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Another labeling strategy to detect GPCR oligomers is to use fluores-
cent ligands, developed, for example, for the binding assays described pre-
viously. They can also help identify dimers or heteromers directly on
native tissues, as no modification of the receptor is required. Albizu and
colleagues used agonists and antagonists to the vasopressin and oxytocin
receptors coupled to fluorophores compatible with TR-FRET and
succeeded in demonstrating the presence of oxytocin receptor dimers or
oligomers in native mammary glands.
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4.2.2.3 New perspectives for oligomerization TR-FRET assays
TR-FRET tools can also be used to study oligomer association/dissociation
processes during internalization and recycling, for example, or other mecha-
nism occurring inside the cells. For example, Rajapakse and colleagues were
able, with TR-FRET microscopy, to study inside the cells protein-protein
interactions that were hardly detectable with traditional microscopy imaging,
with improved speed and sensitivity.
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Unfortunately, contrary to all the
above-mentioned technologies, this one is not yet transposable to HTS.
Of interest, screening for heterodimerization between distinct GPCRs
appears to be promising to understand the role of orphan receptors. Note
that roughly a third of the about 400 nonodorant GPCRs are still orphans.
For example, the orphan receptor GPR50 was shown to heterodimerize
constitutively and specifically with MT1 and MT2 melatonin receptors.
While the association with MT2 had no effect on its function, GPR50
abolished agonist binding and G protein coupling to the MT1 protomer en-
gaged in the heterodimer.
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With the increasing number of GPCRs shown to form heteromers in
heterologous systems, the need for methods to attest their existence in native
tissues has never been greater. Though microscopy techniques have been
used to that aim, it appears now that TR-FRET methods could be an alter-
native with the advantage to potentially permit in the future the use of an
HTS-compatible plate reader on native systems to study GPCR complexes
(
Fig. 7.3
).
4.2.3 TR-FRET screening for specific pharmacological properties
of oligomers
Once a specific heteromer or oligomer has been identified, all the assays
presented previously and many more can be used to determine and charac-
terize its potential, specific pharmacological properties. From a drug devel-
opment point of view, the finding of tissues or pathologies in which
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