Background

Live-cell fluorescence resonance energy transfer (FRET) assays in combination with microscopy and digital imaging technologies are commonly used to monitor the kinetics with which levels of cAMP and Ca²⁺ fluctuate in response to GPCR agonist stimulation. Here, we report an alternative approach that does not use microscopy but that instead enables FRET-based detection of cAMP and Ca²⁺ in a 96-well format using an automated microspectrofluorimeter and monolayers or suspensions of living cells. The protocol uses genetically encoded biosensors so that averaged kinetic data for pharmacological studies can be obtained in real time using a microplate reader under conditions in which GPCR agonists are applied at known concentrations. These FRET assays use cell lines stably expressing the cAMP biosensor H188 (Klarenbeek et al., 2015) or the Ca²⁺ biosensor YC3.60 (Nagai et al., 2004), both of which exhibit superior dynamic range in comparison to earlier FRET reporters. Our protocol describes how H188 or YC3.60 can be expressed stably with recombinant GPCRs so that precise dose-response analysis is achievable. This protocol builds on our published studies, whereby novel dual agonist and triagonist properties of synthetic GPCR agonist peptides (e.g., EP45 and GGP817) were discovered (Chepurny et al., 2018 and 2019).

Using the genetically encoded biosensor Epac1-camps (Nikolaev et al., 2004), we were the first to develop a 96-well microplate FRET assay in which fluctuations of [cAMP] were monitored in monolayers of living cells in real time using a microspectrofluorimeter (FlexStation 3, Molecular Devices) (Chepurny et al., 2009). More recently, we have optimized this approach for purposes of drug discovery in a high-throughput mode. The major technical advance concerns our use of H188 and YC3.60, both of which we find to be highly responsive to cAMP and Ca²⁺, respectively. Importantly, our plate reader FRET assays using H188 or YC3.60 require their high-level expression, either by stable transfection to generate clonal cell lines (Chepurny et al., 2018), or by adenoviral transduction of cell lines (Chepurny et al., 2018 and 2019). Transient transfection is not useful since low-level expression of these biosensors is not compatible with the limited light detection limits of available microplate readers.

For the H188 reporter expressed in our clonal C24 HEK293 cell line, the cAMP-elevating agent forskolin induces a maximal 100% increase of the 485/535 ∆FRET ratio, a response that greatly exceeds the 12% value typically observed using Epac1-camps (Chepurny et al., 2009 and 2018). This C24 clone exhibits high sensitivity (EC₅₀ 500 nM) and excellent Z-score (0.77) when testing forskolin (Chepurny et al., 2018). Calibration of the FRET signal is obtained in assays that use digitonin-permeabilized C24 cells expressing H188 and that are bathed in known concentrations of cAMP (Chepurny et al., 2018). When administering forskolin by its bolus injection into individual wells of a microplate containing monolayers of C24 cells, the time course of adenylyl cyclase activation and resultant cAMP production is monitored with high precision within the first 30 s.

We have also created doubly-transfected HEK293 cells lines that stably express H188 or YC3.60 along with recombinant GPCRs of interest. These GPCRs include glucagon, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), peptide YY (PYY), corticotropin releasing hormone (CRH), and cholecystokinin (CCK) receptors. It is also possible to perform such FRET assays using HEK293 cells that stably express H188 or YC3.60, but are instead transiently transfected with a GPCR. Conversely it is possible to use a cell line that stably expresses a recombinant GPCR so that cells may then be virally transduced with H188 or YC3.60.

Our real-time kinetic assay using H188 and living cells differs from single-endpoint HTR-FRET assays that monitor levels of cAMP in cell lysates. For example, the LANCE^® cAMP assay (Perkin Elmer) uses a Europium-labeled cAMP tracer (FRET donor) in combination with a fluorophore-tagged anti-cAMP antibody (FRET acceptor), both of which are added to a reaction mixture so that the competition of fluorescent cAMP and non-fluorescent cAMP (i.e., endogenous cAMP) for the anti-cAMP binding site on the antibody can be measured. In contrast, H188 is unimolecular in which the cAMP-binding protein Epac1 is flanked by the FRET donor mTurquoise2∆ and the FRET acceptor cp173 Venus-Venus so that a decrease of FRET occurs upon binding cAMP (Klarenbeek et al., 2015). YC3.60 instead uses a Ca²⁺-binding domain of calmodulin (CaM), and the CaM-binding peptide M13, in combination with the FRET donor ECFP∆11, and the FRET acceptor cp173Venus so that an increase of FRET occurs upon binding Ca²⁺ (Nagai et al., 2004). Unlike HTR-FRET assays, H188 and YC3.60 allow live-cell real-time determinations of FRET in the kinetic mode.

Summarized below is a detailed FRET assay protocol that provides additional technical information not previously contained in the methods sections of our prior publications (Chepurny et al., 2009, 2018 and 2019). We also present new information concerning previously unpublished methodology that allows GPCR agonist action to be studied in cells that stably express both a recombinant GPCR and a select FRET biosensor.