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5. Calculating BRET ratio . Data are then represented as a normalized BRET 2 ratio,
which is defined as the BRET ratio for coexpressed Rluc and GFP2 constructs
normalized against the BRET ratio found for the Rluc expression construct alone
in the same experiment:
=
BRET 2 ratio
GFP 2 emission at 515
¼
30 nm
ð
Rluc emission 410
80 nm
Þ
cf
The correction factor, cf, corresponds to (emission at 515
30 nm)/(emission at
410
80 nm) found with the receptor- R luc construct expressed alone in the same
experiment.
BRET-based studies of receptor-receptor interactions are particularly prone to
false-positive signals and require multiple controls. BRET 2 saturation and competition
assays have been developed to extend the information obtained from basic BRET 2 ex-
periments toward a more quantitative and detailed analysis of the BRET 2 signal.
8.3.1 Protocol 1: saturation assay
In titration or saturation BRET 2 experiments, cells are transfected with a constant
amount of BRET 2 -donor in the presence or absence of increasing amounts of the ac-
ceptor. Theoretically, for any specific interaction between the receptor-donor and
receptor-acceptor fusions, the BRET 2 ratio increases hyperbolically as a function
of increasing GFP/Rluc value, to reach an asymptote (saturation) when all donor
molecules are associated with acceptors (BRET max , Fig. 8.1 ). By contrast, in the case
of nonspecific interactions (bystander BRET), a quasi-linear plot is expected or
eventually reaches a plateau for higher values of receptor density. Nevertheless,
the saturation curve should be independent of the total expression level of receptors
and the BRET 2 configuration used. However, BRET max values cannot be used as a
quantitative measure of the relative number of homo/heteromers formed for each
combination because they not only are a function of the dimer numbers but also de-
pend on the distance between the energy transfer partners and their relative orienta-
tion within the receptor complex. BRET 2 saturation curves have been particularly
used with the aim to establish the oligomeric order of receptor complexes, as well
as the proportion of receptors engaged in dimers or oligomers. They are also used
to determine whether ligand-induced BRET 2 signals depend on conformational
changes or association/dissociation of interacting receptors. Also, saturation assay
has been used to compare the relative affinity of receptors for each other and their
probability to form a complex, the so-called BRET 50 , which represents the acceptor/
donor ratio giving 50% of the maximal signal ( Fig. 8.1 ). The BRET 50 value is often
compared between the two different homomer subtypes and their corresponding het-
eromer. Many GPCR-GPCR heteromers show no difference in the relative affinity
between their receptor homomers and their specific heteromers. Furthermore, neither
BRET max nor BRET 50 values may be modified following the agonist activation of
the heteromer, consistent with the general consensus that GPCR homo- and hetero-
merization is often constitutive. In contrast, GPCR-RTK heteroreceptor complexes
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