Biology Reference
In-Depth Information
In the next sections, generic and target-specific TR-FRET methods to
analyze GPCR signaling machinery will be presented, along with what we
consider as good recent examples to illustrate the possibilities and specific-
ities of TR-FRET-based technologies.
3. SCREENING WITH GENERIC METHODS
GPCR signaling consists of a series of spatial and temporal events,
including, of course, the cascade known as the G protein cycle and down-
stream events but also other pathways that are G protein independent. Most
of these signaling cascades are common to many GPCRs, which makes it
worth developing generic methods to measure activation or production
of one of the metabolites in these cascades to screen for GPCR ligands in
HTS. Similar to all screening methods for GPCRs, TR-FRET-based ge-
neric methods must be simple, homogeneous with minimal reagent addi-
tions, nonradioactive, robust, and adaptable to 384- or 1536-well plate
format with robotic automation. But the question is whether to look for
proximal or distal events. Measuring proximal events can reduce false pos-
itives, but most techniques are not sensitive enough to avoid moving down
the signal transduction cascade to amplify the response. In this part, we will
briefly present the TR-FRET-based generic techniques currently available,
and which could offer a really good compromise between sensitivity, detec-
tion of an early event in the GPCR activation cascade, pharmacological in-
formation about the type of ligands or the signaling pathway involved, and in
some cases, more biologically relevant assays.
3.1. Sandwich or competitive assay format
Almost all functional generic assays based on TR-FRET and used for GPCR
HTS are based on two formats: a sandwich format or a competition format,
illustrated in
Fig. 7.2
(see also Ref.
12
).
In a sandwich assay, the TR-FRET signal comes from the proximity of
two antibodies, one labeled with a lanthanide cryptate or chelate as donor of
energy and the second one labeled with a compatible acceptor. The two an-
tibodies recognize the same target but at different regions, one recognizing a
specific modification of the target such as phosphorylated site, for example,
and the second recognizing a part of the protein that remains unmodified.
Thus, the larger the amount of modified target produced, the more the
antibodies bound on the two sites of the target, and thus the more the
TR-FRET signal measured by the proximity of the two fluorophores.
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