Recombinant Pan troglodytes Taste receptor type 2 member 31 (TAS2R31)

What is TAS2R31 and what is its primary biological function?

TAS2R31 belongs to the TAS2R family of G protein-coupled receptors that function as bitter taste receptors in vertebrates. Its primary function is bitter taste perception initiated by responding to agonists and triggering depolarization of taste bud cells . TAS2R31 is part of a five-member subfamily (TAS2R30-46) that can respond to a diverse constellation of compounds including artificial sweeteners like saccharin and acesulfame K, which have aversive bitter aftertastes . Recent research has revealed that TAS2R31 and other bitter taste receptors are also expressed in extra-oral tissues, particularly along the gastrointestinal tract, where they play roles in immune response regulation and metabolic functions .

What are the recommended storage and handling conditions for recombinant TAS2R31?

Recombinant Pan troglodytes TAS2R31 is typically supplied in liquid form containing glycerol . For optimal stability and activity, the protein should be stored at -20°C, and for extended storage, it is recommended to conserve it at -20°C or -80°C . Repeated freezing and thawing is not recommended as it may lead to protein degradation and loss of activity. For short-term use, working aliquots can be stored at 4°C for up to one week . When handling the protein, it's advisable to minimize exposure to room temperature and avoid contamination by using sterile techniques.

What are the common synonyms and related genes for TAS2R31?

TAS2R31 is known by several synonyms in the scientific literature:

• Taste receptor type 2 member 31

• T2R31

• Taste receptor type 2 member 44 (TAS2R44)

• T2R44

The gene nomenclature can sometimes be confusing due to the evolution of naming conventions. The primary gene name is TAS2R31, with TAS2R44 noted as a gene name synonym . Other gene names that may be encountered include TAS2R31, TAS2R44, T2R31, and T2R44 . This variation in nomenclature reflects the evolutionary relationship between these closely related receptor genes and the historical development of their classification.

How does TAS2R31 fit into the evolutionary context of the TAS2R receptor family?

TAS2R31 is part of a large gene family that has undergone significant evolutionary changes across vertebrate lineages. Recent comprehensive phylogenomic analyses have identified 9,291 TAS2Rs from 661 vertebrate genomes, revealing interesting evolutionary patterns . While most vertebrate lineages maintain a relatively stable number of TAS2R genes, amphibians (particularly frogs and salamanders) have experienced a remarkable expansion of TAS2R genes, with approximately 10-fold more receptors than other vertebrates .

TAS2R genes are frequently found in clusters in vertebrate genomes (82% of genes are clustered), which facilitates gene duplication and diversification . In most species with TAS2R genes, these clusters exist in 62% of species, or 76% of species with two or more genes . Amphibians tend to have more clusters and more genes per cluster compared to other vertebrates . The clustering and genomic organization of TAS2R genes appear to play significant roles in their evolutionary dynamics, with both the addition of genes to existing clusters and the creation of new clusters contributing to the expansion of the TAS2R gene family .

What methods are used to assess TAS2R31 receptor function in vitro?

Several methodologies are employed to evaluate TAS2R31 receptor function in vitro:

Calcium Imaging Assays: This technique is commonly used to measure receptor activation by monitoring intracellular calcium flux. TAS2R31-expressing cells are loaded with calcium-sensitive fluorescent dyes, and changes in fluorescence intensity are measured upon agonist application. This method allows for quantitative assessment of receptor activation kinetics and dose-response relationships .

Receptor Expression Systems: For functional characterization, TAS2R31 is typically heterologously expressed in cell lines such as HEK293T cells. The receptor gene is often coupled with a chimeric G protein to enhance signaling efficiency and specificity .

Mutational Analysis: To study structure-function relationships, site-directed mutagenesis is employed to generate receptor variants with specific amino acid substitutions. These mutants are then functionally characterized to determine how sequence variations affect receptor response to bitter compounds .

Ligand Screening: High-throughput screening approaches using libraries of bitter compounds can identify specific agonists for TAS2R31. This typically involves measuring receptor activation in response to a panel of potential ligands at various concentrations .

A comprehensive functional assessment often combines these approaches to characterize receptor pharmacology, including parameters such as EC50 values (effective concentration that produces 50% of maximum response), maximum response amplitudes, and activation/deactivation kinetics.

How does TAS2R31 sequence variation affect bitter taste perception?

Sequence variation in TAS2R31 has been directly linked to differences in bitter taste perception, particularly for artificial sweeteners. Genomic studies have revealed extensive diversity in TAS2R genes, including 34 missense mutations and two nonsense mutations in the TAS2R30-46 subfamily among 60 Caucasian subjects .

Functional analysis of TAS2R31 variants has demonstrated that specific mutations can significantly alter receptor responses to bitter compounds. For instance, in vitro assays have confirmed the functional importance of four TAS2R31 mutations, which had independent effects on receptor response to saccharin and acesulfame K . These findings establish a direct link between genetic variation in TAS2R31 and individual differences in perception of the bitter aftertaste of these artificial sweeteners.

The high linkage disequilibrium (LD) spanning functionally distinct TAS2R loci further complicates the phenotypic effects of TAS2R31 variants. This genetic architecture predicts that bitter taste responses to many compounds will be strongly correlated even when they are mediated by different genes . This may explain why individuals who are sensitive to one bitter compound often show sensitivity to structurally unrelated bitter substances.

What are the implications of TAS2R31 expression in extra-oral tissues?

The discovery of TAS2R31 and other bitter taste receptors in extra-oral tissues, particularly throughout the gastrointestinal tract, has significantly expanded our understanding of their physiological roles beyond taste perception. Research has revealed that these receptors contribute to several important functions:

Immune Regulation: Extra-oral TAS2Rs play crucial roles in regulating innate immune responses. Activation of intestinal bitter taste receptors has been associated with modulation of inflammatory cytokines .

Metabolic Regulation: TAS2Rs in the gut have been implicated in the regulation of metabolic functions. Studies have demonstrated that activation of certain TAS2Rs can influence enteroendocrine hormone release and bile acid metabolism, potentially ameliorating features of metabolic syndrome .

Therapeutic Potential: Emerging evidence suggests that targeting extra-oral bitter taste receptors may have therapeutic applications. Preclinical studies with TAS2R agonists like ARD-101 have shown reduced food intake, decreased body weight, and downregulation of inflammatory cytokines . Such compounds are currently being evaluated in clinical trials for metabolic and inflammatory disorders .

Distribution Patterns: The expression profile of TAS2Rs along the gastrointestinal tract is complex, with noticeable differences in distribution and abundance in different parts of the GI system . This differential expression suggests specialized functions in various segments of the digestive tract.

Understanding the extra-oral roles of TAS2R31 and related receptors opens new avenues for research into metabolic diseases, immune disorders, and novel therapeutic approaches targeting these receptors.

How do genomic organization and clustering affect TAS2R31 evolution?

The genomic organization of TAS2R31 and related genes significantly influences their evolutionary dynamics:

Clustered Organization: TAS2R genes, including TAS2R31, are frequently found in genomic clusters. These clusters exist in 62% of species (or 76% of species with two or more genes), and around 82% of TAS2R genes across species are clustered . This clustered organization facilitates non-allelic homologous recombination, which can lead to gene duplication, loss, and conversion events.

Cluster Size and Distribution: Both the number of clusters and the average number of genes per cluster show strong positive associations with total TAS2R count across vertebrates. Statistical analysis shows significant correlations (for number of clusters: β = 1.1, t = 50.7, p <2e-16; for average genes per cluster: β = 0.88, t = 51.04, p <2e-16; full model adj. r² = 0.92) .

Chromosomal Location: The position of TAS2R genes along chromosomes also affects their evolutionary rates. TAS2R genes are generally closer to the ends of chromosomes (telomeres) in amphibians compared to non-amphibian species (mean distance to chromosome end: 0.148 vs. 0.202, Welch's two-sample t-test: t = −15.25, p = 3.2e-51) . This telomeric location may facilitate higher recombination rates, as clustered genes located closer to telomeres have a higher chance of being duplicated .

Repeat Elements: The regions surrounding TAS2R genes show different patterns of repeat elements that may influence recombination rates. In non-amphibian species, regions near TAS2Rs have significant enrichments of LINEs (p = 0.0073) and loss of SINEs (p = 0.049) . Since LINEs are associated with low recombination rates while SINEs enhance recombination, this may provide a mechanism affecting recombination rates for these gene clusters.

These genomic features collectively contribute to the rapid evolution and diversification of TAS2R gene families, including TAS2R31, through mechanisms of gene duplication, deletion, and conversion.

What potential roles does TAS2R31 play in metabolic disease and therapeutic applications?

Recent research has uncovered intriguing connections between TAS2R31 (and related bitter taste receptors) and metabolic regulation, suggesting potential therapeutic applications:

Metabolic Syndrome Regulation: Activation of TAS2Rs in the gut can remodel enteroendocrine hormone release and bile acid metabolism, potentially ameliorating multiple features of metabolic syndrome . This suggests that TAS2R31 may be involved in metabolic homeostasis beyond its role in taste perception.

Therapeutic Compound Development: Preclinical studies with TAS2R agonists have shown promising results. For example, oral administration of ARD-101, a potential TAS2R agonist, led to reduced food intake, decreased body weight, and downregulated inflammatory cytokines in animal models of obesity and inflammation . These effects appear to be mediated through interactions at the gut level.

Clinical Trials: The potential of compounds targeting TAS2Rs is currently being evaluated in phase 2 clinical trials involving patients with metabolic and inflammatory disorders . This represents a translation of basic research on bitter taste receptors into potential clinical applications.

Research Challenges: Despite significant progress, the precise mechanisms linking TAS2R activation and metabolic syndrome remain incompletely understood . Additional research is needed to fully elucidate the signaling pathways and physiological responses mediated by TAS2R31 and related receptors in metabolic tissues.

These findings highlight the emerging potential of TAS2R31 as a target for therapeutic interventions in metabolic and inflammatory disorders, expanding its significance well beyond its classical role in bitter taste perception.

What approaches can be used to distinguish functions of closely related TAS2R family members?

Distinguishing the specific functions of closely related TAS2R family members presents significant challenges due to their sequence similarity and overlapping ligand specificities. Several methodological approaches can be employed to address this challenge:

Selective Receptor Antagonists: Developing and utilizing compounds that selectively block specific TAS2R subtypes allows researchers to isolate the contribution of individual receptors to observed responses. This pharmacological approach requires extensive screening to identify compounds with high selectivity.

Gene Editing Techniques: CRISPR-Cas9 and other gene editing tools can be used to create knockout or knockdown models for specific TAS2R genes. By selectively eliminating expression of individual receptors, researchers can determine their unique contributions to cellular responses.

Receptor-Specific Antibodies: Generating antibodies with high selectivity for specific TAS2R subtypes enables localization studies to determine tissue-specific expression patterns. This can help identify potential functional specialization based on differential expression.

Heterologous Expression Systems: Expressing individual TAS2R receptors in cell lines that lack endogenous bitter taste receptors allows for isolated functional characterization. Systematic screening of potential ligands against individually expressed receptors can establish receptor-specific activation profiles.

Bioinformatic Analysis of Sequence-Function Relationships: Comparative analysis of sequence variations between closely related TAS2Rs, combined with functional data, can identify key amino acid residues that determine ligand specificity and receptor function.

By combining these approaches, researchers can develop comprehensive profiles of individual TAS2R receptors and distinguish their specific roles in various physiological contexts.

How can researchers optimize expression and purification of recombinant TAS2R31 for structural studies?

Optimizing expression and purification of recombinant TAS2R31 for structural studies presents unique challenges due to its nature as a membrane protein. The following methodological considerations are critical:

Expression System Selection: While E. coli is commonly used for initial production , membrane proteins often require eukaryotic expression systems for proper folding and post-translational modifications. Insect cell systems (Sf9, High Five) or mammalian cells (HEK293, CHO) may provide better yields of properly folded receptor.

Fusion Tags and Constructs: Incorporating stabilizing fusion partners (such as T4 lysozyme, BRIL, or thermostabilized GFP) can enhance expression and stability. Additionally, truncating flexible regions while preserving the core structure may improve crystallization propensity.

Detergent Screening: Systematic screening of detergents is crucial for extracting and maintaining the native conformation of TAS2R31. Mild detergents like DDM, LMNG, or GDN are often effective for G protein-coupled receptors.

Thermostability Engineering: Introducing point mutations to increase thermostability without affecting function can significantly improve the success rate of structural studies. Alanine scanning or directed evolution approaches may identify stabilizing mutations.

Lipid Nanodisc Reconstitution: Incorporating purified receptor into lipid nanodiscs provides a more native-like membrane environment than detergent micelles, potentially preserving functional conformations for structural analysis.

Crystallization and Cryo-EM Considerations: For X-ray crystallography, leveraging lipidic cubic phase crystallization has proven successful for many GPCRs. Alternatively, single-particle cryo-electron microscopy (cryo-EM) may be employed, particularly for complexes with signaling partners.

These methodological approaches must be systematically optimized for TAS2R31, as conditions that work for one membrane protein may not be directly transferable to another, even within the same receptor family.

What are the emerging areas of investigation for TAS2R31 and related bitter taste receptors?

Several promising research directions are emerging for TAS2R31 and related bitter taste receptors:

Systems Biology Approaches: Integrating transcriptomic, proteomic, and metabolomic data to understand how TAS2R31 functions within broader cellular networks could reveal new insights into its regulatory roles and physiological significance.

Single-Cell Analysis: Applying single-cell RNA sequencing and functional imaging techniques to characterize cell-specific expression and activation patterns of TAS2R31 in various tissues could uncover specialized functions in specific cell populations.

Microbiome Interactions: Investigating how TAS2R31 and other taste receptors in the gut interact with the intestinal microbiome may reveal new mechanisms by which these receptors influence metabolism and immunity through microbiota-host communication.

Structural Biology: Determining the three-dimensional structure of TAS2R31 and related receptors would significantly advance our understanding of ligand binding specificity and activation mechanisms, facilitating structure-based drug design for therapeutic applications.

Personalized Medicine Applications: Exploring how genetic variations in TAS2R31 correlate with metabolic phenotypes and response to treatments could lead to personalized therapeutic approaches for metabolic disorders based on individual receptor profiles.

Developmental Regulation: Studying the temporal expression patterns of TAS2R31 during development may reveal previously unappreciated roles in embryonic and postnatal development of sensory and metabolic systems.

These emerging research areas hold promise for expanding our understanding of TAS2R31 beyond its classical role in bitter taste perception, potentially leading to novel therapeutic strategies for metabolic and inflammatory disorders.