Biophysical and functional characterization of the N-terminal domain of the cat T1R1 umami taste receptor expressed in Escherichia coli

Abstract : Umami taste perception is mediated by the heterodimeric G-protein coupled receptors (GPCRs), formed by the assembly of T1R1 and T1R3 subunits. T1R1 and T1R3 subunits are class C GPCRs whose members share common structural homologies including a long N-terminal domain (NTD) linked to a seven transmembrane domain by a short cysteine-rich region. The NTD of the T1R1 subunit contains the primary binding site for umami stimuli, such as L-glutamate (L-Glu) for humans. Inosine-5'-monophosphate (IMP) binds at a location close to the opening of the T1R1-NTD "flytrap", thus creating the observed synergistic response between L-Glu and IMP. T1R1/T1R3 binding studies have revealed species-dependent differences. While human T1R1/T1R3 is activated specifically by L-Glu, the T1R1/T1R3 in other species is a broadly tuned receptor, sensitive to a range of L-amino acids. Because domestic cats are obligate carnivores, they display strong preferences for some specific amino acids. To better understand the structural basis of umami stimuli recognition by non-human taste receptors, we measured the binding of selected amino acids to cat T1R1/T1R3 (cT1R1/cT1R3) umami taste receptor. For this purpose, we expressed cT1R1-NTD in bacteria as inclusion bodies. After purification, refolding of the protein was achieved. Circular dichroism spectroscopic studies revealed that cT1R1-NTD was well renatured with evidence of secondary structures. Using size-exclusion chromatography coupled to light scattering, we found that the cT1R1-NTD behaves as a monomer. Ligand binding quantified by intrinsic tryptophan fluorescence showed that cT1R1-NTD is capable of binding L-amino acids with K-d values in the micromolar range. We demonstrated that IMP potentiates L-amino acid binding onto renatured cT1R1-NTD. Interestingly, our results revealed that IMP binds the extracellular domain in the absence of L-amino acids. Thus, this study demonstrates that the feasibility to produce milligram quantities of cT1R1-NTD for functional and structural studies.
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https://hal-univ-bourgogne.archives-ouvertes.fr/hal-01686203
Contributeur : Csga - Université de Bourgogne <>
Soumis le : mercredi 17 janvier 2018 - 10:30:39
Dernière modification le : jeudi 11 juillet 2019 - 14:10:08

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Christine Belloir, Jimmy Savistchenko, Fabrice Neiers, Andrew J. Taylor, Scott Mcgrane, et al.. Biophysical and functional characterization of the N-terminal domain of the cat T1R1 umami taste receptor expressed in Escherichia coli. PLoS ONE, Public Library of Science, 2017, 12 (10), ⟨10.1371/journal.pone.0187051⟩. ⟨hal-01686203⟩

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