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Communication Dans Un Congrès Année : 2016

Interaction of the human T1R2 taste receptor ligand-binding domain with sweeteners and sweet-tasting proteins

Résumé

Humans perceive sweet-taste through a unique heterodimeric receptor composed of two class C G-protein coupled receptors (GPCR), T1R2 and T1R3. Both of these subunits possess a large extra-cellular domain, composed of the N-terminal domain (NTD) followed by the cysteine-rich domain that links the NTD to the heptahelical transmembrane domain. Cellular assays, molecular docking and site-directed mutagenesis studies have revealed that the NTD of T1R2 (T1R2-NTD) contain the primary binding site for most of the sweet ligands including natural sugars and, artificial and natural sweeteners including sweet-tasting proteins. To elucidate the contribution of T1R2-NTD to sweet tastant binding, we recombinantly expressed human T1R2-NTD in Escherichia coli. Then, we tested T1R2-NTD for its ability to interact with known sweet tasting ligands using intrinsic tryptophan fluorescence. To validate the method, the same experiment was performed with two T1R2-NTD mutants (D278A and E382A) known to abolish the response of the full-length receptor. We then measured the interaction of T1R2-NTD with the two recombinant sweet-tasting proteins, brazzein and monellin, using Bio-Layer Interferometry (BLI). This optical technique analyzes the signal variations in the interference pattern generated from visible light reflected from an optical layer and a biolayer containing the immobilized protein of interest. This method is powerful for studying protein-protein interactions and measuring both affinity constants and kinetic parameters. BLI experiments demonstrated that T1R2-NTD binds these two sweet-tasting proteins with affinities values in agreement with the physiological range. This strategy will be useful to decipher the essential role of T1R2-NTD in the detection of sweet tastants including sweet-tasting proteins.
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Dates et versions

hal-01578246 , version 1 (03-06-2020)

Identifiants

  • HAL Id : hal-01578246 , version 1
  • PRODINRA : 356963
  • WOS : 000386126000042

Citer

Anni Laffitte, Fabrice Neiers, Anne Brockhoff, Wolfgang Meyerhof, Loïc Briand. Interaction of the human T1R2 taste receptor ligand-binding domain with sweeteners and sweet-tasting proteins. The 17. international symposium on Olfaction and taste (ISOT), Jun 2016, Yokohama, Japan. 1 p. ⟨hal-01578246⟩
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