Biology Reference
In-Depth Information
performed using appropriate assay conditions for each receptor. The number of
radioligand binding sites is then plotted against fluorescence or luminescence
values determined in the same sample; and a linear correlation is expected and
obtained. These standard curves can be used to transform fluorescence and
luminescence values into
f
mol
of receptor. Thus, the fluorescence/luminescence
ratios can be transformed into (receptor-GFP
2
)/(receptor-Rluc8) ratios, which
allows to determine accurate BRET
max
and BRET
50
values.
8.
BRET ratio values from Point 4 in
Section 3.1
can be plotted as a function of the
(GFP2-GFP0)/Rluc8 ratio (Point 6 in
Section 3.1
) or (receptor-GFP
2
)/(receptor-
RLuc) ratio as described in Point 7 in
Section 3.1
. Data are fitted using a
nonlinear regression equation assuming a single binding site (GraphPad Prism)
and BRET
max
and BRET
50
values can be determined (
Fig. 8.1
).
8.3.2
Protocol 2: competition assays
BRET
2
displacement experiments can also be performed to shed some light on the
specific nature of a given receptor-receptor interaction. In a BRET
2
competition or
displacement assay, the BRET
2
ratio is measured at a fixed ratio of donor and accep-
tor in the presence of increasing concentrations of a nontagged native partner. Over
the last few years, in order to test the ability of a GPCR to form heteromers and to
further investigate the specificity of BRET signals, competition experiments have
often been carried out. It has been demonstrated that the use of an untagged receptor
X or Y coexpressed with the receptor X-Rluc and receptor Y-GFP2 decreases the
BRET ratio signal as a consequence of the ability of the untagged receptor to interact
with one or both fusion proteins and compete for the complementary BRET fusion
protein. In a GPCR-RTK heteroreceptor complex, a true interacting partner (e.g., an
excess of one of them or a competitive interacting receptor) would be also capable of
reducing the BRET signal of the receptor complex, whereas a noninteracting partner
would not. However, in spite of this theoretically reasonable and until now well-
supported concept that reflects true receptor-receptor interactions (at least for sev-
eral GPCR-GPCR interactions), this does not necessarily represent an unequivocal
interpretation. It could be that, for instance, in the case of GPCR-RTK interaction
analysis, the results will not be in line with the expected idea that a true interacting
partner would be capable of reducing the BRET signal of the receptor complex.
GPCRs are seven helix transmembrane receptors and RTKs are single-helix
transmembrane receptors. The specificity and diversity of the interface interaction
between these two classes of receptor families may allow for a wider plasticity or
diversity of the receptor-receptor interaction. Instead of observing a reduction
of the BRET ratio upon untagged receptor coexpression, we can still observe a
similar or even increased BRET ratio. This behavior could be the result of a
reorganization/reconfiguration of the heteroreceptor complex, where more than
two receptors could be accommodated, sharing different receptor-receptor interface
interactions.
