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Biophysical and functional characterization of the human TAS1R2 sweet taste receptor overexpressed in a HEK293S inducible cell line

ABSTRACT: Sweet taste perception is mediated by a heterodimeric receptor formed by the assembly of the TAS1R2 and TAS1R3 subunits. TAS1R2 and TAS1R3 are class C G-protein-coupled receptors whose members share a common topology, including a large extracellular N-terminal domain (NTD) linked to a seven transmembrane domain (TMD) by a cysteine-rich domain. TAS1R2-NTD contains the primary binding site for sweet compounds, including natural sugars and high-potency sweeteners, whereas the TAS1R2-TMD has been shown to bind a limited number of sweet tasting compounds. To understand the molecular mechanisms governing receptor–ligand interactions, we overexpressed the human TAS1R2 (hTAS1R2) in a stable tetracycline-inducible HEK293S cell line and purified the detergent-solubilized receptor. Circular dichroism spectroscopic studies revealed that hTAS1R2 was properly folded with evidence of secondary structures. Using size exclusion chromatography coupled to light scattering, we found that the hTAS1R2 subunit is a dimer. Ligand binding properties were quantified by intrinsic tryptophan fluorescence. Due to technical limitations, natural sugars have not been tested. However, we showed that hTAS1R2 is capable of binding high potency sweeteners with Kd values that are in agreement with physiological detection. This study offers a new experimental strategy to identify new sweeteners or taste modulators that act on the hTAS1R2 and is a prerequisite for structural query and biophysical studies.

SUBMITTER: Belloir C

PROVIDER: S-EPMC8593021 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Evolution of the sweet taste receptor gene Tas1r2 in bats.

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The Ile191Val is a partial loss-of-function variant of the TAS1R2 sweet-taste receptor and is associated with reduced glucose excursions in humans.

Project description:ObjectiveSweet taste receptors (STR) are expressed in the gut and other extra-oral tissues, suggesting that STR-mediated nutrient sensing may contribute to human physiology beyond taste. A common variant (Ile191Val) in the TAS1R2 gene of STR is associated with nutritional and metabolic outcomes independent of changes in taste perception. It is unclear whether this polymorphism directly alters STR function and how it may contribute to metabolic regulation.MethodsWe implemented a combination of in vitro biochemical approaches to decipher the effects of TAS1R2 polymorphism on STR function. Then, as proof-of-concept, we assessed its effects on glucose homeostasis in apparently healthy lean participants.ResultsThe Ile191Val variant causes a partial loss of function of TAS1R2 through reduced receptor availability in the plasma membrane. Val minor allele carriers have reduced glucose excursions during an OGTT, mirroring effects previously seen in mice with genetic loss of function of TAS1R2. These effects were not due to differences in beta-cell function or insulin sensitivity.ConclusionsOur pilot studies on a common TAS1R2 polymorphism suggest that STR sensory function in peripheral tissues, such as the intestine, may contribute to the regulation of metabolic control in humans.

| S-EPMC8476773 | biostudies-literature

Characterization of the modes of binding between human sweet taste receptor and low-molecular-weight sweet compounds.

Project description:One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2-hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein-coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2-hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling-based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands.

| S-EPMC3335050 | biostudies-literature

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