Variation in the bitter-taste receptor gene TAS2R38, and adiposity in a genetically isolated population in Southern Italy

Objective: Variation in the bitter‐taste receptor gene, TAS2R38 confers the ability to taste 6‐n‐propylthiouracil (PROP). The objective of this study was to relate TAS2R38 haplotypes and PROP‐tasting phenotypes to adiposity in a genetically isolated population. We hypothesized that the nontaster phenotype would be associated with higher BMI and waist circumference (WC) in females, and that dietary restraint would mediate this relationship. Methods and Procedures: Participants were 540 healthy inhabitants of the genetically isolated village of Carlantino in southern Italy who were 15–89 years of age at the time of the study. Haplotype analyses were performed and PROP tasting was assessed using a filter paper method. Height, weight, and WC were measured and restrained eating was assessed using a brief questionnaire. Results: Nontaster females had higher BMI and WC than females who were phenotypic tasters, and this relationship was specific to females with low dietary restraint. Regression analysis showed that BMI declined by 1.7 units across taster groups in females when the model included the PROP by restraint interaction. PROP phenotype was not significantly associated with WC in the regression models. Polymorphisms in TAS2R38 were not associated with BMI or WC in females. Neither TAS2R38 haplotype nor PROP phenotype was strongly related to BMI or WC in males. Discussion: These data support previous findings of a relation between the nontaster phenotype and higher BMI in females that is modified by dietary restraint. Assessment of PROP phenotypes might provide unique information about adiposity that is not captured by haplotype analysis alone.     

Functional diversity of bitter taste receptor TAS2R16 in primates

In mammals, bitter taste is mediated by TAS2R genes, which belong to the large family of seven transmembrane G protein-coupled receptors. Because TAS2Rs are directly involved in the interaction between mammals and their dietary sources, it is likely that these genes evolved to reflect species-specific diets during mammalian evolution. Here, we investigated the sensitivities of TAS2R16s of various primates by using a cultured cell expression system, and found that the sensitivity of each primate species varied according to the ligand. Especially, the sensitivity of TAS2R16 of Japanese macaques to salicin was much lower than that of human TAS2R16, which was supported by behavioural tests. These results suggest the possibility that bitter-taste sensitivities evolved independently by replacing specific amino acid residues of TAS2Rs in different primate species to adapt to food items they use.     

Probenecid inhibits the human bitter taste receptor TAS2R16 and suppresses bitter perception of salicin

Bitter taste stimuli are detected by a diverse family of G protein-coupled receptors (GPCRs) expressed in gustatory cells. Each bitter taste receptor (TAS2R) responds to an array of compounds, many of which are toxic and can be found in nature. For example, human TAS2R16 (hTAS2R16) responds to β-glucosides such as salicin, and hTAS2R38 responds to thiourea-containing molecules such as glucosinolates and phenylthiocarbamide (PTC). While many substances are known to activate TAS2Rs, only one inhibitor that specifically blocks bitter receptor activation has been described. Here, we describe a new inhibitor of bitter taste receptors, p-(dipropylsulfamoyl)benzoic acid (probenecid), that acts on a subset of TAS2Rs and inhibits through a novel, allosteric mechanism of action. Probenecid is an FDA-approved inhibitor of the Multidrug Resistance Protein 1 (MRP1) transporter and is clinically used to treat gout in humans. Probenecid is also commonly used to enhance cellular signals in GPCR calcium mobilization assays. We show that probenecid specifically inhibits the cellular response mediated by the bitter taste receptor hTAS2R16 and provide molecular and pharmacological evidence for direct interaction with this GPCR using a non-competitive (allosteric) mechanism. Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin. Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs. Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function. Finally, we demonstrate that the inhibitory activity of probenecid in cellular experiments translates to inhibition of bitter taste perception of salicin in humans. This work identifies probenecid as a pharmacological tool for understanding the cell biology of bitter taste and as a lead for the development of broad specificity bitter blockers to improve nutrition and medical compliance.     

Functional variant in a bitter-taste receptor (hTAS2R16) influences risk of alcohol dependence

A coding single-nucleotide polymorphism (cSNP), K172N, in hTAS2R16, a gene encoding a taste receptor for bitter beta -glucopyranosides, shows significant association with alcohol dependence (P = .00018). This gene is located on chromosome 7q in a region reported elsewhere to exhibit linkage with alcohol dependence. The SNP is located in the putative ligand-binding domain and is associated with an increased sensitivity to many bitter beta -glucopyranosides in the presence of the N172 allele. Individuals with the ancestral allele K172 are at increased risk of alcohol dependence, regardless of ethnicity. However, this risk allele is uncommon in European Americans (minor-allele frequency [MAF] 0.6%), whereas 45% of African Americans carry the allele (MAF 26%), which makes it a much more significant risk factor in the African American population.     

Variation in the human TAS1R taste receptor genes

We have performed a comprehensive evaluation of single-nucleotide polymorphisms (SNPs) and haplotypes in the human TAS1R gene family, which encodes receptors for sweet and umami tastes. Complete DNA sequences of TAS1R1-, TAS1R2-, and TAS1R3-coding regions, obtained from 88 individuals of African, Asian, European, and Native American origin, revealed substantial coding and noncoding diversity: polymorphisms are common in these genes, and polymorphic sites and SNP frequencies vary widely in human populations. The genes TAS1R1 and TAS1R3, which encode proteins that act as a dimer to form the umami (glutamate) taste receptor, showed less variation than the TAS1R2 gene, which acts as a dimer with TAS1R3 to form the sweet taste receptor. The TAS1R3 gene, which encodes a subunit common to both the sweet and umami receptors, was the most conserved. Evolutionary genetic analysis indicates that these variants have come to their current frequencies under natural selection during population growth and support the view that the coding sequence variants affect receptor function. We propose that human populations likely vary little with respect to umami perception, which is controlled by one major form of the receptor that is optimized for detecting glutamate but may vary much more with respect to sweet perception.     

Selection on the human bitter taste gene, TAS2R16, in Eurasian populations

Bitter taste is one of the most important senses alerting humans to noxious foods. In gatherer communities, sensitivity to bitterness is presumably advantageous because of various noxious plants. TAS2R16 is the gene coding the taste receptor molecules for some of the most common toxins in plants. A previous study of this gene indicated selection has increased the frequency of a derived allele in this gene that arose before the human expansion out of Africa. We have applied a different methodology for detecting selection, the Long Range Haplotype (LRH) analysis, to TAS2R16 in a larger sampling of populations from around the world. The haplotype with the derived alleles at both the functional polymorphism and a polymorphism in the regulatory region of TAS2R16 showed evidence for recent positive selection in most of the Eurasian populations, though the highest selection signal occurs in Mbuti Pygmies, an African hunter-gatherer group. In Eurasia, only populations of Mesopotamia and the southeast coast of China have no signals of selection. The evidence of recent selection found in most Eurasian populations differs from the geographic pattern seen in the earlier study of selection. One can speculate that the difference may result from a gathering lifestyle extending into the most recent 10,000 yrs and the need to recognize newly encountered bitter natural toxins as populations expanded into new environments and the biota changes with the ending of the most recent ice age. Alternatively, the promoter region variant may be a marker for altered function beyond what the derived amino acid allele conferred.     

Insights into the binding of agonist and antagonist to TAS2R16 receptor: a molecular simulation study

The human bitter taste receptors (TAS2Rs) belong to the GPCR family, while the activation mechanism and how TAS2Rs recognise bitter ligands are poorly understood. In this study, 3D structure of TAS2R16 was constructed using homology modelling complemented with molecular dynamics method. Salicin and probenecid were docked to TAS2R16 receptor to investigate the possible activation mechanism of TAS2R16. The results show that salicin and probenecid locate at the binding pocket made up of transmembrane helices TM3, TM5 and TM7, and the second and third extracellular loops ECL2 and ECL3. Structural analysis reveals that the network interactions at the third intracellular loop ICL3 may play a crucial role in stabilising the inactive state of TAS2R16, and structural change in the intracellular region is correlated with the activation of TAS2R16. The binding energies of salicin and probenecid to TAS2R16 are ?152.81 ± 15.09 and ?271.90 ± 26.97 kJ/mol, respectively, indicating that a potential antagonist should have obviously stronger binding affinity.     

Identification and characterization of human taste receptor genes belonging to the TAS2R family

The sense of taste is a chemosensory system responsible for basic food appraisal. Humans distinguish between five primary tastes: bitter, sweet, sour, salty and umami. The molecular events in the perception of bitter taste are believed to start with the binding of specific water-soluble molecules to G-protein-coupled receptors encoded by the TAS2R/T2R family of taste receptor genes. TAS2R receptors are expressed at the surface of taste receptor cells and are coupled to G proteins and second messenger pathways. We have identified, cloned and characterized 11 new bitter taste receptor genes and four new pseudogenes that belong to the human TAS2R family. Their encoded proteins have between 298 and 333 amino acids and share between 23 and 86% identity with other human TAS2R proteins. Screening of a mono-chromosomal somatic cell hybrid panel to assign the identified bitter taste receptor genes to human chromosomes demonstrated that they are located in chromosomes 7 and 12. Including the 15 sequences identified, the human TAS2R family is composed of 28 full-length genes and 16 pseudogenes. Phylogenetic analyses suggest a classification of the TAS2R genes in five groups that may reflect a specialization in the detection of specific types of bitter chemicals.     

Reengineering the ligand sensitivity of the broadly tuned human bitter taste receptor TAS2R14

Background

In humans, bitterness perception is mediated by ~25 bitter taste receptors present in the oral cavity. Among these receptors three, TAS2R10, TAS2R14 and TAS2R46, exhibit extraordinary wide agonist profiles and hence contribute disproportionally high to the perception of bitterness. Perhaps the most broadly tuned receptor is the TAS2R14, which may represent, because of its prominent expression in extraoral tissues, a receptor of particular importance for the physiological actions of bitter compounds beyond taste.

Natural selection and molecular evolution in PTC, a bitter-taste receptor gene

The ability to taste phenylthiocarbamide (PTC) is a classic phenotype that has long been known to vary in human populations. This phenotype is of genetic, epidemiologic, and evolutionary interest because the ability to taste PTC is correlated with the ability to taste other bitter substances, many of which are toxic. Thus, variation in PTC perception may reflect variation in dietary preferences throughout human history and could correlate with susceptibility to diet-related diseases in modern populations. To test R. A. Fisher's long-standing hypothesis that variability in PTC perception has been maintained by balancing natural selection, we examined patterns of DNA sequence variation in the recently identified PTC gene, which accounts for up to 85% of phenotypic variance in the trait. We analyzed the entire coding region of PTC (1,002 bp) in a sample of 330 chromosomes collected from African (n=62), Asian (n=138), European (n=110), and North American (n=20) populations by use of new statistical tests for natural selection that take into account the potentially confounding effects of human population growth. Two intermediate-frequency haplotypes corresponding to "taster" and "nontaster" phenotypes were found. These haplotypes had similar frequencies across Africa, Asia, and Europe. Genetic differentiation between the continental population samples was low (FST=0.056) in comparison with estimates based on other genes. In addition, Tajima's D and Fu and Li's D and F statistics demonstrated a significant deviation from neutrality because of an excess of intermediate-frequency variants when human population growth was taken into account (P<.01). These results combine to suggest that balancing natural selection has acted to maintain "taster" and "nontaster" alleles at the PTC locus in humans.     

Modeling the human PTC bitter-taste receptor interactions with bitter tastants

We employed the first principles computational method MembStruk and homology modeling techniques to predict the 3D structures of the human phenylthiocarbamide (PTC) taste receptor. This protein is a seven-transmembrane-domain G protein-coupled receptor that exists in two main forms worldwide, designated taster and nontaster, which differ from each other at three amino-acid positions. 3D models were generated with and without structural similarity comparison to bovine rhodopsin. We used computational tools (HierDock and ScanBindSite) to generate models of the receptor bound to PTC ligand to estimate binding sites and binding energies. In these models, PTC binds at a site distant from the variant amino acids, and PTC binding energy was equivalent for both the taster and nontaster forms of the protein. These models suggest that the inability of humans to taste PTC is due to a failure of G protein activation rather than decreased binding affinity of the receptor for PTC. Amino-acid substitutions in the sixth and seventh transmembrane domains of the nontaster form of the protein may produce increased steric hindrance between these two α-helices and reduce the motion of the sixth helix required for G protein activation.     

Immunohistochemical detection of TAS2R38 protein in human taste cells

The sense of taste plays an important role in the evaluation of the nutrient composition of consumed food. Bitter taste in particular is believed to serve a warning function against the ingestion of poisonous substances. In the past years enormous progress was made in the characterization of bitter taste receptors, including their gene expression patterns, pharmacological features and presumed physiological roles in gustatory as well as in non-gustatory tissues. However, due to a lack in TAS2R-specifc antibodies the localization of receptor proteins within gustatory tissues has never been analyzed. In the present study we have screened a panel of commercially available antisera raised against human bitter taste receptors by immunocytochemical experiments. One of these antisera was found to be highly specific for the human bitter taste receptor TAS2R38. We further demonstrate that this antibody is able to detect heterologously expressed TAS2R38 protein on Western blots. The antiserum is, however, not able to interfere significantly with TAS2R38 function in cell based calcium imaging analyses. Most importantly, we were able to demonstrate the presence of TAS2R38 protein in human gustatory papillae. Using double immunofluorescence we show that TAS2R38-positive cells form a subpopulation of PLCbeta2 expressing cells. On a subcellular level the localization of this bitter taste receptor is neither restricted to the cell surface nor particularly enriched at the level of the microvilli protruding into the pore region of the taste buds, but rather evenly distributed over the entire cell body.     

A regulatory gene network related to the porcine umami taste receptor (TAS1R1/TAS1R3)

Taste perception plays an important role in the mediation of food choices in mammals. The first porcine taste receptor genes identified, sequenced and characterized, TAS1R1 and TAS1R3, were related to the dimeric receptor for umami taste. However, little is known about their regulatory network. The objective of this study was to unfold the genetic network involved in porcine umami taste perception. We performed a meta‐analysis of 20 gene expression studies spanning 480 porcine microarray chips and screened 328 taste‐related genes by selective mining steps among the available 12 320 genes. A porcine umami taste‐specific regulatory network was constructed based on the normalized coexpression data of the 328 genes across 27 tissues. From the network, we revealed the ‘taste module’ and identified a coexpression cluster for the umami taste according to the first connector with the TAS1R1/TAS1R3 genes. Our findings identify several taste‐related regulatory genes and extend previous genetic background of porcine umami taste.     

Positive selection of a pre-expansion CAG repeat of the human SCA2 gene

A region of approximately one megabase of human Chromosome 12 shows extensive linkage disequilibrium in Utah residents with ancestry from northern and western Europe. This strikingly large linkage disequilibrium block was analyzed with statistical and experimental methods to determine whether natural selection could be implicated in shaping the current genome structure. Extended Haplotype Homozygosity and Relative Extended Haplotype Homozygosity analyses on this region mapped a core region of the strongest conserved haplotype to the exon 1 of the Spinocerebellar ataxia type 2 gene (SCA2). Direct DNA sequencing of this region of the SCA2 gene revealed a significant association between a pre-expanded allele [(CAG)₈CAA(CAG)₄CAA(CAG)₈] of CAG repeats within exon 1 and the selected haplotype of the SCA2 gene. A significantly negative Tajima's D value (−2.20, p < 0.01) on this site consistently suggested selection on the CAG repeat. This region was also investigated in the three other populations, none of which showed signs of selection. These results suggest that a recent positive selection of the pre-expansion SCA2 CAG repeat has occurred in Utah residents with European ancestry.     

Bitter taste study in a sardinian genetic isolate supports the association of phenylthiocarbamide sensitivity to the TAS2R38 bitter receptor gene

Recently, a major locus on chromosome 7q was found in association with the taste sensitivity to phenylthiocarbamide (PTC) in humans. This region contains the TAS2R38 gene that encodes a member of the TAS2R bitter taste receptor family. Three SNPs within this gene demonstrated a strong association with taster status in Utah families and in an additional sample of 85 unrelated individuals. We studied a small isolated village in eastern Sardinia and carried out a genome-wide scan to map the genetic basis of PTC perception in this population. We performed both qualitative and quantitative PTC-taste linkage analysis. Qualitative analysis was carried out by defining a cut-off from the bimodal distribution of the trait and classifying subjects as tasters and non-tasters (75 and 25%, respectively). Linkage analysis on 131 subjects belonging to a unique large multi-generation pedigree comprising 239 subjects confirmed significant evidence for linkage at 7q35 also in our population. Haplotype analyses of the three SNPs inside the PTC gene allowed us to identify only two haplotypes that were associated with the non-taster phenotype (80% AVI homozygous) and to taster phenotype (40% PAV homozygous and 56% PAV/AVI heterozygous). Sex, age and haplotype effect explained 77.2 % of the total variance in PTC sensitivity.     

Positive and negative selection on the human genome.

The distinction between deleterious, neutral, and adaptive mutations is a fundamental problem in the study of molecular evolution. Two significant quantities are the fraction of DNA variation in natural populations that is deleterious and destined to be eliminated and the fraction of fixed differences between species driven by positive Darwinian selection. We estimate these quantities using the large number of human genes for which there are polymorphism and divergence data. The fraction of amino acid mutations that is neutral is estimated to be 0.20 from the ratio of common amino acid (A) to synonymous (S) single nucleotide polymorphisms (SNPs) at frequencies of > or =15%. Among the 80% of amino acid mutations that are deleterious at least 20% of them are only slightly deleterious and often attain frequencies of 1-10%. We estimate that these slightly deleterious mutations comprise at least 3% of amino acid SNPs in the average individual or at least 300 per diploid genome. This estimate is not sensitive to human population history. The A/S ratio of fixed differences is greater than that of common SNPs and suggests that a large fraction of protein divergence is adaptive and driven by positive Darwinian selection.