The role of variation in the TAS2R50 bitter taste receptor gene is unknown, but may influence taste perception and dietary habits. Individuals (n=1171) aged 20 to 29, from the Toronto Nutrigenomics and Health Study, completed a food preference checklist and a semi-quantitative food frequency questionnaire to assess their preference and intake of potentially bitter foods and beverages. DNA was isolated from blood and genotyped for 3 polymorphisms in the TAS2R50 gene (rs2900554 A>C; rs10772397 A>G; rs1376251 A>G). Taste intensity was examined using taste strips infused with 3µg of naringin. The rs2900554 SNP was associated with naringin taste intensity, grapefruit preference and grapefruit intake in females. Homozygotes for the C allele reported the highest frequency of experiencing a high naringin taste intensity, disliking grapefruit and not consuming grapefruit. The rs10772397 and rs1376251 SNPs were associated with disliking grapefruit. These results suggest that naringin may be a ligand for the T2R50 receptor.

The role of variation in the TAS2R50 bitter taste receptor gene is unknown, but may influence taste perception and dietary habits. Individuals (n=1171) aged 20 to 29, from the Toronto Nutrigenomics and Health Study, completed a food preference checklist and a semi-quantitative food frequency questionnaire to assess their preference and intake of potentially bitter foods and beverages. DNA was isolated from blood and genotyped for 3 polymorphisms in the TAS2R50 gene (rs2900554 A>C; rs10772397 A>G; rs1376251 A>G). Taste intensity was examined using taste strips infused with 3µg of naringin. The rs2900554 SNP was associated with naringin taste intensity, grapefruit preference and grapefruit intake in females. Homozygotes for the C allele reported the highest frequency of experiencing a high naringin taste intensity, disliking grapefruit and not consuming grapefruit. The rs10772397 and rs1376251 SNPs were associated with disliking grapefruit. These results suggest that naringin may be a ligand for the T2R50 receptor.

Bitter taste is one of the five basic taste modalities, and is mediated by 25 bitter taste receptors (T2Rs) in humans. How these few receptors recognize a wide range of structurally diverse bitter compounds is not known. To address this question, structural and functional studies on T2Rs are necessary. Quinine is a natural alkaloid and one of the most intense bitter tasting compounds. Previously it was shown that quinine activates T2R4, however, whether T2R4 has only one binding site for quinine, and the amino acids on the receptor involved in binding to quinine remain to be determined. In this study, the ligand binding pocket on T2R4 for quinine was characterized using a combination of approaches. These included molecular model guided site-directed mutagenesis, characterization of the expression of the mutants by flow cytometry, and functional characterization by cell based calcium imaging. Twelve mutations were made in T2R4 and their expression and function were characterized. Results show that the ligand binding pocket of T2R4 for quinine is situated on the extracellular side, and is formed by the residues present on the transmembrane regions TM3, TM4, and extracellular loop regions ECL2 and TM6-ECL3-TM7 interface. Further, this study identified the following amino acids : A90, F91, Y155 N173, T174, Y258 and K270 to play an important role in quinine binding to T2R4. The detailed study of residues interacting with ligand will help in understanding how various ligands interact with T2Rs, and facilitate the pharmacological characterization of potent antagonists or bitter taste blockers. The characterization of novel ligands, including bitter taste blockers will help in dissecting the signaling mechanism(s) of T2Rs, and help in the development of novel therapeutic tools for the food and drug industry.

Magister Scientiae Dentium - MSc(Dent) / Introduction: Dental caries has one the highest incidences in children and the host’s diet may be a major factor in determining susceptibility to the disease. A proposed tool to screen and identify high risk individuals uses a bitter compound 6-n-propylthiouracil (PROP). The goal of this screening tool is to identify mothers and children who are Non-tasters (those who cannot taste PROP) and to educate them about their possible affinity towards sugar substances and its harmful effects on oral and general health. It is suggested that Non-taster children could be prioritized when providing preventative dental treatment. Aim: To validate the use of PROP as a screening tool for determining high caries risk individuals by identifying the taster status of mothers and children, their preference towards sugar, and its impact on their caries status. Methodology: 75 mother/ child pairs were recruited to participate in this study. Caries experience, sugar preference and taster status were determined for all the subjects. Comparisons were made between mothers and their children to find a possible association. Results: Caries experience was greater in individuals who were Non-tasters when compared to Super-tasters. A significant association between taster status and DMFT score was established (p<0.000). A significant association between taster status and sugar preference was established (p<0.000). A positive correlation with regard to taster status, sugar preference and caries experience was observed in mother/child pairs.

Magister Scientiae Dentium - MSc(Dent) / Introduction: Dental caries has one the highest incidences in children and the host’s diet may be a major factor in determining susceptibility to the disease. A proposed tool to screen and identify high risk individuals uses a bitter compound 6-n-propylthiouracil (PROP). The goal of this screening tool is to identify mothers and children who are Non-tasters (those who cannot taste PROP) and to educate them about their possible affinity towards sugar substances and its harmful effects on oral and general health. It is suggested that Non-taster children could be prioritized when providing preventative dental treatment. Aim: To validate the use of PROP as a screening tool for determining high caries risk individuals by identifying the taster status of mothers and children, their preference towards sugar, and its impact on their caries status. Methodology: 75 mother/ child pairs were recruited to participate in this study. Caries experience, sugar preference and taster status were determined for all the subjects. Comparisons were made between mothers and their children to find a possible association. Results: Caries experience was greater in individuals who were Non-tasters when compared to Super-tasters. A significant association between taster status and DMFT score was established (p<0.000). A significant association between taster status and sugar preference was established (p<0.000). A positive correlation with regard to taster status, sugar preference and caries experience was observed in mother/child pairs. Conclusion: Similarities in the mother's and child’s PROP taster status and its association with sugar preference allows such a screening test to identify individuals who are at high risk of developing dental caries. Early identification of mothers who are Non-tasters may allow the introduction of early intervention strategies and assist in the early detection of potentially high-risk children, especially in environments where resources are limited.

Die Bittergeschmacksrezeptoren stellen in der Superfamilie der G-Protein-gekoppelten Rezeptoren eine besondere Gruppe dar. Im Menschen können die 25 Rezeptoren eine große Anzahl unterschiedlichster Bittergeschmacksstoffe detektieren. Diese Substanzen können sowohl schädlich, wie etwa Strychnin, als auch der Gesundheit förderliche Arzneistoffe, wie etwa Chloramphenicol sein. Unter den Bittergeschmacksrezeptoren des Menschen gibt es eine Gruppe von drei Rezeptoren, die besonders viele Bitterstoffe detektieren können. Einer von ihnen ist der Rezeptor hTAS2R10. In dieser Arbeit konnte sowohl experimentell als auch durch computergestützte Modellierung gezeigt werden, dass der hTAS2R10 nur eine Bindungstasche besitzt. Das stimmt mit den bisher ausführlich experimentell und in silico untersuchten Rezeptoren hTAS2R1, -R16, -R38 und -R46 überein. Die für die Agonisteninteraktionen nachweislich wichtigen Transmembrandomänen sind in den bisher untersuchten Bittergeschmacksrezeptoren, wie auch im hTAS2R10, die Transmembrandomänen 3, 5, 6 und 7. Die Untersuchungen zeigten, dass die Bindungstasche des hTAS2R10 in der oberen Hälfte des zum extrazellulären Raum gerichteten Bereichs lokalisiert ist. Insbesondere konnte für die untersuchten Agonisten Strychnin, Parthenolid und Denatoniumbenzoat gezeigt werden, dass die Seitenketten der Aminosäuren in Position 3.29 und 5.40 ausgeprägte agonistenselektive Wechselwirkungen eingehen. Weitere Untersuchungen haben ergeben, dass das weitgefächerte Agonistenspektrum des hTAS2R10 zu Lasten der Sensitivität für einzelne Bitterstoffe geht. Der Vergleich wichtiger Positionen im hTAS2R10, hTAS2R46 und mTas2r105 hat deutlich gemacht, dass sich die Bindungsmodi zwischen diesen Rezeptoren unterscheiden. Dies deutet auf eine getrennte evolutionäre Entwicklung der Bindungseigenschaften dieser Rezeptoren hin. Gleichfalls zeigten die Untersuchungen, dass einige Positionen wie z.B. 7.39 die Funktion aller untersuchten Bittergeschmacksrezeptoren prägen, sich jedoch die genaue Bedeutung im jeweiligen Rezeptor unterscheiden kann. Einzelne dieser Positionen konnten auch bei der Agonisteninteraktion des Rhodopsins und des β2-adrenergen Rezeptors beobachtet werden. Die Ergebnisse dieser Arbeit helfen dabei die Wechselwirkungen zwischen Bitterstoffen und den Bittergeschmacksrezeptoren zu verstehen und geben erste Einblicke in die Entwicklung der Rezeptoren in Hinblick auf ihren Funktionsmechanismus. Diese Erkenntnisse können genutzt werden, um Inhibitoren zu entwickeln, die sowohl ein wichtiges Werkzeug in der Rezeptoranalytik wären, als auch dazu genutzt werden könnten, den unerwünschten bitteren Geschmack von Medikamenten oder gesundheitsfördernden sekundären Pflanzenstoffen zu mindern. Damit könnte ein Beitrag zur Gesundheit der Menschen geleistet werden. / In the Superfamily of G protein-coupled receptors the bitter taste receptors form a notable group. The 25 human receptors are able to detect a large group of structurally diverse bitter compounds. These compounds can be toxic – like strychnine – or have beneficial effects on health – like the pharmacological agent chloramphenicol. Three of these bitter taste receptors show a strikingly broad agonist spectrum. One of them is the hTAS2R10. It was shown empirically and by computational modelling that the hTAS2R10 has only one binding pocket. This agrees with the findings of studies on the bitter taste receptors hTAS2R1, -R16, -R38 and -R46. The domains important for agonist interaction in these receptors, as well as in the hTAS2R10, are the transmembrane domains 3, 5, 6 and 7. The results of this thesis show that the binding pocket of the hTAS210 is located in the upper part of the receptor which points into the direction of the extracellular area. Interestingly, it has been shown for the amino acid side chains in the positions 3.29 and 5.40, that they can interact with the analysed agonists strychnine, parthenolide and denatonium benzoate in an agonist-selective way. Further analyses showed that the broad tuning of the hTAS2R10 goes at the expense of the sensitivity to single agonists. The comparison of crucial positions in the hTAS2R10, hTAS2R46 and the mTas2r105 reveal that these receptors differ in their binding mode. These could be evidence that the binding abilities of these receptors evolved independently. However, the results show that some positions, e.g. 7.39, influence the receptor activity in all analysed receptors, but the function of these positions in the receptors could be different. Some of these positions also have an influence on the agonist-receptor interaction of Rhodopsin and the β2-adrenergic receptor. The findings in this thesis contribute to the knowledge about interaction between bitter receptors and bitter compounds. The results also provide insight into the evolvement of receptor functions. These outcomes can be of use for the development of inhibitors which could serve as analytical tools in taste research. Furthermore, such inhibitors could be used to reduce the bitter taste of medicine and healthy plant compounds and thus increase palatability. This could contribute to improve human well-being.

Der Mensch besitzt ~25 funktionelle Bitterrezeptoren (TAS2R), die für die Wahrnehmung potenziell toxischer Substanzen in der Nahrung verantwortlich sind. Aufgrund der großen genetischen Variabilität der TAS2R-Gene könnte es eine Vielzahl funktionell unterschiedlicher TAS2R-Haplotypen geben, die zu Unterschieden der Bitterwahrnehmung führen. Dies konnte bereits in funktionellen Analysen und sensorischen Studien für einzelne Bitterrezeptoren gezeigt werden. In dieser Arbeit wurden die häufigsten Haplotypen aller 25 Bitterrezeptoren verschiedener Ethnien funktionell charakterisiert. Das Ziel war eine umfassende Aussage über die funktionelle Diversität der TAS2Rs, die die molekulare Grundlage für individuelle Bitterwahrnehmung bildet, treffen zu können. Fehlende Varianten wurden aus genomischer DNA kloniert oder durch gezielte Mutagenese bereits vorhandener TAS2R-Konstrukte generiert. Die funktionelle Analyse erfolgte mittels Expression der TAS2R-Haplotypen in HEK293TG16gust44 Zellen und anschließenden Calcium-Imaging-Experimenten mit zwei bekannten Agonisten. Die Haplotypen der fünf orphanen TAS2Rs wurden mit über hundert Bitterstoffen stimuliert. Durch die gelungene Deorphanisierung des TAS2R41 in dieser Arbeit, wurden für die 21 aktivierbaren TAS2Rs 36 funktionell-unterschiedliche Haplotypen identifiziert. Die tatsächliche funktionelle Vielfalt blieb jedoch deutlich hinter der genetischen Variabilität der TAS2Rs zurück. Neun Bitterrezeptoren wiesen funktionell homogene Haplotypen auf oder besaßen nur eine weltweit vorherrschende Variante. Funktionell heterogene Haplotypen wurden für zwölf TAS2Rs identifiziert. Inaktive Varianten der Rezeptoren TAS2R9, TAS2R38 und TAS2R46 sollten die Wahrnehmung von Bitterstoffen wie Ofloxacin, Cnicin, Hydrocortison, Limonin, Parthenolid oder Strychnin beeinflussen. Unterschiedlich sensitive Varianten, besonders der Rezeptoren TAS2R47 und TAS2R49, sollten für Agonisten wie Absinthin, Amarogentin oder Cromolyn ebenfalls zu phänotypischen Unterschieden führen. Wie für den TAS2R16 bereits gezeigt, traten Haplotypen des funktionell heterogenen TAS2R7 und TAS2R41 ethnien-spezifisch auf, was auf lokale Anpassung und verschiedene Phänotypen hinweisen könnte. Weiterführend muss nun eine Analyse der funktionell-variablen TAS2Rs in sensorischen Tests erfolgen, um ihre phänotypische Relevanz zu prüfen. Die Analyse der funktionsmodulierenden Aminosäurepositionen, z.Bsp. des TAS2R44, TAS2R47 oder TAS2R49, könnte weiterführend zum besseren Verständnis der Rezeptor-Ligand- und Rezeptor-G-Protein-Interaktion beitragen. / Bitter taste perception varies markedly from person to person, due to a high number of polymorphisms present in the 25 known functional bitter receptors (TAS2Rs). These polymorphisms lead to a number of haplotypes for each receptor, which are common in different populations, but vary in frequency. The individual combination of receptor variants seems to determine the person’s sensitivity of bitter perception, as could already be shown for single TAS2Rs. Bitter is an aversive taste quality, indicating the ingestion of harmful substances. Different sensitivity could have an impact on food choice. In order to characterize functional consequences of the genetic diversity, we performed calcium imaging experiments with all main haplotypes for the 25 bitter receptors. The obtained information about receptor properties enables us on the one hand to analyze structure-function relationships and on the other hand gives us the functional diverse candidates to focus on in psychophysical studies. The overall aim is to show genotype-phenotype correlation for bitter taste perception and their impact on food choice and therefore diet and health. Our first aim was to identify agonists for the 5 receptors, which could not be deorphaned in previous screens. We challenged all main haplotypes of these TAS2Rs with 106 bitter compounds and could identify the antibiotic chloramphenicol as agonist for bitter receptor TAS2R41. In total we identified 36 functionally different receptor variants of the 21 deorphaned TAS2Rs. Main haplotypes of nine TAS2Rs were functionally homogeneous while twelve TAS2Rs possessed between two and three functionally heterogeneous receptor variants. In summary the observed functional diversity is not as big as expected. Based on our in vitro findings the shown functional diversity of these twelve bitter receptors might be the molecular basis for individual differences in bitter taste perception and will be further analyzed in psychophysical studies.

In humans, taste is one of the five senses, and helps in the recognition of nutritionally important and potentially harmful substances. It triggers innate behaviour to accept or reject food. Humans can sense five basic tastes, which are sweet, umami, bitter, salt and sour. The receptors that mediate bitter, sweet and umami tastes belong to the G protein-coupled receptor (GPCR) superfamily. A group of three receptors sense sweet and umami tastes, whereas bitter taste is sensed by 25 bitter taste receptors (referred as T2Rs). T2Rs are activated by structurally diverse natural and synthetic bitter compounds. Many common pharmaceutical compounds are bitter in taste and these are effective ligands for T2Rs. Recent finding of T2Rs in extra-oral tissues suggests these receptors are also involved in various physiological and pathophysiological processes. To understand the structure and function of these receptors, studies directed at elucidating their mechanisms of activation, and identification of novel ligands including bitter blockers (antagonists and inverse agonists), are required. To obtain mechanistic insights into the role of the highly conserved, and receptor specific residues, two bitter taste receptors (T2R1 and T2R4) were targeted. In this study, a combination of molecular, biochemical and pharmacological approaches were used to identify the amino acids and motifs, important for T2Rs to switch from inactive to active state. A hydrogen-bonding network between transmembrane (TM) helices 1-2-7 was identified as important for T2R activation. Alanine-scan mutagenesis of intracellular loops (ICLs) 2 and 3 identified T2R regions important for G protein binding, and receptor activation. A pharmacological method was developed, to screen potential bitter blockers for T2Rs. Using this method, three novel bitter blockers, which include two natural antagonists and one synthetic inverse agonist for T2R4, were discovered. The role of expression tags in enhancing T2R4 expression was also pursued. T2R4 expression on the cell surface was increased 2.5 fold, when its N-terminus was tagged with rhodopsin N-terminal 33 residues (Rho33- T2R4 chimera). In conclusion, work carried out provides novel insights into the mechanisms of T2R activation, and in the discovery of bitter blockers for T2R4.

Die Wahrnehmung von Geschmacksempfindungen beruht auf dem Zusammenspiel verschiedener Sinneseindrücke wie Schmecken, Riechen und Tasten. Diese Komplexität der gustatorischen Wahrnehmung erschwert die Beantwortung der Frage wie Geschmacksinformationen vom Mund ins Gehirn weitergeleitet, prozessiert und kodiert werden. Die Analysen zur neuronalen Prozessierung von Geschmacksinformationen erfolgten zumeist mit Bitterstimuli am Mausmodell. Zwar ist bekannt, dass das Genom der Maus für 35 funktionelle Bitterrezeptoren kodiert, jedoch war nur für zwei unter ihnen ein Ligand ermittelt worden. Um eine bessere Grundlage für tierexperimentelle Arbeiten zu schaffen, wurden 16 der 35 Bitterrezeptoren der Maus heterolog in HEK293T-Zellen exprimiert und in Calcium-Imaging-Experimenten funktionell charakterisiert. Die Daten belegen, dass das Funktionsspektrum der Bitterrezeptoren der Maus im Vergleich zum Menschen enger ist und widerlegen damit die Aussage, dass humane und murine orthologe Rezeptoren durch das gleiche Ligandenspektrum angesprochen werden. Die Interpretation von tierexperimentellen Daten und die Übertragbarkeit auf den Menschen werden folglich nicht nur durch die Komplexität des Geschmacks, sondern auch durch Speziesunterschiede verkompliziert. Die Komplexität des Geschmacks beruht u. a. auf der Tatsache, dass Geschmacksstoffe selten isoliert auftreten und daher eine Vielzahl an Informationen kodiert werden muss. Um solche geschmacksstoffassoziierten Stimuli in der Analyse der gustatorischen Kommunikationsbahnen auszuschließen, sollten Opsine, die durch Licht spezifischer Wellenlänge angeregt werden können, für die selektive Ersetzung von Geschmacksrezeptoren genutzt werden. Um die Funktionalität dieser angestrebten Knockout-Knockin-Modelle zu evaluieren, die eine Kopplung von Opsinen mit dem geschmacksspezifischen G-Protein Gustducin voraussetzte, wurden Oozyten vom Krallenfrosch Xenopus laevis mit dem Zwei-Elektroden-Spannungsklemm-Verfahren hinsichtlich dieser Interaktion analysiert. Der positiven Bewertung dieser Kopplung folgte die Erzeugung von drei Mauslinien, die in der kodierenden Region eines spezifischen Geschmacksrezeptors (Tas1r1, Tas1r2, Tas2r114) Photorezeptoren exprimierten. Durch RT-PCR-, In-situ-Hybridisierungs- und immunhistochemische Experimente konnte der erfolgreiche Knockout der Rezeptorgene und der Knockin der Opsine belegt werden. Der Nachweis der Funktionalität der Opsine im gustatorischen System wird Gegenstand zukünftiger Analysen sein. Bei erfolgreichem Beleg der Lichtempfindlichkeit von Geschmacksrezeptorzellen dieser Mausmodelle wäre ein System geschaffen, dass es ermöglichen würde, gustatorische neuronale Netzwerke und Hirnareale zu identifizieren, die auf einen reinen geschmacks- und qualitätsspezifischen Stimulus zurückzuführen wären. / Taste impression is based on the interaction of taste, smell and touch. To evaluate the nutritious content of food mammals possess five distinct taste qualities: sweet, bitter, umami (taste of amino acids), sour and salty. For bitter, sweet, and umami compounds taste signaling is initiated by binding of tastants to G protein-coupled receptors. The interactions of taste stimuli, usually watersoluble chemicals, with their cognate receptors lead to the activation of the G protein gustducin, which, in turn, initiates a signal resulting in the activation of gustatory afferents. However, details of gustatory signal transmission and processing as well as neural coding are only incompletely understood. This is partly due to the property of some tastants to elicit several sensations simultaneously, unspecific effects caused by the temperature, viscosity, osmolarity, and pH of the solvents, as well as by mechanical stimulation of the tongue during stimulus application. The analysis of gustatory processing of taste information are mainly based on mouse models after stimulation with bitter taste stimuli. Even though it is known that the mouse genome codes for 35 bitter taste receptor genes only few of them had been analysed so far. For better understanding and interpretation of animal experiments 16 mouse bitter receptors had been analysed by Calcium Imaging experiments with HEK293T cells. The data reveal that mouse bitter taste receptors are more narrow tuned than human bitter taste receptors, proving that the ligand spectra of murine and human orthologous receptors are not complient. In order to avoid the disturbing effects of solvents and stimulus application on the analysis of gustatory information transfer and processing, I employ an optogenetical approach to address this problem. For this purpose I generated three strains of gene-targeted mice in which the coding regions of the genes for the umami receptor subunit Tas1r1, the sweet receptor subunit Tas1r2 or the bitter taste receptor Tas2r114 have been replaced by the coding sequences of different opsins (photoreceptors of visual transduction) that are sensitive to light of various wavelengths. In these animals I should be able to activate sweet, bitter, or umami signalling by light avoiding any solvent effects. In initial experiments of this project I demonstrated that the various visual opsins indeed functionally couple to taste signal transduction pathway in oocyte expression system, generating basic knowledge and foundation for the generation of the gene-targeted animals. The knockout-knockin strategies have been successfully realized in the case of all three mouse models, revealed by RT-PCR, in situ hybridization and immunohistochemical analysis of taste papillae. All data confirm that the particular taste receptors have been replaced by the different opsins in taste cells. Further analysis concerning the functional consequences of opsin knockin and taste receptor knockout are part of prospective work.

Taste is one of the crucial factors that contributes to shaping eating behaviors and is also one of the leading reasons that affects our preferences to like or dislike some foods that mainly have a bitter taste. Variation in bitter phenotype (tasters and non-tasters) could influence diet quality and in turn body weight, which overall influenced health outcomes. The objective of this study was to identify whether bitter taste phenotype status influences anthropometric measurements, body fat percentage, and eating behaviors (liking and intake) in female college students. In this cross-sectional study (n = 86), female college students aged 18 to 22 from Mississippi State University were classified into one of two groups (taster or non-taster) by means of a taste test of filter paper saturated with the bitter compound 6-n-propylthiouracil (PROP). Adiposity was measured using anthropometric measurements and body composition was measured using bioelectrical impedance analysis. A food liking survey was administrated to identify how much participants liked or disliked various foods and beverages. Dietary intake of total energy intake, macronutrients, fruits, and vegetables were evaluated using the NIH Diet History Questionnaire. Bitter phenotype status was not significantly associated with adiposity indicators; however, it was associated with food liking scores for foods that have bitter and umami tastes such as kale and mushrooms. Additionally, bitter phenotype was associated with dietary intake for total fruits and vitamin C intakes. Ethnic background was the strongest independent variable that was significantly correlated with adiposity indicators and food liking. These results suggested that while bitter taste phenotype may influence eating behaviors in certain foods, it does not affect adiposity indicators and body fat percentage.