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

Greene TA, Alarcon S, Thomas A, Berdougo E, Doranz BJ, Breslin PA, Rucker JB - PLoS ONE (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Integral Molecular, Inc, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
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.

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Non-Gustatory GPCRs are not inhibited by probenecid.HEK-293T cells were transiently transfected with Gα16gust44 and the indicated GPCR receptors. In the case of the endogenously expressed βAR receptor, only Gα16gust44 was transfected. 22 hours post-transfection, calcium influx was measured for cells that were challenged with the indicated ligands in the presence (closed triangles) or absence (open diamonds) of probenecid (1 mM; 1 hour pre-incubation). Probenecid treatment did not attenuate calcium influx upon challenge of (A) CCR5 with 10 nM RANTES, (B) CXCR4 with 10 nM SDF-1α, or (C) βAR with 10 µM isoproterenol.
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pone-0020123-g003: Non-Gustatory GPCRs are not inhibited by probenecid.HEK-293T cells were transiently transfected with Gα16gust44 and the indicated GPCR receptors. In the case of the endogenously expressed βAR receptor, only Gα16gust44 was transfected. 22 hours post-transfection, calcium influx was measured for cells that were challenged with the indicated ligands in the presence (closed triangles) or absence (open diamonds) of probenecid (1 mM; 1 hour pre-incubation). Probenecid treatment did not attenuate calcium influx upon challenge of (A) CCR5 with 10 nM RANTES, (B) CXCR4 with 10 nM SDF-1α, or (C) βAR with 10 µM isoproterenol.

Mentions: We next examined whether probenecid could inhibit the cellular activation of the non-gustatory GPCRs CXCR4 and CCR5 when expressed in HEK-293T cells. The human chemokine receptors CXCR4 and CCR5 are members of the rhodopsin family of GPCRs, which are unrelated to the TAS2Rs and are involved in inflammation, autoimmune disease, and viral infection [32], [33]. We observed no effect on RANTES-mediated calcium mobilization by CCR5 and SDF-1α-mediated calcium mobilization by CXCR4 upon pre-incubation with probenecid (Figure 3A and 3B). We also tested the effect of probenecid on the activity of the β2-adrenergic receptor (βAR), which is endogenously expressed on HEK-293T cells [34]. Stimulation of endogenous βAR co-expressed with Gα16gust44 resulted in an increase in intracellular calcium upon stimulation with a cognate adrenergic ligand (isoproterenol) that was not inhibited by a 1 hr pre-incubation with probenecid (Figure 3C). Since βAR mobilizes calcium in HEK-293T cells only in the presence of transfected Gα16gust44, the inhibitory activity of probenecid is not the result of action through Gα16gust44, which would have led to inhibition of calcium influx. Probenecid also inhibited calcium mobilization of hTAS2R38 in the canine cell line Cf2Th, suggesting that the observed inhibition is not cell line specific (data not shown). While probenecid inhibition of the G protein βγ subunits cannot be directly ruled out with the experiments conducted here, it is unlikely since no effect on calcium mobilization was observed with other GPCRs tested and since probenecid is commonly used to enhance the Ca2+ flux of diverse GPCRs signaling through diverse G protein subunits. Thus, our data suggest that the inhibitory effect of probenecid is specific to the TAS2R receptors and not to downstream cellular components of G protein signaling.


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

Greene TA, Alarcon S, Thomas A, Berdougo E, Doranz BJ, Breslin PA, Rucker JB - PLoS ONE (2011)

Non-Gustatory GPCRs are not inhibited by probenecid.HEK-293T cells were transiently transfected with Gα16gust44 and the indicated GPCR receptors. In the case of the endogenously expressed βAR receptor, only Gα16gust44 was transfected. 22 hours post-transfection, calcium influx was measured for cells that were challenged with the indicated ligands in the presence (closed triangles) or absence (open diamonds) of probenecid (1 mM; 1 hour pre-incubation). Probenecid treatment did not attenuate calcium influx upon challenge of (A) CCR5 with 10 nM RANTES, (B) CXCR4 with 10 nM SDF-1α, or (C) βAR with 10 µM isoproterenol.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101243&req=5

pone-0020123-g003: Non-Gustatory GPCRs are not inhibited by probenecid.HEK-293T cells were transiently transfected with Gα16gust44 and the indicated GPCR receptors. In the case of the endogenously expressed βAR receptor, only Gα16gust44 was transfected. 22 hours post-transfection, calcium influx was measured for cells that were challenged with the indicated ligands in the presence (closed triangles) or absence (open diamonds) of probenecid (1 mM; 1 hour pre-incubation). Probenecid treatment did not attenuate calcium influx upon challenge of (A) CCR5 with 10 nM RANTES, (B) CXCR4 with 10 nM SDF-1α, or (C) βAR with 10 µM isoproterenol.
Mentions: We next examined whether probenecid could inhibit the cellular activation of the non-gustatory GPCRs CXCR4 and CCR5 when expressed in HEK-293T cells. The human chemokine receptors CXCR4 and CCR5 are members of the rhodopsin family of GPCRs, which are unrelated to the TAS2Rs and are involved in inflammation, autoimmune disease, and viral infection [32], [33]. We observed no effect on RANTES-mediated calcium mobilization by CCR5 and SDF-1α-mediated calcium mobilization by CXCR4 upon pre-incubation with probenecid (Figure 3A and 3B). We also tested the effect of probenecid on the activity of the β2-adrenergic receptor (βAR), which is endogenously expressed on HEK-293T cells [34]. Stimulation of endogenous βAR co-expressed with Gα16gust44 resulted in an increase in intracellular calcium upon stimulation with a cognate adrenergic ligand (isoproterenol) that was not inhibited by a 1 hr pre-incubation with probenecid (Figure 3C). Since βAR mobilizes calcium in HEK-293T cells only in the presence of transfected Gα16gust44, the inhibitory activity of probenecid is not the result of action through Gα16gust44, which would have led to inhibition of calcium influx. Probenecid also inhibited calcium mobilization of hTAS2R38 in the canine cell line Cf2Th, suggesting that the observed inhibition is not cell line specific (data not shown). While probenecid inhibition of the G protein βγ subunits cannot be directly ruled out with the experiments conducted here, it is unlikely since no effect on calcium mobilization was observed with other GPCRs tested and since probenecid is commonly used to enhance the Ca2+ flux of diverse GPCRs signaling through diverse G protein subunits. Thus, our data suggest that the inhibitory effect of probenecid is specific to the TAS2R receptors and not to downstream cellular components of G protein signaling.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Integral Molecular, Inc, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus