Bitter Taste Receptors
The molecular basis of bitter taste detection is thought to have evolved for more practical purposes. Plants often produce poisonous secondary metabolites in order to protect themselves from ingestion by predators, and as such mammals, birds, and other animals have evolved the ability to detect which plants and plant material are and are not safe to consume. Bitter taste is detected in humans by ~25 members of the bitter taste receptor (Tas2R) subfamily of G-protein coupled receptors (GPCRs). Since a large proportion of poisonous compounds produced by plants are bitter in taste, the ability to sense bitter taste proved to be advantageous in avoiding harm. However, the correlation between toxicity and bitterness is complicated. Many bitter compounds (such as those found in coffee, beer, and broccoli) are not toxic at concentrations typically consumed, while others even present health benefits such as chemoprotection.
The molecular players responsible for the perception of bitter taste had not been known or understood until the early 2000s: until then it had been hypothesized that there must exist a large family of genes whose products were able to detect bitter compounds, as the chemical entities responsible for evoking bitter taste are structurally diverse (Adler et al. 2000). The first biochemical evidence to prove the existence of these molecules came from Chandrashekar et al. (2000), who used a heterologous expression system to express three candidate taste receptors, mTas2r5 and mTas2r8 from mice, and hTas2R4 from humans, in modified HEK-293 cells. They showed that cells expressing both the mTas2r5 receptor and Gα15 responded specifically to cycloheximide, a compound that is exceptionally aversive to mice, through a G-protein-coupled response resulting in the release of endogenous Ca2+ stores at concentrations similar to the sensitivity of cycloheximide-induced aversion in live mice. Additionally, by assaying a selection of 11 human Tas2Rs, hTas2R4 was found to respond significantly to high levels of denatonium and 6-n-propyl-2-thiouracil and was found to be 70% identical in sequence to the mouse bitter receptor mTas2r8. To determine whether mTas2r5 receptor polymorphisms had any effect on ligand binding or corresponded to the Cyx cycloheximide-tasting locus, three previously characterized cycloheximide taster strain and one nontaster strain mTas2r5 sequences were isolated and compared to the mTas2r5 taster and nontaster strains DBA/2J and C57BL/6 (Chandrashekar et al. 2000). It was found that all the taster strains had the same mTas2r5 alleles as the DBA/2J strain and that all the nontasters harbored the same alleles as those found in the C57BL/6 strain; additionally, the nontaster strains exhibited a change in cycloheximide sensitivity compared to the taster strains, indicating that mTas2r5 is indeed a detector of the bitter ligand cycloheximide. Coupled with the finding that mTas2r5 associates with the taste transduction G-protein gustducin, the authors demonstrated that the Tas2R family of GPCRs is essential in the transduction of bitter taste stimuli.
Evolution, Genetic Regulation, and Location
The dynamic evolution of bitter taste receptors has been documented in the past using comparative genomics and phylogeny-based methods to detect gains and losses across vertebrate, teleost fish, cetacean, and other species. Feng et al. (2014) found evidence of massive losses of Tas2R and Tas1R genes in their analysis of six toothed-whale species and five baleen species, such that all three members of the Tas1R gene family and 10 Tas2R receptor genes were pseudogenized, with the exception of Tas2R16 in three baleen whale species. Massive pseudogenization or absence of bitter taste receptor genes has also been found in teleost fish (Picone et al. 2014). These discoveries are in accordance with the belief that vertebrate bitter taste receptor gene evolution was heavily influenced by environmental factors, namely due to the changing feeding behaviors of animals (Dong et al. 2009). Herbivorous species of animals would most likely encode and express the largest number of bitter taste receptors as their diets consist of many more bitter molecule-containing foods than omnivores or carnivores. As for the major gene losses in aquatic species such as whales and fish several other reasons have been presented, among them (a) that the high concentration of sodium in the ocean would conceal any bitter tastant that could present itself to taste receptor cells in the oral cavity and (b) that engulfing food whole may have rendered their taste perceiving machinery obsolete (Feng et al. 2014). In contrast, lobe-finned fishes such as the coelacanth species Latimeria chalumnae have been the only fish species to date to exhibit a large collection of bitter taste receptors (58) which closer resemble those of vertebrates more than teleost fish. Interestingly, coelacanths not only have the largest repertoire of bitter taste receptors among fish but also among vertebrates, with frogs (49), mice (~36), and humans (~30) rounding out the top four (Picone et al. 2014).
The human bitter taste receptor family consists of 43 Tas2R genes (around 40% of which are pseudogenes), the majority of which are found in two multigene clusters; 10 gene sequences on chromosome 7, and 20 on chromosome 12, while only Tas2R1 is encoded on chromosome 5 (Bachmanov and Beauchamp 2007). Interestingly, the organization of mTas2r sequences in the mouse genome very closely resembles that of humans, where two clusters of mTas2r genes of 10 and 29 sequences are encoded on chromosome 6. The conservation of these motifs has led to the suggestion that the arrangement of Tas2R gene clusters was determined prior to the divergence of primates (Andres-Barquin and Conte 2004).
The Tas2R family of receptors display a low degree of sequence similarity with Class A/rhodopsin-like GPCRs (Di Pizio et al. 2016). As such, they were classified with the frizzled family of GPCRs; however, in most studies they are reported as distant relatives of classical Class A GPCRs. In contrast to the TAS1R family, all Tas2R genes contain no spliceosomal introns. Additionally, Tas2R gene products exhibit short N-terminal extracellular domains and as such are much shorter in length than their Tas1R counterparts (300 amino acids versus ~800 amino acids). Tas2R genes, as with Tas1Rs and salt receptors (epithelial sodium channels or ENaCs), are highly conserved across vertebrates; mouse taste receptor genes in some cases share at least 70% sequence identity with their human counterparts (Chandrashekar et al. 2000).
Oral Bitter Taste Perception
Tas2Rs are almost without exception expressed in α-gustducin containing cells, a Gα protein implicated in the transduction of bitter taste signals (Andres-Barquin and Conte 2004). The involvement of α-gustducin in the transduction of bitter taste signals is crucial for full activation and signaling to occur, as demonstrated through the use of mouse knockout models (Wong et al. 1999). However, lacking the α-gustducin subunit does not limit the potential for GPCR activation as bitter taste potentiation may still occur with the help of other Gα protein subunits expressed in TRCs. This finding has raised the question of whether or not α-gustducin is simply favored due to relative abundance in TRCs, whether different Tas2Rs are selective for particular G-protein subunits to become fully activated, or other biological factors may play a role in their coupling to bitter taste receptors (Behrens and Meyerhof 2009).
Co-localization and mouse knockout studies were performed early on in the elucidation of bitter taste receptor signaling in order to determine the factors necessary for proper signal transduction of bitter taste stimuli. For signaling to occur, the formation of a heterotrimeric G-protein complex between α-gustducin and Gβ3 and Gγ13 occurs the most often, while some trimers are comprised of Gβ1 subunits (Behrens and Meyerhof 2009). Tas2R stimulation and activation of the G-protein heterotrimer leads to the activation of PLCβ2, whose induction causes an increase in cellular levels of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) via the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) (Behrens and Meyerhof 2009). An IP3-dependent increase in intracellular calcium induces the activation of the TRPM5 transient receptor potential channel, whose induction generates a depolarization across the TRC cell membrane. An action potential is then reached and the resulting neurotransmitters released act on taste nerves enervating to the brain. Additionally, the α-gustducin subunit activates phosphodiesterase (PDE) resulting in a decrease in cellular cNMPs, such as cAMP and cGMP; however, the exact reason for which these changes in cNMPs occur is not well known (Behrens and Meyerhof 2009).
Tas2Rs Beyond the Oral Cavity
Although dubbed “bitter taste” receptors, the Tas2R family of GPCRs have in recent years been discovered in a multitude of tissues outside of the oral cavity including the gut, the airway, the heart, the thyroid, the brain, and breast epithelium, among a list of tissues that is rapidly expanding (see Avau and Depoortere 2016; Shaik et al. 2016), where sensing bitterness would not necessarily be associated with poison sensing.
Taste receptors have been shown to reside on the surface of gut endocrine cells and display bitter tastant-mediated contractility (Avau et al. 2015). The bitter tastant denatonium benzoate induced contractions in human gastric smooth muscle through intracellular calcium release and extracellular calcium influx, while intra-gastric denatonium administration caused gastric emptying delay. Additionally, healthy human volunteers who were subjected to intra-gastric denatonium benzoate administration displayed increased hunger satiation and a decrease in tolerance of nutrient volume, suggesting that Tas2Rs are involved in a protective negative feedback loop in the gut, whereby ingestion of bitter and potentially toxic compound causes a decrease in alimentary intake (Avau et al. 2015).
Tas2Rs have been documented on both solitary chemosensory cells and ciliated cells in the airway, and display interesting roles in both innate airway immunity and cell autonomous responses. PLC-dependent calcium release and trigeminal nerve stimulation was observed when a broad-acting stimulant of mTas2rs (denatonium benzoate) was applied to isolated mouse SCCs from nasal epithelium, as was a cessation of breathing upon application to anaesthetized rats (Finger et al. 2003). A different response was observed in human SCCs responsive to denatonium benzoate expressing the bitter taste receptor Tas2R47, where bitter agonist stimulation lead to a “calcium wave” which proceeded through gap junctions to other epithelial cells in the nose and stimulated release of antimicrobial peptides involved in preventing increased bacterial colonization (Lee et al. 2014).
In addition to their role in innate immunity of the upper airway, several studies have elucidated the involvement of Tas2Rs in airway smooth muscle contraction. Deshpande et al. (2010) noted that receptors expressed on airway smooth muscle were not only functional and signaled in a calcium-dependent fashion but were able to induce a higher level of bronchial relaxation than a commercially available β2-agonist. The efficacy of Tas2Rs in comparison to β2-adrenergic-induced bronchodilation has been called into question by some but recognized by the majority as having a bona fide therapeutic potential, perhaps most effectively as a combination therapy with existing β2 agonists.
The expression of Tas2Rs in cancer cells has recently been identified in both breast (Singh et al. 2014) and pancreatic cancer (Gaida et al. 2016). Tas2R4 expression was downregulated by 20–30% in the breast cancer cell lines MDA-MB-231 and MCF-7 when compared to the noncancerous cell line MCF-10A. Functional calcium assays were conducted using quinine, dextromethorphan, and phenylthiocarbamide, showing that although reduced in number, Tas2Rs are functional in breast cancer cells. In pancreatic cancer, Tas2R38 was identified on the surface of lipid droplets, and stimulation of the receptor by phenylthiourea or N-acetyl-dodecanoyl homoserine was found to induce activation of p38 and ERK1/2 while upregulating NFATC1 expression (Gaida et al. 2016). These findings are significant as it could link Tas2Rs with a broad range of disease states, making them possible targets for new cancer therapies.
Bitter taste receptor research has grown steadily since their identification in the early 2000s, and their discovery in tissues outside of the oral cavity is intriguing. Though many aspects of their biology remain a mystery, such as ligand specificity and their therapeutic potential in diseases such as in the airway, their presence in many areas outside of the mouth lend credence to a more important role than previously thought in a multitude of biological processes in humans and other mammals alike.
- Gaida MM, Mayer C, Dapunt U, Stegmaier S, Schirmacher P, Wabnitz GH, et al. Expression of the bitter receptor T2R38 in pancreatic cancer: localization in lipid droplets and activation by a bacteria-derived quorum-sensing molecule. Oncotarget. 2016;7(11):12623–32. doi: 10.18632/oncotarget.7206.PubMedPubMedCentralCrossRefGoogle Scholar