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methods are now available, which include antibodies directed against the
target itself or against a tag fused at the N-terminus of the GPCR target,
self-labeling tags or proteins, or tags labeled by an added enzyme (for review,
see Ref.
46
). The use of orthogonal labeling methods, that is, the combina-
tion of two different tags such as SNAP- and CLIP-tags, or SNAP- and
HALO-tags, is an interesting approach to specifically look at GPCR quater-
nary structures formed by the association of two different subunits, each
carrying a specific tag labeled with one fluorophore (
Fig. 7.3
). For example,
the SNAP- and CLIP-tags strategy was used to demonstrate that members of
the mGluR family can form heteromers and that this association is limited to
strict heterodimers, not dimers or homodimers.
27
4.2.2.2 TR-FRET detection of oligomers of unmodified receptors
Alternatively, the receptor labeling with an antibody directed against the
native protein is also a promising strategy. The main advantage is that no
modification of the target GPCR is necessary, thus avoiding the impact
of molecular modification of the receptor on its functionality and allowing
association studies on primary cultures or native tissues (
Fig. 7.3
). For ex-
ample, with the use of antibodies coupled to TR-FRET-compatible
fluorophores, BILF1, an orphan GPCR involved in the infection of B
lymphocytes by the Epstein-Barr virus, was shown to heterodimerize with
various chemokine receptors, which may lead to an altered response of B
lymphocytes to chemokines after infection.
70
However, this labeling strat-
egy has also some drawbacks. Indeed, the use of bivalent antibodies can
favor and constrain dimers or larger oligomers of GPCR, modifying the
natural tendency of two GPCRs to associate. As an alternative method
to circumvent this problem, the use of monovalent antibody fragments,
typically just a Fab fragment, targeting GPCRs with similarly high affinity
and specificity as classic full-length antibodies, would allow avoiding this
bivalence-induced bias. Monoclonal Fab fragments recognizing several
proteins are already commercially available and are coupled to
fluorophores or enzyme such as horseradish peroxidase for use in ELISA
or Western blot (see, e.g., the Web site of M-fold, a company that
develops Fab on demand). As an example of a Fab targeting a GPCR, a
monomeric Fab fragment was developed against the
b
2-adrenoceptor
and exhibits peculiar pharmacological effects: it showed antagonist-like
properties, whereas the same bivalent antibody exhibited agonist-like
properties.
71
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