Biology Reference
In-Depth Information
BRET
2
assay to incubate first the cells with the selected agonist at different times
ranging in the order of minutes (if it is possible) and then read all together instead
of performing a more quick kinetic analysis with continuous measurements in
view of the fast kinetic delay of substrate when coelenterazine 400a is used
(
Fig. 8.2
).
4.
Calculation and interpretation of the BRET
2
signal. In agonist-induced BRET
2
signaling, the BRET
2
ratio is defined as
ligand-promoted BRET
2
ratio
BRET
ratio (in presence of a ligand)—BRET ratio (in absence of a ligand or in presence
of PBS).
Plot the BRET data against the logarithm of ligand concentration
and analyze it by nonlinear curve fitting (sigmoidal dose-response) using
GraphPad PRISM, which will allow to obtain the concentration eliciting a
half-maximal response (EC50 value). When analyzing the kinetic BRET
2
data, the
BRET
2
signal can be plotted against time to produce kinetic profiles, examples of
which are shown in
Fig. 8.2
. Changes in the time-course profile and apparent
association (or dissociation) rate constants can be calculated from such data.
¼
8.4
DISCUSSION AND NOTE ON CRITICAL PARAMETERS
In spite of the increased understanding of the role of GPCR transactivation by RTK
ligands and vice versa and their existence as heteroreceptor complexes, only few ex-
amples have until now been validated using the BRET
2
methodology (
Borroto-
Escuela, Romero-Fernandez, Mudo, et al., 2012
), most likely due to the particularly
difficult nature of such receptor interactions. We believe that a well-controlled and
carefully analyzed BRET assay has a great potential to identified and/or study
GPCR-RTK heteroreceptor complexes (
Fig. 8.3
). It also seems to have a high value
in investigating the mechanism of action and the pharmacological properties of drugs
acting on these important therapeutic targets. The present work gives a step-by-step
description of the BRET
2
methodology using specific examples, confronting the pros
and cons of various protocols.
BRET
2
assays have been used to study GPCR heteromerization for about a decade
now, and as the field has matured, we have a better understanding of the underlying
technological limitations and an improved ability to interpret BRET data. The method
is particularly suited not only for themolecular characterization of GPCR-RTKhetero-
receptor complexes but also for the screening of new compounds that bind specifically
to these complexes, with the ultimate goal of identifying novel therapeutic drugs.
Progressive improvements and diversification of the BRET-based assay place
this technology among the most powerful methods for the study of GPCR-RTK
heteroreceptor complexes and their conformational changes in living cells. Also,
new perspectives of this methodology could include the monitoring of these interac-
tions at the subcellular level to unravel the molecular mechanisms underlying
GPCR-RTK heteroreceptor complex transactivation/transinhibition processes, in-
ternalization, and signaling.
