Biology Reference
In-Depth Information
show more dynamic features where agonist treatment markedly affects BRET
max
,
BRET
50
, or both these values (
Fig. 8.1
).
Details are shown in the succeeding text for typical BRET
2
assays for detection
and analysis of GPCR-RTK (A2A-FGFR1) heteroreceptor complexes using either
adherent cells or cells in suspension.
1.
Several independent transfections should be performed using a constant amount
of cDNA coding for the BRET donor (10-100 ng, FGFR1-Rluc8) and increasing
quantities of cDNA coding for the BRET acceptor (i.e., 0, 10, 20, 50, 100, 200,
300, 500, and 1000 ng; A2A-GFP2) and sufficient “empty” vector (such as
pCDNA3.1 or any other cloning vector) to bring the total amount of cDNA in the
transfection to 1000 ng/well in a six-well plate.
2.
24 h (preferred option (a), see Point 3 in
Section 3
) or 48 h (preferred option (b),
see Point 3 in
Section 3
) after transfection cells are washed, detached, and
distributed into white opaque 96-well microplates: 40-100
l per well, incubated
as described in the succeeding text (Point 3 in
Section 3.1
) or moved directly to
the BRET ratio measurements.
3.
The cells are preincubated in the absence or presence of agonist/antagonist drugs
if ligand-induced BRET
2
signals will be analyzed. Otherwise, proceed directly to
BRET ratio measurements.
4.
BRET
2
ratio measurements are performed after adding coelenterazine 400a
diluted in HBSS or PBS CaCl
2
/MgCl
2
to each well in order to reach a
final concentration of 5
m
M. BRET
2
ratio readings are preformed using a lumino/
fluorometer that allows sequential integration of luminescence signals detected
with two filter settings (see Point 4 in
Section 3
). The specific BRET
2
ratio is
calculated by subtracting from the mean BRET
2
ratio value above the
m
curve assay in HEK293T27 cells cotransfected with a constant amount of FGFR1-Rluc8
plasmid and increasing amount of the A2AR-GFP2 plasmid. In the current analysis, the
amount of each receptor effectively expressed in transfected cells was monitored for each
individual experiment by correlating both total luminescence and total fluorescence to the
number of receptor-binding sites (biochemical binding analysis) in permeabilized cells
The linear regression equations derived from these data were thus used to convert
fluorescence and luminescence values into femtomoles/mg protein of receptor in order to
obtain accurate values. Cells were preincubated 10 min with vehicle CGS21680 (100 nM) or
FGF-2 (50 ng/ml) or with both CGS21680 and FGF-2 (100 nM and 50 ng/ml, respectively).
The A2AR/FGFR1 curve fitted better to a saturation curve than to a linear regression, F test
(P<
5). The BRET
max
values were significantly
enhanced by combined, CGS21680 and FGF-2 treatment alone versus vehicle or CGS21680
treatment alone and FGF2 treatment alone versus vehicle or CGS21680 treatment alone
(P<0.01). (B) The BRET
50
values were significantly reduced by combined, CGS21680 and
FGF-2 treatment alone versus vehicle (P<
0.001). Data are means
s.e.m. (n¼
0.001).
