Recombinant Mouse Taste receptor type 2 member 113 (Tas2r113)

What is Tas2r113 and how does it fit within the mouse bitter taste receptor family?

Tas2r113 (also known as mGR13, mt2r58, T2R13, and Tas2r13) is a member of the mouse bitter taste receptor family. These receptors belong to the G protein-coupled receptor (GPCR) superfamily and are responsible for detecting bitter compounds. The mouse genome contains approximately 35 putatively functional Tas2r genes, which vary in their expression levels and agonist profiles . Tas2r113 is one of these functional bitter taste receptors with a gene ID of 387345 and an ORF size of 927 bp .

How is Tas2r113 expressed in mouse taste tissues compared to other Tas2r receptors?

While specific data for Tas2r113 expression patterns are not directly provided in the available literature, research on mouse Tas2r receptors generally shows varied expression levels across the posterior papillae of the mouse tongue. Quantitative RT-PCR analyses have demonstrated that all Tas2r genes are expressed in the tongue epithelium, though at different levels. Some receptors, like Tas2r108, Tas2r118, Tas2r126, Tas2r135, and Tas2r137, show abundant expression (reaching ~20% of α-gustducin mRNA levels), while others are expressed at much lower levels . In situ hybridization experiments further confirm these differences at the cellular level, with some receptors being expressed in many taste cells and others in only a few .

What are the known molecular characteristics of Tas2r113?

Tas2r113 is a 927 bp open reading frame encoding a taste receptor from the type 2 family . Like other bitter taste receptors, it likely contains seven transmembrane domains characteristic of GPCRs. The receptor is encoded by the gene ID 387345 and has been referenced in genetic databases including RefSeq (BC140257) and UniGene (Mm.377907) . While specific structural details are not provided in the available literature, mouse bitter taste receptors typically couple with gustducin and other G proteins to initiate downstream signaling cascades when activated by bitter compounds.

What recombinant systems are available for studying Tas2r113 function?

Several recombinant systems are available for studying Tas2r113. A key tool is the AAV-based overexpression system, which allows for the delivery and expression of the Tas2r113 gene in various experimental models. The AAV-m-TAS2R113 (AAV-273723) is available with different serotypes including AAV1, AAV2, AAV3, AAV5, AAV6, AAV8, AAV9, AAV-DJ, AAV-DJ8, and AAV-DJ9 . This recombinant system offers flexibility in promoter selection (CMV default or from 30 different ubiquitous or cell-specific promoters) and optional reporter genes (GFP, CFP, YFP, RFP, or mCherry) .

For in vitro functional studies, heterologous expression systems similar to those used for other bitter taste receptors can be employed. These typically involve transfecting mammalian cell lines such as HEK293T cells with the Tas2r113 gene, along with G protein components like Gα16gust44, which has been shown to couple more efficiently with taste receptors than Gα15 .

How can researchers assess the cell surface expression of Tas2r113 in vitro?

To assess cell surface expression of Tas2r113, researchers can utilize immunocytochemistry techniques similar to those employed for other Tas2r family members. This typically involves adding epitope tags (such as Rho) to the N-terminus of the recombinant receptor and detecting their localization using antibodies in either permeabilized or unpermeabilized cells .

Based on studies with other mouse Tas2r receptors, cell surface expression can be categorized as shown in the following table:

What are the most reliable methods for quantifying Tas2r113 gene expression in tissue samples?

Quantitative RT-PCR (qRT-PCR) represents the most reliable method for quantifying Tas2r113 expression in tissue samples. This approach has been successfully used to measure expression levels of multiple Tas2r genes in mouse taste tissues . When designing qRT-PCR experiments for Tas2r113, researchers should:

• Use appropriate reference genes (such as α-gustducin) for normalization

• Design primers specific to Tas2r113 to avoid cross-amplification with other Tas2r genes

• Validate primers using standard curves and melting curve analysis

• Express results as a percentage of reference gene expression or using the ΔΔCt method

For spatial expression pattern analysis, in situ hybridization provides valuable complementary data by visualizing Tas2r113 expression at the cellular level within taste tissues. This technique allows researchers to determine both the number of cells expressing the receptor and the relative expression level based on signal intensity .

What experimental approaches are most effective for deorphanizing Tas2r113 and identifying its ligands?

For deorphanizing Tas2r113 and identifying its ligands, a calcium imaging-based functional assay in heterologous expression systems has proven most effective for other Tas2r receptors. The methodological approach should include:

• Transfecting HEK293T cells with the Tas2r113 expression construct along with a G protein component such as Gα16gust44, which has shown higher sensitivity than Gα15 for detecting bitter taste receptor activation

• Loading transfected cells with calcium-sensitive dyes like Fluo-4 AM

• Systematically screening a diverse library of bitter compounds (ideally ≥100 compounds) at multiple concentrations

• Measuring changes in intracellular calcium levels (ΔF/F) upon compound application using automated fluorescence plate readers or fluorescence microscopy

• Determining threshold concentrations, EC50 values, and efficacy parameters for active compounds

The choice of G protein is critical, as research on other Tas2r receptors has shown that Gα16gust44 provides higher sensitivity than Gα15, allowing for detection of low-efficacy activators that might be missed in less sensitive systems .

How can researchers determine if Tas2r113 is a specialist or generalist receptor?

To determine whether Tas2r113 functions as a specialist or generalist receptor, researchers need to systematically evaluate its responses to a broad range of bitter compounds. Based on research with other mouse Tas2r receptors, this classification relies on the percentage of test compounds that activate the receptor :

To make this determination for Tas2r113, researchers should:

• Express the receptor in a heterologous system with appropriate G protein coupling

• Screen a diverse library of at least 100 bitter compounds

• Calculate the percentage of compounds that activate the receptor above threshold

• Compare the response profile with other characterized Tas2r receptors

Research on mouse bitter taste receptors suggests that mice have a higher proportion of specialist receptors compared to humans, with only one receptor (Tas2r105) identified as a true generalist .

What parameters should be measured to fully characterize Tas2r113's pharmacological properties?

To fully characterize Tas2r113's pharmacological properties, researchers should measure the following key parameters:

• Efficacy (Emax): The maximal response amplitude (typically expressed as ΔF/F for calcium imaging assays) that indicates the strength of receptor activation by each agonist. This varies substantially among bitter compounds, with some eliciting strong responses (high efficacy) and others weak responses (low efficacy) .

• Potency (EC50): The concentration of agonist required to achieve 50% of the maximal response, providing a measure of the receptor's sensitivity to each compound. For mouse bitter taste receptors, EC50 values can span several orders of magnitude, from submicromolar to millimolar concentrations .

• Threshold concentration: The minimum concentration at which a statistically significant response can be detected, providing information about the detection limit for each compound.

• Specificity profile: Determining whether agonists are selective for Tas2r113 or activate multiple Tas2r family members.

• Antagonist sensitivity: Identifying compounds that can block Tas2r113 activation and characterizing their inhibitory potency (IC50) and mechanism (competitive vs. non-competitive).

• G protein coupling preferences: Evaluating the receptor's ability to signal through different G protein subtypes, which may affect signaling efficacy and downstream responses.

An example data table format for presenting these parameters would be:

How does Tas2r113 expression and function change during development or under pathological conditions?

While specific information about Tas2r113 developmental regulation is not provided in the available literature, research on other Tas2r family members suggests potential developmental and pathological considerations. Investigators studying these aspects should:

• Perform temporal expression analysis using qRT-PCR at different developmental stages from embryonic to adult to determine when Tas2r113 expression initiates and stabilizes

• Examine potential changes in receptor expression under various pathological conditions (inflammation, obesity, diabetes) that might affect taste perception

• Investigate potential extra-gustatory expression of Tas2r113, as some bitter taste receptors show expression in non-taste tissues. For example, research has shown that Tas2r114, which has low expression in taste papillae, exhibits robust expression in testis , suggesting bitter taste receptors may have diverse physiological roles beyond taste sensation

• Consider potential genetic polymorphisms that might affect receptor function, as studies have shown substantial amino acid sequence variations in mouse Tas2r genes between different strains (e.g., C57BL/6 and DBA/2J)

What behavioral assays can be used to validate Tas2r113 function in vivo?

To validate Tas2r113 function in vivo, several behavioral assays can be employed:

• Brief-access taste tests: This gold-standard method measures immediate licking responses to brief presentations of taste solutions. It minimizes post-ingestive effects and provides a direct measure of taste palatability. It has been successfully used to correlate bitter taste receptor properties with avoidance behavior in mice .

• Two-bottle preference tests: Long-term (typically 48-hour) tests where mice choose between water and a test solution. While this provides consumption data, it can be confounded by post-ingestive effects.

• Conditioned taste aversion: Pairing a novel taste with induced malaise to assess if the taste can be detected by the animal.

• Operant taste discrimination: Training mice to discriminate between different taste qualities through reinforcement learning.

For Tas2r113-specific functional validation, researchers should:

• Identify putative ligands of Tas2r113 through in vitro screening

• Test behavioral responses to these ligands in wild-type mice

• Compare responses in Tas2r113 knockout or transgenic overexpression models

• Use AAV-mediated delivery (AAV-m-TAS2R113) to rescue function in knockout models or enhance function in specific taste cell populations

How can researchers determine the structural basis for Tas2r113 ligand specificity?

Determining the structural basis for Tas2r113 ligand specificity requires a multi-faceted approach:

• Homology modeling: Since crystal structures of taste receptors are not readily available, researchers can build homology models based on related GPCRs with solved structures. These models can help predict the three-dimensional arrangement of transmembrane domains and potential ligand binding pockets.

• Site-directed mutagenesis: Systematic mutation of key residues predicted to be involved in ligand binding, followed by functional testing to identify critical amino acids. This approach has been successful in mapping binding sites in other bitter taste receptors.

• Chimeric receptor analysis: Creating chimeric receptors between Tas2r113 and other Tas2r family members with different ligand specificities can help identify domains responsible for ligand recognition.

• Molecular docking simulations: Using computational approaches to predict how ligands interact with the receptor binding pocket, which can guide mutagenesis experiments.

• Structure-activity relationship (SAR) studies: Testing series of structurally related compounds to determine which chemical features are important for receptor activation.

When interpreting results from these approaches, researchers should consider that bitter taste receptors often have multiple binding sites for different ligands, and that agonist binding may induce different conformational changes leading to varied efficacies. Additionally, the substantial sequence variation observed between mouse Tas2r family members (and between mouse strains) suggests that small changes in amino acid sequence can significantly affect ligand specificity .

How does mouse Tas2r113 compare functionally to its orthologs in other species?

A comprehensive functional comparison between mouse Tas2r113 and its orthologs in other species requires consideration of several aspects:

• Sequence conservation: Researchers should perform phylogenetic analysis to identify true orthologs across species and analyze sequence conservation, particularly in transmembrane domains and putative ligand-binding regions.

• Agonist profiles: Comparative functional studies should test the same panel of bitter compounds on Tas2r113 and its orthologs to determine if ligand specificity is conserved. Research on other mouse and human bitter taste receptors has shown that orthologous receptors can have substantially different agonist profiles . For example, PROP activates six mouse Tas2r receptors but only at high threshold concentrations (0.3-1.0 mM), while its human ortholog TAS2R38 is exquisitely sensitive to PROP with an EC50 of 2.1 μM .

• Expression patterns: Comparative analysis of receptor expression across species can provide insights into functional conservation or divergence.

When conducting such comparative studies, researchers should be aware that species-specific Tas2r gene expansions may have resulted in specialized receptors for compounds relevant to particular species' ecological niches .

What evolutionary pressures might have shaped Tas2r113 function in mice?

The evolution of bitter taste receptors, including Tas2r113, is likely driven by several selective pressures:

• Diet adaptation: The mouse bitter taste receptor repertoire may have evolved to detect toxic compounds present in food sources specific to their ecological niche.

• Predator avoidance: Bitter compounds can signal potential toxicity, and receptors may have evolved to detect compounds that are harmful to mice but not necessarily to other species.

• Species-specific gene expansions: The mouse genome contains 35 putatively functional Tas2r genes, suggesting gene duplication events followed by functional diversification. This expansion likely allowed for the detection of a broader range of potentially harmful compounds .

• Specialist vs. generalist strategies: The mouse bitter taste receptor family includes both broadly tuned receptors (generalists) and narrowly tuned receptors (specialists). This mixed strategy may represent an evolutionary optimization between broad toxin detection and specific recognition of particularly relevant compounds .

• Genetic drift and polymorphism: Studies have shown substantial sequence variation in mouse Tas2r genes between different strains, suggesting ongoing genetic diversification . Research has reported that only two of 24 Tas2r genes showed no amino acid sequence differences when C57BL/6 and DBA/2J strains were compared .

When studying Tas2r113 evolution, researchers should consider these broader patterns observed in the taste receptor family while focusing on the specific selective pressures that might have shaped this particular receptor's function.

How can CRISPR-Cas9 gene editing be optimized for studying Tas2r113 function?

CRISPR-Cas9 technology offers powerful approaches for studying Tas2r113 function through precise genetic manipulation:

• Knockout models: Generating Tas2r113-specific knockout mice to assess its contribution to bitter taste perception. When designing guide RNAs, researchers should:

  • Target conserved functional regions of the gene

  • Verify specificity to avoid off-target effects on other Tas2r family members

  • Consider potential compensatory upregulation of other Tas2r genes

• Knock-in models: Creating mice with reporter genes (GFP, tdTomato) fused to Tas2r113 to track expression patterns in vivo, or introducing specific mutations to study structure-function relationships.

• Conditional regulation: Implementing inducible Cre-loxP systems to control Tas2r113 expression temporally or in specific cell populations.

• Base editing and prime editing: Using these newer CRISPR technologies for precise single nucleotide modifications without double-strand breaks, allowing more subtle manipulation of receptor function.

When interpreting results from genetically modified models, researchers should be mindful that even single amino acid changes can significantly affect ligand specificity in taste receptors , making careful design and validation essential.

What single-cell technologies can advance our understanding of Tas2r113 in heterogeneous taste cell populations?

Single-cell technologies offer unprecedented resolution for studying Tas2r113 in the complex cellular environment of taste tissues:

• Single-cell RNA sequencing (scRNA-seq): This technique allows comprehensive profiling of gene expression at single-cell resolution, enabling:

  • Identification of cell populations expressing Tas2r113

  • Characterization of co-expression patterns with other taste receptors and signaling components

  • Discovery of novel cell populations potentially expressing Tas2r113

• Single-cell ATAC-seq: Profiling chromatin accessibility can reveal regulatory mechanisms controlling Tas2r113 expression in different cell types.

• Spatial transcriptomics: Technologies like Visium, MERFISH, or Slide-seq preserve spatial information while measuring gene expression, allowing mapping of Tas2r113-expressing cells within intact taste papillae.

• CyTOF and spectral flow cytometry: These techniques enable multiparameter protein-level analysis of taste cells, allowing correlation of Tas2r113 expression with other cellular markers.

• Patch-seq: Combining patch-clamp electrophysiology with single-cell RNA sequencing links Tas2r113 expression to functional properties of individual taste cells.

These technologies can help resolve questions about heterogeneity in taste cell populations, as studies have shown that different Tas2r receptors are expressed in varying numbers of cells and at different levels , suggesting functional specialization within the bitter taste system.

How might the study of Tas2r113 contribute to understanding non-gustatory roles of bitter taste receptors?

Investigating Tas2r113 in non-gustatory contexts may reveal important physiological functions beyond taste perception:

• Extra-oral expression mapping: Systematic screening of tissues beyond the oral cavity for Tas2r113 expression, building on observations that some Tas2r family members show expression in non-taste tissues, such as Tas2r114 in testis .

• Immune system interactions: Several bitter compounds, including N-acyl homoserine lactones that activate some Tas2r receptors, are involved in bacterial quorum sensing and induce antibacterial responses . Studying whether Tas2r113 plays a role in immune recognition of bacterial compounds could reveal new immunomodulatory functions.

• Metabolic regulation: Recent research has implicated bitter taste receptors in metabolic processes. Investigating Tas2r113 expression and function in metabolic tissues could provide insights into novel regulatory mechanisms.

• Respiratory and gastrointestinal physiology: Bitter taste receptors in these systems often serve as chemosensors for irritants or bacterial products. Determining if Tas2r113 contributes to these functions could expand our understanding of chemical sensing beyond taste.

• Neurodevelopmental roles: Some GPCRs play unexpected roles in neural development. Exploring potential developmental functions of Tas2r113 expression could reveal novel biological functions.

When investigating these potential non-gustatory roles, researchers should employ conditional and tissue-specific knockout approaches to distinguish the contribution of Tas2r113 expressed in different tissues and cell types.