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the extracellular side and the carboxy terminus in the cytosol. The length and
the structure as well as the sequence of amino and carboxy termini largely dif-
fer between receptors.
4
Binding of the agonist at the level of the transmem-
brane domain and/or of the amino terminus promotes conformational
changes that allow the activation of a heterotrimeric G protein, that is, the
exchange from guanosine diphosphate (GDP) to guanosine-5'-triphosphate
(GTP), which will in turn modulate several intracellular signaling pathways.
Several subtypes of heterotrimeric G proteins exist, and each promotes different
signaling events. While primarily thought to activate one specific subtype of G
proteins and downstream signaling cascades, most GPCRs are now described as
complex signaling proteins. Indeed, most of the receptors were reported to ac-
tivate more than one type of G proteins but also to be capable of modulating
signaling pathways independently of the G proteins.
7,8
Moreover, in addition
to the classical agonists and antagonists, synthetic molecules acting as allosteric
modulators of GPCRs have been identified.
9
Furthermore, the formation of
receptor oligomers, that is, the physical interaction between several GPCRs
identical or not, and the participation of GPCRs in signaling complexes,
involving intracellular proteins and other types of membrane proteins, were
proposed to alter the different properties of a given receptor.
10
These concepts
have had a real impact on the understanding of GPCR pharmacology and on
drug discovery aiming at identifying new compounds acting on these receptors.
In the prospect of drug discovery, the complete characterization of a lead
compound requires the analysis of its pharmacological properties as well as its
effect on the target and on the signaling. In addition, off-target effects have
to be assessed to exclude molecules that could have drastic adverse effects. In
the case of GPCRs, a full characterization of molecules requires two types of
HTS-compatible assays: (i) functional assays to determine the effect of com-
pounds in terms of signaling and (ii) mechanistic assays to detect the direct
effects of compounds on their target, for example, binding properties or
oligomerization. TR-FRET was shown to be a suitable method to explore
both aspects in an HTS-compatible format.
2. OVERVIEW OF THE TR-FRET PRINCIPLE AND ITS
ADVANTAGES
Fluorescent techniques represent nowadays the majority of detection
technologies used in HTS, and, among them, FRET, particularly TR-
FRET, has many advantages to offer and thus plays an important role in
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