detection of different GPR37 isoforms

  • Sequence the regions of interest from your experimental system

  • Consider RT-PCR to identify potential splice variants

• Cross-Reactivity Resolution:

  • Perform systematic specificity tests using knockout controls

  • Conduct peptide competition assays with peptides specific to each antibody

  • Implement immunoprecipitation followed by mass spectrometry to identify what each antibody is actually detecting

• Methodological Integration:

  • Combine antibody-based approaches with non-antibody methods (e.g., RNA detection, activity assays)

  • Use tagged GPR37 constructs alongside antibody detection

  • Implement proximity ligation assays to verify protein-protein interactions identified with antibodies

• Biological Context Interpretation:

  • Consider that GPR37's properties can be dramatically altered by interactions with other proteins, such as D2R or syntenin-1

  • Different experimental systems may yield different results based on expression of these interacting partners

  • Tissue-specific factors may influence GPR37 conformation and antibody recognition

When publishing, transparently report contradictory findings rather than selectively presenting data from a single antibody, as these discrepancies often reveal important biological insights about receptor dynamics or modifications.

How can GPR37 antibodies be utilized to investigate GPR37's role in Parkinson's disease pathology?

GPR37 antibodies offer powerful tools for investigating this receptor's role in Parkinson's disease (PD) pathology through multiple experimental approaches:

• Protein Accumulation Studies:

  • GPR37 accumulates in a juvenile form of Parkinson's disease, contributing to stress-induced neuronal cell death

  • Antibodies can quantify GPR37 levels in patient samples versus controls

  • Immunohistochemistry can reveal spatial patterns of accumulation in affected brain regions

• Parkin Interaction Analysis:

  • GPR37 is a substrate of the E3 ubiquitin ligase parkin

  • Co-immunoprecipitation with anti-GPR37 antibodies can assess parkin binding in normal versus pathological conditions

  • Changes in ubiquitination status can be monitored using ubiquitin-specific antibodies in conjunction with GPR37 antibodies

• Stress Response Monitoring:

  • GPR37 upregulation occurs during endoplasmic reticulum stress

  • Antibodies can track changes in GPR37 levels and localization during stress conditions

  • Temporal analysis can reveal whether GPR37 accumulation precedes or follows other pathological markers

• Cell Death Correlation Studies:

  • Dual labeling with GPR37 antibodies and cell death markers

  • Quantify relationships between GPR37 accumulation and neurodegeneration

  • Assess whether neurons with higher GPR37 expression show increased vulnerability

• Therapeutic Intervention Assessment:

  • Evaluate whether potential PD therapeutics affect GPR37 levels or localization

  • Determine if reducing GPR37 accumulation correlates with neuroprotection

  • Monitor changes in GPR37-interacting proteins (like D2R) following treatment

• Animal Model Validation:

  • Compare GPR37 expression patterns between human PD samples and animal models

  • Assess whether interventions targeting GPR37 in animal models affect disease progression

  • Correlate GPR37 levels with behavioral and neuropathological outcomes

By employing GPR37 antibodies in these approaches, researchers can advance understanding of this receptor's involvement in PD pathogenesis and potentially identify new therapeutic targets.

What methodological approaches can be used to study the GPR37-D2R interaction in native tissues?

Investigating the GPR37-D2R interaction in native tissues presents unique challenges compared to heterologous systems but can be approached using several sophisticated methodologies:

• Proximity Ligation Assay (PLA):

  • Uses antibodies against both GPR37 and D2R

  • Generates fluorescent signals only when proteins are within 40nm

  • Provides spatial information about interaction sites in tissue sections

  • Recommended primary antibodies: rabbit anti-GPR37 and mouse anti-D2R for optimal species compatibility

• Co-immunoprecipitation from Native Tissues:

  • Membrane extracts from relevant brain regions (striatum, substantia nigra)

  • Immunoprecipitate with anti-GPR37 antibodies and blot for D2R (or vice versa)

  • Compare with interactions found in heterologous systems

  • Use crosslinking agents to stabilize transient interactions

• FRET/BRET in Primary Neurons:

  • Transfect primary neurons with fluorescent protein-tagged GPR37 and D2R

  • Measure energy transfer as indication of protein proximity

  • Compare interaction efficiency in different neuronal compartments

  • Assess effects of ligand binding on FRET/BRET efficiency

• In Situ Hybridization with Immunohistochemistry:

  • Identify neurons co-expressing GPR37 and D2R mRNA

  • Follow with antibody staining to confirm protein co-expression

  • Quantify co-expression patterns across different brain regions

  • Correlate with functional measures or disease states

• Super-Resolution Microscopy:

  • Apply techniques like STORM or STED using GPR37 and D2R antibodies

  • Achieves resolution below diffraction limit (~20nm)

  • Maps precise co-localization patterns at the membrane level

  • Requires highly specific primary antibodies and appropriate fluorophore-conjugated secondaries

• Functional Impact Assessment:

  • Apply D2R ligands to native tissue preparations while monitoring GPR37 trafficking

  • Measure signaling outcomes (ERK phosphorylation) in tissues with varying GPR37/D2R expression levels

  • Compare pharmacological profiles in tissues from wild-type versus GPR37 knockout animals

  • The ~2-fold change in D2R affinity for [³H]-YM-09151-2 observed in GPR37 knockout mice provides a functional readout

These approaches can be combined to build a comprehensive understanding of GPR37-D2R interactions in physiologically relevant contexts, beyond what can be observed in heterologous expression systems.

How might GPR37 antibodies be used to develop new therapeutic approaches for neurological disorders?

GPR37 antibodies represent valuable tools for therapeutic development in neurological disorders, particularly Parkinson's disease, through several innovative approaches:

• Target Validation and Biomarker Development:

  • Map GPR37 expression patterns across patient cohorts using immunohistochemistry

  • Correlate GPR37 levels or localization with disease progression

  • Identify patient subgroups that might benefit from GPR37-targeted interventions

  • Develop immunoassays to measure soluble GPR37 fragments as potential biomarkers

• Drug Screening Applications:

  • Develop cell-based assays using GPR37 antibodies to detect:

    • Changes in surface expression (particularly important given GPR37's poor membrane trafficking )

    • Receptor internalization following ligand binding

    • Altered interactions with partners like D2R or parkin

  • Screen compound libraries for molecules that normalize GPR37 trafficking or prevent its accumulation

• Therapeutic Antibody Development:

  • Engineer antibodies targeting specific GPR37 epitopes to:

    • Block pathological interactions

    • Enhance degradation of accumulated GPR37

    • Modulate receptor signaling as functional agonists/antagonists

  • Focus on epitopes in the extracellular domain that shares 68% identity between human and mouse to facilitate preclinical testing

• Gene Therapy Monitoring:

  • Use antibodies to assess the efficacy of gene therapies targeting GPR37

  • Monitor expression levels following viral vector delivery

  • Evaluate subcellular distribution of modified GPR37 proteins

  • Assess downstream effects on interacting proteins and pathways

• Precision Medicine Applications:

  • Develop diagnostic antibody panels to characterize patient-specific GPR37 abnormalities

  • Correlate findings with effectiveness of D2R-targeting antipsychotics

  • Guide personalized treatment approaches based on GPR37/D2R interaction profiles

  • Monitor treatment response through sequential tissue or fluid sampling

• Novel Target Exploration Based on Protein Interactions:

  • Investigate the therapeutic potential of modulating GPR37's interaction with syntenin-1

  • Develop compounds that mimic the enhanced surface trafficking observed with syntenin-1 co-expression

  • Explore GPR37/D2R heteromers as targets for region-specific therapeutics

  • The 10-fold increase in GPR37 surface expression observed with syntenin-1 suggests high therapeutic potential

By leveraging GPR37 antibodies in these approaches, researchers can translate fundamental discoveries about this receptor into novel therapeutic strategies for neurological disorders where GPR37 dysfunction plays a role.