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Comprehensive Analysis of Mouse Bitter Taste Receptors Reveals Different Molecular Receptive Ranges for Orthologous Receptors in Mice and Humans*

  • Kristina Lossow
    Affiliations
    Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany
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  • Sandra Hübner
    Affiliations
    Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany
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  • Natacha Roudnitzky
    Affiliations
    Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany
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  • Jay P. Slack
    Affiliations
    Givaudan Flavors Corporation, Cincinnati, Ohio 45216
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  • Federica Pollastro
    Affiliations
    Department of Drug Sciences, University of Eastern Piemonte, 28100 Novara, Italy
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  • Maik Behrens
    Correspondence
    To whom correspondence should be addressed. Tel.:49-33200-88-2545; Fax:49-33200-88-2384;
    Affiliations
    Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany
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  • Wolfgang Meyerhof
    Affiliations
    Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany
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  • Author Footnotes
    * 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.
Open AccessPublished:May 20, 2016DOI:https://doi.org/10.1074/jbc.M116.718544
      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.

      Introduction

      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 (
      • Behrens M.
      • Brockhoff A.
      • Kuhn C.
      • Bufe B.
      • Winnig M.
      • Meyerhof W.
      The human taste receptor hTAS2R14 responds to a variety of different bitter compounds.
      ). However, compounds believed to exert health-beneficial effects such as the antioxidative phytoestrogen genistein from soy (
      • Roland W.S.
      • Vincken J.P.
      • Gouka R.J.
      • van Buren L.
      • Gruppen H.
      • Smit G.
      Soy isoflavones and other isoflavonoids activate the human bitter taste receptors hTAS2R14 and hTAS2R39.
      ), the analgesic drug acetaminophen (
      • Meyerhof W.
      • Batram C.
      • Kuhn C.
      • Brockhoff A.
      • Chudoba E.
      • Bufe B.
      • Appendino G.
      • Behrens M.
      The molecular receptive ranges of human TAS2R bitter taste receptors.
      ), or various polyphenols also taste bitter (
      • Soares S.
      • Kohl S.
      • Thalmann S.
      • Mateus N.
      • Meyerhof W.
      • De Freitas V.
      Different phenolic compounds activate distinct human bitter taste receptors.
      ). 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 (
      • Behrens M.
      • Meyerhof W.
      Bitter taste receptor research comes of age: from characterization to modulation of TAS2Rs.
      ). Vertebrate bitter taste receptors, called taste 2 receptors (TAS2R (human) or Tas2r (murine)),
      The abbreviations used are: TAS2R, human bitter taste receptor class 2; Tas2r, murine bitter taste receptor class 2; FLIPR, fluorometric imaging plate reader; HSV, herpes simplex virus glycoprotein D epitope; PROP, 6-n-propyl-2-thiouracil; PTC, phenylthiocarbamide; qRT-PCR, quantitative RT-PCR.
      are G protein-coupled receptors only remotely related to other classes of this large and enormously versatile receptor family (
      • Adler E.
      • Hoon M.A.
      • Mueller K.L.
      • Chandrashekar J.
      • Ryba N.J.
      • Zuker C.S.
      A novel family of mammalian taste receptors.
      ,
      • Chandrashekar J.
      • Mueller K.L.
      • Hoon M.A.
      • Adler E.
      • Feng L.
      • Guo W.
      • Zuker C.S.
      • Ryba N.J.
      T2Rs function as bitter taste receptors.
      ,
      • Fredriksson R.
      • Lagerström M.C.
      • Lundin L.G.
      • Schiöth H.B.
      The G-protein-coupled receptors in the human genome form five main families: phylogenetic analysis, paralogon groups, and fingerprints.
      ,
      • Matsunami H.
      • Montmayeur J.P.
      • Buck L.B.
      A family of candidate taste receptors in human and mouse.
      ,
      • Meyerhof W.
      Elucidation of mammalian bitter taste.
      ). During evolution the first Tas2r genes appeared in the genomes of bony fish (
      • Grus W.E.
      • Zhang J.
      Origin of the genetic components of the vomeronasal system in the common ancestor of all extant vertebrates.
      ). In higher vertebrates frequent independent expansions and pseudogenization events resulted in differently sized Tas2r gene repertoires (
      • Dong D.
      • Jones G.
      • Zhang S.
      Dynamic evolution of bitter taste receptor genes in vertebrates.
      ). Consequently, the number of putatively functional Tas2r genes varies considerably in vertebrates, ranging from 0 in baleen and tooth whales as well as penguins (
      • Jiang P.
      • Josue J.
      • Li X.
      • Glaser D.
      • Li W.
      • Brand J.G.
      • Margolskee R.F.
      • Reed D.R.
      • Beauchamp G.K.
      Major taste loss in carnivorous mammals.
      ,
      • Zhao H.
      • Li J.
      • Zhang J.
      Molecular evidence for the loss of three basic tastes in penguins.
      ,
      • Zhu K.
      • Zhou X.
      • Xu S.
      • Sun D.
      • Ren W.
      • Zhou K.
      • Yang G.
      The loss of taste genes in cetaceans.
      ,
      • Feng P.
      • Zheng J.
      • Rossiter S.J.
      • Wang D.
      • Zhao H.
      Massive losses of taste receptor genes in toothed and baleen whales.
      ) to more than 50 in Western clawed frogs and 80 in Coelacanth (
      • Behrens M.
      • Korsching S.I.
      • Meyerhof W.
      Tuning properties of avian and frog bitter taste receptors dynamically fit gene repertoire sizes.
      ,
      • Li D.
      • Zhang J.
      Diet shapes the evolution of the vertebrate bitter taste receptor gene repertoire.
      ,
      • Shi P.
      • Zhang J.
      Contrasting modes of evolution between vertebrate sweet/umami receptor genes and bitter receptor genes.
      ,
      • Syed A.S.
      • Korsching S.I.
      Positive Darwinian selection in the singularly large taste receptor gene family of an “ancient” fish, Latimeria chalumnae.
      ). Thus, humans with ∼25 and mice with ∼35 putatively functional members possess average size Tas2r repertoires (
      • Go Y.
      • Satta Y.
      • Takenaka O.
      • Takahata N.
      Lineage-specific loss of function of bitter taste receptor genes in humans and nonhuman primates.
      ). 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 (
      • Conte C.
      • Ebeling M.
      • Marcuz A.
      • Nef P.
      • Andres-Barquin P.J.
      Evolutionary relationships of the Tas2r receptor gene families in mouse and human.
      ). The Tas2r genes of human and mouse occur clustered at few syntenic chromosomal regions (
      • Adler E.
      • Hoon M.A.
      • Mueller K.L.
      • Chandrashekar J.
      • Ryba N.J.
      • Zuker C.S.
      A novel family of mammalian taste receptors.
      ,
      • Conte C.
      • Ebeling M.
      • Marcuz A.
      • Nef P.
      • Andres-Barquin P.J.
      Evolutionary relationships of the Tas2r receptor gene families in mouse and human.
      ,
      • Shi P.
      • Zhang J.
      • Yang H.
      • Zhang Y.P.
      Adaptive diversification of bitter taste receptor genes in mammalian evolution.
      ). 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 (
      • Shi P.
      • Zhang J.
      • Yang H.
      • Zhang Y.P.
      Adaptive diversification of bitter taste receptor genes in mammalian evolution.
      ). 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 (
      • Shi P.
      • Zhang J.
      • Yang H.
      • Zhang Y.P.
      Adaptive diversification of bitter taste receptor genes in mammalian evolution.
      ). 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.
      • Behrens M.
      • Meyerhof W.
      Gustatory and extragustatory functions of mammalian taste receptors.
      ,
      • Chen M.C.
      • Wu S.V.
      • Reeve Jr., J.R.
      • Rozengurt E.
      Bitter stimuli induce Ca2+ signaling and CCK release in enteroendocrine STC-1 cells: role of L-type voltage-sensitive Ca2+ channels.
      ,
      • Janssen S.
      • Laermans J.
      • Verhulst P.J.
      • Thijs T.
      • Tack J.
      • Depoortere I.
      Bitter taste receptors and α-gustducin regulate the secretion of ghrelin with functional effects on food intake and gastric emptying.
      ,
      • Jeon T.I.
      • Zhu B.
      • Larson J.L.
      • Osborne T.F.
      SREBP-2 regulates gut peptide secretion through intestinal bitter taste receptor signaling in mice.
      ,
      • Wu S.V.
      • Chen M.C.
      • Rozengurt E.
      Genomic organization, expression, and function of bitter taste receptors (T2R) in mouse and rat.
      ). However, structure-function analyses of human bitter taste receptors reveal that very few differences in the amino acid sequences of TAS2R can account for the largely deviating agonist spectra (
      • Brockhoff A.
      • Behrens M.
      • Niv M.Y.
      • Meyerhof W.
      Structural requirements of bitter taste receptor activation.
      ). Conversely, human TAS2R paralogs with pronounced amino acid sequence differences can have agonists in common even though they recognize these compounds by different binding modes (
      • Born S.
      • Levit A.
      • Niv M.Y.
      • Meyerhof W.
      • Behrens M.
      The human bitter taste receptor TAS2R10 is tailored to accommodate numerous diverse ligands.
      ).
      The ∼25 human bitter taste receptors (TAS2R) are comparatively well characterized, with agonists identified for 21 of the ∼25 receptors (
      • Behrens M.
      • Brockhoff A.
      • Kuhn C.
      • Bufe B.
      • Winnig M.
      • Meyerhof W.
      The human taste receptor hTAS2R14 responds to a variety of different bitter compounds.
      ,
      • Roland W.S.
      • Vincken J.P.
      • Gouka R.J.
      • van Buren L.
      • Gruppen H.
      • Smit G.
      Soy isoflavones and other isoflavonoids activate the human bitter taste receptors hTAS2R14 and hTAS2R39.
      ,
      • Meyerhof W.
      • Batram C.
      • Kuhn C.
      • Brockhoff A.
      • Chudoba E.
      • Bufe B.
      • Appendino G.
      • Behrens M.
      The molecular receptive ranges of human TAS2R bitter taste receptors.
      ,
      • Soares S.
      • Kohl S.
      • Thalmann S.
      • Mateus N.
      • Meyerhof W.
      • De Freitas V.
      Different phenolic compounds activate distinct human bitter taste receptors.
      ,
      • Chandrashekar J.
      • Mueller K.L.
      • Hoon M.A.
      • Adler E.
      • Feng L.
      • Guo W.
      • Zuker C.S.
      • Ryba N.J.
      T2Rs function as bitter taste receptors.
      ,
      • Born S.
      • Levit A.
      • Niv M.Y.
      • Meyerhof W.
      • Behrens M.
      The human bitter taste receptor TAS2R10 is tailored to accommodate numerous diverse ligands.
      ,
      • Behrens M.
      • Brockhoff A.
      • Batram C.
      • Kuhn C.
      • Appendino G.
      • Meyerhof W.
      The human bitter taste receptor hTAS2R50 is activated by the two natural bitter terpenoids andrographolide and amarogentin.
      ,
      • Brockhoff A.
      • Behrens M.
      • Massarotti A.
      • Appendino G.
      • Meyerhof W.
      Broad tuning of the human bitter taste receptor hTAS2R46 to various sesquiterpene lactones, clerodane and labdane diterpenoids, strychnine, and denatonium.
      ,
      • Bufe B.
      • Breslin P.A.
      • Kuhn C.
      • Reed D.R.
      • Tharp C.D.
      • Slack J.P.
      • Kim U.K.
      • Drayna D.
      • Meyerhof W.
      The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception.
      ,
      • Bufe B.
      • Hofmann T.
      • Krautwurst D.
      • Raguse J.D.
      • Meyerhof W.
      The human TAS2R16 receptor mediates bitter taste in response to β-glucopyranosides.
      ,
      • Kuhn C.
      • Bufe B.
      • Winnig M.
      • Hofmann T.
      • Frank O.
      • Behrens M.
      • Lewtschenko T.
      • Slack J.P.
      • Ward C.D.
      • Meyerhof W.
      Bitter taste receptors for saccharin and acesulfame K.
      ,
      • Dotson C.D.
      • Zhang L.
      • Xu H.
      • Shin Y.K.
      • Vigues S.
      • Ott S.H.
      • Elson A.E.
      • Choi H.J.
      • Shaw H.
      • Egan J.M.
      • Mitchell B.D.
      • Li X.
      • Steinle N.I.
      • Munger S.D.
      Bitter taste receptors influence glucose homeostasis.
      ,
      • Intelmann D.
      • Batram C.
      • Kuhn C.
      • Haseleu G.
      • Meyerhof W.
      • Hofmann T.
      Three TAS2R bitter taste receptors mediate the psychophysical responses to bitter compounds of hops (Humulus lupulus L.) and beer.
      ,
      • Kim U.K.
      • Jorgenson E.
      • Coon H.
      • Leppert M.
      • Risch N.
      • Drayna D.
      Positional cloning of the human quantitative trait locus underlying taste sensitivity to phenylthiocarbamide.
      ,
      • Maehashi K.
      • Matano M.
      • Wang H.
      • Vo L.A.
      • Yamamoto Y.
      • Huang L.
      Bitter peptides activate hTAS2Rs, the human bitter receptors.
      ,
      • Pronin A.N.
      • Tang H.
      • Connor J.
      • Keung W.
      Identification of ligands for two human bitter T2R receptors.
      ,
      • Sainz E.
      • Cavenagh M.M.
      • Gutierrez J.
      • Battey J.F.
      • Northup J.K.
      • Sullivan S.L.
      Functional characterization of human bitter taste receptors.
      ,
      • Thalmann S.
      • Behrens M.
      • Meyerhof W.
      Major haplotypes of the human bitter taste receptor TAS2R41 encode functional receptors for chloramphenicol.
      ). 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 (
      • Behrens M.
      • Korsching S.I.
      • Meyerhof W.
      Tuning properties of avian and frog bitter taste receptors dynamically fit gene repertoire sizes.
      ). 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 (
      • Chandrashekar J.
      • Mueller K.L.
      • Hoon M.A.
      • Adler E.
      • Feng L.
      • Guo W.
      • Zuker C.S.
      • Ryba N.J.
      T2Rs function as bitter taste receptors.
      ). The other receptor is Tas2r108, which is activated by denatonium benzoate and 6-n-propyl-2-thiouracil (PROP) with low potency (
      • Chandrashekar J.
      • Mueller K.L.
      • Hoon M.A.
      • Adler E.
      • Feng L.
      • Guo W.
      • Zuker C.S.
      • Ryba N.J.
      T2Rs function as bitter taste receptors.
      ). 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.

      Discussion

      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 (
      • Behrens M.
      • Foerster S.
      • Staehler F.
      • Raguse J.D.
      • Meyerhof W.
      Gustatory expression pattern of the human TAS2R bitter receptor gene family reveals a heterogenous population of bitter responsive taste receptor cells.
      ), 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 (
      • Matsunami H.
      • Montmayeur J.P.
      • Buck L.B.
      A family of candidate taste receptors in human and mouse.
      ,
      • Caicedo A.
      • Roper S.D.
      Taste receptor cells that discriminate between bitter stimuli.
      ).
      Recently, quantitative expression analyses of rodent Tas2r have been performed in non-gustatory tissues such as testis and heart (
      • Foster S.R.
      • Porrello E.R.
      • Purdue B.
      • Chan H.W.
      • Voigt A.
      • Frenzel S.
      • Hannan R.D.
      • Moritz K.M.
      • Simmons D.G.
      • Molenaar P.
      • Roura E.
      • Boehm U.
      • Meyerhof W.
      • Thomas W.G.
      Expression, regulation and putative nutrient-sensing function of taste GPCRs in the heart.
      ,
      • Xu J.
      • Cao J.
      • Iguchi N.
      • Riethmacher D.
      • Huang L.
      Functional characterization of bitter-taste receptors expressed in mammalian testis.
      ). 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 (
      • Xu J.
      • Cao J.
      • Iguchi N.
      • Riethmacher D.
      • Huang L.
      Functional characterization of bitter-taste receptors expressed in mammalian 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 (
      • Meyerhof W.
      • Batram C.
      • Kuhn C.
      • Brockhoff A.
      • Chudoba E.
      • Bufe B.
      • Appendino G.
      • Behrens M.
      The molecular receptive ranges of human TAS2R bitter taste receptors.
      ,
      • Behrens M.
      • Korsching S.I.
      • Meyerhof W.
      Tuning properties of avian and frog bitter taste receptors dynamically fit gene repertoire sizes.
      ), mouse Tas2r vary in their tuning breadth. Interestingly, this species displays only a single Tas2r, Tas2r105, that functions as a generalist (
      • Meyerhof W.
      • Batram C.
      • Kuhn C.
      • Brockhoff A.
      • Chudoba E.
      • Bufe B.
      • Appendino G.
      • Behrens M.
      The molecular receptive ranges of human TAS2R bitter taste receptors.
      ), exhibiting an extremely broad agonist profile by recognizing >30% of the bitter compound library. Surprisingly, this Tas2r has been reported previously to be highly selective for cycloheximide (
      • Chandrashekar J.
      • Mueller K.L.
      • Hoon M.A.
      • Adler E.
      • Feng L.
      • Guo W.
      • Zuker C.S.
      • Ryba N.J.
      T2Rs function as bitter taste receptors.
      ). Of the 24 bitter compounds tested by Chandrashekar et al. (
      • Chandrashekar J.
      • Mueller K.L.
      • Hoon M.A.
      • Adler E.
      • Feng L.
      • Guo W.
      • Zuker C.S.
      • Ryba N.J.
      T2Rs function as 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 (
      • Chandrashekar J.
      • Mueller K.L.
      • Hoon M.A.
      • Adler E.
      • Feng L.
      • Guo W.
      • Zuker C.S.
      • Ryba N.J.
      T2Rs function as bitter taste receptors.
      ).
      Figure thumbnail gr7
      FIGURE 7Intracellular calcium traces of Tas2r105 (non-taster variant) in HEK293T cells stable expressing Gα15 (continuous line) and Gα16gust44 (dashed line) recorded in FLIPR experiments upon stimulation with six exemplary compounds, indicating that low efficacy activators of Tas2r105 resulted in lower or even absent responses in Gα15-expressing cells. Calibration bar denotes 2000 relative light units (y) and 1 min (x).
      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 (
      • Meyerhof W.
      • Batram C.
      • Kuhn C.
      • Brockhoff A.
      • Chudoba E.
      • Bufe B.
      • Appendino G.
      • Behrens M.
      The molecular receptive ranges of human TAS2R bitter taste receptors.
      ,
      • Thalmann S.
      • Behrens M.
      • Meyerhof W.
      Major haplotypes of the human bitter taste receptor TAS2R41 encode functional receptors for chloramphenicol.
      ), 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. (
      • Nelson T.M.
      • Munger S.D.
      • Boughter Jr., J.D.
      Haplotypes at the Tas2r locus on distal chromosome 6 vary with quinine taste sensitivity in inbred mice.
      ) 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 (
      • Sainz E.
      • Cavenagh M.M.
      • Gutierrez J.
      • Battey J.F.
      • Northup J.K.
      • Sullivan S.L.
      Functional characterization of human bitter taste receptors.
      ,
      • Wong G.T.
      • Gannon K.S.
      • Margolskee R.F.
      Transduction of bitter and sweet taste by gustducin.
      ,
      • Ozeck M.
      • Brust P.
      • Xu H.
      • Servant G.
      Receptors for bitter, sweet, and umami taste couple to inhibitory G protein signaling pathways.
      ).
      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 (
      • Hilliard M.A.
      • Bergamasco C.
      • Arbucci S.
      • Plasterk R.H.
      • Bazzicalupo P.
      Worms taste bitter: ASH neurons, QUI-1, GPA-3, and ODR-3 mediate quinine avoidance in Caenorhabditis elegans.
      ), substances recognized by human and mouse as well. Such examples of agonist overlaps are even more plentiful for vertebrates, ranging from teleostean fish to primates (Refs.
      • Behrens M.
      • Korsching S.I.
      • Meyerhof W.
      Tuning properties of avian and frog bitter taste receptors dynamically fit gene repertoire sizes.
      and
      • Oike H.
      • Nagai T.
      • Furuyama A.
      • Okada S.
      • Aihara Y.
      • Ishimaru Y.
      • Marui T.
      • Matsumoto I.
      • Misaka T.
      • Abe K.
      Characterization of ligands for fish taste receptors.
      ,
      • Wooding S.
      • Bufe B.
      • Grassi C.
      • Howard M.T.
      • Stone A.C.
      • Vazquez M.
      • Dunn D.M.
      • Meyerhof W.
      • Weiss R.B.
      • Bamshad M.J.
      Independent evolution of bitter-taste sensitivity in humans and chimpanzees.
      ,
      • Imai H.
      • Suzuki N.
      • Ishimaru Y.
      • Sakurai T.
      • Yin L.
      • Pan W.
      • Abe K.
      • Misaka T.
      • Hirai H.
      Functional diversity of bitter taste receptor TAS2R16 in primates.
      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 (
      • Behrens M.
      • Korsching S.I.
      • Meyerhof W.
      Tuning properties of avian and frog bitter taste receptors dynamically fit gene repertoire sizes.
      )). Hence, although there is considerable evidence in place for largely overlapping sets of aversive stimuli for many animals (Refs.
      • Behrens M.
      • Korsching S.I.
      • Meyerhof W.
      Tuning properties of avian and frog bitter taste receptors dynamically fit gene repertoire sizes.
      and
      • Oike H.
      • Nagai T.
      • Furuyama A.
      • Okada S.
      • Aihara Y.
      • Ishimaru Y.
      • Marui T.
      • Matsumoto I.
      • Misaka T.
      • Abe K.
      Characterization of ligands for fish taste receptors.
      ,
      • Wooding S.
      • Bufe B.
      • Grassi C.
      • Howard M.T.
      • Stone A.C.
      • Vazquez M.
      • Dunn D.M.
      • Meyerhof W.
      • Weiss R.B.
      • Bamshad M.J.
      Independent evolution of bitter-taste sensitivity in humans and chimpanzees.
      ,
      • Imai H.
      • Suzuki N.
      • Ishimaru Y.
      • Sakurai T.
      • Yin L.
      • Pan W.
      • Abe K.
      • Misaka T.
      • Hirai H.
      Functional diversity of bitter taste receptor TAS2R16 in primates.
      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 (
      • Chandrashekar J.
      • Mueller K.L.
      • Hoon M.A.
      • Adler E.
      • Feng L.
      • Guo W.
      • Zuker C.S.
      • Ryba N.J.
      T2Rs function as bitter taste receptors.
      ). 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 (
      • Bufe B.
      • Breslin P.A.
      • Kuhn C.
      • Reed D.R.
      • Tharp C.D.
      • Slack J.P.
      • Kim U.K.
      • Drayna D.
      • Meyerhof W.
      The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception.
      ). 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 (
      • Bufe B.
      • Hofmann T.
      • Krautwurst D.
      • Raguse J.D.
      • Meyerhof W.
      The human TAS2R16 receptor mediates bitter taste in response to β-glucopyranosides.
      ), 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 (
      • Meyerhof W.
      • Batram C.
      • Kuhn C.
      • Brockhoff A.
      • Chudoba E.
      • Bufe B.
      • Appendino G.
      • Behrens M.
      The molecular receptive ranges of human TAS2R bitter taste receptors.
      ), 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 (
      • Meyerhof W.
      • Batram C.
      • Kuhn C.
      • Brockhoff A.
      • Chudoba E.
      • Bufe B.
      • Appendino G.
      • Behrens M.
      The molecular receptive ranges of human TAS2R bitter taste 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.
      • Meyerhof W.
      • Batram C.
      • Kuhn C.
      • Brockhoff A.
      • Chudoba E.
      • Bufe B.
      • Appendino G.
      • Behrens M.
      The molecular receptive ranges of human TAS2R bitter taste receptors.
      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) (
      • Meyerhof W.
      • Batram C.
      • Kuhn C.
      • Brockhoff A.
      • Chudoba E.
      • Bufe B.
      • Appendino G.
      • Behrens M.
      The molecular receptive ranges of human TAS2R bitter taste 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 (
      • Chen M.C.
      • Wu S.V.
      • Reeve Jr., J.R.
      • Rozengurt E.
      Bitter stimuli induce Ca2+ signaling and CCK release in enteroendocrine STC-1 cells: role of L-type voltage-sensitive Ca2+ channels.
      ,
      • Janssen S.
      • Laermans J.
      • Verhulst P.J.
      • Thijs T.
      • Tack J.
      • Depoortere I.
      Bitter taste receptors and α-gustducin regulate the secretion of ghrelin with functional effects on food intake and gastric emptying.
      ,
      • Jeon T.I.
      • Zhu B.
      • Larson J.L.
      • Osborne T.F.
      SREBP-2 regulates gut peptide secretion through intestinal bitter taste receptor signaling in mice.
      ,
      • Wu S.V.
      • Chen M.C.
      • Rozengurt E.
      Genomic organization, expression, and function of bitter taste receptors (T2R) in mouse and rat.
      ). Whereas in human the sensitivities for PROP and PTC are highly correlated to their activation of TAS2R38 (
      • Bufe B.
      • Breslin P.A.
      • Kuhn C.
      • Reed D.R.
      • Tharp C.D.
      • Slack J.P.
      • Kim U.K.
      • Drayna D.
      • Meyerhof W.
      The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception.
      ,
      • Kim U.K.
      • Jorgenson E.
      • Coon H.
      • Leppert M.
      • Risch N.
      • Drayna D.
      Positional cloning of the human quantitative trait locus underlying taste sensitivity to phenylthiocarbamide.
      ), they are not in mice, suggesting that different mouse Tas2r underlie the responsiveness to these substances (
      • Nelson T.M.
      • Munger S.D.
      • Boughter Jr., J.D.
      Taste sensitivities to PROP and PTC vary independently in mice.
      ). In fact, a polygenic control of PROP sensitivity was suggested (
      • Harder D.B.
      • Whitney G.
      A common polygenic basis for quinine and PROP avoidance in mice.
      ), 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.
      Figure thumbnail gr8
      FIGURE 8Comparisons of ligand-receptor interactions for orthologous human and mouse Tas2r for bitter compounds that activate at least one receptor. A, ligand-receptor responses of one-to-one orthologous bitter taste receptors. B, agonist spectra for species-specific Tas2r clusters and associated Tas2r in the other species. Mice show three such species- or lineage-specific expansions associated with human TAS2R14 (Glires cluster I), TAS2R10 (muroid cluster I), or TAS2R13 (muroid cluster II), whereas humans have only one cluster associated with mouse Tas2r120 and Tas2r136 (anthropoid cluster).
      A good example of this is human TAS2R38 (
      • Biarnés X.
      • Marchiori A.
      • Giorgetti A.
      • Lanzara C.
      • Gasparini P.
      • Carloni P.
      • Born S.
      • Brockhoff A.
      • Behrens M.
      • Meyerhof W.
      Insights into the binding of Phenyltiocarbamide (PTC) agonist to its target human TAS2R38 bitter receptor.
      ,
      • Marchiori A.
      • Capece L.
      • Giorgetti A.
      • Gasparini P.
      • Behrens M.
      • Carloni P.
      • Meyerhof W.
      Coarse-grained/molecular mechanics of the TAS2R38 bitter taste receptor: experimentally validated detailed structural prediction of agonist binding.
      ). 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.
      • Hayakawa T.
      • Suzuki-Hashido N.
      • Matsui A.
      • Go Y.
      Frequent expansions of the bitter taste receptor gene repertoire during evolution of mammals in the Euarchontoglires clade.
      ). 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 (
      • Bufe B.
      • Breslin P.A.
      • Kuhn C.
      • Reed D.R.
      • Tharp C.D.
      • Slack J.P.
      • Kim U.K.
      • Drayna D.
      • Meyerhof W.
      The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception.
      ) (Fig. 9, right panel).
      Figure thumbnail gr9
      FIGURE 9Taxonomic, phylogenetic, and functional relations of bitter taste receptor 38 in Euarchontoglires and Carnivora species. Left panel, taxonomic tree of selected Euarchontoglires and Carnivora species. The tree was generated using the Common Tree software of the Taxonomy Browser on the NCBI website with TreeView software (
      • Huerta-Cepas J.
      • Dopazo J.
      • Gabaldón T.
      ETE: a python environment for tree exploration.
      ). The yellow lines indicate the hypothesized evolutionary origin of TAS2R38 orthologs with low sensitivity PTC recognition and lack of PROP responsiveness. Green lines indicate the assumed evolutionary origin of high sensitivity PTC- and PROP-detecting TAS2R38 orthologs, and the red lines label the origin of PTC/PROP-insensitive TAS2R38 orthologs found in mice. Middle panel, phylogenetic tree of functionally characterized TAS2R38 orthologs. The low PTC-sensitive cat Tas2r38 cDNA (yellow) (
      • Sandau M.M.
      • Goodman J.R.
      • Thomas A.
      • Rucker J.B.
      • Rawson N.E.
      A functional comparison of the domestic cat bitter receptors Tas2r38 and Tas2r43 with their human orthologs.
      ) and the two high PTC- and PROP-sensitive human (
      • Bufe B.
      • Breslin P.A.
      • Kuhn C.
      • Reed D.R.
      • Tharp C.D.
      • Slack J.P.
      • Kim U.K.
      • Drayna D.
      • Meyerhof W.
      The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception.
      ) and chimpanzee (
      • Wooding S.
      • Bufe B.
      • Grassi C.
      • Howard M.T.
      • Stone A.C.
      • Vazquez M.
      • Dunn D.M.
      • Meyerhof W.
      • Weiss R.B.
      • Bamshad M.J.
      Independent evolution of bitter-taste sensitivity in humans and chimpanzees.
      ) TAS2R38 cDNAs, as well as the PTC/PROP-insensitive Tas2r138 cDNAs of mouse (this study), were aligned with AlignX of Vector NTI software. Right panel, comparison of functionally critical residues in selected TAS2R38 orthologs. A subset of species shown in the left panel was analyzed for functionally critical amino acid positions in the corresponding TAS2R38 orthologs. The first three rows refer to amino acid positions found in human PTC/PROP taster and non-taster variants of this receptor. The first position is located in the first intracellular loop (ICP49). The second and third rows specify residues in the sixth and seventh transmembrane domain, respectively, by their position according to Ballesteros-Weinstein nomenclature (
      • Ballesteros J.A.
      • Weinstein H.
      Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors.
      ) followed by functionally important residues in the binding pocket of human TAS2R38 contributing to PROP and PTC activation (
      • Biarnés X.
      • Marchiori A.
      • Giorgetti A.
      • Lanzara C.
      • Gasparini P.
      • Carloni P.
      • Born S.
      • Brockhoff A.
      • Behrens M.
      • Meyerhof W.
      Insights into the binding of Phenyltiocarbamide (PTC) agonist to its target human TAS2R38 bitter receptor.
      ,
      • Marchiori A.
      • Capece L.
      • Giorgetti A.
      • Gasparini P.
      • Behrens M.
      • Carloni P.
      • Meyerhof W.
      Coarse-grained/molecular mechanics of the TAS2R38 bitter taste receptor: experimentally validated detailed structural prediction of agonist binding.
      ). Among these residues were tryptophan 201, serine 260, and phenylalanine 264, with corresponding positions depicted in the 4th (5.46), 5th (6.52), and 6th (6.56) rows.
      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 (
      • Biarnés X.
      • Marchiori A.
      • Giorgetti A.
      • Lanzara C.
      • Gasparini P.
      • Carloni P.
      • Born S.
      • Brockhoff A.
      • Behrens M.
      • Meyerhof W.
      Insights into the binding of Phenyltiocarbamide (PTC) agonist to its target human TAS2R38 bitter receptor.
      ,
      • Marchiori A.
      • Capece L.
      • Giorgetti A.
      • Gasparini P.
      • Behrens M.
      • Carloni P.
      • Meyerhof W.
      Coarse-grained/molecular mechanics of the TAS2R38 bitter taste receptor: experimentally validated detailed structural prediction of agonist binding.
      ). 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 (
      • Marchiori A.
      • Capece L.
      • Giorgetti A.
      • Gasparini P.
      • Behrens M.
      • Carloni P.
      • Meyerhof W.
      Coarse-grained/molecular mechanics of the TAS2R38 bitter taste receptor: experimentally validated detailed structural prediction of agonist binding.
      ). 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 (
      • Bufe B.
      • Breslin P.A.
      • Kuhn C.
      • Reed D.R.
      • Tharp C.D.
      • Slack J.P.
      • Kim U.K.
      • Drayna D.
      • Meyerhof W.
      The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception.
      ,
      • Wooding S.
      • Bufe B.
      • Grassi C.
      • Howard M.T.
      • Stone A.C.
      • Vazquez M.
      • Dunn D.M.
      • Meyerhof W.
      • Weiss R.B.
      • Bamshad M.J.
      Independent evolution of bitter-taste sensitivity in humans and chimpanzees.
      ). 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 (
      • Marchiori A.
      • Capece L.
      • Giorgetti A.
      • Gasparini P.
      • Behrens M.
      • Carloni P.
      • Meyerhof W.
      Coarse-grained/molecular mechanics of the TAS2R38 bitter taste receptor: experimentally validated detailed structural prediction of agonist binding.
      ). 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 (
      • Sandau M.M.
      • Goodman J.R.
      • Thomas A.
      • Rucker J.B.
      • Rawson N.E.
      A functional comparison of the domestic cat bitter receptors Tas2r38 and Tas2r43 with their human orthologs.
      ,
      • Lei W.
      • Ravoninjohary A.
      • Li X.
      • Margolskee R.F.
      • Reed D.R.
      • Beauchamp G.K.
      • Jiang P.
      Functional analyses of bitter taste receptors in domestic cats (Felis catus).
      ) 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 (
      • Shi P.
      • Zhang J.
      • Yang H.
      • Zhang Y.P.
      Adaptive diversification of bitter taste receptor genes in mammalian evolution.
      ) seems, at least in most cases, not to be true.
      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 (
      • Behrens M.
      • Meyerhof W.
      Gustatory and extragustatory functions of mammalian taste receptors.
      ,
      • Prandi S.
      • Bromke M.
      • Hübner S.
      • Voigt A.
      • Boehm U.
      • Meyerhof W.
      • Behrens M.
      A subset of mouse colonic goblet cells expresses the bitter taste receptor Tas2r131.
      ), 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 (
      • Shi P.
      • Zhang J.
      • Yang H.
      • Zhang Y.P.
      Adaptive diversification of bitter taste receptor genes in mammalian evolution.
      ). 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 (
      • Meyerhof W.
      Elucidation of mammalian bitter taste.
      ). 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 (
      • Born S.
      • Levit A.
      • Niv M.Y.
      • Meyerhof W.
      • Behrens M.
      The human bitter taste receptor TAS2R10 is tailored to accommodate numerous diverse ligands.
      ), 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).
      Figure thumbnail gr10
      FIGURE 10Sequence comparison of muroid cluster I. Upper panel, amino acid sequence alignment of muroid cluster I receptors. The alignment was created using the AlignX program of the Vector NTI software (Life Technologies). Residues are labeled according to the degree of conservation: yellow, identical; green, conserved; blue, identical in at least half of the sequences. The transmembrane domains (TM) are labeled by red boxes and numbered. The orientation in the lipid bilayer is shown by arrows (arrowheads point toward the extracellular site). Intracellular (ICL) and extracellular loops (ECL) are indicated and numbered. Amino acid positions demonstrated to reside in the ligand binding pocket of human TAS2R10 are indicated by stars. Red stars highlight amino acid positions with pronounced agonist selectivity, and black stars contribute to ligand binding and binding pocket constitution (
      • Born S.
      • Levit A.
      • Niv M.Y.
      • Meyerhof W.
      • Behrens M.
      The human bitter taste receptor TAS2R10 is tailored to accommodate numerous diverse ligands.
      ). Lower panel, comparison of functionally important receptor positions. The six receptors belonging to muroid cluster I were compared for their conservation at positions experimentally proven to contribute to agonist binding in human TAS2R10, a member of this cluster. Receptor residues are indicated by their position according to the Ballesteros-Weinstein numbering system (
      • Ballesteros J.A.
      • Weinstein H.
      Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors.
      ). Red-labeled residues reside in positions exhibiting pronounced agonist selectivity in TAS2R10; other positions were demonstrated to constitute the receptor binding site (
      • Born S.
      • Levit A.
      • Niv M.Y.
      • Meyerhof W.
      • Behrens M.
      The human bitter taste receptor TAS2R10 is tailored to accommodate numerous diverse ligands.
      ).
      Figure thumbnail gr11
      FIGURE 11Selected concentration-response functions of cells transiently transfected with DNA for orthologous mouse and human Tas2r. Functions are based on calcium traces acquired by FLIPR recordings. Changes in fluorescence (ΔF/F) were plotted semilogarithmically versus agonist concentrations for sequence-related human and mouse Tas2r, including one-to-one orthologs (A and B), members of the muroid (C and D), or anthropoid cluster (E and F).
      Another hypothesis about the development of species-specific Tas2r gene clusters concerns tuning breadth rather than individual agonist spectra (
      • Meyerhof W.
      • Batram C.
      • Kuhn C.
      • Brockhoff A.
      • Chudoba E.
      • Bufe B.
      • Appendino G.
      • Behrens M.
      The molecular receptive ranges of human TAS2R bitter taste receptors.
      ). 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 (
      • Matsuo R.
      Role of saliva in the maintenance of taste sensitivity.
      ) 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 (
      • Sainz E.
      • Cavenagh M.M.
      • Gutierrez J.
      • Battey J.F.
      • Northup J.K.
      • Sullivan S.L.
      Functional characterization of human bitter taste receptors.
      ,
      • Wong G.T.
      • Gannon K.S.
      • Margolskee R.F.
      Transduction of bitter and sweet taste by gustducin.
      ,
      • Ozeck M.
      • Brust P.
      • Xu H.
      • Servant G.
      Receptors for bitter, sweet, and umami taste couple to inhibitory G protein signaling pathways.
      ,
      • Ming D.
      • Ruiz-Avila L.
      • Margolskee R.F.
      Characterization and solubilization of bitter-responsive receptors that couple to gustducin.
      ,
      • Ruiz-Avila L.
      • McLaughlin S.K.
      • Wildman D.
      • McKinnon P.J.
      • Robichon A.
      • Spickofsky N.
      • Margolskee R.F.
      Coupling of bitter receptor to phosphodiesterase through transducin in taste receptor cells.
      ), 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 (
      • Behrens M.
      • Korsching S.I.
      • Meyerhof W.
      Tuning properties of avian and frog bitter taste receptors dynamically fit gene repertoire sizes.
      ) 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 (
      • Lush I.E.
      • Holland G.
      The genetics of tasting in mice. V. Glycine and cycloheximide.
      ) and by Tas2r105 knock-out mice, which demonstrated loss of nerve responses and behavioral aversion to this translational inhibitor (
      • Mueller K.L.
      • Hoon M.A.
      • Erlenbach I.
      • Chandrashekar J.
      • Zuker C.S.
      • Ryba N.J.
      The receptors and coding logic for bitter taste.
      ). 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) (
      • Lush I.E.
      The genetics of tasting in mice. III. Quinine.
      ,
      • Nelson T.M.
      • Munger S.D.
      • Boughter Jr., J.D.
      Taste sensitivities to PROP and PTC vary independently in mice.
      ,
      • Harder D.B.
      • Whitney G.
      A common polygenic basis for quinine and PROP avoidance in mice.
      ,
      • Boughter J.D.
      • Harder D.B.
      • Capeless C.G.
      • Whitney G.
      Polygenic determination of quinine aversion among mice.
      ) or sucrose octaacetate (Soa) (
      • Capeless C.G.
      • Whitney G.
      • Azen E.A.
      Chromosome mapping of Soa, a gene influencing gustatory sensitivity to sucrose octaacetate in mice.
      ,
      • Bachmanov A.A.
      • Li X.
      • Li S.
      • Neira M.
      • Beauchamp G.K.
      • Azen E.A.
      High-resolution genetic mapping of the sucrose octaacetate taste aversion (Soa) locus on mouse chromosome 6.
      ,
      • Lush I.E.
      • Hornigold N.
      • King P.
      • Stoye J.P.
      The genetics of tasting in mice. VII. Glycine revisited, and the chromosomal location of Sac and Soa.
      ), yet the cognate Tas2r genes remained unknown. The close genetic linkage of sucrose octaacetate and strychnine sensitivity (
      • Lush I.E.
      The genetics of tasting in mice. II. Strychnine.
      ) 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 (
      • Nelson T.M.
      • Munger S.D.
      • Boughter Jr., J.D.
      Haplotypes at the Tas2r locus on distal chromosome 6 vary with quinine taste sensitivity in inbred mice.
      ), likely explaining the inability of DBA/2J mice to taste sucrose octaacetate, strychnine, and brucine (
      • Boughter Jr., J.D.
      • Whitney G.
      Behavioral specificity of the bitter taste gene Soa.
      ,
      • Lush I.E.
      The genetics of tasting in mice. II. Strychnine.
      ,
      • Shingai T.
      • Beidler L.M.
      Response characteristics of three taste nerves in mice.
      ), which are all activators of Tas2r117.
      Recently, it was reported that solitary chemosensory cells in the anterior nasal epithelium express Tas2r and their signaling molecules (
      • Tizzano M.
      • Gulbransen B.D.
      • Vandenbeuch A.
      • Clapp T.R.
      • Herman J.P.
      • Sibhatu H.M.
      • Churchill M.E.
      • Silver W.L.
      • Kinnamon S.C.
      • Finger T.E.
      Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals.
      ) respond to bacterial quorum-sensing N-acyl homoserine lactones (
      • Tizzano M.
      • Gulbransen B.D.
      • Vandenbeuch A.
      • Clapp T.R.
      • Herman J.P.
      • Sibhatu H.M.
      • Churchill M.E.
      • Silver W.L.
      • Kinnamon S.C.
      • Finger T.E.
      Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals.
      ,
      • Sbarbati A.
      • Tizzano M.
      • Merigo F.
      • Benati D.
      • Nicolato E.
      • Boschi F.
      • Cecchini M.P.
      • Scambi I.
      • Osculati F.
      Acyl homoserine lactones induce early response in the airway.
      ). 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 (
      • Lee R.J.
      • Xiong G.
      • Kofonow J.M.
      • Chen B.
      • Lysenko A.
      • Jiang P.
      • Abraham V.
      • Doghramji L.
      • Adappa N.D.
      • Palmer J.N.
      • Kennedy D.W.
      • Beauchamp G.K.
      • Doulias P.T.
      • Ischiropoulos H.
      • Kreindler J.L.
      • et al.
      T2R38 taste receptor polymorphisms underlie susceptibility to upper respiratory infection.
      ), 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 (
      • Williams P.
      Quorum sensing, communication, and cross-kingdom signalling in the bacterial world.
      ,
      • Zhang L.H.
      • Dong Y.H.
      Quorum sensing and signal interference: diverse implications.
      ). 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 (
      • Lee R.J.
      • Xiong G.
      • Kofonow J.M.
      • Chen B.
      • Lysenko A.
      • Jiang P.
      • Abraham V.
      • Doghramji L.
      • Adappa N.D.
      • Palmer J.N.
      • Kennedy D.W.
      • Beauchamp G.K.
      • Doulias P.T.
      • Ischiropoulos H.
      • Kreindler J.L.
      • et al.
      T2R38 taste receptor polymorphisms underlie susceptibility to upper respiratory infection.
      ). It would be interesting to know if this also applies to those strains of mice harboring the less sensitive variant of Tas2r105 (
      • Chandrashekar J.
      • Mueller K.L.
      • Hoon M.A.
      • Adler E.
      • Feng L.
      • Guo W.
      • Zuker C.S.
      • Ryba N.J.
      T2Rs function as bitter taste receptors.
      ).
      In light of findings showing that Tas2r are present in organs that are not at all or only partially accessible to xenobiotics, such as brain (
      • Ansoleaga B.
      • Garcia-Esparcia P.
      • Llorens F.
      • Moreno J.
      • Aso E.
      • Ferrer I.
      Dysregulation of brain olfactory and taste receptors in AD, PSP and CJD, and AD-related model.
      ,
      • Garcia-Esparcia P.
      • Schlüter A.
      • Carmona M.
      • Moreno J.
      • Ansoleaga B.
      • Torrejón-Escribano B.
      • Gustincich S.
      • Pujol A.
      • Ferrer I.
      Functional genomics reveals dysregulation of cortical olfactory receptors in Parkinson disease: novel putative chemoreceptors in the human brain.
      ,
      • Dehkordi O.
      • Rose J.E.
      • Fatemi M.
      • Allard J.S.
      • Balan K.V.
      • Young J.K.
      • Fatima S.
      • Millis R.M.
      • Jayam-Trouth A.
      Neuronal expression of bitter taste receptors and downstream signaling molecules in the rat brainstem.
      ,
      • Singh N.
      • Vrontakis M.
      • Parkinson F.
      • Chelikani P.
      Functional bitter taste receptors are expressed in brain cells.
      ), testes (
      • Xu J.
      • Cao J.
      • Iguchi N.
      • Riethmacher D.
      • Huang L.
      Functional characterization of bitter-taste receptors expressed in mammalian testis.
      ,
      • Li F.
      • Zhou M.
      Depletion of bitter taste transduction leads to massive spermatid loss in transgenic mice.
      ,
      • Voigt A.
      • Hübner S.
      • Lossow K.
      • Hermans-Borgmeyer I.
      • Boehm U.
      • Meyerhof W.
      Genetic labeling of Tas1r1 and Tas2r131 taste receptor cells in mice.
      ), thyroid (
      • Clark A.A.
      • Dotson C.D.
      • Elson A.E.
      • Voigt A.
      • Boehm U.
      • Meyerhof W.
      • Steinle N.I.
      • Munger S.D.
      TAS2R bitter taste receptors regulate thyroid function.
      ), and urethra (
      • Deckmann K.
      • Filipski K.
      • Krasteva-Christ G.
      • Fronius M.
      • Althaus M.
      • Rafiq A.
      • Papadakis T.
      • Renno L.
      • Jurastow I.
      • Wessels L.
      • Wolff M.
      • Schütz B.
      • Weihe E.
      • Chubanov V.
      • Gudermann T.
      • Klein J.
      • Bschleipfer T.
      • Kummer W.
      Bitter triggers acetylcholine release from polymodal urethral chemosensory cells and bladder reflexes.
      ), 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 (
      • Correia J.N.
      • Conner S.J.
      • Kirkman-Brown J.C.
      Non-genomic steroid actions in human spermatozoa: “persistent tickling from a laden environment”.
      ). Although Tas2r114 is expressed in gustatory tissue at rather low levels, its RNA is abundantly present in testis (
      • Xu J.
      • Cao J.
      • Iguchi N.
      • Riethmacher D.
      • Huang L.
      Functional characterization of bitter-taste receptors expressed in mammalian testis.
      ). Mouse spermatids and spermatozoa respond to bitter compounds with calcium signaling in an α-gustducin-dependent manner (
      • Xu J.
      • Cao J.
      • Iguchi N.
      • Riethmacher D.
      • Huang L.
      Functional characterization of bitter-taste receptors expressed in mammalian testis.
      ). Genetic ablation of bitter receptor cells in mice causes massive spermatid loss (
      • Li F.
      • Zhou M.
      Depletion of bitter taste transduction leads to massive spermatid loss in transgenic mice.
      ). 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.

      Author Contributions

      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.

      Acknowledgments

      We thank Elke Chudoba, Josefine Würfel, Florian Padberg, Lisa Oldorff, Julia Freydank, Alexandra Semmler, and Eva-Katharina Hage (Nuthetal) for expert technical assistance.

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