Biology Reference
In-Depth Information
molecules, ranging from 1.5 to 6 nm, and the fact that QDs are semiconductor nano-
crystals. Thus, it is not a genetically coded protein that can be synthesized by living
cells and must therefore be added.
Several of these variants of the BRET technology have or can be utilized to pro-
vide evidence for receptor-receptor interactions in living cells. In line with the huge
potential of BRET technology, bimolecular fluorescence complementation (
Cabello
et al., 2009
) and bimolecular luminescence complementation (see
Vidi, Ejendal,
Przybyla, & Watts, 2011
) have recently been developed and combined with BRET
to study more complex receptor-receptor interactions in higher-order receptor olig-
omers. Several studies continue to improve the potential use of other luciferases and
new coelenterazine derivatives have been developed with brighter (ViviRenTM) or
extended (EnduRenTM) light emission. Furthermore, the development of the BRET
3
opens the door to the characterization of receptor-receptor interaction
in vivo
.
8.2
SETTING UP A BRET ASSAY
Performing a BRET assay to investigate a potential GPCR-RTK interaction involves
several steps:
1. Selection and generation of the donor/acceptor and substrate combination
.
Generation of the two proteins of interest genetically fused with either donor
luciferase protein or acceptor fluorescent protein depending of the chosen BRET
approach (see
Table 8.1
) at either the N- or the C-terminus. For a membrane-
linked receptor, it is more intuitive to fuse the donor/acceptor protein to the
intracellular C-terminal tail of the receptor. A large proportion of GPCRs and
RTKs require the presence of a N-terminal signal peptide for correct cell surface
expression. This signal is susceptible to proteolytic cleavage and therefore
precludes the use of the N-terminal position for the fusion. To avoid to lose
the N-terminal moiety after cleavage of the peptide signal, the only alternative is
to insert the cDNA of the fluorescent protein downstream of the peptide
signal. While this has been done successfully with smaller tags (e.g., myc and
HA), it does not seem to be as practical with larger cDNAs.
2. Design and validation of the receptor of interest, including suitable controls
. For
a given BRET assay, the most adequate control protein has to be determined
since the inclusion of a negative/positive control is crucial to determine the
specificity of the studied interaction. As a positive control, one can generate a
double-fused chimeric protein where the donor and acceptor proteins have been
linked together. Also, when studying GPCR-RTK interactions, the use of the
corresponding homodimeric pair of receptors can be considered as positive
control(s). Thus, for both GPCRs and RTKs, it has been extensively documented
that they can exist as homodimers. In the case of RTKs, this process takes place
upon agonist-induced receptor activation. As negative controls, receptors
presenting a similar topology and subcellular localization as the receptor of
