Recombinant Mouse Taste receptor type 2 member 135 (Tas2r135)

Where is Tas2r135 expressed in mouse tissues?

Tas2r135 exhibits a complex expression pattern across multiple tissues, extending well beyond the oral cavity. The receptor has been detected in the respiratory tract, particularly in airways and lungs, and shows variable expression throughout the gastrointestinal tract .

Through quantitative real-time PCR (qRT-PCR) analyses, researchers have determined that of the 35 mouse Tas2rs, nine transcripts (including Tas2r135) are present in the airways, and four (including Tas2r135) in the lungs . Within the gastrointestinal tract, the expression varies significantly by region - with notable differences between the stomach, duodenum, jejunum, ileum, and colon . Unlike some other taste receptors like Tas2r131, which appears in goblet cells in the colon and Paneth cells in the ileum, Tas2r135 shows a distinct expression pattern that suggests specialized functions in different tissues .

How does Tas2r135 differ from other mouse taste receptors?

Tas2r135 (previously also known as T2r38 or Tas2r35) possesses distinctive characteristics compared to other mouse bitter taste receptors. It belongs to a specific genomic cluster with Tas2r143 and Tas2r126, with all three genes positioned adjacently without any intervening genes . This genomic arrangement facilitated their simultaneous deletion in gene knockout studies.

From a functional perspective, Tas2r135 responds to specific bitter compounds, distinct from the ligand profiles of other Tas2r family members. Its expression pattern overlaps partially with other Tas2rs in taste receptor cells (TRCs), but the degree of overlap varies, creating a complex mosaic of bitter taste reception . Unlike some other taste receptors, Tas2r135 expression extends beyond gustatory tissues to respiratory and gastrointestinal systems, suggesting broader physiological roles .

What are the unexpected findings from the Tas2r143/Tas2r135/Tas2r126 triple knockout mouse model?

The generation of Tas2r143/Tas2r135/Tas2r126 triple knockout (Tas2r TKO) mice using CRISPR/Cas9 gene-editing has revealed surprising results that challenge prevailing hypotheses about bitter taste receptor function outside the oral cavity .

Most notably, despite the common assumption that bitter taste receptors mediate bronchodilation responses to bitter compounds, the study found that:

• TAS2R135 or TAS2R126 agonists either completely failed to induce relaxation of pre-contracted airways in both wild-type and Tas2r TKO mice

• In cases where these agonists did induce relaxation, they did so dose-dependently but with identical effects in both wild-type and knockout mice

These unexpected findings strongly suggest that, contrary to previous beliefs, these specific Tas2r receptors are not required for bitter tastant-induced bronchodilation in mice. This contradicts earlier research hypotheses and necessitates a reevaluation of the mechanisms behind bitter compound-induced airway relaxation .

The study authors concluded that these results "question the involvement of TAS2Rs in bitter tastant-induced bronchodilation and argue for the need to re-examine the roles of TAS2Rs in many other systems using genetic approaches" .

How might differences in Tas2r135 expression patterns explain contradictory functional findings?

The variable expression patterns of Tas2r135 across different tissues may potentially explain some contradictory findings regarding its function. Quantitative analysis has shown that the number and expression level of Tas2r genes profoundly vary along the alimentary canal, with significant differences between the stomach and other regions .

Several factors may contribute to these contradictions:

• Cell-type specificity: Tas2r expression appears to be restricted to specific cell subtypes within tissues. For example, Tas2r131 is expressed in a subset of mucin-producing goblet cells in the colon and deep-crypt Paneth cells in the ileum . If Tas2r135 follows similar patterns of cell-specific expression, experimental approaches that analyze whole tissues might mask important functional differences.

• Regional specialization: The differential expression of Tas2r135 across gastrointestinal regions suggests region-specific functions that may respond differently to the same stimuli .

• Ligand sensitivity: Different tissues might exhibit varied threshold sensitivities to Tas2r135 agonists, potentially explaining why some functional responses occur independently of the receptor's presence.

• Compensatory mechanisms: In Tas2r TKO mice, other signaling pathways might compensate for the lack of these receptors, particularly in functions like bronchodilation that appear to proceed normally in their absence .

These variations highlight the importance of tissue-specific and cell-type-specific analyses when investigating Tas2r135 function.

What molecular mechanisms might explain Tas2r135-independent responses to bitter compounds?

The discovery that bitter tastant-induced bronchodilation proceeds normally in Tas2r143/Tas2r135/Tas2r126 knockout mice necessitates consideration of alternative molecular mechanisms. Several possibilities exist:

• Other Tas2r family members: With 35 Tas2r genes in mice, functional redundancy among family members could explain the persistence of responses. Some bitter compounds might activate multiple receptors, allowing response preservation even when specific receptors are deleted .

• Non-Tas2r GPCRs: Other G protein-coupled receptors might recognize bitter compounds, particularly at the higher concentrations often used in physiological studies.

• Direct ion channel interactions: Some bitter compounds may directly interact with ion channels, bypassing GPCR signaling entirely. This could explain the dose-dependent relaxation observed with certain agonists in both wild-type and knockout mice .

• Metabolic alterations: Bitter compounds might influence cellular metabolism through mechanisms independent of taste receptor activation, potentially affecting contractile properties of smooth muscle cells.

• Mitochondrial effects: Some bitter compounds may directly impact mitochondrial function, altering calcium handling or cellular energetics in ways that affect bronchial smooth muscle contraction.

The Tas2r TKO mouse model provides an invaluable tool for dissecting these potential mechanisms, as it allows researchers to definitively determine which responses require these receptors and which proceed through alternative pathways .

What are the optimal methods for producing recombinant mouse Tas2r135 protein?

The production of recombinant mouse Tas2r135 protein requires specialized techniques due to the challenges associated with membrane protein expression. Based on current protocols, the following methodology has proven effective:

• Expression system selection: E. coli has been successfully used for expressing full-length mouse Tas2r135 (1-312 amino acids) . The protein can be fused with an N-terminal His-tag to facilitate purification.

• Vector design: Ensure the coding sequence is codon-optimized for the expression system and includes appropriate promoter elements for efficient transcription.

• Expression conditions: Optimize temperature, induction timing, and inducer concentration to maximize protein yield while maintaining proper folding. Lower temperatures (16-25°C) often favor proper membrane protein folding.

• Extraction and purification: Use appropriate detergents for membrane protein solubilization followed by immobilized metal affinity chromatography (IMAC) to purify the His-tagged protein.

• Storage: After purification, the protein is typically lyophilized and can be stored as a powder. For working solutions, reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and add glycerol (final concentration 5-50%) for long-term storage at -20°C/-80°C .

To verify proper protein production, assess purity using SDS-PAGE (should exceed 90%) and consider functional assays to confirm the protein retains its ability to bind known ligands .

How can CRISPR/Cas9 be applied to create Tas2r knockout mouse models?

The successful generation of Tas2r143/Tas2r135/Tas2r126 triple knockout mice demonstrates an effective CRISPR/Cas9 methodology for creating Tas2r gene deletions. The approach involves:

• sgRNA design: Select guide RNAs with high specificity scores using algorithms like those at http://crispr.mit.edu/. For the Tas2r cluster deletion, researchers selected two sgRNAs with the highest scores for each gene and modified them to be 18 nucleotides long by removing two nucleotides from the end far from PAM to improve specificity .

• Vector construction: Clone the designed sgRNAs into appropriate vectors such as pSpCas9(BB)-2A-PuroA (available from Addgene) .

• Validation in cell lines: Test the genomic editing efficiency of the sgRNAs in cell lines (e.g., NIH3T3) before proceeding to animal models .

• T7 promoter addition: Add T7 promoter to sgRNA templates by PCR amplification for in vitro transcription .

• Microinjection preparation: For mouse models, prepare Cas9 mRNA and sgRNAs for microinjection into zygotes .

• Embryo transfer: After microinjection, transfer blastocysts into pseudo-pregnant females .

• Genotyping: Validate successful gene deletion through genomic PCR and sequencing .

• Functional validation: Confirm the effectiveness of the knockout through functional assays, such as taste tests with specific agonists for the deleted receptors .

This approach successfully deleted all three clustered genes simultaneously, creating a valuable model for studying their functions .

What quantitative methods can reliably detect Tas2r135 expression in tissue samples?

Due to the generally low expression levels of taste receptors in extraoral tissues, specialized quantitative methods are required to reliably detect Tas2r135 expression:

• Quantitative real-time PCR (qRT-PCR): This is the most widely used method for quantifying Tas2r mRNA expression. Due to the low expression levels of Tas2rs, higher input RNA amounts (approximately 10 ng cDNA per well) are recommended . The PCR should consist of a 45-cycle amplification with primers annealing at 60°C. Expression levels should be calculated using the 2^-ΔCt method and normalized against housekeeping genes like β-actin .

• Expression threshold determination: Transcripts with Ct values >35 are typically considered low precision and often counted as no expression, since a 35-cycle PCR reaction should amplify even a single transcript to a measurable level .

• Primer specificity: Due to sequence similarities among Tas2r family members, highly specific primers are essential. Previously validated primers for mouse Tas2rs have been described in the literature and should be used for consistency .

• Tissue preparation: Careful microdissection of specific tissue regions may be necessary to detect region-specific expression patterns, particularly in the gastrointestinal tract where expression varies significantly between regions .

• Single-cell approaches: For detecting expression in specific cell types, single-cell RNA sequencing or in situ hybridization techniques may provide more accurate information than whole-tissue analysis .

These methods have successfully identified differential expression patterns of Tas2r135 across multiple tissues and organs .

How should researchers reconcile contradictory findings about Tas2r135 function across different studies?

When facing contradictory findings about Tas2r135 function, researchers should adopt a systematic approach to reconciliation:

• Methodology comparison: Carefully analyze differences in experimental approaches between studies. The most notable contradiction involves bitter tastant-induced bronchodilation, where pharmacological studies suggested Tas2r involvement, but genetic knockout models demonstrated Tas2r-independent mechanisms . This highlights the importance of distinguishing between correlation and causation - the presence of receptors in a tissue does not necessarily indicate their involvement in all responses to potential ligands.

• Model system evaluation: Consider differences between in vitro cellular models, ex vivo tissue preparations, and in vivo animal models. Each system may reveal different aspects of Tas2r135 function due to variations in receptor expression, signaling pathway integrity, and compensatory mechanisms.

• Dose-response analysis: Examine concentration ranges used in different studies. At higher concentrations, bitter compounds may activate multiple receptors or non-receptor mechanisms, obscuring Tas2r135-specific effects.

• Genetic background consideration: Even within the same species, strain differences can affect receptor expression and function. Documenting the exact genetic background used in each study is essential for proper comparison.

• Temporal dynamics: Consider whether acute responses might differ from chronic adaptations, particularly in knockout models where compensatory mechanisms may develop over time.

• Integrative analysis: Rather than dismissing contradictory findings, researchers should attempt to develop comprehensive models that explain how these apparently discrepant results might coexist within a more complex understanding of bitter sensing mechanisms .

What statistical approaches are most appropriate for analyzing Tas2r135 expression data across tissues?

Due to the complex expression patterns of Tas2r135 across diverse tissues and the often low and variable expression levels, specialized statistical approaches are recommended:

These approaches have successfully revealed the variable expression of Tas2r135 and other family members across tissues, showing region-specific patterns that suggest specialized functions .

How can researchers distinguish between direct and indirect effects of Tas2r135 activation in physiological studies?

Differentiating between direct effects of Tas2r135 activation and indirect downstream consequences presents a significant challenge in physiological studies. The following methodological approaches can help make this distinction:

• Genetic knockout controls: The Tas2r143/Tas2r135/Tas2r126 triple knockout mouse model provides a crucial tool for determining whether observed responses to bitter compounds require these specific receptors . As demonstrated in airway relaxation studies, comparing responses in wild-type and knockout animals can reveal surprising Tas2r-independent mechanisms .

• Selective agonists/antagonists: Employing compounds with high specificity for Tas2r135 versus those affecting multiple Tas2rs or other pathways can help isolate receptor-specific effects.

• Tissue-specific knockouts: Beyond global knockout models, tissue-specific receptor deletion can help identify which effects are mediated by receptors in specific cell types.

• Time-course analysis: Direct receptor-mediated effects typically occur rapidly, while indirect effects may emerge with delay. Detailed temporal analysis of responses can help distinguish these patterns.

• Signaling pathway inhibitors: Systematic blocking of different downstream signaling components can help trace the pathway from receptor activation to physiological effect.

• Heterologous expression systems: Reconstituting Tas2r135 signaling in cell systems lacking other Tas2rs can confirm direct activation by specific compounds.

• Dose-response relationships: Different concentration thresholds for various effects may indicate distinct mechanisms. The dose-dependent but receptor-independent relaxation observed with some bitter compounds exemplifies this distinction .

The discovery that bitter tastant-induced bronchodilation proceeds normally in Tas2r TKO mice illustrates the value of these approaches in challenging assumptions about direct receptor involvement in physiological responses .

What promising therapeutic applications might emerge from Tas2r135 research?

Despite the unexpected findings regarding airway relaxation, Tas2r135 research suggests several potential therapeutic directions:

• Respiratory disorders: While the Tas2r TKO studies challenge the direct involvement of these receptors in bronchodilation , other respiratory functions might still be modulated through Tas2r135. The expression of this receptor in lung tissues suggests possible roles in immune response, mucus secretion, or epithelial function that could be therapeutically relevant.

• Gastrointestinal applications: The variable expression of Tas2r135 throughout the gastrointestinal tract suggests potential roles in digestive function, nutrient sensing, or gut immunity. Targeted activation or inhibition might influence gut motility, secretion, or inflammatory processes.

• Immune modulation: The presence of taste receptors in various immune cell populations suggests potential immunomodulatory applications. If Tas2r135 influences immune cell function, selective targeting could affect inflammatory or allergic responses.

• Cancer therapeutics: Some research has linked taste receptor mutations to cancer susceptibility . Understanding how Tas2r135 might influence cell proliferation or apoptosis could reveal novel therapeutic targets for specific cancers.

• Diagnostic applications: Expression patterns of Tas2r135, particularly if altered in disease states, might serve as diagnostic or prognostic biomarkers for certain conditions.

• Nutritional interventions: Better understanding of how bitter compounds interact with extraoral taste receptors might lead to dietary approaches for managing conditions like obesity, diabetes, or inflammatory bowel disease.

Importantly, the Tas2r TKO mouse model provides a valuable tool for rigorously testing these potential applications .

What unresolved questions persist regarding Tas2r135 signaling pathways?

Despite significant advances, several critical questions about Tas2r135 signaling remain unanswered:

• Signal transduction mechanisms: While gustatory Tas2r signaling involves G-proteins and PLCβ2 , the precise signaling cascades activated by Tas2r135 in extraoral tissues remain poorly characterized. Different cell types may employ distinct downstream pathways.

• Ligand specificity: The complete profile of compounds that activate Tas2r135 under physiological conditions has not been fully determined, particularly for endogenous ligands that might be present in extraoral tissues.

• Receptor trafficking and localization: How Tas2r135 is transported to specific cellular compartments in different tissue types, and whether this affects signaling capabilities, remains largely unknown.

• Cross-talk with other receptors: Potential interactions between Tas2r135 and other signaling systems, including other taste receptors, nutrient sensors, or immune receptors, warrant investigation.

• Transcriptional regulation: The mechanisms controlling the highly variable expression of Tas2r135 across different tissues and cell types remain to be elucidated.

• Functional redundancy: Given the inability of Tas2r143/Tas2r135/Tas2r126 deletion to affect bitter compound-induced bronchodilation , the extent of functional redundancy among taste receptors and other sensing mechanisms requires further exploration.

• Physiological roles: The fundamental question of why Tas2r135 is expressed in extraoral tissues, particularly in specific cell types like Paneth cells or subsets of respiratory epithelial cells, remains central to understanding its biological significance.

Addressing these questions will require integrated approaches combining genetic, molecular, cellular, and physiological methodologies.