Recombinant Rat Taste receptor type 2 member 105 (Tas2r105)

What is Taste receptor type 2 member 105 (Tas2r105) and what is its function?

Tas2r105 is a bitter taste receptor belonging to the type 2 taste receptors (T2Rs) family, which are members of the G-protein-coupled receptors (GPCRs) superfamily. This receptor plays a crucial role in the detection and avoidance of potentially harmful bitter compounds. Functionally, Tas2r105 has been characterized as a generalist receptor with an exceptionally broad agonist profile, responding to approximately 35% of tested bitter compounds in comprehensive screening studies .

The primary function of Tas2r105 is to mediate the recognition of bitter toxins, enabling animals to avoid potentially harmful substances. Research has demonstrated that Tas2r105 is particularly important for the detection of cycloheximide, as evidenced by studies with Tas2r105 knockout mice that show decreased avoidance responses to this compound . Beyond its role in taste perception, emerging evidence suggests Tas2r105 may serve important physiological functions in extraoral tissues, potentially contributing to immune and digestive responses .

Where is Tas2r105 expressed in rodents?

Particularly notable is the expression of Tas2r105 in the gastrointestinal tract, specifically in the small intestinal villus and crypts . This extraoral expression suggests broader physiological roles beyond taste perception, potentially including immune surveillance, regulation of digestive processes, or detection of bacterial metabolites. The relative expression levels of Tas2r105 compared to other bitter taste receptors vary across tissues, with some studies showing moderately high expression in taste papillae compared to certain other Tas2r genes .

What compounds activate Tas2r105 and at what concentrations?

Tas2r105 exhibits one of the broadest agonist profiles among mouse bitter taste receptors, responding to 45 different bitter substances (35% of tested compounds) in comprehensive screening studies . Key agonists include:

• Cycloheximide - Tas2r105 shows particular sensitivity to this compound, with studies confirming its crucial role in cycloheximide detection

• N-acyl homoserine lactones - Compounds involved in bacterial quorum sensing that exclusively activate Tas2r105

• Denatonium benzoate - A broadly detected bitter compound that activates Tas2r105

• Quinine dihydrochloride - Activates Tas2r105 at threshold concentrations between 3.0-10 μM

• PROP (propylthiouracil) - Activates multiple mouse Tas2rs including Tas2r105

• Yohimbine - Detected by Tas2r105 at a threshold concentration of approximately 0.3 mM

• Saccharin - Activates Tas2r105 at a threshold concentration of 1.0 mM

The potency and efficacy of these compounds vary considerably. For instance, concentration-response analyses reveal that Tas2r105 detects quinine at relatively low concentrations (3-10 μM), while higher concentrations of saccharin (1.0 mM) are required for activation .

How does the signaling cascade for Tas2r105 function?

Tas2r105, like other bitter taste receptors, utilizes the canonical taste signaling pathway involving several key components. When a bitter ligand binds to Tas2r105, the receptor undergoes conformational changes that activate associated G-proteins, particularly gustducin. The activated G-protein then triggers the following signaling cascade:

• Activation of phospholipase C (PLC) β2, which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol triphosphate (IP3) and diacylglycerol (DAG)

• IP3 binds to IP3 receptors on the endoplasmic reticulum, triggering release of Ca²⁺ from intracellular stores

• Increased intracellular Ca²⁺ activates the transient receptor potential channel M5 (TRPM5)

• TRPM5 activation leads to cation influx, resulting in membrane depolarization

• Membrane depolarization stimulates the release of neurotransmitters such as ATP and acetylcholine

This signaling pathway has been demonstrated through studies using heterologous expression systems with different G-protein subunits. Research indicates that Tas2r105 responses are more robust when expressed in cells with Gα16gust44 compared to cells expressing only Gα15, suggesting important considerations for experimental design when studying this receptor .

What methodologies are most effective for generating and validating Tas2r105 knockout models?

The generation of Tas2r105 knockout models has been successfully accomplished using CRISPR/Cas9 gene-editing techniques. Based on published methodologies, the following protocol has proven effective:

• Design of sgRNAs targeting the Tas2r105 coding region, with careful consideration of potential off-target effects. Multiple sgRNAs may be used to target different regions of the gene

• Microinjection of a mixture containing Cas9 and sgRNA mRNAs into the cytoplasm of mouse zygotes

• Screening of founder mice for genetic mutations at target sites using T7 Endonuclease 1 (T7EN1) assays

• Confirmation of mutations by DNA sequencing to identify frameshift mutations that disrupt protein function

• Breeding of heterozygous mice to establish homozygous knockout lines

For validation of knockout models, a multi-faceted approach is recommended:

• Molecular validation:

  • Quantitative RT-PCR to confirm absence or significant reduction of Tas2r105 mRNA expression

  • Immunostaining to verify absence of Tas2r105 protein in taste buds and other tissues

• Functional validation:

  • Two-bottle preference tests to assess behavioral responses to known Tas2r105 agonists, particularly cycloheximide, quinine, and denatonium benzoate

  • Calcium imaging of isolated taste cells to confirm loss of responses to Tas2r105-specific agonists

• Specificity controls:

  • Assessment of expression and function of other taste signaling components (e.g., GNAT3, PLCB2) to ensure specific effects on Tas2r105

  • Evaluation of responses to non-bitter tastants to confirm selective effects on bitter taste perception

How can Tas2r105 function be assessed in heterologous expression systems?

Heterologous expression of Tas2r105 in cell lines provides a powerful approach for characterizing receptor function. Based on published methodologies, the following protocol has proven effective:

• Expression system selection:

  • HEK293T cells have been successfully used for Tas2r105 expression

  • Coexpression with chimeric G-proteins (particularly Gα16gust44) significantly enhances signal detection compared to Gα15 alone

• Functional assay optimization:

  • Calcium imaging using fluorescent calcium indicators (e.g., Fluo-4) provides sensitive detection of receptor activation

  • Plate-based fluorometric assays enable high-throughput screening of potential ligands

  • Response normalization to positive controls (e.g., ATP or ionomycin) improves data reliability

• Agonist characterization:

  • Concentration-response curves with various dilutions of test compounds (typically 0.01 μM to 10 mM)

  • Calculation of threshold concentrations and EC50 values to quantify potency

  • Assessment of efficacy through maximum response amplitude

• Important considerations:

  • Cell surface expression of bitter taste receptors can be challenging; inclusion of export sequences may improve trafficking

  • Receptor responses may be influenced by the specific G-protein coupling; systematic comparison of different G-proteins is recommended

  • Vehicle controls and mock-transfected cells are essential for distinguishing specific receptor-mediated responses from non-specific effects

What is the role of Tas2r105 in extraoral tissues and how can it be studied?

The extraoral expression of Tas2r105, particularly in intestinal tissues, suggests physiological functions beyond taste perception . Current evidence points to several potential roles:

• Detection of bacterial metabolites and quorum sensing molecules:

  • Tas2r105 responds to N-acyl homoserine lactones involved in bacterial quorum sensing

  • This may enable host monitoring of bacterial populations in the gut lumen

• Regulation of innate immune responses:

  • Extraoral bitter taste receptors have been implicated in type 2 immune responses to helminth infections in the intestinal tract

  • They may contribute to antibacterial responses in various tissues

To effectively study these extraoral functions, several methodologies are recommended:

• Tissue-specific expression analysis:

  • Quantitative RT-PCR to measure Tas2r105 mRNA levels in different tissues

  • In situ hybridization to localize expression to specific cell types within tissues

  • Single-cell RNA sequencing to identify the complete repertoire of cells expressing Tas2r105

• Functional analysis:

  • Ex vivo calcium imaging of isolated cells or tissue explants from extraoral sites

  • Organoid cultures from intestinal tissues to study Tas2r105 function in a physiologically relevant system

  • Cell type-specific knockout models using conditional Cre-loxP systems to selectively delete Tas2r105 in extraoral tissues while preserving gustatory function

• Physiological readouts:

  • Measurement of immune mediator production in response to Tas2r105 agonists in extraoral tissues

  • Assessment of bacterial clearance and host defense mechanisms in Tas2r105 knockout models

  • Intestinal motility and secretion studies to investigate potential roles in digestive function

How does Tas2r105 compare with human bitter taste receptors?

Comparative analysis of Tas2r105 with human bitter taste receptors reveals important species-specific differences in receptor function and agonist profiles:

These comparative analyses provide valuable insights into the evolution of bitter taste perception and can guide the selection of appropriate animal models for studying specific aspects of bitter taste biology.

What are the challenges in expressing and purifying recombinant Tas2r105 for structural studies?

Expressing and purifying GPCRs, including Tas2r105, for structural studies presents numerous challenges that require specialized approaches:

• Expression challenges:

  • Poor membrane trafficking and cell surface expression in heterologous systems

  • Protein instability and aggregation due to hydrophobic transmembrane domains

  • Low expression yields, particularly in eukaryotic expression systems required for proper folding and post-translational modifications

• Purification challenges:

  • Maintaining receptor stability in detergent solutions during extraction from membranes

  • Preserving native conformation and ligand-binding properties throughout purification

  • Achieving sufficient purity and homogeneity for crystallization or cryo-EM studies

• Recommended strategies based on GPCR structural biology approaches:

  • Fusion protein approaches (e.g., T4 lysozyme or BRIL insertions) to enhance expression and crystallization properties

  • Thermostabilizing mutations identified through alanine scanning or directed evolution

  • Nanobody or antibody fragment co-crystallization to stabilize specific conformations

  • Lipid cubic phase crystallization to maintain a membrane-like environment

  • Expression screening in multiple systems (bacterial, yeast, insect, mammalian) to identify optimal conditions

  • Detergent screening to identify conditions that maintain receptor stability and function

• Validation of purified receptor:

  • Ligand binding assays to confirm retention of native binding properties

  • Thermal stability assays (e.g., CPM or differential scanning fluorimetry) to assess protein stability

  • Size-exclusion chromatography and multi-angle light scattering to evaluate monodispersity

While no published structures of Tas2r105 are available based on the search results, these approaches represent the current state-of-the-art for GPCR structural biology and would be applicable to Tas2r105 structural studies.

What methods can differentiate between the functions of Tas2r105 and other bitter taste receptors with overlapping agonist profiles?

Differentiating between the functions of Tas2r105 and other bitter taste receptors with overlapping agonist profiles requires a multi-faceted approach. The following methodologies have proven effective:

• Genetic approaches:

  • Gene-specific knockout models targeting Tas2r105 alone or in combination with other Tas2r genes

  • Selective rescue experiments in knockout backgrounds to confirm specific receptor functions

  • Comparative phenotyping of single vs. multiple receptor knockouts to assess functional redundancy

• Pharmacological approaches:

  • Identification of receptor-selective agonists through comprehensive screening

  • For Tas2r105, cycloheximide and N-acyl homoserine lactones show high selectivity

  • Dose-response analyses to identify concentration ranges where compounds selectively activate specific receptors

• In vitro discrimination techniques:

  • Heterologous expression of individual receptors for direct comparison of agonist profiles

  • Competition assays between selective and non-selective agonists to characterize binding interactions

  • Use of different G-protein coupling systems that may differentially impact receptor activation patterns

• Data analysis approaches:

  • Principal component analysis of response profiles to identify receptor-specific signatures

  • Hierarchical clustering of agonist responses to group receptors with similar profiles

  • Calculation of selectivity indices to quantify the relative specificity of compounds for different receptors

The table below compares agonist profiles for Tas2r105 and selected other bitter taste receptors based on available data:

Note: ++ indicates high selectivity; + indicates activation with threshold concentration in parentheses where available; - indicates no activation

What are the most sensitive methods for detecting Tas2r105 expression in different tissues?

Detecting Tas2r105 expression, particularly in extraoral tissues where expression levels may be low, requires sensitive and specific methodologies. Based on current research approaches, the following methods are recommended:

• Transcript detection methods:

  • Quantitative real-time PCR (qRT-PCR) with carefully designed primers spanning exon junctions to ensure specificity

  • Digital PCR for absolute quantification of low-abundance transcripts

  • RNA-Seq and single-cell RNA-Seq for comprehensive transcriptome analysis and cellular localization

  • In situ hybridization with RNAscope or similar high-sensitivity techniques for spatial localization within tissues

  • Reverse transcription droplet digital PCR (RT-ddPCR) for absolute quantification of low-abundance transcripts

• Protein detection methods:

  • Immunohistochemistry or immunofluorescence with validated antibodies

  • Proximity ligation assay (PLA) for increased sensitivity and specificity

  • Western blotting with signal amplification methods for low-abundance proteins

  • Mass spectrometry-based proteomics for unbiased detection and quantification

• Functional detection approaches:

  • Calcium imaging in isolated cells or tissue explants using Tas2r105-selective agonists

  • Reporter gene assays with Tas2r105 promoter constructs to monitor transcriptional activity

  • CRISPR-based transcriptional reporters (e.g., CRISPRa/i) to monitor endogenous gene expression

• Critical considerations:

  • Inclusion of appropriate positive controls (e.g., taste papillae) and negative controls

  • Validation with multiple independent methods to confirm expression

  • Use of Tas2r105 knockout tissues as specificity controls for antibody-based methods

  • Careful primer and probe design to avoid cross-reactivity with other Tas2r family members

The sensitivity and specificity of these methods vary considerably. Based on published studies, qRT-PCR combined with in situ hybridization provides a robust approach for detecting Tas2r105 expression, with good correlation between these methods observed in taste tissues .

How can Tas2r105 research contribute to understanding extraoral chemosensory mechanisms?

Research on Tas2r105 expression and function in extraoral tissues has significant implications for understanding broader chemosensory mechanisms beyond taste perception. Key contributions include:

• Gastrointestinal chemosensing:

  • Tas2r105 expression in small intestinal villus and crypts suggests potential roles in nutrient sensing, gut hormone secretion, or microbiome interactions

  • Studies in knockout models can reveal specific physiological consequences of Tas2r105 signaling in digestive processes

• Host-microbe interactions:

  • The ability of Tas2r105 to detect N-acyl homoserine lactones (bacterial quorum sensing molecules) suggests roles in monitoring bacterial populations

  • This may contribute to host defense mechanisms or microbiome regulation

• Innate immunity:

  • Bitter taste receptors in other tissues have been implicated in type 2 immune responses to helminth infections

  • Understanding Tas2r105's role in immune surveillance may reveal novel host defense mechanisms

• Translational applications:

  • Identification of Tas2r105-mediated pathways may reveal novel therapeutic targets for gastrointestinal disorders

  • Understanding species differences between rodent and human bitter receptors can improve translation of preclinical findings

Future research directions should focus on:

• Tissue-specific conditional knockout models to delineate the physiological roles of Tas2r105 in different systems

• Identification of endogenous agonists that may activate Tas2r105 in extraoral contexts

• Characterization of downstream signaling pathways that may differ from canonical taste signaling in other tissues

• Investigation of potential interactions between Tas2r105 and other chemosensory or immune receptors

What are the most promising experimental models for studying Tas2r105 function in vivo?

Several experimental models have proven valuable for investigating Tas2r105 function in vivo, each with specific advantages for addressing different research questions:

• Genetically modified mouse models:

  • CRISPR/Cas9-generated Tas2r105 knockout mice enable direct assessment of receptor function in various physiological contexts

  • Cluster knockouts targeting multiple Tas2r genes (e.g., Tas2r106/Tas2r104/Tas2r105/Tas2r114) help address functional redundancy among related receptors

  • Conditional knockout models using tissue-specific Cre drivers allow investigation of Tas2r105 function in specific cell populations

  • Reporter knockin lines expressing fluorescent proteins under Tas2r105 promoter control facilitate identification and isolation of Tas2r105-expressing cells

• Behavioral assays:

  • Two-bottle preference tests provide a robust measure of taste perception and avoidance behaviors

  • Brief-access lick tests offer more refined analysis of immediate taste responses

  • Conditioned taste aversion paradigms can assess the aversive properties of Tas2r105 agonists

  • Operant conditioning approaches to quantify motivation and discrimination capabilities

• Ex vivo tissue preparations:

  • Isolated taste buds or taste cells for calcium imaging and electrophysiological recordings

  • Intestinal organoids derived from wild-type or Tas2r105 knockout mice for studying extraoral functions

  • Tissue explants from various organs expressing Tas2r105 for functional studies

• In vivo physiological measurements:

  • Gustatory nerve recordings to assess taste signaling at the peripheral nervous system level

  • Intravital microscopy of labeled Tas2r105-expressing cells to monitor responses in living tissues

  • Metabolic phenotyping to assess potential roles in energy homeostasis

  • Immune challenge models to investigate contributions to host defense mechanisms

Each model system has specific strengths and limitations. Integration of multiple approaches is recommended for comprehensive characterization of Tas2r105 function across different physiological contexts.