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Récepteurs gustatifs humains : étude des relations structure-fonction

Abstract : Sweet, umami and bitter taste detectors are membrane receptors that belong to the family of G-protein coupled receptors (GPCRs). They are characterized by the existence of a hydrophobic transmembrane domain (TMD) and an activation mechanism that involves a heterotrimeric G protein.Human has 25 bitter taste receptors TAS2R. These receptors belong to class A GPCRs. Their architecture consists of a TMD structured in 7 a-helix which form the orthosteric binding site of bitter molecules. The umami taste receptor is a heterodimer composed of the TAS1R1 and TAS1R3 subunits, while the TAS1R2 and TAS1R3 subunits form the sweet taste receptor. Each subunits belongs to the class C GPCRs and shares a common architecture, consisting of a large extracellular N-terminal domain (NTD) connected to the TMD by a cysteine-rich region (CRR). However, the relative contribution of each subunit to the heterodimeric receptors function remains largely unknown.Because of their amphipathic nature, GPCRs are extremely difficult to study from a biochemical point of view. Characterizing their interactions and determining their structures are important issues and a real challenge for the next years. In this work, we have developed different expression systems to understand molecular mechanisms that govern receptor-ligand interactions.For the study of umami taste, the TAS1R1 and TAS1R3 NTDs were produced in E. coli bacteria as inclusion bodies and then folded in vitro. By combining biochemical approaches and functional cell assays, we have shown that inosine-5'-monophosphate (IMP), an umami flavor enhancer, binds to TAS1R3-NTD and acts synergistically with sucralose and neotame.Regarding the sweet taste receptor, the subunit (full size) of TAS1R2 was overexpressed in a tetracycline inducible cell line HEK293S. Solubilization and purification protocol allows to obtain functional TAS1R2 receptor. Far-UV circular dichroism spectroscopy analysis revealed that TAS1R2 is well folded. Multiangle light scattering coupled with gel filtration showed that TAS1R2 was predominantly present in dimeric form. Interactions with sugar ligands measured by intrinsic fluorescence revealed micromolar range affinities in agreement with cellular assays and the sweetening powers of molecules.In parallel, to study bitter receptors, we designed different TAS2R14 receptor expression vectors in order to improve receptor expression and target to the plasma membrane. We showed that use of the QBI SP163 sequence upstream of the translational initiation codon, associated with the signal peptide of the rat somatostatin 3 in the N-terminal position and the FLAG tag in the C-terminal position, increase the functional response of the receptor to bitter ligands both in terms of amplitude and sensitivity. This plasmid construct represents a promising tool to help identify some orphaned TAS2R agonists.
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https://hal.inrae.fr/tel-03244299
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Submitted on : Tuesday, June 1, 2021 - 10:42:12 AM
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Christine Belloir (dupont). Récepteurs gustatifs humains : étude des relations structure-fonction. Alimentation et Nutrition. Université de Bourgogne Franche-Comté, 2019. Français. ⟨tel-03244299⟩

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