TAS2R7 Antibody

What is TAS2R7 and what is its physiological role?

TAS2R7 (Taste Receptor Type 2 Member 7) is a G protein-coupled receptor that functions as a gustducin-coupled receptor implicated in the perception of bitter compounds. It is primarily expressed in subsets of taste receptor cells of the tongue and palate epithelium, exclusively in gustducin-positive cells . TAS2R7 plays a crucial role in bitter taste perception in the oral cavity and also functions in sensing the chemical composition of gastrointestinal content. Recent research has identified TAS2R7 as a unique metal cation receptor that responds to a broad range of divalent and trivalent salts, including zinc, calcium, magnesium, copper, manganese, and aluminum, which contributes to the bitter taste perception of these metal ions .

What are the key structural and functional characteristics of TAS2R7?

TAS2R7 is a membrane-bound G protein-coupled receptor with seven transmembrane domains. The protein has 318 amino acids with molecular weight of approximately 36-37 kDa . Functionally, TAS2R7 signals through PLCB2 and the calcium-regulated cation channel TRPM5 . Molecular modeling and mutagenesis analysis have identified specific residues, particularly H94, that are critical for metal ion binding and receptor activation . The receptor demonstrates selective responses to divalent and trivalent salts but not to monovalent salts like KCl, suggesting a specific tuning mechanism for higher-valence cations .

What are the optimal conditions for using TAS2R7 antibodies in Western blot applications?

For Western blot applications using TAS2R7 antibodies, the following methodological considerations are important:

• Sample preparation: Cell lysates from relevant cell lines (e.g., HL-60, K562) have been successfully used to detect TAS2R7 .

• Dilution range: Recommended dilutions typically range from 1:500 to 1:2000 for Western blot applications . For specific antibodies like that from Novus Biologicals, a concentration of 1.0 μg/ml has been recommended .

• Detection system: Standard secondary antibody detection systems compatible with rabbit IgG should be used.

• Protein size: The expected molecular weight for detection is approximately 36-37 kDa .

• Storage conditions: Most antibodies should be stored at -20°C for long-term storage to maintain reactivity, with aliquoting recommended to avoid freeze-thaw cycles .

How can I validate the specificity of a TAS2R7 antibody for my research?

Validating TAS2R7 antibody specificity requires multiple approaches:

• Positive controls: Use cell lines known to express TAS2R7, such as taste receptor cells or transfected cell lines (e.g., HEK293 cells transiently transfected with TAS2R7) .

• Negative controls: Include mock-transfected cells or cells known not to express TAS2R7.

• Immunostaining validation: Compare immunostaining patterns with expected cellular localization (membrane-bound for TAS2R7) .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to confirm binding specificity.

• Molecular techniques: Consider validating with complementary techniques such as RT-PCR to confirm expression at the mRNA level.

• Knockdown experiments: siRNA knockdown of TAS2R7 should reduce antibody signal if the antibody is specific.

What functional assays can be used to study TAS2R7 activity in conjunction with antibody-based detection?

Several functional assays can be employed alongside antibody-based detection:

• Calcium mobilization assay: This assay monitors TAS2R7 activation by measuring intracellular calcium release. HEK293 cells transfected with TAS2R7 along with a coupling chimeric G protein (Gα16-gust44) can be used, and receptor activation monitored using calcium-sensitive dyes .

• Dose-response experiments: These can determine the sensitivity of TAS2R7 toward various ligands, including metal ions. Studies have shown TAS2R7 responds to metal ions in a dose-dependent manner with varying EC50 values (Al2(SO4)3: 39±15 μM; CuSO4: 1.04±0.36 mM; ZnSO4: 3.36±0.14 mM; MgCl2: 6.07±1.07 mM; CaCl2: 5.27±0.50 mM; MnCl2: 6.59±1.73 mM) .

• Immunofluorescence assays: These can be used to visualize receptor localization in cells using specific antibodies .

• Molecular modeling and mutagenesis: Site-directed mutagenesis can be used to identify critical residues involved in ligand binding and receptor activation, complementing antibody studies .

How do different metal ions interact with TAS2R7, and what experimental approaches can elucidate these interactions?

Research has shown that TAS2R7 interacts differently with various metal ions, with distinct potency and efficacy profiles:

• Metal ion sensitivity profile: TAS2R7 demonstrates highest sensitivity to aluminum sulfate (EC50: 39±15 μM), followed by copper sulfate, zinc sulfate, magnesium chloride, calcium chloride, and manganese chloride .

• Experimental approaches:

  • Dose-response analysis: Systematic testing with varying concentrations of metal ions can establish sensitivity profiles.

  • Calcium-free assays: Performing assays in calcium-free conditions can eliminate potential interference from calcium in the assay buffer.

  • Anion effect studies: Comparing responses to different salt forms (e.g., ZnSO4 vs. ZnCl2) can determine if anions affect potency or efficacy .

  • Molecular docking: Computational approaches can predict binding sites and interaction modes. For example, electrostatic potential calculations using APBS and molecular surface generation with water probes can model metal ion interactions .

  • Site-directed mutagenesis: Mutating specific residues (e.g., H94) can validate predicted binding sites .

How does TAS2R7 differ from other bitter taste receptors in terms of ligand specificity and signal transduction?

TAS2R7 shows distinctive characteristics compared to other bitter taste receptors:

• Narrowly tuned receptor: Unlike many TAS2Rs that respond to multiple bitter compounds, TAS2R7 appears to be relatively selective, responding primarily to metal ions and cromolyn (at high concentrations, EC50: 5.9 mM) .

• Metal ion sensitivity: TAS2R7 is unique in responding to multiple metal ions, a property shared among GPCRs only with the calcium-sensing receptor (CaSR) .

• Signal transduction: Like other bitter taste receptors, TAS2R7 signals through PLCB2 and TRPM5 , but its specific downstream pathways when activated by metal ions versus organic compounds might differ.

• Experimental approach considerations:

  • Comparative analysis: Side-by-side testing of TAS2R7 with other TAS2Rs using the same assay conditions.

  • Pathway inhibitor studies: Using inhibitors of different signaling components to elucidate specific pathways activated by TAS2R7.

  • Receptor chimeras: Creating chimeric receptors between TAS2R7 and other TAS2Rs to identify domains responsible for specific ligand interactions.

What are the challenges in developing highly specific TAS2R7 antibodies, and how can these be addressed?

Developing specific TAS2R7 antibodies presents several challenges:

• Sequence homology: TAS2Rs share sequence similarities, which can lead to cross-reactivity. Careful epitope selection from unique regions is crucial.

• Membrane protein challenges: As a seven-transmembrane protein, TAS2R7 has limited exposed regions for antibody targeting, often requiring use of synthetic peptides rather than full-length protein for immunization.

• Validation complexity: Confirming specificity requires multiple approaches including testing in different tissues and cell types.

• Potential solutions:

  • Epitope mapping: Systematic analysis to identify unique regions within TAS2R7.

  • Recombinant expression: Expression of specific domains for antibody generation.

  • Affinity purification: Two-step purification against target peptide and cross-absorption against related TAS2R peptides.

  • Advanced validation: Using CRISPR/Cas9 knockout models to confirm specificity.

  • Monoclonal development: Development of monoclonal antibodies against specific epitopes for increased specificity.

What is the potential role of TAS2R7 in cancer biology, and how can TAS2R7 antibodies contribute to this research?

Emerging research suggests potential roles for bitter taste receptors including TAS2R7 in cancer:

• Expression patterns: Studies indicate that TAS2Rs are expressed in various cancerous cell lines and tissues, with some being predominantly downregulated in cancerous compared to non-cancerous samples .

• Prognostic significance: Higher expression levels of TAS2Rs in primary cancerous cells and tissues have been associated with improved prognosis in humans .

• Anti-cancer effects: Receptor-specific, agonist-mediated activation of TAS2Rs has been shown to induce various anti-cancer effects, including decreased cell proliferation, migration, and invasion, as well as increased apoptosis .

• Research applications of TAS2R7 antibodies:

  • Expression profiling: Antibodies can be used to assess TAS2R7 expression levels in different cancer types.

  • Prognostic markers: TAS2R7 detection might serve as a prognostic marker given the association between TAS2R expression and cancer outcomes.

  • Therapeutic target validation: Antibodies can help validate TAS2R7 as a potential therapeutic target.

  • Mechanism studies: Immunoprecipitation using TAS2R7 antibodies can identify interacting partners in cancer cells.

How can TAS2R7 genetic variations be studied in relation to disease susceptibility, and what role do antibodies play in this research?

Research on TAS2R genetic variations and disease susceptibility represents an emerging field:

• Genetic association studies: Variations in TAS2R genes, including TAS2R7, have been studied in relation to cancer risk, though findings remain inconclusive .

• Functional consequences: Genetic variations in TAS2Rs might affect receptor function, potentially impacting tissue-specific functions relevant to carcinogenesis .

• Role of antibodies in variant research:

  • Expression analysis: Antibodies can be used to assess whether genetic variants affect protein expression levels.

  • Localization studies: Immunohistochemistry can determine if variants affect receptor trafficking and localization.

  • Functional studies: Combined with genetic data, antibody-based detection can link genotype to phenotype through functional analyses.

  • Methodology considerations: When studying variants, researchers should use antibodies targeting conserved regions not affected by the variations under investigation.

What are the methodological considerations for studying TAS2R7 in extra-oral tissues, particularly in relation to pathological conditions?

Investigating TAS2R7 in extra-oral tissues requires specific methodological approaches:

• Tissue-specific expression verification:

  • Use validated TAS2R7 antibodies for immunohistochemistry to confirm expression in the tissue of interest.

  • Complement with RT-PCR or RNA-seq to verify transcript expression.

  • Include positive controls (taste tissue) and negative controls.

• Function in non-taste contexts:

  • Develop tissue-specific functional assays that may differ from taste cell assays.

  • Consider cell-type specific differences in signaling components.

• Disease context considerations:

  • Compare expression between normal and pathological tissues using consistent methodology.

  • Account for potential changes in receptor localization or post-translational modifications in disease states.

  • Consider environmental factors (e.g., pH changes in tumor microenvironments) that might affect antibody binding or receptor function.

• Technical challenges and solutions:

  • Low expression levels: Use high-sensitivity detection methods or signal amplification.

  • Background staining: Optimize blocking conditions and use highly specific antibodies.

  • Tissue heterogeneity: Consider single-cell approaches or laser capture microdissection.

  • Functional verification: Combine antibody detection with functional assays to confirm not just expression but activity in the tissue of interest.

How can TAS2R7 antibodies be utilized in high-throughput screening for novel ligands or therapeutic compounds?

TAS2R7 antibodies can facilitate high-throughput screening through several approaches:

• Cell-based screening platforms:

  • Develop stable cell lines expressing TAS2R7 that can be validated with antibodies.

  • Use antibodies to confirm receptor expression levels across screening plates.

  • Employ antibody-based detection methods as secondary validation for hits identified in primary functional screens.

• Binding assays:

  • Develop competitive binding assays using labeled antibodies.

  • Use antibodies in proximity-based assays (e.g., BRET, FRET) to detect conformational changes upon ligand binding.

• Technical considerations:

  • Select antibodies recognizing extracellular domains for live-cell applications.

  • Validate that antibody binding doesn't interfere with ligand interaction sites.

  • Consider developing conformation-specific antibodies that recognize active or inactive states of the receptor.

What are the latest advances in understanding the structural biology of TAS2R7, and how can antibodies contribute to this field?

Recent advances in TAS2R7 structural biology include:

• Homology modeling: Studies have employed homology modeling based on related GPCRs, such as the 5-HT2C serotonin receptor, to predict TAS2R7 structure .

• Binding site identification: Molecular modeling and mutagenesis have identified potential binding sites for metal ions and other ligands .

• Antibody contributions to structural studies:

  • Conformational antibodies: Development of antibodies recognizing specific conformational states can provide insights into receptor dynamics.

  • Crystallization aids: Antibodies can be used as crystallization chaperones to stabilize the receptor for structural studies.

  • Epitope mapping: Systematic epitope mapping with antibodies can validate predicted structural elements.

  • Structure-function relationships: Combining antibody binding studies with functional assays can link structural elements to receptor function.

• Methodological approach:

  • Generate panel of antibodies against different domains.

  • Use antibody accessibility studies to validate topology models.

  • Employ antibodies in cross-linking studies to identify spatial relationships between domains.

The field of TAS2R7 structural biology remains developing, with antibodies playing a crucial role in validating computational models and advancing understanding of this important taste receptor.