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.
Highlights
Taste detection is mediated by a single heteromeric receptor composed of two G-protein coupled receptors (GPCRs), named TAS1R2 and TAS1R3[1,2,3,4,5,6]
The codon-optimized human TAS1R2 (hTAS1R2) gene was overexpressed in the order of few hundreds of micrograms using the tetracycline-inducible HEK293S GnTI- cell line
We demonstrated the ability of the detergent lauryl maltose neopentyl glycol (LMNG) to efficiently extract and solubilize hTAS1R2, maintaining its functional activity
Summary
Taste detection is mediated by a single heteromeric receptor composed of two G-protein coupled receptors (GPCRs), named TAS1R2 (taste receptor type 1, member 2) and TAS1R3 (taste receptor type 1, member 3)[1,2,3,4,5,6]. One binding site is located in the TAS1R3-VFT module, where natural sugars (sucrose, fructose and glucose) and the chlorodeoxysugar sucralose have been found to bind[13,18] Another binding site is located in TAS1R3-TMD, where the sweeteners cyclamate and neohesperidin dihydrochalcone and the sweet taste inhibitors lactisole and gymnemic acid b ind[19,20,21,22,23]. The relative contribution of the two subunits constituting the heterodimeric human TAS1R2/TAS1R3 receptor remains largely unknown This lack of knowledge is partly due to difficulties in preparing sufficient quantities of functional VFT domains suitable for ligand binding assays and biophysical analysis. This study provides new insights into the molecular determinants of sweet taste perception and opens ways to screen new sweet tasting compounds or taste modulators
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