Biology Reference
In-Depth Information
and the plasmid encoding for the WT-GPCR protomer. BRET measurements are
performed over time (up to 20-30 min) after incubation with a RTK-specific
agonist or upon combined agonist treatment (RTK and GPCR agonist).
1.
HEK-293T cells are prepared and transfected using a constant ratio amount of
cDNA coding for the BRET RTK-donor and RTK-acceptor. This ratio should be
selected from a previously performed BRET saturation assay. Otherwise,
different plasmid DNA concentrations should be tested to determine the optimal
ratio of receptor-donor to receptor-acceptor that gives the highest BRET
2
signal
following receptor activation. Also, a constant amount of the untagged GPCR
protomer and sufficient “empty” vector (such as pCDNA3.1 or any other cloning
vector) will be used to maintain equal total amount of cDNA per well during
transfection.
2.
The next day, cells are harvested and dispensed in 50-100
l of cell suspension
containing 20,000 to 30,000 cells into a white opaque 96-microplate in HEPES-
buffered DMEM without phenol red.
3. For agonist-induced dose-response BRET:
24 h after plating cells are incubated
with different concentrations of the selected receptor agonist or PBS alone
(basal control condition) for a fixed time. Then, coelenterazine 400a solution at
a final concentration of 5
m
M is added and the BRET
2
signal is measured
(
Fig. 8.2
). The agonist and substrate can be added manually or injected if the plate
reader is equipped with built-in online injectors.
For kinetic BRET
experiments:
cells are incubated for different time periods with a fixed
concentration of the selected receptor agonist or PBS alone (basal control
condition). Then, coelenterazine 400a solution at a final concentration of 5
m
Mis
added and the BRET
2
signal is measured. We highly recommend when using
m
FIGURE 8.2—Cont'd induced FGFR1/FGFR1 homodimer formation by means of BRET
2
analysis. HEK293T27 cells were transiently cotransfected at a constant ratio (1:1:1) with
5-HT1A, FGFR1-Rluc8, and FGFR1-GFP2. (A) A concentration-response curve with FGF-2
was performed on the development of the BRET
2
signal from the FGFR1 homodimer in the
HEK293T27 cells. The cells were transiently cotransfected at a constant ratio (1:1:1) of
5-HT1A, FGFR1-Rluc8, and FGFR1-GFP2 and treated with the agonist ligands for 5 min
before BRET
2
measurement. Treatment with 8-OH-DPAT (with two different concentrations:
50 and 250 nM) shifted the curves of the BRET
2
signal to the left, which indicate an enhanced
potency of combined treatment with FGF2 and the 5-HT1A agonist versus FGF-2 treatment
alone to promote FGFR1 homodimer formation. (B) The kinetics of the FGFR1-Rluc/FGFR1-
GFP2 interaction after FGF2 treatment and its modulation by 8-OHDPAT was also studied in
transiently transfected HEK293T27 cells using the BRET
2
assay to study the FGFR1
homodimer over a period of 20 min. FGF-2 and the combined FGF-2 and 8-OH-DPAT
treatments showed no clear-cut changes of the BRET
2
value over the 8 min period. However,
the combined treatment had a weak tendency to increase the BRET
2
signal over time, whereas
the FGF2 alone treatment had a markedly tendency to decrease the BRET
2
signal over time.
