Recombinant Gorilla gorilla gorilla Taste receptor type 2 member 39 (TAS2R39)

What is TAS2R39 and what is its role in primate physiology?

TAS2R39 is a member of the bitter taste receptor family (TAS2Rs), which consists of 25 functional receptors in humans. These receptors belong to the G protein-coupled receptor (GPCR) superfamily. The primary evolutionary role of TAS2R39, like other bitter taste receptors, is to detect potentially harmful substances in food and protect the organism from ingesting toxic compounds . The gene encoding TAS2R39 is located on chromosome 7 (7q34) in humans and is structurally notable for being an intron-less gene, belonging to a relatively small group of single-exon genes .

Beyond its protective function in taste perception, TAS2R39 appears to play additional physiological roles. Research indicates it may be involved in regulating enterohormones which affect food intake, though interestingly, unlike some other bitter taste receptors, TAS2R39 activation has been linked to increased food intake in some studies . In the respiratory system, evidence suggests it may participate in allergic rhinitis congestion processes and inflammatory cytokine stimulation, though more research is needed to fully elucidate these functions .

Where is TAS2R39 expressed in tissue samples?

While initially identified in oral tissues, TAS2R39 has been detected in multiple extraoral locations, making it an interesting subject for researchers investigating taste receptor function beyond the mouth. Expression studies have confirmed TAS2R39 presence in:

• Gastrointestinal tissues, particularly the colon

• Respiratory system, including bronchi and nasal mucosa

• Vascular tissues, including arteries

• Skin cells

• Nervous system, specifically in cells of the choroid plexus

• Reproductive tissues, including myometrial cells

• Immune cells, such as lung macrophages

More recent evidence from databases like the Human Protein Atlas has expanded this list to include the pancreas, spleen, brain, testes, and ovaries . It's important for researchers to note that TAS2R39 expression levels are generally low across these tissues, which presents technical challenges for detection and functional studies . When comparing oral expression specifically, TAS2R39 is one of the bitter taste receptors expressed in lower quantities compared to other TAS2R family members .

What are the known agonists and antagonists for TAS2R39?

TAS2R39 was considered an orphan receptor until 2009 when its first ligands were discovered . Since then, numerous compounds have been identified that activate this receptor, primarily plant-derived substances. The major classes of TAS2R39 agonists include:

Table 1: Major Classes of TAS2R39 Agonists

Regarding antagonists, fewer compounds have been identified. The most notable TAS2R39 antagonists come from the flavanone group:

• 6,3'-dimethoxyflavanone (strong inhibition)

• 4'-fluoro-6-methoxyflavanone (strong inhibition)

• 6-methoxyflavanone (weaker inhibition)

Structure-activity relationship studies have revealed that a methoxy group at position 6 of the A ring is mandatory for antagonistic activity, along with the absence of a double bond in the C ring .

How conserved is TAS2R39 across primate species?

Evolutionary analysis of TAS2R39 and related taste receptors provides important insights into dietary adaptation across primates. Unlike some TAS2R pseudogenes that show substantial diversification across species, TAS2R39 displays a relatively high degree of conservation among primates, suggesting its maintained functional importance throughout evolution .

Comparative genomic studies between human TAS2R39 and its homologs in other primates like chimpanzee, gorilla, orangutan, gibbon, and more distant relatives like mouse lemur, reveal conservation of key functional domains . This conservation contrasts with the pattern seen in some other bitter taste receptor genes, which have undergone lineage-specific pseudogenization events .

The higher conservation of TAS2R39 compared to some other taste receptor genes might reflect the importance of detecting specific classes of bitter compounds that remained relevant to primate diets throughout evolutionary history. This conservation makes gorilla TAS2R39 particularly valuable for comparative studies with human TAS2R39 .

What are the methodological challenges in functional characterization of TAS2R39?

Researchers working with TAS2R39 face several significant technical challenges:

How do structural modifications of flavonoids impact TAS2R39 activation?

Structure-activity relationship studies have revealed several critical features that determine how flavonoids and isoflavones interact with TAS2R39:

• Glycosylation effects: While glycosylation inhibits the activation of TAS2R14, TAS2R39 activation is preserved, though often requiring higher ligand concentrations . This differential response to glycosylation provides a useful tool for differentiating between these receptors in functional assays.

• C-ring structural requirements: Alterations to the C-ring skeletal structure of isoflavonoids generally do not prevent TAS2R39 activation, though such modifications can affect both potency and efficacy . This tolerance to C-ring modifications suggests a relatively accommodating binding pocket.

• Hydroxyl group importance: For compounds to function as TAS2R39 ligands, substitutes—preferably hydroxyl groups—are obligatory . The position of these hydroxyl groups contributes significantly to binding affinity.

• Galloyl group significance: For catechins, the presence of a galloyl group creates important bonds for ligand-receptor interaction, as demonstrated by the higher affinity of epicatechin gallate and epigallocatechin gallate compared to their non-galloylated counterparts .

• Antagonist requirements: For flavanones to function as TAS2R39 antagonists, a methoxy group at position 6 of the A ring is mandatory, and the C ring must lack a double bond . This specific structural requirement provides insight into the receptor's binding mechanisms.

These structure-activity relationships offer critical insights for researchers designing selective agonists or antagonists and provide fundamental knowledge about the receptor's binding site characteristics.

What is the relationship between TAS2R39 activation and hormonal regulation?

Emerging evidence suggests TAS2R39 plays a complex role in hormonal regulation, particularly regarding enterohormones that control appetite and food intake:

• Protein YY (PYY) modulation: Specific activation of TAS2R39 has been shown to increase PYY secretion, though interestingly, this does not appear to decrease food intake as might be expected . This contrasts with the effect of other bitter taste receptors like TAS2R5, which increases GLP-1 secretion and decreases food intake in animal models.

• Increased food intake paradox: Contrary to what might be predicted for bitter taste receptors, which evolved as warning systems against potentially toxic substances, a combination of ligands preferential to TAS2R39 has been observed to increase food intake in experimental models . This suggests TAS2R39 may have evolved specialized functions distinct from the general aversive response to bitter compounds.

• Tissue-specific effects: The hormonal effects of TAS2R39 likely vary by tissue, with different signaling pathways and physiological outcomes in the gut compared to other locations. Researchers must consider tissue context when interpreting TAS2R39 activation studies.

This complex relationship between TAS2R39 activation and hormonal regulation presents an intriguing area for future research, particularly given the current interest in targeting taste receptors for metabolic disorders and appetite regulation.

How can researchers distinguish between TAS2R39 and TAS2R14 activation given their overlapping ligand profiles?

The significant overlap in ligand specificity between TAS2R39 and TAS2R14 presents a methodological challenge for researchers. Several strategies can be employed to differentiate between activation of these receptors:

• Selective agonists: Use compounds that selectively activate TAS2R39 but not TAS2R14, such as:

  • Epigallocatechin (from green tea)

  • Acetylgenistin, genistin, glycitin, and malonyl genistin (from soybeans)

  • Acacetin, 5,2′-dihydroxyflavone, gardenin A, genkwanin, gossypetin, 6-methoxyflavonol, and 4′-hydroxyflavanone

• Glycosylation test: Utilize the differential response to glycosylation, where glycosylated compounds maintain TAS2R39 activation but lose TAS2R14 activation .

• Selective antagonists: Apply the identified selective antagonists of TAS2R39 (6,3'-dimethoxyflavanone and 4'-fluoro-6-methoxyflavanone) to block responses and determine the contribution of each receptor .

• Receptor knockout models: Develop cell lines with CRISPR-Cas9 mediated knockout of either TAS2R39 or TAS2R14 to isolate receptor-specific responses.

• Dose-response analysis: Careful dose-response characterization can help distinguish between receptors, as many compounds show different potencies for TAS2R39 versus TAS2R14.

• Computational modeling: Molecular docking and binding simulations can predict ligand specificity and help design experiments to differentiate between the receptors.

These approaches, particularly when used in combination, allow researchers to parse the specific contributions of TAS2R39 versus TAS2R14 in experimental systems.

What are the optimal storage and handling conditions for recombinant Gorilla gorilla gorilla TAS2R39?

Proper storage and handling of recombinant TAS2R39 protein is critical for maintaining activity in experimental applications. Based on available product information, researchers should observe the following recommendations:

• Storage temperature: Store recombinant TAS2R39 at -20°C for regular use, or at -80°C for extended storage periods .

• Buffer composition: The protein is typically provided in a Tris-based buffer with 50% glycerol, optimized specifically for TAS2R39 stability .

• Freeze-thaw considerations: Repeated freezing and thawing should be avoided as this can lead to protein denaturation and loss of function . Instead, prepare small working aliquots during initial thawing.

• Working aliquots: Store working aliquots at 4°C for up to one week to minimize freeze-thaw cycles .

• Concentration considerations: The protein is typically supplied at a specific concentration (e.g., 50 μg per vial), and researchers should calculate dilutions carefully to maintain consistent experimental conditions .

• Temperature transitions: Allow frozen protein to thaw gradually on ice rather than at room temperature to maintain structural integrity.

• Handling precautions: As with all recombinant proteins, use sterile technique when handling to prevent contamination, and avoid vigorous vortexing which can cause protein denaturation.

Following these guidelines will help ensure experimental reproducibility when working with recombinant TAS2R39 protein.

What expression systems are recommended for producing functional TAS2R39?

The successful expression of functional TAS2R39 requires careful consideration of the expression system. Based on current research practices with bitter taste receptors, the following systems are recommended:

• Mammalian cell lines: HEK293 or HEK293T cells are commonly used for GPCR expression due to their human-derived cellular machinery for proper protein folding and post-translational modifications. These cells can be transiently or stably transfected with TAS2R39 expression constructs.

• Inducible expression systems: Given that overexpression of some GPCRs can be toxic to cells, inducible expression systems like Tet-On may be advantageous for TAS2R39 production.

• Fusion constructs: To improve expression, trafficking, and detection, TAS2R39 can be expressed as a fusion protein with tags such as:

  • Rhodopsin or 5-HT3 receptor N-terminal sequences to enhance membrane targeting

  • Fluorescent proteins (e.g., GFP) for visualization and localization studies

  • Epitope tags (e.g., FLAG, HA) for antibody detection and purification

• Co-expression considerations: For functional studies, co-expression with appropriate G proteins (typically Gα16gust44, a chimeric G protein) is essential to couple receptor activation to measurable calcium responses.

• Viral expression systems: For difficult-to-transfect cells or for in vivo studies, adenoviral or lentiviral expression systems carrying the TAS2R39 gene can provide efficient delivery and expression.

When expressing gorilla TAS2R39 specifically, researchers should consider codon optimization for the expression system of choice, as codon usage bias can significantly affect protein production levels.

What functional assays can be used to measure TAS2R39 activation?

Several functional assays can be employed to measure TAS2R39 activation in response to potential ligands:

• Calcium mobilization assays: The most common approach for bitter taste receptor functional studies involves measuring intracellular calcium release using:

  • Fluorescent calcium indicators (Fluo-4 AM, Fura-2 AM)

  • Calcium-sensitive bioluminescent proteins (aequorin)

  • Genetically encoded calcium indicators (GCaMP)

  These assays are typically performed in heterologous expression systems co-expressing TAS2R39 with a promiscuous G protein to couple receptor activation to calcium release.

• cAMP assays: While bitter taste receptors primarily couple to calcium signaling, measuring changes in cAMP levels can provide complementary information about receptor coupling efficiency.

• β-arrestin recruitment: BRET or FRET-based assays measuring β-arrestin recruitment can assess receptor activation and subsequent desensitization.

• Receptor internalization: Fluorescently tagged TAS2R39 can be monitored for internalization following agonist exposure using confocal microscopy or flow cytometry.

• Electrophysiological methods: For certain cell types, particularly neurons, patch-clamp recordings can measure electrical responses following TAS2R39 activation.

• Downstream signaling assays: Depending on the cell type, measuring activation of ERK1/2, NFAT, or other downstream effectors can provide insight into TAS2R39 signaling pathways.

• Label-free systems: Technologies such as dynamic mass redistribution (DMR) can monitor receptor activation without the need for artificial labels or reporters.

For all these assays, appropriate controls are essential, including positive controls (known agonists), negative controls (vehicle), and specificity controls (selective antagonists or receptor knockout models).

What approaches are effective for studying TAS2R39 expression and function in extraoral tissues?

Investigating TAS2R39 in extraoral tissues presents unique challenges due to low expression levels and complex cellular environments. Effective approaches include:

• Gene expression analysis:

  • Quantitative RT-PCR with validated primers specific for TAS2R39

  • RNAscope in situ hybridization for spatial resolution of mRNA expression

  • Single-cell RNA sequencing to identify specific cell populations expressing TAS2R39

• Protein detection:

  • Immunohistochemistry or immunofluorescence with validated antibodies

  • Western blotting of membrane fractions

  • Proximity ligation assay for detecting protein-protein interactions in tissue

• Functional studies in primary cells:

  • Isolation of primary cells from tissues of interest

  • Calcium imaging in response to TAS2R39 agonists

  • siRNA or CRISPR-mediated knockdown to confirm specificity

• Ex vivo tissue preparations:

  • Organ bath studies with TAS2R39 agonists and antagonists

  • Precision-cut tissue slices for maintaining tissue architecture

  • Measurement of tissue-specific responses (e.g., contractility, secretion)

• In vivo approaches:

  • Tissue-specific conditional knockout models

  • Administration of selective TAS2R39 agonists and measurement of physiological responses

  • In vivo calcium imaging in genetically modified animals expressing calcium indicators in TAS2R39-expressing cells

When studying extraoral TAS2R39 expression specifically in gorilla tissues, ethical considerations and sample availability become significant limitations, making comparative genomic approaches and in vitro models particularly valuable.

How can evolutionary analysis inform TAS2R39 research?

Evolutionary analysis provides valuable context for TAS2R39 research, helping to identify conserved functional regions and species-specific adaptations:

• Comparative genomics approaches:

  • Sequence alignment of TAS2R39 across primates and other mammals to identify conserved domains

  • Analysis of selection pressure (dN/dS ratios) to detect regions under positive or purifying selection

  • Examination of species-specific polymorphisms that may correlate with dietary adaptations

• Ancestral sequence reconstruction:

  • Computational methods to infer ancestral TAS2R39 sequences

  • Functional testing of reconstructed ancestral receptors to trace the evolution of ligand specificity

• Population genetics analysis:

  • Study of TAS2R39 polymorphisms within species, particularly humans and great apes

  • Correlation of genetic variants with phenotypic differences in bitter taste perception

  • Identification of potential signatures of selection in different populations

• Structural biology integration:

  • Homology modeling of TAS2R39 from different species

  • Molecular docking studies to compare ligand binding across species variants

  • Prediction of functional consequences of species-specific amino acid substitutions

• Ecological correlation:

  • Analysis of TAS2R39 sequence in relation to species' dietary patterns

  • Investigation of potential co-evolution with plant defensive compounds

By comparing TAS2R39 from Gorilla gorilla gorilla with human and other primate variants, researchers can gain insights into the functional significance of specific receptor domains and potentially identify selective pressures related to dietary adaptations .