Time-Resolved Förster Resonance Energy Transfer-Based Technologies to Investigate G Protein-Coupled Receptor Machinery: High-Throughput Screening Assays and Future Development - Progress in Molecular Biology and Translational Science - page 299

(TTR) tetramers have been developed by Alhamadsheh and colleagues. 68
TTR is a tetrameric protein that transports thyroxine and retinol in the
blood, but whose dissociation leads to amyloid cardiomyopathy. Using a
fluorescence polarization-based high-throughput screen to detect small
molecules that bind to the thyroxine-binding pocket of TTR under
physiological conditions, the authors have identified molecules that stabilize
the dimer-dimer interface and increase the activation energy for tetramer
dissociation, thus rescuing cardiomyocytes from TTR proteotoxicity
without causing cytotoxicity. TR-FRET-based technologies are under
development to investigate these aspects of GPCR oligomerization.
4.2.2.1 TR-FRET detection of oligomers with tag insertion
Besides classical biochemical methods to identify oligomers or larger com-
plexes, such as coimmunoprecipitation or functional complementation,
which are hardly compatible with HTS and can induce biases in the results
by promoting association, resonance energy transfer approaches offer many
advantages to screen for GPCR-specific interactions. The Tag-lite Ò GPCR
dimer assay from Cisbio combines many of these advantages, especially the
possibilities to work on protein interactions occurring specifically at the cell
surface, on living cells, and in a 96- or 384-well plate format. In this assay,
studied GPCR subunits are labeled with cell-impermeable fluorophores
compatible with TR-FRET, typically terbium cryptate as a donor on
one subunit and a green or a red fluorescent molecule as an acceptor on
the potential partner subunit ( Fig. 7.3 ). Because energy transfer occurs only
when the two labeled subunits are at a distance of less than 100 ˚ , which is
about twice the diameter of a GPCR transmembrane domain, only two re-
ceptors in direct physical interaction are compatible with FRET emission,
and especially with a high signal-to-noise ratio when measured in a time-
resolved manner. This assay has been validated for both class-A and class-
C GPCRs. For example, Maurel and colleagues used GPCR tagged at their
N-terminus with a SNAP-tag, and showed that, whereas mGluRs assemble
into strict dimers, the GABA B receptors spontaneously form dimers of
heterodimers. 26 Alvarez-Curto and colleagues also studied cell-surface
M3 muscarinic acetylcholine receptors fused with a SNAP-tag, and together
with other techniques, TR-FRET allowed them to show that this receptor
exist as dimeric or oligomeric complexes at the surface of cells and that this
organization is regulated by ligand binding. 69
This technology relies, of course, on the possibility to specifically label
the GPCR target with fluorophores. As previously mentioned, several