Biology Reference
In-Depth Information
1.
Before running a BRET
2
displacement assay, it is highly recommended to
conduct a classical BRET
2
saturation experiment as described in
Section 3.1
.
Using around 50-60% of the (GFP2-GFP20)/Rluc8 BRET
max
values in the
saturation experiment will give a better idea of the amount of donor and acceptor
needed for transfection in the BRET competition experiments.
2.
Perform several independent transfections using a constant ratio amount of
cDNA coding for the BRET
2
donor and acceptor (as selected in Point 1 in
Section 3.2
). Increasing amounts of the untagged receptor are used together with
sufficient “empty” vector (such as pCDNA3.1 or any other cloning vector) to
keep equal total amount of cDNA/well.
3.
Incubate the cells and measure the BRET
2
ratio (see Point 4 in
Section 3
).
4.
Calculate BRET
2
ratios (see Point 5 in
Section 3
) and plot them as a function of
the expression of native receptor determined by binding experiments or as a
function of the total untagged cDNA amount employed (
Fig. 8.1
).
8.3.3
Protocol 3: kinetics and dose-response assays
In addition to monitoring constitutive receptor-receptor interactions, BRET
2
can
be used to measure ligand-dependent induction of facilitatory or inhibitory recep-
tor-receptor interactions and also to follow the kinetics of these interactions in real
time. Indeed, ligand modulation of the BRET
2
signal may also prove the specificity
of the examined interaction. Until now, for most GPCR heteromers studied by
BRET, homomerization and heteromerization appear to occur constitutively and in-
dependently of receptor activation state. However, the dependence of the energy
transfer process on the distance between the donor and acceptor fused to the recep-
tors and on their relative orientation suggests that movements within receptor com-
plexes as a consequence of ligand-induced conformational changes may result in
detectable changes in FRET/BRET signals. This does not necessarily mean changes
in the association-dissociation between the receptors.
Many interesting examples where specific ligand-induced conformational
changes translate into changes in BRET
2
signal were observed in most studies on
RTK-RTK interactions and on GPCR-RTK heteroreceptor complexes (
Romero-
Fernandez et al., 2011
). This illustrates the usefulness of the BRET
2
technology
as a readout to characterize GPCR-RTK heteroreceptor complexes and their phar-
macological features. This is in addition to the more specific outcome of opening up
novel avenues for GPCR-RTK drug discovery.
As an example, this protocol describes the use of BRET
2
to measure the kinetics
and dose-responses of the agonist-promoted FGFR1 activation (homodimerization)
and the effects of combined and single agonist treatment with 8-OH-DPAT and
FGF2 on FGFR1 homodimerization in cells containing FGFR1-5-HT1A heterore-
ceptor complexes (
Fig. 8.2
). For this type of experiments where we intend to test
how the GPCR-RTK heteroreceptor complexes may induce modulation of the
ligand-dependent BRET
2
RTK homodimerization signal, cells are cotransfected at
a fixed ratio with a plasmid coding for the BRET donor (RTK-Rluc8) in the presence
of a single concentration of the plasmid encoding the BRET acceptor (RTK-GFP2)
