GALR3 Antibody

What is GALR3 and what are its primary biological functions?

GALR3 is a G protein-coupled receptor that acts as a receptor for the neuropeptide galanin and spexin-1. The receptor functionally couples to intracellular effectors through distinct signaling pathways . GALR3 is involved in modulating several physiological processes including cognition/memory, sensory/pain processing, hormone secretion, and feeding behavior . Its expression pattern and signaling capabilities suggest involvement in fine-tuning nervous system responses . The protein is expressed as 1.4-, 2.4-, and 5-kb transcripts in various tissues, with significant expression reported in the brain, testis, adrenal gland, and pancreas .

How does GALR3 expression vary across different tissue types?

GALR3 has been detected in multiple tissue types with varying expression levels. The receptor is predominantly expressed in the brain, testis, adrenal gland, and pancreas . In colorectal tissues, GALR3 immunoreactivity has been observed in the cell membrane and cytoplasm of epithelial cells (including enterocytes and goblet cells) of the large intestine. Additionally, GALR3 expression has been detected in intestinal immune/stromal cells, myenteric plexuses, and smooth muscle cells . Research has also documented GALR3 expression in human skin, as evidenced by Western blot analysis showing a band at approximately 37 kDa (slightly lower than the predicted 40 kDa) .

What structural features distinguish GALR3 from other galanin receptors?

GALR3 exhibits specific structural differences compared to other galanin receptors, particularly GALR2, which affect ligand selectivity and function. Key differences include:

• Amino acid variations in the transmembrane helix 3 (TM3) domain, where Phe 103, Phe 106, and His 110 in GALR2 correspond to Leu 100, Tyr 103, and Tyr 107 in GALR3

• Differences in the TM5 domain affecting ligand interactions, particularly with certain peptides like Qu-SPX

• Variations in N-terminal length between GALR3 and GALR2, which influence receptor response to ligands

These structural differences explain the selective binding properties of GALR3 and its distinct functional profile compared to other galanin receptors.

What criteria should researchers use when selecting a GALR3 antibody for their experiments?

When selecting a GALR3 antibody, researchers should consider:

• Target epitope location: Antibodies targeting different regions of GALR3 may yield different results. For example, some commercial antibodies target synthetic peptides within the C-terminus (aa 300 to C-terminus) , while others target different epitopes.

• Validated applications: Confirm the antibody has been validated for your specific application (Western blot, IHC, ELISA). For instance, certain GALR3 antibodies are specifically tested and validated for immunohistochemistry on paraffin-embedded sections at concentrations of 4-6 μg/ml .

• Species reactivity: Most commercial GALR3 antibodies are validated for human samples , so cross-reactivity with other species should be verified before use.

• Clonality: Consider whether a polyclonal (offering broader epitope recognition) or monoclonal (higher specificity) antibody is more appropriate. Many commercial GALR3 antibodies are goat polyclonal antibodies .

• Positive controls: Select antibodies with documented positive controls. For example, human skin lysate has been used as a positive control for GALR3 Western blot applications .

How can researchers validate the specificity of GALR3 antibodies?

To validate GALR3 antibody specificity, researchers should implement the following approach:

• Knockout controls: Similar to strategies used for validating GalR1 and GalR2 antibodies, use tissue or cells from GALR3 knockout models when available to confirm absence of signal .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to block specific binding sites. A reduction in signal indicates specific binding.

• Multiple antibody validation: Use at least two antibodies targeting different epitopes of GALR3 to confirm consistent localization patterns.

• Correlation with mRNA expression: Compare antibody detection patterns with mRNA expression data, though it's important to note that discrepancies between GALR gene expression and protein immunoexpression have been observed in colorectal cancer tissues .

• Western blot analysis: Verify that the observed band matches the predicted molecular weight of GALR3 (approximately 40 kDa), although some antibodies detect GALR3 at slightly lower weights (e.g., 37 kDa) .

• Negative controls: Include appropriate negative controls by omitting the primary antibody or using isotype controls to assess non-specific binding.

What are the recommended protocols for using GALR3 antibodies in Western blotting?

For optimal Western blot results with GALR3 antibodies, researchers should follow these guidelines:

• Sample preparation: Prepare human tissue lysates in appropriate buffers such as RIPA buffer. For example, human skin lysate at 35 μg/mL has been successfully used .

• Antibody concentration: Use GALR3 antibodies at concentrations of 1-3 μg/mL for Western blot applications . Some commercial antibodies have shown optimal results at 2 μg/mL .

• Detection method: The ECL (enhanced chemiluminescence) technique has been successfully used for visualizing GALR3 bands .

• Expected molecular weight: The predicted band size for GALR3 is approximately 40 kDa, though observed bands around 37 kDa have been reported . This discrepancy could be due to post-translational modifications or alternative splicing.

• Loading control: Include appropriate loading controls such as β-actin (ACTB) to normalize protein expression levels .

• Membrane blocking: Use 5% non-fat milk or BSA in TBST for blocking non-specific binding sites.

• Incubation conditions: Incubate with primary antibody overnight at 4°C for optimal results.

What protocol is recommended for GALR3 immunohistochemistry on formalin-fixed paraffin-embedded tissues?

For immunohistochemistry of GALR3 in formalin-fixed paraffin-embedded (FFPE) tissues:

• Tissue preparation: Fix tissues in formalin and embed in paraffin using standard protocols. Cut sections at 4-6 μm thickness.

• Antigen retrieval: Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0).

• Blocking: Block endogenous peroxidase activity with 3% hydrogen peroxide and block non-specific binding with appropriate serum.

• Antibody concentration: Apply GALR3 antibody at 4-6 μg/mL concentration for optimal staining in FFPE sections .

• Incubation time: Incubate with primary antibody overnight at 4°C.

• Detection system: Use appropriate secondary antibody and visualization system compatible with the host species of the primary antibody (typically anti-goat detection systems for goat polyclonal GALR3 antibodies).

• Counterstaining: Counterstain with hematoxylin to visualize tissue architecture.

• Controls: Include both positive controls (tissues known to express GALR3, such as brain or intestinal tissue) and negative controls (primary antibody omitted).

How should GALR3 antibodies be optimized for ELISA applications?

For ELISA applications using GALR3 antibodies:

• Antibody dilution: Commercial GALR3 antibodies have been recommended at dilutions as high as 1:32,000 for ELISA applications , though optimal dilution should be determined empirically for each specific assay format.

• Antigen coating: For direct ELISA, coat plates with the target antigen (synthetic GALR3 peptide) at concentrations ranging from 1-10 μg/mL.

• Blocking buffer: Use 2-5% BSA or non-fat milk in PBS-T to minimize background.

• Sample preparation: Prepare cell or tissue lysates using non-denaturing buffers to maintain the native conformation of GALR3.

• Detection system: Use appropriate enzyme-conjugated secondary antibodies (e.g., HRP-conjugated anti-goat IgG for goat polyclonal primary antibodies).

• Standard curve: Generate a standard curve using known quantities of recombinant GALR3 protein or synthetic peptide.

• Validation: Confirm specificity by comparing signals from samples with known differential expression of GALR3.

How does GALR3 expression correlate with colorectal cancer progression and prognosis?

Research on GALR3 expression in colorectal cancer (CRC) has revealed significant associations with disease progression and patient outcomes:

• Differential expression: GalR1 and GalR3 immunoreactivities are stronger in cancer cells compared to epithelial cells of the unchanged mucosa of the large intestine .

• Prognostic significance: Low immunoexpression of GalR3 protein has been correlated with poor prognosis in CRC patients, suggesting its potential utility as a prognostic biomarker .

• Expression discrepancies: Interestingly, GalR gene expression in colorectal cancer databases and normal adjacent tissue does not always correlate with the immunoexpression of the GalR proteins . This discrepancy highlights the complexity of GALR3 regulation and the importance of protein-level analysis.

• Cellular localization: GALR3 immunolocalization in CRC is confined to the cell membrane and cytoplasm of colorectal cancer cells, similar to its localization in normal epithelial cells (enterocytes and goblet cells) .

• Stromal expression: GALR3 is also expressed in intestinal immune/stromal cells, myenteric plexuses, and smooth muscle cells, suggesting potential roles in the tumor microenvironment beyond cancer cell signaling .

These findings suggest that GALR3 expression analysis may contribute to improved patient stratification and potentially identify novel therapeutic targets in colorectal cancer.

What molecular features determine ligand selectivity between GALR2 and GALR3?

The molecular basis for ligand selectivity between GALR2 and GALR3 has been elucidated through detailed structural and functional studies:

Key findings include:

• TM3 domain differences: The substitution of specific amino acids in the TM3 domain (Leu 100Phe, Tyr 103Phe, Tyr 107His) of GALR3 to match GALR2 residues significantly enhances interactions with specific ligands, particularly those containing Asn 5 and Ala 7 residues .

• TM5 domain contribution: The TM5 domain of GALR2 plays a crucial role in recognizing Pro 13 residues in ligands, as demonstrated by GALR3/2 [TM5] chimeric receptors showing stronger responses to [P 13]-SPX compared to wild-type GALR3 .

• Combined effects: Double and triple mutations in the TM3 domain of GALR3 show more pronounced effects than single mutations, indicating cooperative interactions between these residues in ligand binding .

• N-terminal effects: The longer N-terminal portion of GALR3 compared to GALR2 affects ligand responses, as demonstrated by chimeric receptors GALR3/2a and GALR3/2b showing decreased responses to SPX and Qu-SPX .

These structural insights explain the molecular basis for selective ligand interactions and could guide the development of subtype-specific agonists or antagonists for galanin receptors.

What techniques can be used to study GALR3 signaling pathways in neuronal cells?

To investigate GALR3 signaling pathways in neuronal cells, researchers can employ several complementary techniques:

• Calcium imaging: Monitor intracellular Ca²⁺ levels in response to GALR3 activation using fluorescent calcium indicators (Fura-2, Fluo-4) to assess coupling to Gq proteins.

• cAMP assays: Measure changes in intracellular cAMP levels following GALR3 stimulation to evaluate Gi/Go protein coupling, using either ELISA-based methods or FRET-based biosensors.

• Chimeric receptor studies: Generate GALR2/GALR3 chimeric constructs to identify domains critical for specific signaling pathways, similar to the approach used in ligand selectivity studies .

• Phosphorylation assays: Assess activation of downstream signaling molecules (ERK, Akt, etc.) using phospho-specific antibodies and Western blotting.

• Receptor mutagenesis: Introduce point mutations in key residues identified in structural studies (e.g., TM3 or TM5 domains) to evaluate their impact on signal transduction .

• BRET/FRET assays: Monitor protein-protein interactions between GALR3 and various G proteins or β-arrestins to characterize signaling biases.

• Electrophysiology: Record changes in neuronal activity (membrane potential, ion currents) in response to GALR3 agonists using patch-clamp techniques.

• Transcriptional reporter assays: Measure activation of relevant transcription factors (CREB, NFκB) downstream of GALR3 activation.

• Single-cell RNA sequencing: Profile transcriptional changes in neuronal populations following GALR3 stimulation to identify regulated genes and pathways.

• Optical biosensors: Employ genetically encoded biosensors to visualize spatiotemporal dynamics of second messengers in living neurons.

Why might there be discrepancies between GALR3 mRNA expression and protein detection?

Discrepancies between GALR3 mRNA and protein expression levels, as observed in colorectal cancer tissues , can result from several biological and technical factors:

• Post-transcriptional regulation: MicroRNAs and RNA-binding proteins may regulate GALR3 mRNA stability or translation efficiency, leading to differential protein expression despite similar mRNA levels.

• Protein stability and turnover: Differences in protein half-life or degradation rates between tissues or disease states can cause protein levels to diverge from mRNA abundance.

• Alternative splicing: GALR3 may undergo alternative splicing, producing transcript variants that aren't detected by standard mRNA quantification methods but affect protein expression. The presence of multiple transcript sizes (1.4-, 2.4-, and 5-kb) supports this possibility .

• Epitope accessibility: In certain cellular contexts, protein modifications or interactions may mask antibody epitopes, resulting in underestimation of protein levels despite high mRNA expression.

• Sensitivity differences: Methods for detecting mRNA (qPCR, RNA-seq) often have different sensitivity thresholds compared to protein detection methods (Western blot, IHC).

• Tissue heterogeneity: In complex tissues, bulk measurements may not reflect cell type-specific expression patterns, particularly if GALR3 is expressed in minority cell populations.

• Technical variations: Differences in sample preparation, fixation methods, or antibody performance can contribute to apparent discrepancies between protein and mRNA measurements.

Researchers should consider these factors when interpreting GALR3 expression data and ideally use complementary approaches to validate findings.

What are common pitfalls when detecting GALR3 in tissue samples by immunohistochemistry?

Common challenges and pitfalls in GALR3 immunohistochemistry include:

• Background staining: Non-specific binding of GALR3 antibodies can produce false-positive signals. To minimize this, optimize blocking conditions (duration, blocking agent) and antibody concentrations.

• Epitope masking: Formalin fixation can cross-link proteins and mask epitopes. Ensure proper antigen retrieval using optimized protocols (heat-induced epitope retrieval in appropriate buffers).

• Specificity issues: Cross-reactivity with other galanin receptors (GALR1, GALR2) may occur due to sequence homology. Validate antibody specificity using appropriate controls.

• Variable expression levels: GALR3 expression may be low in certain tissues, requiring sensitive detection methods. Consider using amplification systems like tyramide signal amplification.

• Membrane protein detection challenges: As a G protein-coupled receptor, GALR3 is a membrane protein that may require special fixation and permeabilization protocols for optimal detection.

• Post-mortem changes: Autolysis and protein degradation in post-mortem samples can affect GALR3 detection. Minimize post-mortem interval and use appropriate fixation methods.

• Subcellular localization variability: GALR3 may localize to both membrane and cytoplasmic compartments , requiring careful interpretation of staining patterns.

• Tissue processing artifacts: Overfixation, inadequate dehydration, or poor paraffin infiltration can create artifacts that interfere with accurate GALR3 detection.

For valid results, researchers should include appropriate positive and negative controls, carefully optimize staining protocols, and consider using multiple antibodies targeting different epitopes of GALR3.

How can researchers distinguish between specific and non-specific bands in GALR3 Western blots?

To differentiate between specific and non-specific bands in GALR3 Western blots:

• Molecular weight verification: Compare observed bands to the predicted molecular weight of GALR3 (40 kDa) . Note that the observed band may be slightly lower (e.g., 37 kDa) due to post-translational modifications or proteolytic processing .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide. Specific bands should be reduced or eliminated while non-specific bands remain unchanged.

• Multiple antibodies: Use different antibodies targeting distinct epitopes of GALR3. Specific bands should be detected by multiple antibodies.

• Positive controls: Include samples with confirmed GALR3 expression (e.g., human skin lysate , brain tissue ) as positive controls.

• Knockout/knockdown controls: When available, include samples from GALR3 knockout models or cells with GALR3 siRNA knockdown. Specific bands should be absent or reduced in these samples.

• Loading gradient: Run a dilution series of your sample to verify that band intensity correlates with protein concentration, which is expected for specific interactions.

• Recombinant protein: Include purified recombinant GALR3 as a standard to confirm band size and antibody reactivity.

• Detection method optimization: Adjust exposure times to optimize visualization of specific bands while minimizing background.

• Sample preparation variations: Test different lysis buffers and conditions, as GALR3 detection may be sensitive to specific extraction methods.

How can GALR3 antibodies be used to investigate receptor internalization and trafficking?

GALR3 antibodies can be valuable tools for studying receptor internalization and trafficking through several methodological approaches:

• Live-cell imaging: Use fluorescently labeled GALR3 antibodies (that recognize extracellular epitopes) to track receptor movement in real-time following ligand stimulation.

• Pulse-chase experiments: Label surface GALR3 with antibodies at 4°C (to prevent internalization), then warm cells to 37°C in the presence or absence of ligands to track internalization kinetics.

• Co-localization studies: Perform double immunofluorescence with GALR3 antibodies and markers of various cellular compartments (early endosomes, recycling endosomes, lysosomes) to map trafficking pathways.

• Surface biotinylation assays: Combine surface protein biotinylation with GALR3 immunoprecipitation to quantify changes in receptor surface expression over time.

• ELISA-based internalization assays: Develop cell-based ELISAs using GALR3 antibodies to quantify changes in surface receptor levels following various treatments.

• Antibody feeding assays: Label surface receptors with antibodies, allow internalization, then detect internalized antibodies after stripping remaining surface-bound antibodies.

• Receptor recycling studies: Use GALR3 antibodies to track the reappearance of internalized receptors at the cell surface following ligand removal.

• Super-resolution microscopy: Apply techniques like STORM or PALM with GALR3 antibodies to visualize receptor clustering and organization at the nanoscale level.

These approaches can provide insights into the regulatory mechanisms controlling GALR3 signaling and may identify novel targets for therapeutic intervention.

What is known about the role of GALR3 in colorectal cancer progression and how can antibodies advance this research?

GALR3 appears to have significant implications in colorectal cancer biology, and antibody-based studies have provided key insights:

• Expression patterns: GALR3 shows stronger immunoreactivity in cancer cells compared to normal epithelial cells of the large intestine . This differential expression suggests potential roles in cancer cell biology.

• Prognostic biomarker potential: Low immunoexpression of GALR3 protein correlates with poor prognosis in CRC patients , suggesting its utility as a prognostic biomarker.

• Subcellular localization: GALR3 is localized to both cell membrane and cytoplasm in colorectal cancer cells , which may indicate altered trafficking or signaling in cancer contexts.

GALR3 antibodies can advance colorectal cancer research through:

• Tissue microarray analysis: Large-scale screening of GALR3 expression across patient cohorts to validate prognostic associations and identify patient subgroups.

• Functional studies: Combining GALR3 antibodies with proliferation, invasion, and apoptosis assays to elucidate receptor functions in cancer cells.

• Therapeutic targeting evaluation: Developing and testing function-blocking antibodies that might inhibit GALR3-mediated signaling in cancer cells.

• Circulating tumor cell detection: Using GALR3 antibodies to identify and characterize CTCs as potential liquid biopsy markers.

• Drug response prediction: Correlating GALR3 expression levels with response to specific chemotherapeutic agents to identify predictive biomarkers.

• Tumor microenvironment studies: Investigating GALR3 expression in stromal and immune cells within the tumor microenvironment to understand its broader roles in cancer biology.

Future research should focus on clarifying the mechanisms by which GALR3 influences cancer progression and evaluating its potential as a therapeutic target or biomarker.

How can researchers apply GALR3 antibodies to investigate interactions with other galanin receptor subtypes?

GALR3 antibodies can be powerful tools for studying interactions and functional relationships between different galanin receptor subtypes:

• Co-immunoprecipitation: Use GALR3 antibodies to pull down receptor complexes, followed by detection of GALR1 or GALR2 to identify heterodimeric interactions.

• Proximity ligation assay (PLA): Apply PLA using antibodies against GALR3 and other receptor subtypes to visualize and quantify receptor proximity (<40 nm) in situ, providing evidence for potential interactions.

• FRET/BRET analysis: Combine antibody-based detection with fluorescence or bioluminescence resonance energy transfer techniques to study dynamic receptor interactions in living cells.

• Double immunofluorescence: Perform co-localization studies of GALR3 with GALR1 or GALR2 in tissues or cells to identify anatomical sites where receptor interactions might occur.

• Sequential immunoprecipitation: Deplete GALR3 from samples using specific antibodies, then assess how this affects detection of other receptor subtypes to identify receptor populations that exist as heteromeric complexes.

• Competition binding assays: Use GALR3 antibodies in combination with labeled ligands to study how receptor interactions affect ligand binding properties.

• Cross-linking studies: Apply chemical cross-linkers followed by immunoprecipitation with GALR3 antibodies to stabilize and identify transient receptor complexes.

• Single-molecule imaging: Combine GALR3 antibodies with super-resolution microscopy techniques to visualize individual receptor molecules and their dynamic associations.

These approaches can provide insights into the physiological relevance of receptor heteromerization and potentially identify novel therapeutic targets for conditions involving dysregulated galanin signaling.