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3.2. G protein activation and secondary messengers
Upon ligand binding, the receptor undergoes conformational changes lead-
ing to a stabilized, active form, responsible for initiating the signaling cas-
cade, which occurs mainly by the activation of the heterotrimeric G protein.
3.2.1 Protein activation and exchange of GDP for GTP
When the G protein cycle is initiated, it results in the exchange of GDP with
GTP in the G
a
subunit, but unfortunately, no TR-FRET-based assay to
measure GTP binding is yet available. However, a method was developed
by Perkin Elmer based on the time-resolved measurement of the fluorescence
of a europium chelate (no acceptor fluorophore is present in this case).
15
Very
similar to what is classically done with radiolabeled [
35
S]GTP
g
S, upon agonist
binding to the GPCR expressed in the membranes, the G proteins are acti-
vated and incorporate the nonhydrolyzable europium-labeled GTP analog.
After washing or filtration steps to remove unbound labeled GTP, the fluo-
rescence is measured. Because it is measured in a time-resolved manner, the
signal is higher than with classical fluorophores, avoiding background fluores-
cence from the membranes, for example, and it represents a powerful alter-
native to radioisotopic assays. However, this assay is not widely used in
HTS yet.
3.2.2 Heterotrimeric G protein subfamilies
Following G protein activation and dissociation (or rearrangement), the
G
a
-GTP on one side and the G
bg
subunits on the other modulate several ef-
fectors. Gproteins have been classified into four subfamilies, namely, Gs, Gi/o,
Gq/11, and G12/13, based on the structural similarity of their
a
subunits and
on the type of modulatory response they induce.
16
Each GPCR preferentially
couples to one subfamily of G protein, thus stimulating preferentially one sig-
naling cascade. TR-FRET-based assays are being developed to investigate
each of G protein signaling pathway, offering a catalog of assays for screening
activation of each GPCR.
3.2.3 Screening for Gs and Gi/o signaling pathways
Both Gs and Gi/o families modulate adenylate cyclase activity, leading, re-
spectively, to an increase and a decrease in the intracellular concentration of
3
0
,5
0
-cyclic adenosine monophosphate (cAMP). Two main TR-FRET-
based assays are currently available to detect cAMP level variations in cells,
both based on a competition format: the HTRF-based cAMP detection kit
from Cisbio Bioassays
17
and the LANCE
Ò
cAMP detection kit from Perkin
Elmer
18
(see
Table 7.1
). Both assays rely on the use of acceptor-labeled
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