Biology Reference
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Upon addition of a competitive compound, the fluorescent ligand is
displaced, and hence the FRET signal decreases. Accordingly, the ligand-
binding HTRF
Ò
assay requires the labeling of the target GPCR specifically
at the cell surface.
4.1.2 TR-FRET binding assays using GPCRs targeting antibodies
GPCR labeling with HTRF
Ò
-compatible fluorophores can be achieved
using several strategies. The first strategy is to use a fluorescent antibody that
recognizes the native receptor or a tag sequence inserted at the N-terminus
of the receptor. Antibodies have the advantage of being easily labeled with
any fluorophore. Such antibodies were successfully used to perform HTRF
binding assays in cell lines expressing an HA-tagged vasopressin receptor
V1a
44
or an HA-tagged complement 5A receptor C5AR,
45
for example.
In both studies, anti-HA antibodies were labeled with an HTRF-
compatible acceptor fluorophore, Alexa647 or AlexaFluor488, and applied
on cells expressing the receptor of interest. The fluorescent ligands, either a
europium cryptate derivative of a V1a antagonist or a terbium chelate-
labeled C5R purified protein, and the compounds to be tested were then
applied. Once the binding equilibrium is reached, the HTRF signal is di-
rectly recorded without any washing steps. The data reported support the
robustness of the assay (compatible with 384-well plates) and its accuracy
is comparable to that of radioactive assays.
44,45
Of interest, if antibodies
directed against the native protein are available, the HTRF binding assay
could be performed directly from native tissues, which would provide
information regarding the pharmacological properties of the screened
compounds in the native environment of the target (
Fig. 7.3
).
4.1.3 TR-FRET binding assays using innovative strategies to label GPCRs
A second strategy to label the target GPCR is to use self-labeling tags and,
more efficiently, self-labeling proteins (for review, see Ref.
46
). In short,
these self-labeling techniques consist in fusing a suicide enzyme that will re-
act with a fluorescent substrate leading to the formation of a covalent bond
between the fluorescent moiety and the tag. These suicide enzymes include
the SNAP-tag
Ò
,
47
the CLIP-tag
Ò
,
48
and the Halotag
Ò
technologies.
49
The
ACP-tag technology is an additional self-labeling technique where the co-
valent transfer of fluorescent substituents from derivatized coenzyme A to
the ACP-tagged fusion proteins is catalyzed by AcpSynthase.
50
While all
these techniques could be used in theory to develop HTS-compatible
HTRF binding assay, only assays based on the SNAP-tag
Ò
, CLIP-tag
Ò
,
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