GPR37 Antibody, FITC conjugated

What is GPR37 and what are its primary biological functions?

GPR37, also known as the Parkin-associated endothelin receptor-like receptor (PAELR), is an orphan G protein-coupled receptor predominantly expressed in the nervous system. GPR37 functions as a receptor for the neuroprotective and glioprotective factor prosaposin (also known as sulfated glycoprotein-1) and its active fragment prosaptide . Its primary biological function involves mediating neuroprotective and glioprotective effects in the central nervous system. When activated by prosaposin or prosaptide, GPR37 triggers a signaling cascade including receptor endocytosis followed by ERK phosphorylation . This signaling pathway has been shown to protect primary astrocytes against oxidative stress, suggesting a crucial role in cellular defense mechanisms within the brain . Additionally, GPR37 has been implicated in potential interactions with the dopaminergic system through its association with the D2 dopamine receptor, which may have implications for neurological disorders affecting dopamine signaling .

How specific is the GPR37 Antibody, FITC conjugated for human samples?

The GPR37 Antibody, FITC conjugated is highly specific for human samples as it is generated against a peptide sequence (114-133AA) from the Human Prosaposin receptor GPR37 protein . Testing confirms reactivity with human samples, making it reliable for research applications focused on human cells and tissues . While the antibody is raised specifically against the human sequence, it may exhibit cross-reactivity with rat samples as indicated in product specifications . This cross-species reactivity is not unexpected given the evolutionary conservation of GPCRs across mammalian species. For researchers planning experiments with non-human samples, predicted reactivity extends to mouse and rabbit models, though validation experiments are advisable before proceeding with extensive studies in these species . When working with human brain tissue or cell cultures, the antibody provides specific labeling of the receptor, especially in regions with known GPR37 expression such as oligodendrocytes, neurons in specific brain regions, and certain astrocyte populations. Specificity can be validated through appropriate controls, including peptide competition assays or using samples from GPR37 knockout models.

What are the primary applications for GPR37 Antibody, FITC conjugated?

The GPR37 Antibody, FITC conjugated is suitable for a diverse range of experimental applications in neuroscience and cell biology research:

The FITC conjugation makes this antibody particularly valuable for multi-color immunofluorescence studies, flow cytometry analysis of cell surface expression, and monitoring the trafficking dynamics of GPR37 in live cell imaging experiments . Given GPR37's interesting trafficking properties and poor plasma membrane expression in most cell types, this antibody serves as an important tool for investigating the regulatory mechanisms controlling GPR37 surface expression and internalization in response to ligands like prosaposin and prosaptide .

How can surface expression of GPR37 be enhanced for functional studies?

Enhancing the surface expression of GPR37 represents a significant challenge in functional studies due to its inherent trafficking defects. Research has identified three independent approaches that can dramatically improve GPR37 plasma membrane localization:

• N-terminal truncation: Removing the first 210 amino acids of the GPR37 N-terminus significantly enhances receptor trafficking to the plasma membrane, nearly as effectively as removing the entire N-terminus . This finding suggests that specific regulatory elements within the N-terminal domain may act as retention signals that normally constrain surface expression.

• Co-expression with specific GPCRs: Surface expression of GPR37 increases substantially when co-expressed with either the adenosine A2A receptor (A2AR) or dopamine D2 receptor (D2R) . This co-expression approach not only enhances trafficking but may also recapitulate physiologically relevant receptor complexes, as GPR37 can physically associate with D2R as demonstrated through co-immunoprecipitation experiments .

• Co-expression with PDZ scaffold proteins: Specifically, syntenin-1 interaction with the GPR37 C-terminus results in dramatically increased surface expression in heterologous cell systems like HEK-293 cells . This finding highlights the importance of PDZ scaffold interactions in regulating the subcellular distribution of GPR37.

When designing experiments to study GPR37 function, researchers should consider implementing one or more of these approaches to overcome the trafficking limitations, while acknowledging that modifications may potentially alter some aspects of receptor function. The FITC-conjugated antibody provides an excellent tool for quantifying these enhancements in surface expression through techniques like flow cytometry or quantitative immunofluorescence microscopy .

What is the relationship between GPR37 and prosaposin signaling pathways?

The relationship between GPR37 and prosaposin represents a sophisticated signaling system with important neuroprotective implications. When prosaposin or its active fragment prosaptide binds to GPR37, a specific G protein-coupled signaling cascade is initiated . This cascade exhibits the following characteristics:

• G protein coupling specificity: GPR37 signaling through prosaposin is pertussis toxin-sensitive, indicating primary coupling to Gi/o proteins that inhibit adenylyl cyclase activity . This is evidenced by the observed inhibition of forskolin-stimulated cAMP production following receptor activation.

• ERK phosphorylation cascade: Ligand binding to GPR37 triggers receptor endocytosis, which is followed by activation of the ERK (extracellular signal-regulated kinase) phosphorylation pathway . This represents a key downstream effector mechanism that mediates many of the neuroprotective effects of prosaposin.

• GTPγS binding stimulation: Receptor activation promotes increased binding of 35S-GTPγS, confirming functional G protein coupling and activation .

• Neuroprotective outcomes: Both prosaposin and prosaptide protect primary astrocytes against oxidative stress through GPR37-dependent mechanisms, as demonstrated by the attenuation of these protective effects following siRNA-mediated knockdown of endogenous astrocytic GPR37 .

This signaling relationship has significant implications for understanding endogenous neuroprotective mechanisms and developing potential therapeutic approaches for conditions involving oxidative stress and neurodegeneration. Researchers can use the FITC-conjugated GPR37 antibody to track receptor dynamics following ligand stimulation, including surface expression changes, internalization patterns, and potential co-localization with downstream signaling components .

How does the interaction between GPR37 and D2 dopamine receptor impact experimental design?

The interaction between GPR37 and the D2 dopamine receptor (D2R) has significant implications for experimental design, particularly in studies investigating dopaminergic signaling. Co-immunoprecipitation experiments have demonstrated that GPR37 can robustly associate with D2R, with truncated GPR37 showing even stronger association than the full-length receptor . This physical interaction produces functional consequences that researchers must consider:

• Altered ligand binding properties: The GPR37-D2R interaction modestly alters D2R's affinity for both agonists and antagonists . This phenomenon necessitates careful pharmacological characterization in experimental systems where both receptors are expressed.

• Enhanced GPR37 surface trafficking: Co-expression with D2R increases GPR37 plasma membrane expression . When designing experiments to study GPR37 in isolation, this effect must be controlled for, or alternatively, leveraged to achieve sufficient surface expression.

• Potential heteromeric signaling: The physical association suggests the possibility of heteromeric signaling complexes with unique pharmacological and signaling properties distinct from either receptor alone. This may require specialized approaches to distinguish heteromer-specific signaling from individual receptor contributions.

• Physiological relevance: Since both receptors are co-expressed in certain neuronal populations, particularly in the dopaminergic system, experimental models should consider this native context. Brain regions with significant co-expression represent important targets for investigation using the FITC-conjugated GPR37 antibody .

Researchers investigating GPR37 function in dopaminergic systems should design controls that account for potential D2R interactions, possibly including D2R antagonists to distinguish direct GPR37 effects from those mediated through D2R modulation. Additionally, analyzing the co-localization of these receptors using the FITC-conjugated GPR37 antibody alongside D2R-specific antibodies can provide valuable insights into their functional relationship in various experimental systems .

What are the optimal fixation and permeabilization protocols for GPR37 immunostaining?

Optimizing fixation and permeabilization protocols is critical for successful GPR37 immunostaining, particularly given its unique trafficking characteristics and predominant intracellular localization in many cell types . Based on the available research data, the following protocol recommendations apply when using the FITC-conjugated GPR37 antibody:

For cell cultures (ICC applications):

• Fixation: 4% paraformaldehyde in PBS for 15-20 minutes at room temperature provides optimal preservation of GPR37 epitopes while maintaining cellular architecture . Over-fixation should be avoided as it can mask epitopes, particularly for membrane proteins like GPR37.

• Permeabilization: 0.1-0.2% Triton X-100 in PBS for 5-10 minutes is generally sufficient for accessing intracellular GPR37 pools . For more selective plasma membrane versus intracellular staining comparisons, researchers can use 0.1% saponin, which permeabilizes plasma membranes while preserving intracellular membrane structures.

• Blocking: 5-10% normal serum (from a species unrelated to the antibody host) with 1% BSA in PBS for 30-60 minutes reduces non-specific binding .

• Antibody dilution: For ICC applications, a 1:100-500 dilution of the FITC-conjugated antibody is recommended .

For tissue sections (IHC applications):

• Fixation: For frozen sections, brief post-fixation with 2-4% paraformaldehyde for 10 minutes is recommended . For paraffin sections, antigen retrieval becomes essential due to extensive cross-linking during processing.

• Antigen retrieval: For paraffin sections, heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 10-20 minutes can significantly improve staining intensity .

• Permeabilization: 0.2-0.3% Triton X-100 in PBS for 15-20 minutes is typically required for tissue sections .

• Antibody dilution: For IHC-F applications, a 1:100-500 dilution is recommended, while IF applications may require a more concentrated 1:50-200 dilution .

Researchers should note that GPR37's membrane trafficking dynamics may be influenced by experimental conditions, and optimization might be necessary for specific experimental systems .

How can potential artifacts in GPR37 trafficking studies be minimized?

When studying GPR37 trafficking, several potential artifacts can confound results, particularly given GPR37's known trafficking defects and tendency to accumulate intracellularly . To minimize these artifacts and ensure reliable data:

• Optimize expression levels: Excessive overexpression of GPR37 can lead to protein aggregation, endoplasmic reticulum (ER) stress, and aberrant trafficking patterns that don't reflect physiological conditions . Using inducible expression systems or promoters of appropriate strength can help maintain expression at more physiological levels.

• Control temperature conditions: GPR37 trafficking is temperature-sensitive, with reduced surface expression at 37°C compared to lower temperatures in some cell types . Consistent temperature control during experiments is essential, and researchers should consider examining trafficking at both physiological and reduced temperatures.

• Include appropriate positive controls: When examining trafficking interventions, include known enhancers of GPR37 surface expression as positive controls:

  • N-terminally truncated GPR37 constructs

  • Co-expression with D2R or A2AR

  • Co-expression with syntenin-1

• Use complementary detection methods: Combine multiple approaches to assess surface expression:

  • Flow cytometry for quantitative population analysis

  • Surface biotinylation followed by Western blotting

  • Live-cell imaging with non-permeabilizing conditions

  • Subcellular fractionation to separate membrane from intracellular pools

• Validate with endogenous expression systems: Where possible, confirm findings from heterologous expression systems with studies of endogenous GPR37 in relevant cell types such as neurons or glial cells .

• Consider ligand-induced effects: The presence of prosaposin or prosaptide can induce receptor endocytosis, potentially altering steady-state distribution . Control for potential ligands in culture media or tissue preparation.

By implementing these strategies, researchers can minimize artifacts and obtain more physiologically relevant insights into GPR37 trafficking dynamics using the FITC-conjugated antibody for detection .

What controls should be included when studying GPR37-D2R interactions?

When investigating interactions between GPR37 and the D2 dopamine receptor (D2R), comprehensive controls are essential to establish specificity and functional relevance. The following controls should be incorporated into experimental designs:

For co-immunoprecipitation studies:

• Negative control receptors: Include co-immunoprecipitation experiments with unrelated GPCRs that should not interact with either GPR37 or D2R . Candidates include serotonin receptors (e.g., 5-HT1A) or adrenergic receptors that are expressed in similar brain regions.

• Truncation controls: Compare interaction strength between full-length GPR37 and N-terminally truncated GPR37 with D2R, as truncation has been shown to enhance the interaction .

• Antibody controls: Perform reverse co-immunoprecipitation (pulling down with anti-D2R and blotting for GPR37) to confirm the interaction bidirectionally .

• Peptide competition: For the FITC-conjugated GPR37 antibody, include controls where the immunoprecipitation is performed in the presence of excess immunizing peptide to confirm specificity .

For functional interaction studies:

• D2R pharmacological controls: Include selective D2R agonists (quinpirole) and antagonists (sulpiride) to pharmacologically isolate D2R contributions to observed effects .

• Signal transduction pathway inhibitors: Use pertussis toxin to block Gi/o-mediated signaling, which affects both receptors, and specific MEK inhibitors to block the ERK phosphorylation pathway downstream of GPR37 activation .

• siRNA knockdown controls: Perform selective knockdown of either GPR37 or D2R to determine the contribution of each receptor to observed phenotypes .

For co-localization imaging studies:

• Single-transfection controls: Image cells expressing only GPR37 or only D2R to establish baseline localization patterns .

• Co-localization quantification: Apply rigorous quantitative co-localization analysis using metrics such as Pearson's or Mander's coefficients, rather than relying on visual assessment alone .

• Subcellular markers: Include markers for relevant subcellular compartments (plasma membrane, endoplasmic reticulum, Golgi, endosomes) to precisely define where interaction occurs .

By systematically implementing these controls, researchers can establish with confidence whether observed GPR37-D2R interactions are specific, functionally relevant, and correctly localized within cellular compartments .

How should variations in GPR37 surface expression between different cell types be interpreted?

Variations in GPR37 surface expression between different cell types represent a complex but informative aspect of this receptor's biology that requires careful interpretation:

• Cell-type specific trafficking machinery: Different cell types express varying levels of trafficking proteins that influence GPR37 surface expression. Particularly relevant are PDZ-domain scaffolding proteins like syntenin-1, which dramatically enhance GPR37 surface expression . When comparing expression across cell types, researchers should consider cataloging the expression of known GPR37 trafficking partners.

• Co-receptor expression profiles: The presence of interacting receptors, particularly A2AR and D2R, significantly impacts GPR37 surface levels . Variations between cell types may reflect differences in the expression of these co-receptors rather than intrinsic differences in GPR37 processing. A comprehensive analysis should include characterization of relevant co-receptor expression.

• N-terminal processing differences: Since N-terminal truncation significantly enhances GPR37 surface expression, cell-type variations may reflect differences in proteolytic processing of the receptor . Researchers should consider examining the molecular weight profiles of GPR37 across cell types to identify potential processing differences.

• Physiological significance: Rather than viewing poor surface expression as merely a technical challenge, it likely represents a physiologically relevant regulatory mechanism. Neurons and glial cells that naturally express GPR37 may possess specialized machinery for regulating its surface expression in response to specific stimuli or developmental stages .

• Methodological considerations: When quantifying surface expression differences, researchers should employ multiple complementary techniques:

The FITC-conjugated GPR37 antibody is particularly well-suited for flow cytometry and immunofluorescence approaches, allowing quantitative assessment of surface expression across different cell types .

How do truncation states of GPR37 affect interpretation of antibody-based detection methods?

The truncation state of GPR37 significantly impacts antibody-based detection methods, requiring careful consideration during experimental design and data interpretation:

• Epitope accessibility variations: The FITC-conjugated GPR37 antibody targets a peptide sequence from Human Prosaposin receptor GPR37 protein (114-133AA) . In N-terminally truncated forms of GPR37, particularly those missing the first 210 amino acids that enhance surface expression, this epitope may be eliminated or its conformation altered . Researchers must verify whether their truncated constructs retain the target epitope.

• Subcellular distribution differences: Full-length GPR37 predominantly localizes intracellularly with punctate staining patterns, while N-terminally truncated GPR37 shows significantly enhanced plasma membrane expression . This fundamental difference in distribution patterns affects quantitative comparisons:

• Western blot interpretation challenges: Truncated forms will show different molecular weights on Western blots, requiring careful consideration when interpreting bands. Additionally, the glycosylation state may differ between full-length and truncated forms, further complicating molecular weight interpretations .

• Functional correlations: When interpreting antibody-based trafficking studies, researchers should correlate observations with functional readouts like signaling responses to prosaposin/prosaptide . Truncated forms may show enhanced surface expression but potentially altered signaling properties.

• Validation strategies: For studies employing both full-length and truncated GPR37, multiple validation approaches are advisable:

  • Use antibodies targeting different epitopes (C-terminal versus N-terminal)

  • Employ epitope tags (FLAG, HA) placed at different positions in the receptor

  • Perform parallel studies with fluorescent protein fusions

  • Correlate antibody detection with functional assays

The FITC-conjugated GPR37 antibody remains a valuable tool for these studies, but researchers must carefully document the exact constructs used and their relationship to the antibody's target epitope to enable proper interpretation of results .