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The chemical senses of olfaction and taste are well developed in fish and play a vital role in its various activities such as navigation, mate recognition, and food detection. The small teleost fish Astyanax mexicanus consists of interfertile river‐dwelling and cave‐dwelling populations, referred to as "surface fish" and "cavefish" respectively. An important anatomical feature of cavefish is the lack of eyes leading them to be referred to as blind fish and suggesting an enhanced functional role for other senses such as taste. In this study, we characterize the expression of bitter taste receptors (T2Rs or Tas2Rs) in A. mexicanus and investigate their functionality in a heterologous expression system. The genome database of A. mexicanus (ensemble and NCBI) showed 7 Tas2Rs, among these Tas2R1, Tas2R3, Tas2R4, and Tas2R114 are well characterized in humans and mice but not in A. mexicanus. Therefore, the 4 Tas2Rs were selected for further analysis and their expression in A. mexicanus was confirmed by in situ hybridization and RT‐PCR in early developmental stages. These Tas2Rs are expressed in various oral and extraoral organs (liver, fins, jaws, and gills) in A. mexicanus, and Tas2R1 has maximum expression and is localized throughout the fish body. Using the heterologous expression of A. mexicanus T2Rs in HEK293T cells coupled with cell‐based calcium mobilization assays, we show that A. mexicanus T2Rs are activated by commonly used fish food and known bitter agonists, including quinine. This study provides novel insights into the extraoral expression of T2Rs in A. mexicanus and suggests their importance in extraoral food detection. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 122, 537-544. Abstract The human bitter taste receptors (T2Rs) belong to the G-protein coupled receptor (GPCR) superfamily. T2Rs share little homology with the large subfamily of Class A G-protein coupled receptors, and their mechanisms of activation are poorly understood. Guided by biochemical and molecular approaches, we identified two conserved amino acids Gly281.46 and Ser2857.47 present on transmembrane (TM) helices, TM1 and TM7, which might play important roles in T2R activation. Previously, it was shown that naturally occurring Gly511.46 mutations in the dim light receptor, rhodopsin, cause autosomal dominant retinitis pigmentosa, with the mutants severely defective in signal transduction. We mutated Gly281.46 and Ser2857.47 in T2R4 to G28A, G28L, S285A, S285T, and S285P, and carried out pharmacological characterization of the mutants. No major changes in signaling were observed upon mutation of Gly281.46 in T2R4. Interestingly, S285A mutant displayed agonist-independent activity (approximately threefold over basal wild-type T2R4 or S285T or S285P). We propose that Ser2857.47 stabilizes the inactive state of T2R4 by a network of hydrogen-bonds connecting important residues on TM1-TM2-TM7. We compare and contrast this hydrogen-bond network with that present in rhodopsin. Thus far, S285A is the first constitutively active T2R mutant reported, and gives novel insights into T2R activation. [ABSTRACT FROM AUTHOR]

Highlights • Bitter peptide fraction had more positive charges, but less umami peptide residues. • HPH peptide fractions significantly inhibited quinine bitterness. • The least bitter peptide fractions had the most bitter-inhibitory effect. • HPH peptides are the blockers of human T2R4, T2R7, and T2R14. • HPH fraction is a rich T2R antagonist source. Abstract Bitter sensation is mediated by various bitter taste receptors (T2Rs), thus T2R antagonists are actively explored. Our objective was to look for novel T2R blockers in hen protein hydrolysate (HPH). We screened the least bitter HPH fractions using electronic tongue, and analyzed their peptide sequences and calcium mobilization in HEK293T cells expressing T2Rs. The results showed that the HPH fractions with higher bitterness intensity had higher hydrophobicity, more hydrophobic amino acids, and more positively charged peptides, but fewer known umami peptides. The peptide fractions from the least bitter HPH fraction significantly inhibited quinine bitterness (P < 0.05), and also significantly inhibited quinine- or diphenhydramine-dependent calcium mobilization of HEK293T cells expressing human T2R4, T2R7, or T2R14 (P < 0.05). Among them, the first eluted (least bitter) peptide fraction showed the strongest bitter-inhibitory effect. In conclusion, HPH peptides are the blockers of T2R4, T2R7, and T2R14. [ABSTRACT FROM AUTHOR]

Bitter taste perception in humans is believed to act as a defense mechanism against ingestion of potential toxic substances. Bitter taste is perceived by 25 distinct bitter taste receptors (T2Rs) which belong to the family of G protein-coupled receptors (GPCRs). In the overall context of the role of membrane lipids in GPCR function, we show here that T2R4, a representative member of the bitter taste receptor family, displays cholesterol sensitivity in its signaling function. In order to gain further insight into cholesterol sensitivity of T2R4, we mutated two residues Tyr114 3.59 and Lys117 3.62 present in the cholesterol recognition amino acid consensus (CRAC) motif in T2R4 with alanines. We carried out functional characterization of the mutants by calcium mobilization, followed by cholesterol depletion and replenishment. CRAC motifs in GPCRs have previously been implicated in preferential cholesterol association. Our analysis shows that the CRAC motif represents an intrinsic feature of bitter taste receptors and is conserved in 22 out of 25 human T2Rs. We further demonstrate that Lys117, an important CRAC residue, is crucial in the reported cholesterol sensitivity of T2R4. Interestingly, cholesterol sensitivity of T2R4 was observed at quinine concentrations in the lower mM range. To the best of our knowledge, our results represent the first report addressing the molecular basis of cholesterol sensitivity in the function of taste receptors. [ABSTRACT FROM AUTHOR]

Abstract: The bitter taste receptors (T2Rs) belong to the G protein-coupled receptor (GPCR) superfamily. In humans, bitter taste sensation is mediated by 25 T2Rs. Structure–function studies on T2Rs are impeded by the low-level expression of these receptors. Different lengths of rhodopsin N-terminal sequence inserted at the N-terminal region of T2Rs are commonly used to express these receptors in heterologous systems. While the additional sequences were reported, to enhance the expression of the T2Rs, the local structural perturbations caused by these sequences and its effect on receptor function or allosteric ligand binding were not characterized. In this study, we elucidated how different lengths of rhodopsin N-terminal sequence effect the structure and function of the bitter taste receptor, T2R4. Guided by molecular models of T2R4 built using a rhodopsin crystal structure as template, we constructed chimeric T2R4 receptors containing the rhodopsin N-terminal 33 and 38 amino acids. The chimeras were functionally characterized using calcium imaging, and receptor expression was determined by flow cytometry. Our results show that rhodopsin N-terminal 33 amino acids enhance expression of T2R4 by 2.5-fold and do not cause perturbations in the receptor structure. [Copyright &y& Elsevier]

Abstract: Humans are capable of sensing five basic tastes which are sweet, sour, salt, umami and bitter. Of these, bitter taste perception provides protection against ingestion of potentially toxic substances. Bitter taste is sensed by bitter taste receptors (T2Rs) that belong to the G-protein coupled receptors (GPCRs) superfamily. Humans have 25 T2Rs that are expressed in the oral cavity, gastrointestinal (GI) neuroendocrine cells and airway cells. Electrophysiological studies of the brain neurons show that the neurons are able to respond to different tastants. However, the presence of bitter taste receptors in brain cells has not been elucidated. In this report using RT-PCR, and immunohistochemistry analysis we show that T2Rs are expressed in multiple regions of the rat brain. RT-PCR analysis revealed the presence of T2R4, T2R107 and T2R38 transcripts in the brain stem, cerebellum, cortex and nucleus accumbens. The bitter receptor T2R4 was selected for further analysis at the transcript level by quantitative real time PCR and at the protein level by immunohistochemistry. To elucidate if the T2R4 expressed in these cells is functional, assays involving G-protein mediated calcium signaling were carried out. The functional assays showed an increase in intracellular calcium levels after the application of exogenous ligands for T2R4, denatonium benzoate and quinine to these cultured cells, suggesting that endogenous T2R4 expressed in these cells is functional. We discuss our results in terms of the physiological relevance of bitter receptor expression in the brain. [Copyright &y& Elsevier]

Abstract: Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, the molecular targets or receptors in humans for these bitter peptides were poorly characterized and least understood. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. In addition, we tested the ability of peptide inhibitors of the blood pressure regulatory protein, angiotensin converting enzyme (ACE) to activate T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium was measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Among the peptides examined, the bitter tri-peptide Phe-Phe-Phe (FFF), is the most potent in activating T2R1 with an EC50 value in the micromolar range. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The molecular models showed that the bitter peptides bind within the same binding pocket on the receptor. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2 forming a cap like structure on the binding pocket. [Copyright &y& Elsevier]