Construction of a bioluminescence-based assay for bitter taste receptors (TAS2Rs)

Abstract

In humans, a family of 25 bitter taste receptors (TAS2Rs) mediates bitter taste perception. A common approach to characterize bitter causative agents involves expressing TAS2Rs and the appropriate signal transducers in heterologous cell systems, and monitoring changes in the intracellular free calcium levels upon ligand exposure using a fluorescence-based modality, which typically suffers from a low signal window, and is susceptible to interference by autofluorescence, therefore prohibiting its application to screening of plant or food extracts, which are likely to contain autofluorescent compounds. The aim of this study is to develop and validate a bioluminescence-based intracellular calcium release assay for TAS2Rs that has a better assay performance than a fluorescence-based assay. Furthermore, the bioluminescence-based assay enabled the evaluation of TAS2R agonists within an autofluorescent matrix, highlighting its potential utility in the assessment of the bitterness-inducing properties of plant or food fractions by the food industry. Additionally, improvement to the bioluminescence-based assay for some TAS2Rs was achieved by altering their N-terminal signal sequences, leading to signal window enhancement. Altogether, the bioluminescence-based TAS2R assay can be used to perform functional studies of TAS2Rs, evaluate TAS2R-modulating properties of autofluorescent samples, and facilitate the discovery of compounds that can function as promising bitter taste modulators.

Enhancement of transcription efficiency by TAR-Tat system increases the functional expression of human olfactory receptors

“…sensitivity of the TAS2R assay system [31]. Similar to the TAS2R study, this study elucidated that increasing the cell surface expression of hOR is effective in detecting the clear responses of hORs.…”

“…The cell surface expression of GPCRs is essential for their functional analysis using heterologous cells. For instance, Tan et al optimized N-terminal signal sequences to increase the cell surface expression of the bitter taste receptor (TAS2R) and succeeded in improving the sensitivity of the TAS2R assay system [ 31 ]. Similar to the TAS2R study, this study elucidated that increasing the cell surface expression of hOR is effective in detecting the clear responses of hORs.…”

Non-Volatile Compounds Involved in Bitterness and Astringency of Pulses: A Review

“…Then, cytoplasmic calcium binds a fluorescent indicator (Fluo-4 or Fura-2) [51] or a genetically encoded calcium biosensor (GCaMP) [59,60]. More recently, a bioluminescence-based assay was developed for TAS2R to measure calcium mobilization using mt-clytin II, a calcium-sensitive photoprotein, and the luminophore coelenterazine [61]. The calcium signal is measured through fluorescence or luminescence using a fluorometric imaging microplate reader (FlexStation or FLIPR).…”

“…The calcium signal is measured through fluorescence or luminescence using a fluorometric imaging microplate reader (FlexStation or FLIPR). The response of TAS2R depends on the concentration of bitter compounds and leads to different signal amplitudes [28,58,61]. Then, the AT (activation threshold) is determined, corresponding to the lowest concentration for which fluorescence is observed.…”

Inhibitors of human bitter taste receptors from the five-flavour berry, Schisandra chinensis

“…Multigene expression vectors containing cDNA encoding respective human TAS2R receptors along with mt-clytin II and the Gα16-gust44 chimera were constructed as described by Tan & Seetoh. 35 On day 0, 293AD cells (Cell Biolabs Inc.) were seeded on a 384-well white flat-bottom µCLEAR® cell culture microplate (Greiner Bio-One, Kremsmünster, Austria) at 13 200 cells per well and incubated for at least 18 h. On day 1, the respective expression vectors were transiently transfected into 293AD cells using ViaFect (Promega, Madison, WI, USA) for heterologous expression. On day 2, the growth media in the plate was discarded and the cells were incubated in the dark with 40 µL of 10 µM coelenterazine h (CAS 50909-86-9, AAT Bioquest, Sunnyvale, CA, USA) per well at room temperature (27 °C) for 4 h. Subsequently, the luminescence assay was performed using an automated fluorometric imaging plate reader (FLIPR) (Molecular Devices, San Jose, CA, USA).…”