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 288

cAMP and donor-labeled anti-cAMP antibody, with the difference that, in
the HTRF Ò assay, the donor is a cryptate of lanthanide, whereas in the
LANCE Ò assay, it is a lanthanide chelate. The first one has, for example,
been validated both for Gs-coupled receptors such as the b 2-adrenergic re-
ceptor or the histamine H2 receptor and for Gi/o-coupled receptors such as
the histamine H3 receptor. 28,29 Both assays have been reported as more
sensitive technologies compared to reporter gene techniques, for example,
with concentrations below 10 fmol cAMP/well being quoted. 30 Moreover,
a study by Jean and colleagues recently showed that the sensitivity of these
methodologies could enable the detection of cAMP in the native cerebral
tissues of rats. 31 Knowing how strongly cell phenotypes can influence
many aspects of GPCR pharmacology, studying unmodified receptors in
their native environment would be the optimal assay to screen for, and
methodologies that enable this have to be developed. Another approach,
developed by Martikkala and colleagues and also based on a competition
assay, uses the quenching resonance energy transfer technique (QRET)
instead of TR-FRET to measure changes in cAMP levels in HEK293 cells
overexpressing either b 2-adrenergic or d -opioid receptors (DOP). 24 In this
assay, soluble quenchers reduce the signal of unbound europium-labeled
cAMP in solution, whereas the antibody-bound fraction is fluorescent. It is
an HTS-compatible method and has the advantage of using only a single-
label probe.
It has to be emphasized that screening Gs-coupled receptors is generally
straightforward, whereas screening Gi/o-coupled receptors in cAMP assays
can be considerably more difficult. To maximize the inhibition signal
(reflecting the inhibition of cAMP production by adenylyl cyclase), it is of-
ten necessary to stimulate adenylyl cyclase with forskolin (a direct activator
of adenylyl cyclase) or a Gs-coupled GPCR expressed in the same cells, and
this has to be carefully titrated during optimization of the assay so as not to
miss out on nonpotent and partial agonists, for example.
3.2.4 Screening for Gq/11 signaling pathway
Gq/11 family of G proteins modulates phospholipase C (PLC), which pro-
motes the production of inositol 1,4,5-trisphosphate (IP3) that interacts with
the inositol-3-phosphate or ryanodine receptors (ligand-gated Ca 2 þ chan-
nels) located in the endoplasmic and/or sarcoplasmic reticulum, resulting in
the transient release of calcium ions into the cytosol. One of the most widely
used methods to screen for GPCR ligand is to detect this calcium ion release,
usually with fluorescent probes changing their emission properties when