Vertebrate Bitter Taste Receptors: Keys for Survival in Changing

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Vertebrate Bitter Taste Receptors: Keys for Survival in Changing Environments Maik Behrens, and Wolfgang Meyerhof J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04835 • Publication Date (Web): 25 Dec 2016 Downloaded from http://pubs.acs.org on December 27, 2016

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Journal of Agricultural and Food Chemistry

Perspectives

Vertebrate Bitter Taste Receptors: Keys for Survival in Changing Environments

Maik Behrens and Wolfgang Meyerhof German Institute of Human Nutrition Potsdam-Rehbruecke, Dept. Molecular Genetics Correspondence: Dr. Maik Behrens, German Institute of Human Nutrition PotsdamRehbruecke, Dept. Molecular Genetics, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany. Fax: +49 33200 88 2384; Phone: +49 33200 88 2545; e-mail: [email protected]

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Abstract

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Research on bitter taste receptors has made enormous progress during the recent years. While

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in the early period after the discovery of this highly interesting receptor family special

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emphasis was placed on the deorphanization of mainly human bitter taste receptors, the

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research focus has shifted to sophisticated structure-function analyses, the discovery of small

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molecule interactors, and the pharmacological profiling of non-human bitter taste receptors.

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These findings allowed novel perspectives on e.g. evolutionary and ecological questions that

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have arisen and that are discussed.

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Key words

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Bitter taste perception; G protein-coupled receptors

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Introduction

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The ability of vertebrates to sense bitterness is thought to be important for the avoidance of

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potentially toxic compounds occurring frequently in nature, although a clear correlation

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between bitterness and toxicity is lacking.1 The detection of these substances is mediated by

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G protein-coupled receptors belonging to the taste 2 receptor (TAS2R) family that are present

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in specialized taste receptor cells located on the tongue and in the oral cavity. Following their

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discovery in the year 2000,2-4 enormous progress has been made including the functional

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characterization, the establishment of intra- and extraoral expression patterns, the

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determination of structure-function relationships and other biochemical as well as cell

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biological details. More recently, the identification of bitter taste receptor repertoires of a

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larger collection of vertebrates and the acquisition of the agonist profiles detected by some of

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these receptors allowed better insights in the evolutionary processes shaping these highly

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interesting proteins. However, the answers to many of the early questions resulted in new, so

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far unanswered questions, which need to be addressed in the future. Rather than reviewing all

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aspects of bitter taste research, the present article will highlight only some of the past

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developments and achievements in the field and how they shaped current views and, likely,

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future research directions.

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Bitter taste receptors

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The human bitter taste receptor repertoire- The enormous variety of bitter substances is

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detected by G protein-coupled receptors of the taste 2 receptor (gene symbol = TAS2R

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(human), Tas2r (mouse)) family. The first functionally characterized receptors, the mouse

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Tas2r105 (mT2R5), mouse Tas2r108 (mT2R8), and human TAS2R4 (hT2R4) were shown to

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respond to one or maximally two of 55 diverse bitter compounds used for functional 3 ACS Paragon Plus Environment


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screening, suggesting that bitter taste receptor genes could be specialized for the detection of

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distinct agonists.3 Although the subsequently deorphaned human TAS2R16 was demonstrated

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to respond to numerous chemically closely related β-D-glucopyranosides,5 the pronounced

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specificity of this receptor again pointed towards a narrow range of substances detected by

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these proteins raising the obvious question of how can so few receptors facilitate the detection

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of almost countless and chemically diverse bitter agonists? A reasonable solution to this

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problem came from the observation that human TAS2Rs are able to form homo- and

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heterodimers with each other in vitro and, since the possible combinations appeared

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unrestricted, 325 homo- and heterodimeric receptors could exist.6 However, it is still

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unknown if bitter taste receptor heterodimers contribute to a broadening of the detectable

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agonist spectrum as it was not possible to identify functional consequences of the

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oligomerization despite considerable efforts.6 To date, all reported bitter taste receptor

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responses in vitro can be ascribed to monomeric or homodimeric receptors. The discovery of

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the much broader tuning properties of the human TAS2R14, which responded to about a

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quarter of the tested compounds7 hinted at another possible solution for the apparent

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discrepancy between receptor number and the plethora of bitter tastants, as some receptors

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may contribute to the overall bitter taste profile of humans more than others. Indeed, after the

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deorphanization of 21 of the ~25 putative functional human bitter taste receptors,8,9 it appears

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that the number of TAS2Rs is fully sufficient to facilitate the detection of that many bitter

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substances. In general, the human TAS2Rs can be categorized into 4 groups, the 3 receptor

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“generalists” with extensive agonist spectra comprising of TAS2R10, TAS2R14, and

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TAS2R46, each able to respond to about one-third of the bitter substances (their combined

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activities suffice for the detection of about half of the bitter substances tested so far), a

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number of narrowly tuned receptor “specialists” that detect few bitter compounds, the

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intermediately tuned receptors representing the majority, as well as two receptors, the


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TAS2R165 and TAS2R38,10 which exhibit pronounced selectivity for defined classes of

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chemicals (Fig. 1).

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TAS2R gene variants- Shortly after the discovery of human TAS2R genes it was recognized

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that numerous genetic polymorphisms of these genes exist with high frequencies in the human

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population.11,12 Some of the TAS2R variants resulting from these polymorphisms were

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subsequently shown to affect the function of the corresponding receptors contributing to

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individual bitter taste perception. Whereas some of the genetic variations result in the

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complete loss of receptor function due to incapacitating changes of the receptors’ polypeptide

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chains10,13,14 or the genomic deletion of entire TAS2R genes,15-18 other variants exhibit more

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subtle changes leading to reduced receptor responsiveness.19 The best investigated genetic

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polymorphism in a TAS2R gene affects the receptor TAS2R38.12 The two major alleles occur

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with rather similar frequencies in most populations and determine the ability to taste the

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synthetic bitter substances phenylthiocarbamide (PTC) and 6-n-propyl-thiouracil (PROP).

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The functional taster variant exhibits 3 amino acid sequence differences at positions 49, 262,

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and 296 compared to the non-functional non-taster variant.14 Whereas the taster variant,

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TAS2R38-P49A262V296

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TAS2R38A49V262I296 shows no response in vitro.10 Also natural compounds activating human

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TAS2R38 are plentiful and may thus influence food choice20 and innate immunity, since

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TAS2R38 has been reported to respond to bacterial quorum sensing molecules and is

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implicated in pathogen defense reactions.21 Other variations resulting in non-functional bitter

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taste receptors affect TAS2R9 (missense mutation),13 TAS2R46 (nonsense mutation),11,16

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TAS2R43 and TAS2R45 (whole gene deletions).15-18 Additional TAS2R variants affect the

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receptors TAS2R16,19 TAS2R31 (former gene symbol TAS2R44), and TAS2R43,15,17

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however, these receptors do not lose their function completely. A highly interesting case is

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presented by receptor TAS2R16, which occurs as a low-sensitive variant with high frequency

confers

exquisite

sensitivity

for

PTC

and

PROP,

the



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in some areas of the African continent, whereas outside of Africa exclusively the high-

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sensitive variant is found.19 Since this receptor responds to various cyanogenic β-D-

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glucopyranosides such as amygdalin from bitter almonds and linamarin from manioc, it was

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suggested that the low-sensitive variant dominates in regions with an elevated malaria risk,

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because a lower sensitivity for bitter vegetables containing cyanogenic-β-D-glucopyranosides

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in humans could exert antimalarial activity causing protective sickle cell-like symptoms.19 A

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recent report, however, challenged the regional correlation between the occurrence of low-

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sensitive TAS2R16 alleles and malaria risk.22 Among the mentioned functional

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polymorphisms two have been associated with non-gustatory TAS2R functions. Whereas the

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non-functional TAS2R9V187 is associated with an elevated diabetes mellitus risk, which could

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be attributable to its expression in enteroendocrine L-cells secreting blood glucose regulating

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incretin hormones,13 the non-functional TAS2R38-A49V262I296, whose expression in human

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sinonasal epithelia was detected, correlated with an increased frequency of upper-airway

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infections.21,23 As more and more reports on extraoral expression of TAS2Rs emerge, it

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appears likely that TAS2R-polymorphisms have profound physiological consequences apart

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from perceptual differences.

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Structure-function analyses- The thorough characterization of human TAS2Rs, on the one

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hand raised questions about the architecture of the binding pockets that enable these receptors

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to accommodate so many diverse bitter substances, yet maintaining an astonishing degree of

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specificity, and, on the other hand provided the basis for careful structure-function analyses.

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Consequently, in the recent years several studies have been devoted to elucidate structural

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features of TAS2Rs involved in agonist activation. As these studies were subject of detailed

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reviews24-26 only some facets of the findings shall be presented here. Already the first detailed

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structure-function study devoted to one of the broadly tuned human bitter taste receptors, the

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TAS2R46, found an answer to the question whether large ligand profiles may require the 6 ACS Paragon Plus Environment

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existence of multiple binding pockets rather than relying on a single binding site. By a

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combination of functional calcium-mobilization assays, extensive site-directed mutagenesis as

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well as in silico homology modeling and ligand docking experiments, it was shown that

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agonists interact with the receptor in a single orthosteric binding pocket with overlapping, but

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individual contact points.27 Recently, a subsequent study found evidence that agonists before

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entering the orthosteric binding pocket transiently occupy a vestibular binding site, which

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may act as a “specificity filter” for agonists.28 In light of the complex and concentrated

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mixtures of chemicals to which TAS2Rs are exposed during eating this seems to represent an

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appealing mechanism to enhance detection accuracy. In another study investigating the

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likewise broadly tuned human TAS2R10 two intriguing observations were presented.29

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Firstly, it was demonstrated that several amino acid residues located in the binding pocket of

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this receptor were highly agonist selective, supporting the interaction with some agonists,

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while perturbing optimal interaction with other agonists, suggesting that this receptor is

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optimized to interact with many agonists at the expense of potentially higher affinities for

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individual agonists (Fig. 2). Secondly, the finding that the binding mode for the toxic alkaloid

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strychnine in TAS2R10 differs from that of the same molecule in TAS2R46 indicates that the

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ability of different TAS2Rs to respond to the same bitter substances is not necessarily the

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result of conserved pharmacological features “inherited” from common ancestral bitter

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receptors, but rather evolved independently during evolution. Moreover, the above mentioned

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studies agree with structure-function analyses of other TAS2Rs such as the chemical class-

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specific TAS2R1630 and TAS2R3831 with respect to the location of the orthosteric binding

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pocket rather deeply buried in the upper one-third of the transmembrane domain area (Fig. 3),

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although the involved transmembrane domains may slightly differ among these TAS2Rs.

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Other reports suggested a more pronounced involvement of extracellular loops in ligand

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binding of TAS2R4,32 TAS2R31, and TAS2R4333 and it remains to be seen whether these

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residues indeed contribute to the formation of the orthosteric binding site or rather indicate the 7 ACS Paragon Plus Environment


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general presence of vestibular sites in TAS2Rs. Comparing the location of binding sites of

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TAS2Rs with those of class A GPCRs it is eminent to stress that similarities prevail25 and

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hence, despite low overall sequence homology of TAS2Rs with other GPCRs, the structures

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and functional principles of TAS2Rs are far less exotic than initially thought.

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Tas2r repertoires of other vertebrates- From an evolutionary perspective the bitter taste

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receptor gene family represents a rather recent addition to the GPCR superfamily traceable

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back to teleostean fish (bony fish), but absent in cartilaginous fish such as elephant sharks.34

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Even though the history of bitter taste receptor genes is not as long as those for many other

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GPCRs, their evolution has been more dynamic leading to rapid diversification of the Tas2r

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genes. This is indicated by both, substantial sequence variation among Tas2r paralogs and

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considerable differences in the sizes of Tas2r gene repertoires among vertebrates, which range

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from 0-1 putatively functional genes in penguins35 and cetaceans (including e.g. whales)36-40

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over ~25 in humans41 to almost 80 in the coelacanth.42 Not surprisingly, the number of

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pseudogenes is also subject to intense variation. Some hypotheses that could explain the

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considerable variability of the numbers of potentially intact bitter receptors have been

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formulated and may help to understand why humans fit right in between the extremes,

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although throughout human history dietary habits, including the acceptance of bitter food

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items, were clearly influenced by changing sociocultural factors as well (for reviews see43,44).

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One such hypothesis is that a low number of intact bitter taste receptor genes indicates

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inferior bitter tasting abilities or even the complete loss of the sense of taste. Indeed, some

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animals that swallow their prey whole such as dolphins and other cetaceans have lost all or

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almost all of their taste receptors. Similarly, it has been speculated that chickens, which do not

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possess a functional sweet receptor and carry only 3 intact bitter taste receptor genes in the

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genome, have inferior tasting abilities (for a review see45).


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Several recent reports addressed the relationship between the numbers of bitter taste receptor

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genes in broader set of vertebrates with the corresponding dietary habits.37,39,46,47 In general, it

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seems that herbivores, who more frequently encounter bitter substances than carnivores

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possess more Tas2r genes. Whereas some studies found a positive correlation between diet

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and Tas2r gene numbers,37,39,46 other studies failed to obtain significant differences.47 Several

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reasons may exist for a somewhat skewed relationship between dietary habits and the number

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of bitter taste receptors. Firstly, at least in some herbivore species the tolerance for the

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consumption of bitter plant constituents may result from improved degradation mechanisms

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that have co-evolved.48 Secondly, there is not a strict correlation between bitterness and

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toxicity1 and therefore some variability in the receptor numbers may not immediately affect

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the chances for survival of species in particular in highly specific habitats. Thirdly, some

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bitter substances have even beneficial health effects, e.g. in cases of infections with worms or

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other pathogens, which would suggest a role of Tas2rs in active seeking behavior for

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medicinal plants49,50 and therefore a selective benefit beyond nutritional needs appears likely.

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Fourthly, and related to the last point, it is still a matter of debate whether the vertebrates’

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bitter sensing system has some discriminative capacity (c.f.51 and references therein) and thus,

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some bitter substances could be tolerated, while others lead to rejection behavior. If

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discrimination among bitter substances is possible and, in turn, connected with specialized

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Tas2rs for, e.g. rejection, the simple counting of functional Tas2rs would insufficiently

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describe dietary preferences. Fifthly, and perhaps most importantly- bitter taste receptor

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expression is not restricted to the oral cavity, an ever growing number of non-gustatory

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tissues were reported indicating roles beyond taste (for recent reviews see23,52,53). While some

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of the expression sites such as the gastrointestinal tract may indicate an interaction with food

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derived xenobiotics analogous to the role of Tas2rs in the oral cavity, their expression in other

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tissues such as respiratory tract (for a recent review see23) , brain,54-57 mast cells58 and white



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blood cells,59-62 testis (for a recent review see63), or heart,64,65 to name just a few are difficult

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to correlate at present with dietary habits.

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Some of the above speculations were nourished by the fact that the knowledge about the

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functions of bitter taste receptors were strongly human biased since no comprehensive

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analyses of other vertebrate Tas2r was performed until recently. The functional

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characterization of a number of non-human Tas2rs shed some light on the functional

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relationships among Tas2rs of different clades. Whereas previous studies concentrated on the

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characterization of single or few Tas2rs from other species such as rodents, fish, and primates,

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recently more comprehensive analyses were published on avian, amphibian,66 carnivores67,68

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and mouse (Fig. 4).69

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An important outcome of the characterization of chicken and turkey Tas2rs was that very

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small bitter taste receptor repertoires represented by the 3 chicken and 2 turkey receptors do

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not necessarily indicate inferior bitter tasting abilities.66 It was shown that the Tas2rs of

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chicken and turkey are on average very broadly tuned and therefore, their low number is at

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least partially compensated by tuning breadth. On the other hand, a large number of Tas2rs as

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in the cases of mice and the Western clawed-frog X. tropicalis apparently allows the

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development of highly specialized receptors.66,69 The Tas2r repertoire of the domestic cat (F.

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catus) is until now the only functionally characterized bitter taste receptor repertoire within

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the order of carnivores67,68 and exhibits, albeit a relative small Tas2r gene number with 12

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potentially intact genes, similar characteristics possessing broadly tuned as well as

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intermediate and narrowly tuned receptors.67 The analyses of mouse Tas2rs revealed more

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interesting details. On the one hand, it was demonstrated that among the 35 putatively

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functional receptors only a single receptor can be considered broadly tuned, whereas more

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narrowly tuned receptors exist. On the other hand and most surprisingly, it was reported that

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orthologous receptors are not functionally conserved.69 In fact, for none of the compared

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mouse and human orthologs an unambiguous functional conservation could be demonstrated 10 ACS Paragon Plus Environment

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indicating that even receptor pairs whose sequence was well conserved after the split of

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rodent and primate lineages contribute to diversification of bitter recognition rather than the

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detection of common agonists.69

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An interesting possibility to investigate the evolutionary development of bitter taste receptor

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genes results from the availability of functional data on a large number of Tas2rs and detailed

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structure-function analyses on selected reference receptors. Combining such data Lossow and

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colleagues69 were able to conclude that species specific Tas2r gene expansions generated

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diversified receptor arrays by permutation of few critical positions located in the ligand

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binding pockets of the Tas2r. This represents a highly efficient way to generate different

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agonist selectivities with a limited number of mutations. Moreover, such comparative data can

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be used to trace changes in receptor specificities over a range of species with a limited

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number of functional data.

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Bitter taste receptor gene expression- In the mammalian oral cavity Tas2r genes are

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expressed in a specific subpopulation of type II taste receptor cells (TRCs), which do not

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overlap with those TRCs that express sweet or umami taste receptors.70 It has been a matter of

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debate whether the bitter TRCs represent uniform sensors for bitter substances expressing all

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Tas2r genes in every cell or whether they form a heterogeneous population where each bitter

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TRC expresses only subsets of them. On the one hand in situ hybridization data with Tas2r

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probe mixtures2 as well as sophisticated functional complementation experiments in

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genetically modified mouse models71 were interpreted in support of a uniform bitter TRC

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population in rodents, on the other hand independent in situ hybridization experiments using

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multiple probes4 and elaborate in vivo stimulation protocols on lingual slices of rats72 pointed

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to a heterogeneous bitter TRC population. Comprehensive analyses of Tas2r mRNAs in

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lingual tissues of human51 and mouse69 supported the existence of a heterogeneous bitter TRC

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population. Since a non-homogeneous bitter TRC population would be a prerequisite for a 11 ACS Paragon Plus Environment


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possible discrimination among different bitter compounds, these findings have important

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implications. Although a number of studies investigated the bitter discriminatory capacity of

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mammals, a final answer to this question is still lacking as contrasting results were obtained

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(cf.51 and references therein). Whereas it was demonstrated that indeed all Tas2r genes are

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expressed in gustatory tissues of the oral cavity of humans and mice51,69 and hence, support a

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function as taste receptors, more specialized expression profiles seem to exist in extraoral

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tissues. Whereas 7 of the 35 putative functional mouse Tas2rs and about half of the 25 human

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TAS2Rs are expressed in cardiac tissue,65 the occurrence of these receptors in respiratory and

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gastrointestinal epithelia seems to be more heterogeneous as evident by the differential

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responses seen upon stimulation with different bitter compounds in rodent experiments.23,73

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As some indications for nutrient-dependent regulation of Tas2r gene expression in

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gastrointestinal tissues have accumulated,65,74,75 these expression profiles may exhibit

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dynamic changes. It has to be mentioned that only few reports provided direct evidence on the

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cell type(s) expressing Tas2rs in gastrointestinal tissues74-76 and hence, the heterogeneity of

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bitter responsive cells could be even higher than assumed. The intriguing observation that

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Tas2r genes expressed in cardiac tissue are clustered together on the corresponding

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chromosomes,65 suggests that common regulatory elements in these loci exist orchestrating

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tissue-specific expression. However, the frequent lack of identified cell types and the

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substantial overlap in agonist profiles identified for the individual Tas2rs does currently not

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allow conclusions about the biological meaning of the specific arrays of receptors found in

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non-gustatory tissues.

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Future directions- Despite the incredible gain in knowledge about bitter taste receptors

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over the last 16 years open questions remained or have emerged in the course of the research.

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While the architecture of the binding pockets of the broadly tuned TAS2Rs is quite well

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understood, it is not clear how a narrow tuning breadth is achieved. Hence, structure-function 12 ACS Paragon Plus Environment

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experiments on narrowly tuned TAS2Rs and subsequent comparison with TAS2Rs possessing

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large agonist panels are important to understand which mechanism(s) govern limited agonist

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spectra. The prediction of bitter taste receptor structures today relies exclusively on homology

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modeling with template structures that derive from GPCRs with low amino acid homology.

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Hence, the corresponding models cannot be considered to represent high resolution structures.

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Such structures would greatly improve the prediction of novel agonists, but also help to

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design selective antagonists, which are urgently needed for basic research as well as for use in

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pediatric medicinal formulations. Therefore, attempts to obtain experimental structures for at

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least some of the TAS2Rs would significantly accelerate research in this area. Another rather

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poorly understood process concerns the ligand-induced conformational changes occurring

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during receptor activation. To date just a single study has been devoted to investigate the

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mechanism of bitter taste receptor activation at the example of human TAS2R1.77 It would be

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important to intensify research devoted to the activation mechanism of TAS2Rs as the gained

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knowledge could aid rational design of small molecule modulators.

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Only recently the relationship between the level of TAS2R38 mRNA in taste cells and human

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bitter perception was established.78 As the expression strength of genes could be modulated

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by epigenetic mechanisms and food items have been associated with epigenetic modifications

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(for a review see79), it seems warranted to devote research efforts to study this interesting

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field, especially since TAS2R gene expression is not limited to the oral cavity.

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Bitter taste receptors cannot longer be seen solely as taste receptors, because their expression

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and functional roles in other tissues such as the respiratory epithelia, the gastrointestinal tract,

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testis, brain, heart, make them prime targets for drug design. Clarification of the role(s) that

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bitter taste receptors play outside the gustatory system is of outmost importance for many

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open and urgent questions. Whereas some of the activities exerted by the activation of bitter

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taste receptors in epithelia such as the gastrointestinal tract or the airways might be related to

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xenobiotic detection as well, other tissues such as brain or heart are not directly accessible by 13 ACS Paragon Plus Environment


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bitter compounds from the outside environment. Here, the detection of yet unknown

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endogenously formed ligands might be conceivable. It would be a high priority to identify

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such endogenous ligands for bitter taste receptors and to elucidate the function of this

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detection system. Moreover, studies aiming at the evolutionary sequence of tissues acquiring

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bitter compound responsiveness would not only allow to identify the original function of

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bitter taste receptors in vertebrates, it would also shed light on the interdependence of food

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resources and the evolutionary development of bitter taste receptor pharmacology.53 Of

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course, if bitter taste receptors have dominant functions aside from taste, the existence of a

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gene sharing-like mechanism like that proposed for lens crystalline genes

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impact the evolutionary flexibility of the Tas2r gene family.

80

would greatly

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304

Bitter compounds

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Bitter substances occur plentiful in nature and cover a wide variety of chemicals that can

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differ in size, polarity, and chemical structures (cf.81 and references therein). Whereas many

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bitter substances represent plant, fungal, or animal metabolites, other rich sources of bitter

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compounds are chemical processes occurring during cooking, fermentation, or chemical

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syntheses (Fig. 5). At present, it is impossible to judge how many bitter compounds may exist

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in nature. Current data including synthetic compounds document over 680 bitter substances

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based on published functional receptor screenings and information about perceptual properties

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of chemicals.82 It appears reasonable to assume that the number of identified bitter substances

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will increase considerably with time and may exceed one thousand easily. It is important to

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note that the term “bitter” for the taste of these substances cannot be simply extended to

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species other than humans. While there is usually a good overlap between substances that

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represent aversive stimuli to other species and their bitter taste in humans, differences should

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be anticipated. These differences could be due to different bitter taste receptor gene 14 ACS Paragon Plus Environment

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repertoires shaped to meet the corresponding ecological niches of the species, as well as

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alternative routes leading to orosensorically-mediated aversive reactions such as irritants

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activating TRP channels residing in the oral cavity83,84 or compounds eliciting a dry, tightened

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mouthfeel called astringency.85,86 Not all substances that taste bitter to humans have been

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matched with one of the 25 human TAS2Rs in vitro.8 One explanation for this unanticipated

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outcome could be technical issues preventing the deorphanization of the remaining 4 orphan

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human TAS2Rs which could possess the necessary recognition spectra to close this gap.

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Another possibility is the existence of TAS2R-independent bitter detection routes. Indeed,

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direct interaction of cell permeable bitter compounds with intracellular signaling

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molecules87,88 as well as alternative receptors such as the nicotinic acetylcholine receptor,

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which has been identified in taste receptor cells of rodents89 have been proposed.

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Bitter substances exert a wide range of pharmacological activities, which include, but are not

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limited to acute toxic effects.90 One of the most infamous toxic bitter compounds is the

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alkaloid strychnine from the seeds of the Strychnos nux-vomica tree that acts as glycin

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receptor antagonist leading91 to muscular convulsions. Less known, but with a similar mode

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of action, is the sesquiterpene lactone picrotoxinin from the seeds of the plant Anamirta

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cocculus, which inhibits GABAA-receptors92 again resulting in potentially deadly

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convulsions. Interestingly, both compounds occur in the corresponding plants mixed with

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structurally closely related substances called brucin and picrotin, respectively, which are

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nevertheless less potent bitter compounds7,93 demonstrating pronounced selectivity of the

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corresponding bitter receptors. Also the bitter substance (-)-α-thujone, the psychotropic

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component of the liqueur absinthe, exerts its incapacitating effect via the inhibition of

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GABAA and 5-HT receptors.94 Glycin-, GABAA-, and 5-HT-receptors are members of the

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neurotransmitter-gated ion channels of the Cys-loop receptor family (for a review see95) and

342

hence, unrelated to Tas2rs belonging to the GPCR-family. Therefore, the protective

343

interaction of these substances with Tas2rs evolved independently. Other bitter substances 15 ACS Paragon Plus Environment


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344

possess rather beneficial effects as they act as antimalarial agents in case of artemisinin96 or

345

can be used as analgesic, anti-inflammatory, and antipyretic drug such as D-(-)-salicin from

346

willow bark.97 As different as the pharmacological activities are, the chemical classes of bitter

347

compounds span a wide spectrum including amino acids and peptides, amines and amides,

348

esters and lactones, ketones, fatty acids, phenols, alkaloids, metal ions, N-heterocyclic

349

compounds, crown ethers, azacycloalkanes as well as urea and related substances (for a more

350

detailed list see81 and references therein).

351

Somewhat surprisingly, naturally occurring bitter substances do not appear to activate the

352

receptors better than synthetic compounds despite their presumed co-evolution. Among the

353

compounds that taste most bitter to humans we find the plant metabolite amarogentin as well

354

as the synthetic substance denatonium benzoate indicating that maximal bitterness may not be

355

a particular strong natural selector for human receptor-substance co-evolution. Since the

356

number of potent natural bitter compounds, which activate TAS2Rs already at low

357

concentrations, exceeds the number of potent synthetic substances,8 one could speculate that

358

the dominant trait affecting the evolution of the system is detection sensitivity. The number of

359

human bitter taste receptors activated by natural or synthetic bitter compounds (cf.8,9,69)

360

appears quite similar with 17 TAS2Rs being responsive to natural compounds and 20

361

receptors responding to synthetic compounds. Sixteen receptors were actually activated by

362

both. This is also true for individual compound receptor combinations. Whereas the synthetic

363

substance diphenidol with 15 cognate receptors activates the largest number of human

364

TAS2Rs, the natural substance quinine is able to elicit responses from 9 receptors. Moreover,

365

there are numerous examples for both, natural and synthetic chemicals that activate several

366

different TAS2Rs.8 Evidence is still missing to demonstrate if the number of cognate bitter

367

receptors determines the perceived bitterness of a given compound. Since overall bitter

368

chemicals stimulate their various cognate TAS2Rs with different potencies and efficacies it is

369

likely that some receptors contribute more to the perceived bitterness of a compound than 16 ACS Paragon Plus Environment

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370

others. Recent research started to address the question whether the ability of certain bitter

371

compounds to activate many bitter receptors is intrinsic to the substance or dependent on the

372

individual bitter taste receptor repertoire of a biological species and whether specific chemical

373

features identify broad acting bitter substances compared to compounds with a limited

374

spectrum of responding bitter taste receptors. It was shown that some substances such as the

375

antibiotic chloramphenicol, which activates 9 human TAS2Rs,8,9 also activated all 3 chicken,

376

all 2 turkey, 1 of 3 tested zebra finch, and 4 of 6 tested frog Tas2rs66 as well as two mouse

377

TAS2Rs,69 similarly diphenidol, which acted most broadly on human TAS2Rs,8 activated

378

chicken (3 of 3), turkey (2 of 2), zebra finch (2 of 3), frog (1 of 6),66 and mouse Tas2rs (6 of

379

34).69 According to in silico analyses of molecular properties of broadly versus narrowly

380

acting bitter substances it is suggested that, among other descriptors, small and globular

381

substances behave rather promiscuous, whereas large and flat molecules tend to be bitter taste

382

receptor selective.98

383

Not all molecules that bind to TAS2Rs act as agonists, rather some represent antagonists. The

384

first receptor selective but rather broad acting antagonist was discovered in a high throughput

385

screening devoted to discover small molecules able to block the bitter off-taste of the artificial

386

sulfonyl amide sweeteners saccharin and acesulfame K.99 It was demonstrated that the

387

compound 4-(2,2,3-trimethylcyclopentyl)butanoic acid, also known as GIV3727, potently

388

inhibited the activation of the two dominant bitter receptors for saccharin’s and acesulfame

389

K’s bitterness, the TAS2R31 and TAS2R43, by a competitive mode of action. Soon

390

thereafter, the first natural bitter inhibitors were identified.100 These compounds were

391

strikingly similar to bona fide agonists of the receptor TAS2R46, however, they did not

392

activate,

393

hydroxydihydrocostunolide and 3β-hydroxypelenolide, occur in the same plants that produce

394

TAS2R46 activating sesquiterpen lactones, a previously unanticipated level of complexity for

395

the bitterness of plants became evident. Moreover, all inhibitors, GIV3727 as well as the

but

rather

inhibited

the

receptor.

Since

these

substances,

3β-



Page 18 of 41

396

natural bitter blockers, surprisingly exhibited a bivalent feature by acting as bitter inhibitors

397

on some TAS2Rs and as bitter agonists on others. After these initial reports a growing number

398

of bitter blockers were identified (e.g.32,101,102), indicating that such molecules may not be

399

rare.

400

Future directions- There are many open questions concerning bitter substances that

401

remain to be answered in the future. One of these is the existence of common chemical (core-)

402

structures that identify bitter substances. This question was already asked, and partially

403

answered, by Tancredi and colleagues many years before bitter taste receptor genes were

404

identified.103 Based on previous models predicting that the common core structure of sweet

405

molecules would consist of a hydrogen bond donor and –acceptor site at a distance of about

406

3Å,104,105 it was suggested that bitter compounds have a similar structure but with a reverse

407

orientation.103 Whereas this model indeed explained why D- and L-enantiomers of some

408

amino acids elicit pronounced sweet or bitter taste, the finding that so many differently tuned

409

TAS2Rs exist in human argues for a more complex interaction pattern. For two of the 25

410

receptors, the TAS2R16 and the TAS2R38, such common chemical structures, namely the β-

411

D-glucopyranose moiety and the isothiocyanate/thiourea moiety, respectively, were

412

identified.5,10 However, other receptors seem to respond to a larger variety of chemicals.

413

There are several possible explanations for the difficulties associated with the identification of

414

common pharmacophores for TAS2Rs: Firstly, recent evidence was provided that not only

415

bitter agonists can be found in nature but also antagonists. Since the majority of reports so far

416

relied on assays measuring receptor activation in vitro and not ligand binding, the

417

identification of the chemical core structure required for binding to the receptors without

418

leading to activation was usually not implemented. Recent publications using competitive

419

approaches making use of agonists/antagonist mixtures32,99-102,106,107 could close this gap since

420

classical binding assays suffer from the rather low affinities of most bitter compound-receptor

421

interactions and, as a consequence, no such data exist. Secondly, some bitter receptor binding 18 ACS Paragon Plus Environment

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422

sites may actually be composed of sub-sites, which interact with subsets of chemically diverse

423

bitter agonists. An indication for agonists based on multiple distinct chemical scaffolds was

424

recently provided for in silico predicted novel TAS2R14 agonists107 and could apply to other

425

receptors as well. Of course, this obscures the identification of individual pharmacophores

426

considerably.

427

Most likely only some (if at all) of the identified bitter activators of present day’s bitter taste

428

receptors are identical to those that shaped the evolution of the receptors. Major vertebrate

429

classes were already formed prior to the neophyticum/upper Cretaceous period when

430

angiosperm plants, which dominate our planet since then, started to conquer the earth.

431

Clearly, the early development of nowadays bitter receptors was not driven by bitter

432

substances originating from “modern” plants, but rather by toxic compounds from other

433

sources. Also climate and habitat changes that early humans faced when they started to

434

migrate out of Africa obscure the bitter substance-receptor co-evolution because plants

435

growing ~2 million years ago in Africa dominantly shaped our TAS2R gene repertoire.

436

Hence, it is anticipated that important contributions on the structures of relevant bitter

437

substances may come from botanical and paleo botanical experts who join the field of bitter

438

taste research. Finally, the relationship between bitterness and toxicity is less tight than

439

frequently assumed.1 It would be highly relevant to investigate this question from an

440

ecological point of view to see if bitterness is mostly indicating potential toxins or if it is also

441

guiding seeking behavior for therapeutic substances in case of illness49,50 and whether plants

442

may not only synthesize toxins to defend herbivores but also, as a kind of chemical

443

mimicry,108 non-toxic substances.

444

The occurrence of natural bitter receptor inhibitors raises the question of how abundant such

445

molecules in nature are and if the existence of such modulatory compounds has influenced the

446

evolution bitter taste receptor gene repertoires. It may well be that the expansion of bitter taste

447

receptor genes was not dominantly influenced by the need to recognize more and more bitter 19 ACS Paragon Plus Environment


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448

substances, but rather to avoid as much as possible completely overlapping activities of

449

activators and inhibitors with potentially fatal outcomes.100 In the future large screening

450

campaigns to identify more natural bitter inhibitors may help to clarify this question.

451


Page 21 of 41


452

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Page 34 of 41

758

Figure captions

759

Figure 1. The contribution of differently tuned bitter taste receptors to overall bitter

760

perception. More than 100 bitter substances were screened for their activation of 25 human

761

TAS2Rs. The fraction of substances detected by receptors with broad, intermediate, narrow,

762

and chemical group-specific (Group spec.) detection spectra is indicated. The human TAS2Rs

763

belonging to the four groups are indicated.

764

Figure 2. The binding pocket of human TAS2R10. Residues that were demonstrated to be

765

critical for agonist activation in the binding pocket of the human bitter taste receptor

766

TAS2R10 are shown. Three residues with pronounced agonist selectivity are depicted as bold

767

stick representations. Other residues contributing to general agonist activation are indicated as

768

think sticks. The center sphere indicates that sufficient space to accommodate large agonists is

769

available in the binding pocket.

770

Figure 3. Schematic of a TAS2R embedded in the plasma membrane with bound agonist. The

771

seven transmembrane domains connected by 3 extracellular and 3 intracellular loops of a

772

TAS2R are indicated by cylinders and connecting lines. The amino terminus points to the

773

extracellular site, the carboxy terminus (not shown) is located at the intracellular site. The

774

approximate ligand binding site in the upper third of the transmembrane domain helices is

775

indicated by a strychnine molecule (red).

776

Figure 4. Bitter taste receptor gene repertoires and tuning breadths. The bitter taste receptor

777

repertoires of seven species which have been functionally characterized are shown. The

778

putatively functional receptors are indicated by black squares and the adjacent number. The

779

tuning widths of the receptors are indicated by the sizes of the spheres and the fraction of test

780

compounds that activated the corresponding receptor is provided in percent. The receptor

781

symbols are given. The information for turkey, chicken, zebra finch, and Western clawed frog 34 ACS Paragon Plus Environment

Page 35 of 41


782

were derived from;66 the information for cat were derived from;67 the mouse data were taken

783

from;69 the information for human were compiled from.8,69

784

Figure 5. Chemical structures and sources of bitter substances are highly diverse. The

785

diversity of bitter compound structures range from small inorganic salts to complex organic

786

substances. The compounds originate from biological to abiotic sources and processes related

787

to food production.

788



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