Abstract
Taste signaling is a complex process that is linked to obesity and its associated metabolic syndromes. The sweet taste is mediated through a heterodimeric G protein coupled receptor (GPCR) in a species-specific manner and at multi-tissue specific levels. The sweet receptor recognizes a large number of ligands with structural and functional diversities to modulate different amplitudes of downstream signaling pathway(s). The human sweet-taste receptor has been extremely difficult to study by biophysical methods due to the difficulty in producing large homogeneous quantities of the taste-receptor protein and the lack of reliable in vitro assays to precisely measure productive ligand binding modes that lead to activation of the receptor protein. We report here a multimodal high throughput assay to monitor ligand binding, receptor stability and conformational changes to model the molecular ligand-receptor interactions. We applied saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR) complemented by differential scanning calorimetry (DSC), circular dichroism (CD) spectroscopy, and intrinsic fluorescence spectroscopy (IF) to characterize binding interactions. Our method using complementary NMR and biophysical analysis is advantageous to study the mechanism of ligand binding and signaling processes in other GPCRs.
Highlights
The human sweet taste receptor is a heterodimeric complex of the proteins T1R2 and T1R3.The complex is a member of the G-protein-coupled receptor class (GPCR); which share a common design of a seven transmembrane heptahelical domain with an extracellular N-terminus and intracellular C-terminus
We show that combination of intrinsic fluorescence spectroscopy, circular dichroism spectroscopy and saturation transfer difference spectroscopy can be useful in evaluating detailed molecular changes at the receptor level while monitoring ligand binding
We report here the results of experiments using multimodal biophysical techniques that probe the interaction of human and mouse T1R2 amino-terminal domain (ATD) with small ligands that are known to elicit a sweet-taste response
Summary
The human sweet taste receptor is a heterodimeric complex of the proteins T1R2 and T1R3. The complex is a member of the G-protein-coupled receptor class (GPCR); which share a common design of a seven transmembrane heptahelical domain with an extracellular N-terminus and intracellular C-terminus. The class C GPCR share a common structure of a large amino-terminal domain (ATD), which serves as the principle ligand-binding domain, followed. Molecules 2018, 23, 2531 by a short cysteine-rich domain (CRD) tied to the transmembrane domain (TMD) and intracellular. The sweet-taste receptor has been shown to bind a large ensemble of molecules such as sugars, artificial sweeteners, sweet-tasting proteins and some D-amino acids that mediate the sweet taste response (Figure 1A). Regions of the complex that bind specific ligands include the ATD of human (h)
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