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 289

bound to Ca 2 þ (FLIPR calcium assay fromMolecular Device is probably the
most widely used). 32 Cisbio Bioassays recently developed an IP-One HTRF
competitive assay based on the detection of the intracellular accumulation of a
metabolite of IP3, D -myo-inositol monophosphate (IP1), thanks to lithium
chloride (LiCl), a known inhibitor of inositol monophosphatase (IMPase)
which degrades IP1 into inositol. 21 The IP-One HTRF assay takes advantage
of measuring the final step in the IP cascade, allowing it to accumulate in the
cell and to be measured without requiring a kinetic readout. An advantage of
this assay for HTS over others is that it can be used with endogenously or het-
erologously expressed GPCRs, in either adherent or suspension cells, in the
1536-well plate format, to quantify the activity of agonists, antagonists, and
inverse agonists. Of note, this last category of ligands cannot be seen via a cal-
cium assay, for example, as increases in intracellular basal Ca 2 þ concentration
are not observed in cells expressing constitutively active Gq-coupled
receptors.
3.2.5 Screening for G12/13 signaling pathway
The G12/13 family of G proteins activates Rho, a family of small GTPase,
causing the downstream activation of the c-Jun N-terminal kinase (JNK)
pathway. To our knowledge, no generic TR-FRET-based assay is actually
available to screen for metabolites of this G12/13 pathway in an HTS format.
3.3. Later signaling events
3.3.1 Screening for bg subunits activated pathways
Upon agonist GPCR activation, in parallel to the G protein a subunit-
activated cascades, the bg subunits of the G protein have been demonstrated
to also modulate several effectors such as phospholipase or ion channels, but,
again to our knowledge, no HTS-compatible TR-FRET-based assay is
available to detect such events. In the case of a specific pathway, some me-
tabolites can be titrated with an HTS-compatible TR-FRET assay, such as
insulin, cortisol, or cyclic guanosine monophosphate (cGMP), 12 , but this has
to be implemented for a specific characterized GPCR target. Another pos-
sibility is to measure the GPCR-induced phosphorylation of some cellular
substrates that occurs during the activation of a particular GPCR. These
phosphorylation events could be analyzed in a sandwich format assay using
a specific labeled antibody directed against the phosphorylated protein.
However, such an assay is target specific and has to be developed for each
GPCR. In this respect, for example, a Ras activation assay using time-
resolved fluorescence was recently developed. 25 Ras is the prototypical