Characterization of the beta-D-glucopyranoside binding site of the human bitter taste receptor hTAS2R16.
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ABSTRACT:
SUBMITTER: Sakurai T
PROVIDER: S-EPMC2934701 | biostudies-literature | 2010 Sep
REPOSITORIES: biostudies-literature
Publications
Characterization of the beta-D-glucopyranoside binding site of the human bitter taste receptor hTAS2R16.
Sakurai Takanobu T Misaka Takumi T Ishiguro Masaji M Masuda Katsuyoshi K Sugawara Taishi T Ito Keisuke K Kobayashi Takuya T Matsuo Shinji S Ishimaru Yoshiro Y Asakura Tomiko T Abe Keiko K
The Journal of biological chemistry 20100706 36
G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point m ...[more]
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Project description: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 ?-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.
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Project description:An important question in taste research is how 25 receptors of the human TAS2R family detect thousands of structurally diverse compounds. An answer to this question may arise from the observation that TAS2Rs in general are broadly tuned to interact with numerous substances. Ultimately, interaction with chemically diverse agonists requires architectures of binding pockets tailored to combine flexibility with selectivity. The present study determines the structure of hTAS2R binding pockets. We focused on a subfamily of closely related hTAS2Rs exhibiting pronounced amino acid sequence identities but unique agonist activation spectra. The generation of chimeric and mutant receptors followed by calcium imaging analyses identified receptor regions and amino acid residues critical for activation of hTAS2R46, -R43, and -R31. We found that the carboxyl-terminal regions of the investigated receptors are crucial for agonist selectivity. Intriguingly, exchanging two residues located in transmembrane domain seven between hTAS2R46, activated by strychnine, and hTAS2R31, activated by aristolochic acid, was sufficient to invert agonist selectivity. Further mutagenesis revealed additional positions involved in agonist interaction. The transfer of functionally relevant amino acids identified in hTAS2R46 to the corresponding positions of hTAS2R43 and -R31 resulted in pharmacological properties indistinguishable from the parental hTAS2R46. In silico modeling of hTAS2R46 allowed us to visualize the putative mode of interaction between agonists and hTAS2Rs. Detailed structure-function analyses of hTAS2Rs may ultimately pave the way for the development of specific antagonists urgently needed for more sophisticated analyses of human bitter taste perception.
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