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The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor.

Winnig M, Bufe B, Kratochwil NA, Slack JP, Meyerhof W - BMC Struct. Biol. (2007)

Bottom Line: Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore.This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor activation by neohesperidin dihydrochalcone and cyclamate.Some of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are involved in the binding of allosteric modulators in other class C GPCRs, suggesting a general role of these amino acid positions in allosterism and pointing to a common architecture of the heptahelical domains of class C GPCRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: German Institute of Human Nutrition Potsdam-Rehbruecke, Department of Molecular Genetics, Arthur-Scheunert Allee 114-116, 14558 Nuthetal, Germany. marcel.winnig.mw@axxam.com

ABSTRACT

Background: Differences in sweet taste perception among species depend on structural variations of the sweet taste receptor. The commercially used isovanillyl sweetener neohesperidin dihydrochalcone activates the human but not the rat sweet receptor TAS1R2+TAS1R3. Analysis of interspecies combinations and chimeras of rat and human TAS1R2+TAS1R3 suggested that the heptahelical domain of human TAS1R3 is crucial for the activation of the sweet receptor by neohesperidin dihydrochalcone.

Results: By mutational analysis combined with functional studies and molecular modeling we identified a set of different amino acid residues within the heptahelical domain of human TAS1R3 that forms the neohesperidin dihydrochalcone binding pocket. Sixteen amino acid residues in the transmembrane domains 2 to 7 and one in the extracellular loop 2 of hTAS1R3 influenced the receptor's response to neohesperidin dihydrochalcone. Some of these seventeen residues are also part of the binding sites for the sweetener cyclamate or the sweet taste inhibitor lactisole. In line with this observation, lactisole inhibited activation of the sweet receptor by neohesperidin dihydrochalcone and cyclamate competitively, whereas receptor activation by aspartame, a sweetener known to bind to the N-terminal domain of TAS1R2, was allosterically inhibited. Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore.

Conclusion: From our data we conclude that we identified the neohesperidin dihydrochalcone binding site at the human sweet taste receptor, which overlaps with those for the sweetener cyclamate and the sweet taste inhibitor lactisole. This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor activation by neohesperidin dihydrochalcone and cyclamate. Some of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are involved in the binding of allosteric modulators in other class C GPCRs, suggesting a general role of these amino acid positions in allosterism and pointing to a common architecture of the heptahelical domains of class C GPCRs.

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Identification of additional determinants in TAS1R3 critical for responsiveness to cyclamate and lactisole. (A) Concentration-dependent inhibition of calcium responses to 10 mM aspartame by lactisole in HEK293T-G16Gust44 cells cotransfected with DNA for wild type hTAS1R2/hTAS1R3 (filled circle, solid line), C801I7.39 (open diamond, short dashed line), Y699L5.60 (open circle, dash-dot-dotted line), Y699F5.60 (filled triangle up, medium dashed line), or W775A6.48 (filled hexagon, dash-dotted line) and hTAS1R2 DNA. (B) Concentration-dependent responses of HEK293T-G16Gust44 cells to cyclamate cotransfected with DNA for hTAS1R3 (filled circle, solid line), S726A5.39 (open triangle up, medium dashed line), C801I7.39 (open diamond, short dashed line), or W775A6.48 (filled hexagon, dash-dotted line) and hTAS1R2.
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Figure 4: Identification of additional determinants in TAS1R3 critical for responsiveness to cyclamate and lactisole. (A) Concentration-dependent inhibition of calcium responses to 10 mM aspartame by lactisole in HEK293T-G16Gust44 cells cotransfected with DNA for wild type hTAS1R2/hTAS1R3 (filled circle, solid line), C801I7.39 (open diamond, short dashed line), Y699L5.60 (open circle, dash-dot-dotted line), Y699F5.60 (filled triangle up, medium dashed line), or W775A6.48 (filled hexagon, dash-dotted line) and hTAS1R2 DNA. (B) Concentration-dependent responses of HEK293T-G16Gust44 cells to cyclamate cotransfected with DNA for hTAS1R3 (filled circle, solid line), S726A5.39 (open triangle up, medium dashed line), C801I7.39 (open diamond, short dashed line), or W775A6.48 (filled hexagon, dash-dotted line) and hTAS1R2.

Mentions: Since our results indicate a partial overlap of the NHDC binding site with those for lactisole and cyclamate, we next asked, if any of the newly identified residues also affect the sweet receptor's responses to these compounds. To this end, we tested all mutant hTAS1R3 subunits by coexpression with hTAS1R2 in HEK293T-G16Gust44 cells for their sensitivity to lactisole and cyclamate (Additional file 1). While we essentially confirmed the findings of Jiang et al. [14,15] that amino acids in positions 6363.28, 6373.29, 6403.32, 6413.33, 721ex2, 7235.36, 7295.42, 7305.43, 7335.46, 7786.51, 7796.52, 7826.55, 790ex3, and 7987.36 of hTAS1R3 contribute to the sensitivity of the sweet receptor to cyclamate and/or lactisole, we found additional amino acids in the heptahelical domain of hTAS1R3 that altered the responses of the sweet receptor to the two compounds. The mutant receptor W775A6.48 failed to respond to lactisole (Fig. 4A), suggesting that this residue is crucial for the sensitivity of the sweet receptor to the inhibitor. The mutants C801I7.39 (IC50 = 0.3 ± 0.01 mM), Y699L4.60 (IC50 = 0.3 ± 0.01 mM), and Y699F4.60 (IC50 = 0.5 ± 0.01 mM) showed three- to fivefold reduced responses to lactisole compared to the wild type receptor (IC50 = 0.1 ± 0.01 mM). Thus, we conclude that the amino acid positions Y6994.60 and C8017.39 also contribute to the inhibition of the sweet receptor by lactisole. The mutants C801I7.39 and W775A6.48 could not be activated at any tested concentration of cyclamate indicating the importance of these residues for the sweet receptor's responsiveness to cyclamate (Fig. 4B, Additional file 1). In addition, the mutant receptor S726A5.39 displayed more than fivefold lower sensitivity to cyclamate (EC50 > 10 mM) than the wild type receptor (EC50 = 1.9 ± 0.1 mM), suggesting that all three residues contribute to the receptor's ability to be activated by cyclamate.


The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor.

Winnig M, Bufe B, Kratochwil NA, Slack JP, Meyerhof W - BMC Struct. Biol. (2007)

Identification of additional determinants in TAS1R3 critical for responsiveness to cyclamate and lactisole. (A) Concentration-dependent inhibition of calcium responses to 10 mM aspartame by lactisole in HEK293T-G16Gust44 cells cotransfected with DNA for wild type hTAS1R2/hTAS1R3 (filled circle, solid line), C801I7.39 (open diamond, short dashed line), Y699L5.60 (open circle, dash-dot-dotted line), Y699F5.60 (filled triangle up, medium dashed line), or W775A6.48 (filled hexagon, dash-dotted line) and hTAS1R2 DNA. (B) Concentration-dependent responses of HEK293T-G16Gust44 cells to cyclamate cotransfected with DNA for hTAS1R3 (filled circle, solid line), S726A5.39 (open triangle up, medium dashed line), C801I7.39 (open diamond, short dashed line), or W775A6.48 (filled hexagon, dash-dotted line) and hTAS1R2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Identification of additional determinants in TAS1R3 critical for responsiveness to cyclamate and lactisole. (A) Concentration-dependent inhibition of calcium responses to 10 mM aspartame by lactisole in HEK293T-G16Gust44 cells cotransfected with DNA for wild type hTAS1R2/hTAS1R3 (filled circle, solid line), C801I7.39 (open diamond, short dashed line), Y699L5.60 (open circle, dash-dot-dotted line), Y699F5.60 (filled triangle up, medium dashed line), or W775A6.48 (filled hexagon, dash-dotted line) and hTAS1R2 DNA. (B) Concentration-dependent responses of HEK293T-G16Gust44 cells to cyclamate cotransfected with DNA for hTAS1R3 (filled circle, solid line), S726A5.39 (open triangle up, medium dashed line), C801I7.39 (open diamond, short dashed line), or W775A6.48 (filled hexagon, dash-dotted line) and hTAS1R2.
Mentions: Since our results indicate a partial overlap of the NHDC binding site with those for lactisole and cyclamate, we next asked, if any of the newly identified residues also affect the sweet receptor's responses to these compounds. To this end, we tested all mutant hTAS1R3 subunits by coexpression with hTAS1R2 in HEK293T-G16Gust44 cells for their sensitivity to lactisole and cyclamate (Additional file 1). While we essentially confirmed the findings of Jiang et al. [14,15] that amino acids in positions 6363.28, 6373.29, 6403.32, 6413.33, 721ex2, 7235.36, 7295.42, 7305.43, 7335.46, 7786.51, 7796.52, 7826.55, 790ex3, and 7987.36 of hTAS1R3 contribute to the sensitivity of the sweet receptor to cyclamate and/or lactisole, we found additional amino acids in the heptahelical domain of hTAS1R3 that altered the responses of the sweet receptor to the two compounds. The mutant receptor W775A6.48 failed to respond to lactisole (Fig. 4A), suggesting that this residue is crucial for the sensitivity of the sweet receptor to the inhibitor. The mutants C801I7.39 (IC50 = 0.3 ± 0.01 mM), Y699L4.60 (IC50 = 0.3 ± 0.01 mM), and Y699F4.60 (IC50 = 0.5 ± 0.01 mM) showed three- to fivefold reduced responses to lactisole compared to the wild type receptor (IC50 = 0.1 ± 0.01 mM). Thus, we conclude that the amino acid positions Y6994.60 and C8017.39 also contribute to the inhibition of the sweet receptor by lactisole. The mutants C801I7.39 and W775A6.48 could not be activated at any tested concentration of cyclamate indicating the importance of these residues for the sweet receptor's responsiveness to cyclamate (Fig. 4B, Additional file 1). In addition, the mutant receptor S726A5.39 displayed more than fivefold lower sensitivity to cyclamate (EC50 > 10 mM) than the wild type receptor (EC50 = 1.9 ± 0.1 mM), suggesting that all three residues contribute to the receptor's ability to be activated by cyclamate.

Bottom Line: Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore.This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor activation by neohesperidin dihydrochalcone and cyclamate.Some of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are involved in the binding of allosteric modulators in other class C GPCRs, suggesting a general role of these amino acid positions in allosterism and pointing to a common architecture of the heptahelical domains of class C GPCRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: German Institute of Human Nutrition Potsdam-Rehbruecke, Department of Molecular Genetics, Arthur-Scheunert Allee 114-116, 14558 Nuthetal, Germany. marcel.winnig.mw@axxam.com

ABSTRACT

Background: Differences in sweet taste perception among species depend on structural variations of the sweet taste receptor. The commercially used isovanillyl sweetener neohesperidin dihydrochalcone activates the human but not the rat sweet receptor TAS1R2+TAS1R3. Analysis of interspecies combinations and chimeras of rat and human TAS1R2+TAS1R3 suggested that the heptahelical domain of human TAS1R3 is crucial for the activation of the sweet receptor by neohesperidin dihydrochalcone.

Results: By mutational analysis combined with functional studies and molecular modeling we identified a set of different amino acid residues within the heptahelical domain of human TAS1R3 that forms the neohesperidin dihydrochalcone binding pocket. Sixteen amino acid residues in the transmembrane domains 2 to 7 and one in the extracellular loop 2 of hTAS1R3 influenced the receptor's response to neohesperidin dihydrochalcone. Some of these seventeen residues are also part of the binding sites for the sweetener cyclamate or the sweet taste inhibitor lactisole. In line with this observation, lactisole inhibited activation of the sweet receptor by neohesperidin dihydrochalcone and cyclamate competitively, whereas receptor activation by aspartame, a sweetener known to bind to the N-terminal domain of TAS1R2, was allosterically inhibited. Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore.

Conclusion: From our data we conclude that we identified the neohesperidin dihydrochalcone binding site at the human sweet taste receptor, which overlaps with those for the sweetener cyclamate and the sweet taste inhibitor lactisole. This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor activation by neohesperidin dihydrochalcone and cyclamate. Some of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are involved in the binding of allosteric modulators in other class C GPCRs, suggesting a general role of these amino acid positions in allosterism and pointing to a common architecture of the heptahelical domains of class C GPCRs.

Show MeSH
Related in: MedlinePlus