Recombinant Mouse Taste receptor type 2 member 124 (Tas2r124)

Basic Research Questions

• What is Tas2r124 and what is its primary function in mouse physiology?

Tas2r124 (taste receptor type 2 member 124) is one of approximately 35 putatively functional bitter taste receptors in mice. It belongs to the T2R family of G protein-coupled receptors responsible for detecting bitter compounds. Tas2r124 is also known by several synonyms including T2R24, mGR24, mt2r50, and T2R124 .

Functionally, Tas2r124 plays a role in bitter taste signal transduction, contributing to the detection and avoidance of potentially harmful bitter compounds. In comprehensive functional analyses of mouse bitter taste receptors, Tas2r124 has been identified as one of the receptors that contribute to the mouse's ability to recognize diverse bitter toxins .

• How is Tas2r124 expressed in different mouse tissues and what is its expression pattern?

While taste receptors have traditionally been thought to be expressed exclusively in taste buds, research has demonstrated broader expression patterns. In mouse gustatory tissue, Tas2r124 shows low to moderate expression levels compared to other Tas2rs . Interestingly, Tas2r124 and Tas2r113 demonstrate highest expression in mouse testis, suggesting potential non-gustatory functions .

In taste buds, quantitative RT-PCR and in situ hybridization experiments have shown that all mouse Tas2r genes, including Tas2r124, are expressed in the epithelium of the posterior tongue, though at varying levels . Some receptors like Tas2r108, Tas2r118, and Tas2r126 show abundant expression, while others display lower expression levels .

Additionally, inflammation can affect Tas2r124 expression. In an LPS-induced inflammation model mimicking bacterial infection, Tas2r124 expression peaks on day 5 after LPS injection, suggesting its involvement in inflammatory responses .

• What is known about the structure and genomic organization of Tas2r124?

Mouse Tas2r124 is encoded by the Tas2r124 gene located on mouse chromosome 6 . The gene contains an open reading frame (ORF) of 930 bp . Like other Tas2r genes, it is located in a taste receptor gene cluster, with multiple Tas2r genes organized in proximity to each other .

The Tas2r genes, including Tas2r124, occur clustered at few syntenic chromosomal regions . The majority of bitter taste receptor genes located on mouse chromosome 6 occur in clusters of species-specific bitter taste receptor genes, which likely arose from gene duplications after the divergence of primate and rodent lineages .

In mice, the T2R family displays significant genetic polymorphism, with studies showing that haplotypes at the Tas2r locus on distal chromosome 6 vary with quinine taster status . These variations can affect the function and ligand specificity of the receptors.

• What are the known ligands for Tas2r124 and how was its activation profile determined?

The comprehensive functional analysis of mouse bitter taste receptors revealed the agonist profile of Tas2r124 through heterologous expression assays . Researchers challenged 34 mouse bitter taste receptors, including Tas2r124, with 128 prototypical bitter substances in a heterologous expression system to identify cognate compounds .

While the search results don't specify the exact ligands for Tas2r124 individually, the study identified cognate agonists for 21 of the 34 examined receptors. Mouse taste receptors, like their human counterparts, vary greatly in their breadth of tuning, ranging from very broadly to extremely narrowly tuned receptors .

The activation profile was determined using calcium imaging assays in cells expressing the recombinant receptors . This method allows researchers to measure intracellular calcium responses to various bitter compounds, which indicates receptor activation.

Advanced Research Questions

• How does inflammation affect Tas2r124 expression and function?

Inflammation significantly impacts Tas2r124 expression. In an inflammation model mimicking bacterial infection using lipopolysaccharide (LPS) treatment, researchers found that Tas2r124 expression is upregulated . Specifically, elevated expression of Tas2r124 peaked on day 5 after LPS injection, similar to the expression pattern of Tas2r108 and Trpm5 .

This inflammation-induced upregulation suggests that Tas2r124 may play a role in immune or inflammatory responses beyond its traditional role in taste perception. The study demonstrated that LPS-induced inflammation results in the expression of more than 20 mouse Tas2rs being significantly up-regulated by at least 2-fold .

Using single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), researchers found that inflammation increases the chromatin accessibility of many Tas2rs, including potentially Tas2r124, indicating an epigenetic mechanism for heightened bitter taste reception under disease conditions .

• What experimental approaches are most effective for studying Tas2r124 activation in vitro?

For in vitro studies of Tas2r124 activation, several experimental approaches have proven effective:

• Heterologous Expression Systems: Expressing recombinant Tas2r124 in cell lines such as HEK293 or CHO cells allows for controlled studies of receptor function . The receptor can be expressed using various viral vectors, including AAV (adeno-associated virus) systems .

• Calcium Imaging Assays: Since taste receptors are G protein-coupled receptors that trigger calcium release upon activation, calcium imaging using fluorescent indicators is a reliable method for measuring Tas2r124 activation . This approach enables researchers to screen multiple compounds for receptor activation.

• Co-expression with Signaling Components: For optimal function in heterologous systems, Tas2r124 should be co-expressed with taste signaling components like gustducin or other G proteins .

• Cell-based Functional Assays: Measuring downstream signaling events such as cAMP production, IP3 generation, or ERK phosphorylation can provide additional insights into receptor activation mechanisms .

• Electrophysiological Recordings: Patch-clamp recordings from cells expressing Tas2r124 and appropriate ion channels can measure receptor-induced currents directly .

• How do polymorphisms in Tas2r124 affect bitter taste perception in different mouse strains?

Mouse bitter taste receptors, including Tas2r124, display significant genetic variation between strains, which can affect taste perception . The Tas2r locus on distal chromosome 6, where Tas2r124 is located, contains haplotypes that vary with quinine taster status .

Studies using BXD/Ty recombinant inbred (RI) mouse strains mapped the major quantitative trait locus (QTL) for taste sensitivity to quinine hydrochloride (QHCl) to a ~5 Mb interval on chromosome 6 that includes 24 genes encoding T2Rs . These Tas2rs display 307 coding region single nucleotide polymorphisms (SNPs) between C57BL/6J (quinine-sensitive) and DBA/2J (quinine-insensitive) strains .

Individual RI lines contain exclusively either C57BL/6J or DBA/2J Tas2r alleles at this locus, and lines containing C57BL/6J Tas2r alleles are more sensitive to QHCl than lines containing DBA/2J alleles . While the search results don't specify Tas2r124 polymorphisms specifically, its location in this gene cluster suggests it likely contributes to strain differences in bitter taste perception.

• What is the role of Tas2r124 in non-gustatory tissues and what are the implications for broader physiological functions?

Tas2r124 shows expression in non-gustatory tissues, particularly high expression in mouse testis , suggesting functions beyond taste perception. This broader expression pattern is consistent with emerging evidence that bitter taste receptors have physiological roles outside the mouth.

In non-gustatory tissues, bitter taste receptors including potentially Tas2r124 may function as:

• Chemical Sensors: Detecting toxins or microbial products in various tissues.

• Immune Modulators: Responding to inflammatory signals and potentially regulating immune responses, as suggested by the upregulation of Tas2r124 following LPS administration .

• Smooth Muscle Regulators: Some bitter taste receptors have been shown to relax detrusor smooth muscle (DSM) in both human and mouse bladders, with activation by compounds like chloroquine, denatonium, and quinine . While Tas2r124 wasn't specifically identified in this role, the study demonstrated that multiple TAS2Rs exist in human and mouse DSM.

• Reproductive Function: The high expression in testis suggests a potential role in reproductive biology or sperm function .

These non-gustatory functions expand our understanding of bitter taste receptors as versatile chemosensors throughout the body, potentially involved in homeostatic regulation and defensive responses against harmful compounds.

Methodological Questions

• What are the recommended protocols for expressing and purifying recombinant Tas2r124?

Based on available data from commercial recombinant Tas2r124 products and typical approaches for membrane protein production:

• Expression Systems:

  • E. coli: Can be used for partial expression (typically used for cytoplasmic domains)

  • Pichia pastoris (yeast): Suitable for full-length expression of membrane proteins

  • Baculovirus: Efficient for expression of GPCRs in insect cells

  • Mammalian cells: Provides native-like post-translational modifications

• Purification Approaches:

  • Recombinant Tas2r124 is typically supplied in liquid form containing glycerol for stability

  • Purity standards for research applications aim for >90% purity

• Storage and Stability:

  • Store at -20°C for short-term, or -80°C for long-term storage

  • For working aliquots, store at 4°C for up to one week

  • Repeated freezing and thawing is not recommended

For experimental applications, viral vector systems like AAV (adeno-associated virus) are available for delivering and expressing Tas2r124 in target cells . These can be obtained with various serotypes (AAV1, AAV2, AAV3, AAV5, AAV6, AAV8, AAV9, AAV-DJ, AAV-DJ8, AAV-DJ9) and promoters (CMV or tissue-specific) .

• How can researchers effectively measure Tas2r124 activation in experimental settings?

Several methodologies are effective for measuring Tas2r124 activation:

• Calcium Imaging:

  • The most common approach for taste receptors

  • Cells expressing Tas2r124 are loaded with calcium-sensitive fluorescent dyes

  • Receptor activation triggers calcium release, measured as changes in fluorescence intensity

  • This method was successfully used to identify cognate compounds for mouse bitter taste receptors

• Electrophysiological Recording:

  • Direct measurement of taste receptor cell responses using patch-clamp techniques

  • Can be performed on isolated taste cells or heterologous expression systems

  • Provides high temporal resolution of receptor activation kinetics

• Biochemical Assays:

  • Measurement of second messengers like IP3 or cAMP

  • Analysis of downstream signaling pathway activation (e.g., ERK phosphorylation)

  • These provide indirect but quantifiable measures of receptor activation

• Cell-Based Reporter Assays:

  • Construction of cells co-expressing Tas2r124 and reporter constructs (luciferase, GFP)

  • Allows high-throughput screening of potential ligands

  • Can be designed to measure various stages of the signaling cascade

• Inhibitor Studies:

  • Using specific inhibitors like gallein (G protein βγ moiety inhibitor) or U73122 (Plcβ2 inhibitor) can help confirm Tas2r-mediated signaling pathways

When applying these techniques, researchers should consider using appropriate controls, including cells without Tas2r124 expression and known bitter receptor agonists and antagonists to validate the specificity of responses.

• What are the most effective approaches for studying Tas2r124 function in vivo?

For in vivo studies of Tas2r124 function, researchers can employ several complementary approaches:

• Genetic Modification Strategies:

  • Knockout models: Selective deletion of Tas2r124 using CRISPR/Cas9 or traditional gene targeting

  • Knockin models: Introduction of reporter genes (e.g., GFP) under the control of the Tas2r124 promoter to track expression

  • Conditional expression systems: Tissue-specific or temporally controlled expression of Tas2r124

• Behavioral Assays:

  • Brief-access taste tests to measure aversive responses to bitter compounds

  • Two-bottle preference tests to assess taste preferences

  • These assays can compare responses between wild-type and Tas2r124-modified mice

• Functional Imaging:

  • In vivo calcium imaging of taste receptor cells in transgenic mice expressing calcium indicators

  • Can be performed on isolated taste buds or the gustatory epithelium

• Gustatory Nerve Recordings:

  • Electrophysiological recordings from chorda tympani or glossopharyngeal nerves in response to taste stimuli

  • Can measure integrated responses from taste receptor cells expressing Tas2r124

• AAV-Mediated Expression:

  • Targeted delivery of Tas2r124 using AAV vectors with tissue-specific promoters

  • Allows for spatially and temporally controlled expression studies

• Inflammation Models:

  • LPS-induced inflammation to study Tas2r124 upregulation in inflammatory conditions

  • Analysis of taste function in disease models to understand physiological relevance

These approaches can be combined to provide a comprehensive understanding of Tas2r124 function in intact biological systems, linking molecular mechanisms to physiological outcomes and behavior.

• How does Tas2r124 compare functionally with other bitter taste receptors in the mouse genome?

Tas2r124 is one of approximately 35 putatively functional bitter taste receptors in mice, each with distinct expression patterns and ligand specificities . Comprehensive functional analysis reveals several important comparisons:

• Expression Levels:

  • Quantitative RT-PCR shows that Tas2r124 has moderate expression in taste tissues compared to highly expressed receptors like Tas2r108, Tas2r118, and Tas2r126

  • Tas2r124 shows notably high expression in mouse testis, alongside Tas2r113, suggesting specialized non-gustatory functions

• Tuning Properties:

  • Mouse Tas2rs vary greatly in their breadth of tuning, ranging from very broadly to extremely narrowly tuned receptors

  • Compared to humans, mice possess fewer broadly tuned receptors and more narrowly tuned receptors, suggesting greater specialization

  • While specific data on Tas2r124's tuning breadth isn't provided in the search results, the comprehensive analysis identified cognate compounds for 21 of 34 examined receptors

• Genomic Organization:

  • Tas2r124 is part of a taste receptor gene cluster on mouse chromosome 6

  • This cluster contains receptors that likely arose from gene duplications after the divergence of primate and rodent lineages

  • The genes in Tas2r clusters are strongly co-regulated, as shown by scATAC-seq studies revealing shared chromatin accessibility patterns

• Response to Inflammation:

  • Tas2r124 expression peaks on day 5 after LPS injection, similar to Tas2r108 and Trpm5

  • This expression pattern differs from other Tas2rs like Tas2r102, Tas2r110, Tas2r116, and Tas2r137, which peak on day 3 after LPS injection

This comparative analysis suggests that Tas2r124 has evolved specific roles within the mouse bitter taste receptor repertoire, potentially with specialized functions both in taste perception and in non-gustatory tissues.