Oligomerization of Sweet and Bitter Taste Receptors - Methods in Cell Biology - page 232

FIGURE 13.3
Analysis of TAS2R homo- and hetero-oligomerization by bioluminescence resonance
energy transfer (BRET). (A) Two receptors bearing C-terminal GFP 2 and Rluc moieties,
respectively, are coexpressed in a mammalian cell line, for example, HEK293T cells.
Administration of the coelenterazine DeepBlueC will lead to light emission from the luciferase
at 370-450 nm. If the GFP 2 is brought into close proximity by protein-protein interactions
of the respective receptors, it can be excited and light emission at 500-530 nm can be
detected. (B) Graph showing BRET results obtained with TAS2R10-Rluc and all other
TAS2R-GFP 2 . The sweet taste receptor heteromer (TAS1R2-Rluc

TAS1R3-GFP 2 ) serves
as a positive control, TAS2R10-Rluc alone as a negative control (dashed line). Almost all
combinations show BRET ratios (light emission at 500-530 nm over light emission at
370-450 nm) higher than the negative control, which is indicative of receptor oligomerization.
Only TAS2R10-Rluc with TAS2R4-GFP 2 and TAS2R45-GFP 2 , respectively, have lower values.
Data represent means and standard deviation from two experiments carried out in duplicate.

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When both BRET donor and acceptor molecules are brought in close proximity to
each other through protein-protein interactions of the receptors, an energy transfer
from the luciferase to the GFP 2 can occur, resulting in detectable fluorescence of
GFP 2 ( Fig. 13.3 ).
BRET has been successfully employed for the analysis of dimerization of several
GPCRs including TAS1Rs ( Jiang et al., 2004 ). But while the reading was in general
being performed in 96-well plates and luminescent plate readers, the cells had been
grown and transfected in larger cell culture dishes and only then had the cells been
transferred to 96-well plates for BRET assay reading. We have adapted the procedure
for higher throughput by directly growing the cells in 96-well plates and performing
the BRET assay in the same 96-well plate.
Therefore, modified HEK293T cells are seeded at 10% confluence in white,

clear-bottomed, poly- D -lysine-coated 96-well plates. After 48 h, at

50% conflu-
ence, cells are transfected with the plasmids encoding for the BRET fusion proteins
(one receptor-Rluc and one receptor-GFP 2 construct) using a lipofection reagent
according to the manufacturer's recommendation. Cotransfection of two plasmids