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

Belloir, Christine; Brulé, Marine; Tornier, Lucie; Neiers, Fabrice; Briand, Loı̈c

doi:10.1038/s41598-021-01731-3

Cited by 15 publications

(20 citation statements)

References 72 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Interestingly, the measured K d values for all tested compounds were lower than the predicted values deduced from the half maximal effective concentration (EC 50 ) values from the human TAS1R2/TAS1R3 receptor measured using a cellular-based assay or from sensory experiments conducted on human panels ( Table 1 ). These K d values are lower than those measured for the whole hTAS1R2 subunit [ 26 ]. This may be due to the impact of the presence of TMD.…”

Section: Discussioncontrasting

confidence: 62%

“…Combining various biophysical approaches and NMR, hTAS1R2-VFT was demonstrated to interact with the artificial sweetener neotame with a K d value in the micromolar range [ 25 ]. More recently, the whole hTAS1R2 subunit overexpressed in a HEK293S inducible cell line was capable of binding high-potency sweeteners with K d values that are in agreement with physiological detection [ 26 ].…”

Section: Introductionmentioning

confidence: 95%

See 1 more Smart Citation

Functional Characterization of the Venus Flytrap Domain of the Human TAS1R2 Sweet Taste Receptor

Laffitte

Belloir

Neiers

et al. 2022

IJMS

Self Cite

View full text Add to dashboard Cite

The human sweet taste receptor is a heterodimeric receptor composed of two distinct G-protein-coupled receptors (GPCRs), TAS1R2 and TAS1R3. The TAS1R2 and TAS1R3 subunits are members of a small family of class C GPCRs whose members share the same architecture, comprising a Venus Flytrap (VFT) module linked to the seven transmembrane domains (TMDs) by a short cysteine-rich region (CRR). The VFT module of TAS1R2 contains the primary binding site for most of the sweet-tasting compounds, including natural sugars and artificial and natural sweeteners. However, cellular assays, molecular docking and site-directed mutagenesis studies have revealed that the VFT, CRR and TMD of TAS1R3 interact with some sweeteners, including the sweet-tasting protein brazzein. The aim of this study was to better understand the contribution of TAS1R2-VFT in the binding of sweet stimuli. To achieve this, we heterologously expressed human TAS1R2-VFT (hTAS1R2-VFT) in Escherichia coli. Circular dichroism spectroscopic studies revealed that hTAS1R2-VFT was properly folded with evidence of secondary structures. Using size-exclusion chromatography coupled with light scattering, we found that hTAS1R2-VFT behaves as a monomer. Ligand binding quantified by intrinsic tryptophan fluorescence showed that hTAS1R2-VFT is capable of binding sweet stimuli with Kd values, in agreement with physiological detection. Furthermore, we investigated whether the impact of point mutations, already shown to have deleterious effects on cellular assays, could impact the ability of hTAS1R2-VFT to bind sweet ligands. As expected, the ligand affinities of hTAS1R2-VFT were drastically reduced through the introduction of single amino acid substitutions (D278A and E382A) known to abolish the response of the full-length TAS1R2/TAS1R3 receptor. This study demonstrates the feasibility of producing milligram quantities of hTAS1R2-VFT to further characterize the mechanism of binding interaction and perform structural studies.

show abstract

Section: Discussioncontrasting

confidence: 62%

Section: Introductionmentioning

confidence: 95%

Functional Characterization of the Venus Flytrap Domain of the Human TAS1R2 Sweet Taste Receptor

Laffitte

Belloir

Neiers

et al. 2022

IJMS

Self Cite

View full text Add to dashboard Cite

show abstract

“…Due to these distinct structural features and mandatory dimerization, the class C GPCRs have been the most complex of the GPCRs in terms of understanding their activation mechanism [ 31 , 32 , 33 , 34 , 35 ]. Using several methods such as crystallization [ 30 ], lipid cubic phase [ 36 ], and most commonly single particle Cryo-EM, structures of over 20 human class C GPCRs have been solved to date [ 37 ], comprising metabotropic glutamate receptors (mGluR1–5,mGluR7) [ 36 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ], gamma-aminobutyric acid receptors (GABA

1 and GABA

2) [ 23 , 24 , 47 ], calcium-sensing receptors (CaS) [ 48 , 49 , 50 ], the extra-cellular domain of taste receptors (TAS1R1–TAS1R3) [ 51 , 52 , 53 , 54 , 55 ], and orphan receptors (GPR158, GPR179, GPR156) [ 56 , 57 , 58 , 59 , 60 ]. Similarly to other GPCR structures, class C GPCR structures are solved with inclusion of cholesterol or cholesteryl hemisuccinate (CHS) to the detergent mix during crystallization and recently, Cryo-EM ( Table 1 ).…”

Section: Introductionmentioning

confidence: 99%

Cholesterol in Class C GPCRs: Role, Relevance, and Localization

et al. 2023

View full text Add to dashboard Cite

G-protein coupled receptors (GPCRs), one of the largest superfamilies of cell-surface receptors, are heptahelical integral membrane proteins that play critical roles in virtually every organ system. G-protein-coupled receptors operate in membranes rich in cholesterol, with an imbalance in cholesterol level within the vicinity of GPCR transmembrane domains affecting the structure and/or function of many GPCRs, a phenomenon that has been linked to several diseases. These effects of cholesterol could result in indirect changes by altering the mechanical properties of the lipid environment or direct changes by binding to specific sites on the protein. There are a number of studies and reviews on how cholesterol modulates class A GPCRs; however, this area of study is yet to be explored for class C GPCRs, which are characterized by a large extracellular region and often form constitutive dimers. This review highlights specific sites of interaction, functions, and structural dynamics involved in the cholesterol recognition of the class C GPCRs. We summarize recent data from some typical family members to explain the effects of membrane cholesterol on the structural features and functions of class C GPCRs and speculate on their corresponding therapeutic potential.

show abstract

“…However, this is likely due in part to the difficulty in obtaining defined dose-response top asymptotes for these sweeteners in the DMR assay. Potency (EC 50 ) values of sweet taste receptor agonists are known to vary by 2- to 10-folds from laboratory to laboratory and with different experimental conditions ( Palmer, 2019 ; Servant et al, 2020 ; Ahmad and Dalziel, 2020 ; Belloir et al, 2021 ). The potency values reported in this study fall within this range.…”

Section: Discussionmentioning

confidence: 99%

A Dynamic Mass Redistribution Assay for the Human Sweet Taste Receptor Uncovers G-Protein Dependent Biased Ligands

et al. 2022

View full text Add to dashboard Cite

The sweet taste receptor is rather unique, recognizing a diverse repertoire of natural or synthetic ligands, with a surprisingly large structural diversity, and with potencies stretching over more than six orders of magnitude. Yet, it is not clear if different cell-based assays can faithfully report the relative potencies and efficacies of these molecules. Indeed, up to now, sweet taste receptor agonists have been almost exclusively characterized using cell-based assays developed with overexpressed and promiscuous G proteins. This non-physiological coupling has allowed the quantification of receptor activity via phospholipase C activation and calcium mobilization measurements in heterologous cells on a FLIPR system, for example. Here, we developed a novel assay for the human sweet taste receptor where endogenous G proteins and signaling pathways are recruited by the activated receptor. The effects of several sweet taste receptor agonists and other types of modulators were recorded by measuring changes in dynamic mass redistribution (DMR) using an Epic® reader. Potency and efficacy values obtained in the DMR assay were compared to those results obtained with the classical FLIPR assay. Results demonstrate that for some ligands, the two assay systems provide similar information. However, a clear bias for the FLIPR assay was observed for one third of the agonists evaluated, suggesting that the use of non-physiological coupling may influence the potency and efficacy of sweet taste receptor ligands. Replacing the promiscuous G protein with a chimeric G protein containing the C-terminal tail 25 residues of the physiologically relevant G protein subunit Gαgustducin reduced or abrogated bias.

show abstract

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

Cited by 15 publications

References 72 publications

Functional Characterization of the Venus Flytrap Domain of the Human TAS1R2 Sweet Taste Receptor

Functional Characterization of the Venus Flytrap Domain of the Human TAS1R2 Sweet Taste Receptor

Cholesterol in Class C GPCRs: Role, Relevance, and Localization

A Dynamic Mass Redistribution Assay for the Human Sweet Taste Receptor Uncovers G-Protein Dependent Biased Ligands

Contact Info

Product

Resources

About