Biology Reference
In-Depth Information
concentration for coelenterazine). BRET signals are measured immediately after
substrate addition using a BRET reader capable of measuring light emitted at
donor and acceptor wavelengths in a quasi-simultaneous manner. The use of the
BRET method to study GPCR-RTK interactions goes further than the simple
measurement of energy transfer between the donor and acceptor pair together
with the use of proper positive and negative controls. If a BRET signal is
observed, the proper cellular localization and function of the fusion proteins have
to be verified. If the functional validation step is successful, the BRET signal can
be studied further to evaluate its specificity. In fact, several assays have been
developed with the aim of providing evidence for the existence of specific
receptor-receptor interactions and to discriminate between genuine physical
interactions between the GPCR and the RTK versus a random collision due to
overexpression of the receptor pairs. Therefore, in addition to the classical
negative control used, BRET saturation, competition, and ligand-promoted
(kinetic and dose-response) assays have been developed. In addition, each of
these assays can shed light on the stoichiometry, conformation, and dynamic
changes that may take place in GPCR-RTK heteroreceptor complexes.
5. Analysis of the BRET signal
. The BRET signal or BRET ratio is defined as the
light signal of the acceptor emission relative to the light signal of the donor
emission. This proportion is corrected for the background signal due to the
overlap of donor emission at the acceptor wavelength, always determined in
parallel for cells expressing the donor alone. Then, the BRET net value is
calculated by subtracting this BRET background ratio from the BRET ratio
obtained in cells coexpressing the two partners. The amount of donor can be
estimated from the maximal luciferase values measured separately after the
BRET reading. The amount of acceptor has to be determined in an independent
reading by recording fluorescence values of the acceptor pair in a fluorometer
(using a black microplate to avoid light scattering). The calculated acceptor/
donor ratio can be used to compare different experiments. To obtain acceptor/
donor ratios that correspond to real protein quantities, luciferase and fluorescence
values have to be converted into protein amounts using independently established
standard correlation curves with real protein quantities (e.g., determined in
radioligand binding experiments for each receptor fusion protein).
8.3
METHODS AND DETAILED PROTOCOLS
In recent years, a number of excellent papers have well documented and provided
detailed discussions on different forms and approximations to the BRET assay
methodology and its straightforward use to study receptor-receptor interactions
mainly focused on GPCR-GPCR interactions (
Ayoub & Pfleger, 2010; Hamdan,
Percherancier, Breton, &Bouvier, 2006
; Marullo &Bouvier, 2007). However, in this
field, works describing in detail the potential use of BRET methodology to study
GPCR-RTK heteroreceptor complexes and their dynamics are still missing.
