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* This study was supported by the Federal Ministry for Education and Research and the Ministry for Science, Research, and Culture of the state of Brandenburg, Germany, as well as the European Union's Seventh Framework Programme for research, technological development, and demonstration (SynSignal Grant 613879). J. P. S. is an employee of Givaudan Flavors Corp. This article contains supplemental Tables 1S–3S.
One key to animal survival is the detection and avoidance of potentially harmful compounds by their bitter taste. Variable numbers of taste 2 receptor genes expressed in the gustatory end organs enable bony vertebrates (Euteleostomi) to recognize numerous bitter chemicals. It is believed that the receptive ranges of bitter taste receptor repertoires match the profiles of bitter chemicals that the species encounter in their diets. Human and mouse genomes contain pairs of orthologous bitter receptor genes that have been conserved throughout evolution. Moreover, expansions in both lineages generated species-specific sets of bitter taste receptor genes. It is assumed that the orthologous bitter taste receptor genes mediate the recognition of bitter toxins relevant for both species, whereas the lineage-specific receptors enable the detection of substances differently encountered by mice and humans. By challenging 34 mouse bitter taste receptors with 128 prototypical bitter substances in a heterologous expression system, we identified cognate compounds for 21 receptors, 19 of which were previously orphan receptors. We have demonstrated that mouse taste 2 receptors, like their human counterparts, vary greatly in their breadth of tuning, ranging from very broadly to extremely narrowly tuned receptors. However, when compared with humans, mice possess fewer broadly tuned receptors and an elevated number of narrowly tuned receptors, supporting the idea that a large receptor repertoire is the basis for the evolution of specialized receptors. Moreover, we have demonstrated that sequence-orthologous bitter taste receptors have distinct agonist profiles. Species-specific gene expansions have enabled further diversification of bitter substance recognition spectra.
The plethora of natural compounds that taste bitter for humans comprises numerous chemicals with pharmacological activities that can make them powerful toxins, such as the alkaloids strychnine and colchicine or the sesquiterpene lactone picrotoxinin (
). To avoid ingestion of bitter substances that would pose a threat to organisms, efficient recognition and rejection mechanisms have developed throughout the animal kingdom. In bony vertebrates (Euteleostomi), the avoidance of bitter compounds is centered on taste receptors that detect potentially harmful substances with high accuracy and adequate sensitivity (
). The human genome not only contains fewer intact TAS2R genes than the mouse genome but also a larger number of pseudogenes (11 in human versus 7 in mice). This has been interpreted as a sign of relaxed selective constraints on the human TAS2R gene repertoire (
). The majority of bitter taste receptor genes located on human chromosome 12 and mouse chromosome 6, respectively, occur in clusters of species-specific bitter taste receptor genes, which likely arose from gene duplications after the divergence of primate and rodent lineages. It has been speculated that these lineage-specific Tas2r recognize toxic bitter substances of particular relevance for the corresponding species (
). In contrast, the majority of Tas2r genes located on human chromosomes 5 and 7 and mouse chromosomes 2 and 15, respectively, exhibit a one-to-one orthology, suggesting that they developed prior to the divergence of primate and rodent lineages and enable the recognition of bitter substances equally important to humans and mice (
). If the above hypothesis is true, human and mouse should share Tas2r with conserved agonists, namely the one-to-one orthologs, and possess others with cognate bitter substances mostly relevant to one of the two species. In fact, when interpreting the data from rodent behavioral experiments, it is frequently argued that the murine Tas2r with highest sequence identity are true functional orthologs of their human counterparts, recognizing the same bitter compounds (cf. Refs.
). Collectively, these data indicated that humans have three very broadly tuned TAS2R “generalists” and eight receptor “specialists” that are narrowly tuned. Moreover, they have two TAS2R for compounds sharing structural motifs as well as eight moderately tuned receptors. Recently, the bitter taste receptor gene repertoires of chicken, turkey, and zebra finch, as well as the Western clawed frog, have been analyzed functionally. These studies revealed that narrowly tuned Tas2r are found only in species with larger Tas2r gene numbers, such as frog and zebra finch, whereas the three chicken and two turkey receptors are all broadly tuned (
). In mice, agonists have been reported for only two of the 35 putatively functional Tas2r, leaving the receptive range of the mouse Tas2r repertoire uncharacterized. For Tas2r105, an inhibitor of mRNA translation, cycloheximide, has been identified as a specific and potent agonist (
). Thus, the scarcity of data on the functional properties of mouse Tas2r does not provide clear insight into the extent of functional orthology or whether species-specific Tas2r gene expansions have indeed resulted in specialized Tas2r for bitter compounds of species-specific relevance.
To close this gap in knowledge we investigated whether the putatively functional murine Tas2r respond to an array of 128 bitter substances in a functional heterologous expression assay. Behavioral experiments were also performed to correlate the tuning properties of mouse Tas2r with avoidance behavior of the animals assessed by brief-access taste tests.
In our present work we performed a comprehensive analysis of the mouse Tas2r repertoire. In particular, we deorphaned the majority of mouse Tas2r, allowing comparisons of the pharmacological profiles with their well characterized human counterparts as well as with the plethora of behavioral data from previous sensory experiments. Although, in general, our results agreed well with observations in other species, some findings, such as functional differences among mouse and human bitter taste receptor orthologs, required some adjustment of firm beliefs in light of these data.
By in situ hybridization and qRT-PCR experiments we monitored the expression of mouse Tas2r genes in taste epithelium and compared their expression levels. Our data indicate that, similar to results obtained previously with human TAS2R (
), mouse Tas2r are indeed all expressed in gustatory tissue, confirming a role in bitter taste perception. Moreover, the variation in expression levels and numbers agree with the existence of a heterogeneous bitter taste receptor cell population in mouse (
). In mouse testis the highest expression was observed for Tas2r113 and Tas2r124, which showed low to moderate expression levels in gustatory tissue (Fig. 1A). Moreover, one of the Tas2r genes with the lowest expression in lingual papillae, Tas2r114, exhibited robust expression in testis (
). The data suggest that Tas2r gene regulation in taste papillae differs from that in other tissues.
Central to this work was the deorphanization and functional characterization of mouse Tas2r by heterologous expression. We identified agonists for 21 of the 35 putatively functional mouse Tas2r. The number of identified agonists per receptor revealed that, like human and frog bitter taste receptors (
) for activation of Tas2r105, we used 19 in the present study and found that, in addition to cycloheximide, denatonium, quinine, PROP, and yohimbine also stimulated Tas2r105-transfected cells (FIGURE 3, FIGURE 5). This discrepancy appears to be due to differences in experimental methodologies. Heterologous expression analysis of Tas2r105 in HEK293T cells stably expressing Gα15 or Gα16gust44 stimulated with selected agonists indicated that low efficacy activators of Tas2r105 result in lower or even absent responses in Gα15-expressing cells (Fig. 7). Therefore, the Gα16gust44 cell system shows higher sensitivity than the Gα15-based assay (
Mice like humans show similar proportions of moderately tuned Tas2r responsive to >3–10% of the chemicals and of Tas2r specialists recognizing less than 3% of the compounds. However, the fact that we discovered activators for only 60% of the mouse Tas2r, whereas 84% of the human TAS2R were deorphaned with a comparable set of bitter chemicals previously (
), suggests that mice have a higher proportion of specialist receptors relative to humans.
For 48 of the 128 compounds we failed to find a sensitive Tas2r, and for 13 Tas2r we were unable to find any bitter agonist. Low receptor expression or lack of cell surface localization in the heterologous cells as a general cause for the observed failure to identify agonists for these receptors is unlikely because transfection rates, expression levels, and cell membrane localization were not generally correlated between the groups of orphan or deorphaned Tas2r (Fig. 4 and Table 2). The inability to deorphan more Tas2r could be due to non-functional receptor variants generated by single nucleotide polymorphisms in the coding region. Nelson et al. (
) report that only two of 24 Tas2r genes showed no amino acid sequence differences when C57BL/6 and DBA/2J strains were compared. These changes in the Tas2r sequences could potentially affect ligand response profiles. Further, a lack of or inefficient G protein coupling might be another confounding feature (
The question of whether animals may recognize rather similar or different arrays of substances eliciting aversive behavior (e.g. bitter taste in human) cannot be answered conclusively as of today. Of course, one needs to assume that substances occur in nature that represent relevant toxins for some species and therefore require their recognition by bitter taste receptors, whereas other species may never encounter them and hence do not rely on receptors detecting these compounds. Answering this question would require the screening of bitter taste receptor repertoires from different species with compound libraries not preselected for their taste in humans. Thus far, such experiments have not been published, and we are aware that by screening mainly substances that taste bitter to humans our compound library was not unbiased. Indeed, some of the agonists we identified as activating mouse Tas2r, but that failed to activate human TAS2R (see below), suggest a substantial but not complete overlap among the bitter taste receptor agonists of both species. Nevertheless, from the bulk of available data, it appears that large overlaps among aversive (bitter) substances exist throughout the animal kingdom. Examples for such overlapping “bitter worlds” are plentiful and extend even to invertebrates possessing phylogenetically unrelated receptors expressed in different (neuronal) cell types. For example the nematode Caenorhabditis elegans shows aversive behavior to quinine, denatonium, and chloroquine (
and this work). In comparison, fewer reports have identified compounds that result in aversive behavior in other species but fail to activate human bitter taste perception (some compounds presented in this work (see below) and perhaps nicotine, for which we have not found a human TAS2R but a chicken Tas2r has been identified (
and this work), the assumption of the existence of large groups of species-selective bitter compounds is, even though valid, hypothetical. Nevertheless, we propose that the majority of those Tas2r that remain orphaned represent specialist receptors for compounds that are not contained in our substance library.
However, not only the number of agonists differed considerably among the receptors, but also the efficacies and potencies of the substances interacting with the various mouse receptors deviated. The highest efficacies were observed for cycloheximide (ΔF/F = 1.23 ± 0.20), denatonium saccharide (ΔF/F = 1.06 ± 0.22), and amarogentin (ΔF/F = 0.96 ± 0.24) at Tas2r105, suggesting that this receptor is critical for the recognition of these compounds in vivo when they are present in appropriate concentrations (Fig. 3 and supplemental Table 2S, A). Other compounds such as cucurbitacins B, D, E, and I demonstrate more than 10-fold lower efficacy at Tas2r105 (supplemental Table 2S, A). For the recognition of the cucurbitacins in vivo, Tas2r114 may be more relevant because they activated this receptor with substantially higher efficacies. Some substances, such as diphenidol and phenanthroline, activated their cognate receptors with similar efficacies. Thus, their overall bitterness is less likely to be dependent on a single Tas2r.
The potencies of bitter compounds also deviated largely across compounds for the same as well as different Tas2r (Table 1 and supplemental Table 2S, B). The highest potency with a threshold concentration of 10 nm and an EC50 concentration of 0.3 ± 0.2 μm was observed for cycloheximide at Tas2r105 (Fig. 3), confirming the dominant role of this receptor for the exquisite cycloheximide sensitivity of mice (
). Other agonists activated the receptor with at least ∼10-fold lower potencies, together spanning a concentration range of about 4 orders of magnitude. Most substances showed very low potencies, in the millimolar concentration range, for their cognate Tas2r. One of these substances is PROP, which activated six receptors at thresholds of only 0.3–1.0 mm (Table 1). Thus, Tas2r138 did not respond to PROP, whereas the orthologous human receptor TAS2R38 is exquisitely sensitive to PROP, showing an EC50 concentration of 2.1 μm (
). The β-d-glucopyranosides arbutin, helicin, phenyl-β-d-glucopyranoside, and d-(−)salicin all activated the receptor Tas2r126, with the highest observed threshold concentrations between 10 and 30 mm. However, in contrast to human TAS2R16 (
), the mouse Tas2r126 recognition pattern is not limited to β-d-glucopyranosides. Hence, mice appear to lack Tas2r that detect common structural configurations such as those detected by human TAS2R16 and TAS2R38.
In some cases, compounds that activated multiple Tas2r displayed similar potencies. For example, the seven receptors Tas2r105, Tas2r108, Tas2r115, Tas2r126, Tas2r137, Tas2r140, and Tas2r144 are activated by quinine at concentrations between 3.0 and 10 μm. However, for other compounds the concentrations required to activate different Tas2r are staggered. A good example for this is the artificial sweetener saccharin, which activates Tas2r135, Tas2r105, Tas2r109, and Tas2r144 with threshold concentrations of 0.1, 1.0, 3.0, and 10 mm, respectively. Hence, it is conceivable that increasing concentrations of saccharin in vivo result in a graded bitter response involving one to four Tas2r. In total, like human TAS2R (
), mouse Tas2r displayed threshold concentrations for bitter chemicals spanning 6 orders of magnitude.
The results of the present study allow a systematic comparison of the agonist spectra of mouse and human bitter taste receptors. To provide an even broader basis for such comparisons, we subjected human TAS2R to a screening with numerous substances not tested previously. These new agonist data did not change the classification of human TAS2R in broadly, moderately, or narrowly tuned receptors (
Our data further revealed that mice and humans detect a similar set of bitter compounds. Of the 128 substances used to challenge both the mouse and human bitter taste receptors, 80 (63%) activated mouse Tas2r and 98 (77%) human TAS2R, of which 72 substances (56%) stimulated bitter taste receptors in both species. We identified eight compounds that were selective for mouse Tas2r (shown in bold on Table 1), whereas 26 substances specifically stimulated human TAS2R (Ref.
and supplemental Table 2S, C). Twenty-two (17%) test substances activated neither mouse nor human Tas2r, probably because higher concentrations would be required to evoke Tas2r responses.
The ability of individual bitter compounds to activate multiple bitter taste receptors also varied between mouse and humans. Whereas quinine activated similar numbers of mouse (seven receptors) and human Tas2r (nine receptors), diphenidol stimulated more than twice as many bitter taste receptors in humans (15 receptors) as in mice (six receptors) (
). Vice versa, other substances such as PROP, with one main receptor in human, TAS2R38, acts more broadly on mouse Tas2r, being an agonist for six receptors. Thus, the response patterns of mouse and human bitter taste receptors are heterogeneous.
An important question is whether one-to-one orthologous Tas2r represent functional orthologs, e.g. show identical or at least similar agonist profiles. Although four of the one-to-one orthologous pairs could not be compared because the human, mouse, or both Tas2r remained orphan, this comparison was possible for seven orthologous pairs (Fig. 8A). Of these receptor pairs, Tas2r108 and its human ortholog TAS2R4 exhibited the highest degree of overlap in their set of agonists. Of the 18 bitter compounds for this pair, we observed that one-third of them activated both receptors. The substances capable of activating both receptors did not show apparent structural similarities. Only one of the 12 bitter substances was commonly recognized by Tas2r138 and TAS2R38. It is remarkable that the two prototypical agonists for the human TAS2R38, PROP and PTC, are not activators of the orthologous mouse receptor Tas2r138, which has been frequently, but erroneously, assumed in the past (
), which is in good agreement with our observation that PROP activated six Tas2r (Table 1). Other pairs of orthologs share few or even not a single agonist (Fig. 8A) demonstrating that, in general, orthologous Tas2r have largely distinct agonist profiles. The little overlap is probably at the level of chance and is not unexpected given the broad tuning of Tas2r. In fact, statistical analyses (4-fold χ2 test) confirmed that the number of common agonists for all but one pair (TAS2R4/Tas2r108) of the one-to-one orthologs did not exceed chance levels. Although we cannot exclude the possibility that additional common bitter agonists for these one-to-one orthologous receptor pairs exist in nature, it seems that these receptors also contribute to species-specific bitter substance recognition.
). A comparison of the data from structure-function analyses, amino acid sequence homologies, and the pharmacological properties of TAS2R38 orthologues suggests that the receptor was modified differently during the evolution of Euarchontoglires, a clade including primates and rodents (cf. Ref.
). Although primate TAS2R38 acquired sensitive PTC responsiveness as well as activation by PROP, this was not the case for the rodent ortholog Tas2r138 (Fig. 9, left and middle panels). A comparison of functionally critical residues in selected TAS2R38 orthologs of the Euarchontoglires clade revealed that all of them invariantly exhibit amino acid residues characteristic for the human taster variant TAS2R38-PAV or a variation thereof (TAS2R38-PAI), which has been experimentally validated for full PTC/PROP responsiveness (
Previous in vitro mutagenesis experiments combined with functional heterologous expression assays have revealed several functionally important residues in the binding pocket of human TAS2R38 that contribute to PROP and PTC activation (
). Among these residues were tryptophan 201 (5.46), serine 260 (6.52), and phenylalanine 264 (6.56). With one exception, the mutation of serine 260 to alanine, which resulted in unimpaired responsiveness to PROP but not PTC, all modifications resulted in severely reduced activation of the mutated receptors (
). In particular, exchanging tryptophan in position 201 for leucine or phenylalanine caused severely reduced PTC responsiveness and, practically, a loss of activation by PROP. Intriguingly, Trp-5.46 is found only in the haplorrhine primate clade including human and chimpanzee, which exhibit exquisitely PTC- as well as PROP-sensitive TAS2R38 receptors (
). Hence, it seems that PTC/PROP-sensitive TAS2R38 evolved within the Primate order in the haplorrhine branch. Moreover, the residue at position 6.52 also showed a strict separation among the compared clades. In this case, all but the Catarrhini, which carry a serine residue at this position, have a phenylalanine (or valine in the case of Jaculus jaculus, belonging to the jerboa) at this position. Strikingly, this position affected PTC and PROP recognition in vitro as well, with serine being the preferential residue for the activation by both substances (
). Finally, position 6.56 differs among the Catarrhini, which exhibit a phenylalanine residue, and the other species with different, dominantly hydrophobic residues at this position. As experimental evidence suggests the requirement of phenylalanine at this position for full PTC/PROP responsiveness and rodent receptors differ in all three mentioned positions from the human and chimpanzee counterparts, we concluded this to be the underlying reason that the mouse receptor and perhaps all rodent receptors are incapable of interacting with PTC or PROP. This indicates that a functional divergence occurred prior to the separation of the rodent and primate lineages at the beginning of the earlier cretaceous period. Nevertheless, as the cat Tas2r38 ortholog shows insensitive PTC responsiveness and no PROP responses (
) we assumed the existence of a common ancestral TAS2R38 ortholog permissive for PTC/PROP responses. Of course, the existence of further critical positions for PTC/PROP responses cannot be ruled out and may contribute significantly. This example indicates that pharmacological diversification occurs also among the group of one-to-one orthologous receptors. Therefore, the hypothesis that orthologous receptors may recognize bitter compounds important for both species (
The persistence of these genes intact in the genomes of mice and humans and not pseudogenized could indicate that common, yet still unknown, bitter substances that pose or have posed a severe threat to the survival of both organisms throughout evolution could exist for these one-to-one orthologs. Alternatively, the corresponding receptors may fulfill another and dominant function beyond bitter taste perception by recognizing endogenous and well conserved agonists. As the number of reports on extragustatory expression of bitter taste receptors is ever increasing (
), this hypothesis does not seem too farfetched. Lastly, the structure of these receptors may have allowed the rapid evolution of binding sites tailored to recognize compounds for the specific needs of mice or humans. In that case, the unique ability of bitter taste receptors to dynamically adapt their functions to the nutritional requirements of organisms may be more important than the fixation of pharmacological properties.
In contrast to the one-to-one orthologous receptor pairs, it is assumed that lineage-specific expansions possibly generated Tas2r critical for the recognition of bitter substances encountered only in the concerned species (
). If this were true, then the cluster of amplified Tas2r in one species should recognize more compounds than the related single Tas2r in the other species, some of which should be species-specific.
Glires cluster I consists of 11 mouse Tas2r and human TAS2R14, which is the most broadly tuned TAS2R in humans. In the course of our experiments, we found agonists for eight of the 11 mouse Tas2r, and three remained orphan. Of the 64 agonists activating the Tas2r of Glires cluster I, the majority, namely 38 of them, were specific for human TAS2R14; 11 activated human TAS2R14 and several mouse Tas2r, whereas only 15 substances were specific for the mouse Tas2r of this group. For muroid cluster I, which includes one broadly tuned human receptor together with five mouse Tas2r, of which we deorphaned two receptors, as well as muroid cluster II with 1 human TAS2R and three mouse Tas2r, we found similar results (Fig. 8B). In contrast to clusters showing an expansion of mouse Tas2r, in Glires cluster II/anthropoid cluster, the mouse paralogs Tas2r136 and Tas2r120 linked with eight human TAS2R. Of these receptors, mouse Tas2r136, as well as human TAS2R19 and TAS2R45, is an orphan receptor and cannot be compared. Only a single compound of the 54 activators for this cluster was specific for a murine Tas2r, and two substances activated Tas2r in both species (Fig. 8B). Remarkably, the remaining 51 chemicals were selective for the human TAS2R of this cluster.
Thus, the hypothesis that lineage-specific expansions generate Tas2r for species-specific bitter chemicals is not generally supported by our data. For example, only five of 26 human-specific compounds are recognized by members of anthropoid cluster, whereas most of the human-specific compounds are recognized by the three most broadly tuned receptors: TAS2R10, TAS2R14, and TAS2R46.
However, the bitter taste receptor gene expansions contribute to a broadening of the overall agonist profiles, which may be particularly important for the more narrowly tuned mouse receptors and, hence, may account for the fact that more frequent gene expansions occurred in mice. In fact, a closer look at the amino acid sequence of receptors of murine cluster I showed a general tendency of higher amino acid sequence homologies in intracellularly oriented transmembrane domain parts and intracellular loops compared with extracellular transmembrane domain regions and extracellular loops, which has been recognized previously (
). A detailed comparative analysis of receptor positions that constitute the binding pockets of murine cluster I receptors suggests that diversification of agonist spectra has occurred (Fig. 10). All receptors of murine cluster 1 containing human TAS2R10, which has been subjected to detailed structure-function analyses (
), exhibited a different combination of amino acid residues at positions showing pronounced agonist selectivity and, hence, their corresponding putative agonist spectrum. The concentration-response relationships for selected groups of human and mouse Tas2r indicate that the activation properties of sequence-related human and mouse Tas2r differ more substantially than is evident from sole comparisons of their agonist profiles (Fig. 11). Sequence orthologs such as TAS2R38/Tas2r138 or TAS2R1/Tas2r119 can or cannot recognize the same compounds (Fig. 11, A and B). However, even if they do so, the potencies and efficacies differ substantially (Fig. 11B). The same pronounced differences are also seen in the case of members of muroid cluster I (Fig. 11, C and D) or representatives of Glires cluster II/anthropoid cluster (Fig. 11, E and F).
Another hypothesis about the development of species-specific Tas2r gene clusters concerns tuning breadth rather than individual agonist spectra (
). Because both of the single human TAS2R corresponding to mouse Tas2r gene clusters (Glires cluster I and muroid cluster I) are extraordinarily broadly tuned receptors, one could assume that the gene expansion in the rodent lineage resulted in the development of multiple specialized receptors arising from a broadly tuned ancestral receptor or that the broad tuning of the ancestral receptor was maintained by the derived Tas2r to cover an even larger chemical space. Our results strongly suggest that multiple broadly tuned receptors were not generated but rather that a specification of several receptors occurred (Table 1).
Our analyses of bitter compounds and the corresponding mouse Tas2r for avoidance behavior revealed that in some but not all cases the sensitivity of Tas2r responses measured in vitro matched the concentration range of the substance in vitro. Whether these differences could be due to perireceptor events (
) not mimicked in our in vitro assays remains to be determined. The data suggest that receptor threshold values can, in some cases, predict bitterness avoidance of mice. However, it appears that for other bitter chemicals other possible factors such as interaction with the oral mucosa or saliva may reduce their potency of inducing aversion. However, in view of different G protein coupling of Tas2r in vitro and in vivo (
), the data on the behavioral experiments agree reasonably well with the data from the receptor assays on a qualitative level and in some cases also on a quantitative level. In the case of the substance colchicine, which showed a 100-fold higher potency when eliciting avoidance behavior in vivo compared with the receptor assays, other explanations need to be taken into account. Either the “best” receptor has not been discovered, or alternative recognition mechanisms exist that do not rely on Tas2r. However, the ability of colchicine to activate three human, one chicken, one turkey, and one frog receptor (
) suggests that Tas2r-dependent detection mechanisms likely exist for this compound.
Molecular genetics can shed light on the importance of specific Tas2r for taste-relevant behavior. The importance of Tas2r105 for cycloheximide recognition is illuminated by strain-specific differences leading to the identification of a chromosomal locus mediating cycloheximide sensitivity (
). However, the avoidance of denatonium, PROP, and quinine was not altered in these mice. Because all three substances activated four to six other Tas2r, it is conceivable that they suffice to evoke avoidance of those compounds. Strain-specific recognition extends to other bitter compounds, leading to the identification of chromosomal regions critical for the detection of the quinine (Qui) (
) agrees well with our finding that only a single receptor, Tas2r117, responded to both compounds. Intriguingly, the Tas2r117 sequence was fully intact in C57BL/6 mice used for our analyses, but it contains missense mutations and small deletions in the DBA/2J strain (
). Our experiments revealed that mouse Tas2r105, as well as human TAS2R1, TAS2R10, and TAS2R14, is sensitive to various N-acyl homoserine lactones. We failed, however, to monitor N-acyl homoserine lactone responses for TAS2R38 (
), which may be explained by the use of different experimental methodologies. The ability to detect quorum-sensing molecules contributes to environmental adaptions and influences the behavior of eukaryotic organisms (
). Humans homozygous for the non-taster allele of TAS2R38 are reported to be more susceptible to upper respiratory tract infections by Gram-negative bacteria than individuals carrying the taster variant of this receptor (
), we also examined whether hormones could function as Tas2r activators. We found that progesterone stimulated Tas2r114 and Tas2r110. This steroid hormone is expressed in ovaries (corpus luteum), the adrenal glands, and testicular Leydig cells. It also has major effects on human sperm motility (
). Further studies are needed to elucidate the role of this and other Tas2r in testicular function.
Taken together the work presented here sheds light on the evolutionary dynamics that acted on the bitter receptor repertoires of vertebrates, resulting in the development of highly versatile G protein-coupled receptors capable of adapting to various lifestyles and habitats.
K. L. designed the research, constructed vectors for functional expression, performed the calcium imaging analysis, immunocytochemistry, and brief-access tests, and prepared the manuscript. S. H. carried out qRT-PCR, prepared in situ hybridization probes, and performed corresponding experiments. N. R. conducted phylogenetic tree and statistical analysis. J. P. S. contributed to the design of the study. F. P. isolated and/or repurified several sequiterpene lactones. M. B. and M. W. designed research and prepared the manuscript. All authors read and approved of the final version of the manuscript.
We thank Elke Chudoba, Josefine Würfel, Florian Padberg, Lisa Oldorff, Julia Freydank, Alexandra Semmler, and Eva-Katharina Hage (Nuthetal) for expert technical assistance.