Biology Reference
In-Depth Information
Consequently, we have used here the A2A-FGFR1 and 5-HT1A-FGFR1 heterore-
ceptor complexes as examples to illustrate the potential use of different BRET
2
assay
formats to monitor GPCR-RTK interactions in living cells.
We chose the BRET
2
variant assay with Rluc8 as a donor and GFP2 as an accep-
tor with improved spectral separation of the donor and acceptor emission peaks. This
implies less bleed through at the acceptor emission maximum and lower background.
Also, the rationale for the use of mutated Rluc (Rluc8, GenBank: EF446136) instead
of the nonmutated Rluc is mainly based on the fact that Rluc8 gives a higher quantum
yield compared to the nonmutated Rluc. Therefore, we do not need to overexpress
the luciferase fusion receptor that is detrimental for interaction specificity.
Performing a BRET assay to investigate a potential GPCR-RTK interaction and,
in more general terms, any receptor-receptor interaction involves several steps:
1. Generation and validation of BRET fusion constructs
. The two receptors of
interest must be genetically fused to either
Renilla
luciferase (Rluc) or GFP
variants at either the N- or C-terminus. The choice of the fusion protein (N- vs.
C-terminus) depends on the nature of the studied proteins. For RTK and GPCR, it
is, for instance, more advisable to fuse the donor and acceptor protein to the
C-terminal tail (see Point 1 in
Section 2
). It is important to consider preexisting
information concerning specific domains of the receptor of interest, like, for
example, the tyrosine kinase domains in RTK and their potential binding sites to
regulatory/adaptor proteins. Also, posttranslational modification sites, like
palmitoylation at the C-terminal of some class A GPCR, can induce a restricted
conformation of the C-terminal tail of the receptor, and longer polylinker spacers
should be used in order to unmask the donor/acceptor protein and give it a proper
orientation. Finally, it is important to ensure that the insertion of the donor/
acceptor protein does not interfere with the proper folding of the receptor and
insurance of a correct functionality and localization deserves a particular
attention. Listed in the succeeding text are the five categories of constructs
generated for this study: (1) BRET acceptor constructs: 5-HT1A-GFP2 and A2A-
GFP2 for expression of both the 5-HT1A and A2A receptors, C-terminally tagged
with GFP2 (PerkinElmer, Sweden). (2) BRET donor constructs: FGFR1-Rluc8
and 5-HT1A-Rluc8 for expression of the FGFR1 and 5-HT1A, C-terminally
tagged with Rluc8. (3) Positive control: BRET fusion construct for expression of
the Rluc8-GFP2 fusion protein. (4) Negative control BRET constructs:
pcDNA3.1-Rluc8 and pGFP2 for expression of Rluc8 and GFP2 alone,
respectively. (5) Specificity controls: 5-HT2A-GFP2 and D2mutantR-Rluc8 for
expression of other GPCRs were C-terminally tagged with either Rluc8 or GFP2.
Each fusion construct was tested for detectable luminescence (using a
luminometer following addition of coelenterazine 400a or DeepBlueC
TM
substrate) or fluorescence (using a fluorometer following direct laser excitation).
The fusion receptor was also validated with respect to their function by means of
radioligand binding assay and/or gene reporter assays to ensure that the addition
of the donor or acceptor molecule has not altered ligand affinity/efficacy/
