Biology Reference
In-Depth Information
heteromerization represents an important mechanism for modulating and integrating
the physiological functions of GPCRs and RTKs. This can take place at different
stages of the receptor's life: biosynthesis, ligand binding, G protein activation, de-
sensitization, internalization, and degradation. Also GPCR-RTK heteroreceptor
complexes allow flexibility in terms of programming of spatially controlled signal-
ing pathways and/or increasing signaling gain (
Fuxe et al., 2010
).
The existence of conformation-specific GPCRs present in the GPCR-RTK het-
eroreceptor complexes may offer unique opportunities to modulate pathophysiology
driven by growth factors. It is likely that long-term and global inhibition of RTK in
associated diseases will not be well tolerated due to the role of RTK signaling in nor-
mal physiology (
Andrae, Gallini, & Betsholtz, 2008; Takeuchi & Ito, 2011
). There-
fore, GPCR-RTK partnership may lead to novel therapeutic strategies based on
specific blockade of RTK signaling via GPCR transinhibition by antagonists and/
or inverse agonists of the corresponding GPCR (Pyne & Pyne, 2011). Such strategies
are less likely to produce side effects than approaches based on direct RTK inhibi-
tion. This could potentially extend the repertoire of biological actions of a large num-
ber of compounds and drugs that bind to GPCRs. Combinations of RTK inhibitors
and GPCR-specific ligands that reduce G protein or
b
-arrestin function may be an
effective way of abrogating signaling from these heteroreceptor complexes.
In order to progress in our understanding of the functional role of GPCR-RTK het-
eroreceptor complexes as well as their potential role as therapeutic targets, we need to
continuously look for methods and technologies that can be used to demonstrate and
evaluate such heteroreceptor complexes and their receptor-receptor interactions. The
bioluminescence resonance energy transfer (BRET) technology has emerged as a pow-
erful and straightforward biophysical technique for studying receptor heteromers and
heteroreceptor complexes and their receptor-receptor interactions. The present study
focuses on recent work illustrating the power of BRET
2
for the study of GPCR-RTK
interactions, using A2A-FGFR1 (
Flajolet et al., 2008
) and 5-HT1A-FGFR1 (
Borroto-
Escuela et al., 2013; Borroto-Escuela, Romero-Fernandez, Mudo, et al., 2012
)
heteroreceptor complexes as examples. We highlight the current ways in which the
BRET-based methodology is being used to establish the existence of GPCR-RTK het-
eroreceptor complexes and their specificity. Furthermore, we describe howBRETmay
be used to establish the involvement of a bidirectional cross-communication mecha-
nism between GPCRs and RTKs.
8.1
THE METHOD PRINCIPLE
BRET is a natural phenomenon found in some marine species (for instance, in the sea
pansy, Renilla reniformis), resulting from the nonradiative energy transfer between a
luminescent donor (Renilla luciferase—Rluc) and a fluorescent acceptor protein
(green fluorescent protein—GFP). Originally developed to study the interactions
of circadian clock proteins in bacteria (
Xu, Piston, & Johnson, 1999
), BRET has sub-
sequently been applied to study receptor-receptor interactions in living cells. It has
