FRET Analysis of cAMP Signaling

Forster resonance energy transfer (FRET) analysis of cAMP production was performed in HEK293 cells transiently transfected with plasmids encoding PTH1R-WT or mutant, and the Epac1^CFP/YFP FRET-based biosensor of intracellular cAMP^{68 (link)}. Cells plated on poly-D-lysine–coated glass coverslips were mounted in Attofluor cell chambers (Life Technologies); maintained in Hepes buffer containing 150 mM NaCl, 20 mM Hepes, 2.5 mM KCl, and 1 mM CaCl2, as well as 0.1% bovine serum albumin (BSA) (pH 7.4); and transferred on an in-verted Nikon Ti-E equipped with an oil immersion 40× numerical aperture (NA) 1.30 Plan Apo objective and a moving stage (Nikon Corporation). Recordings were performed at room temperature in single cells upon perfusion of agonist ligand or buffer for ~2 min (indicated by horizontal bar in graph) and during a subsequent washout period. The CFP and YFP fluorescent groups were excited with 440- and 514-nm lasers (Melles Griot), respectively. Fluorescence data were extracted using Nikon Element Software (Nikon Corpo- ration), and calculated as corrected CFP/YFP fluorescent ratios^{68 (link)}. Data were normalized to the FRET response induced by forskolin (10 μM) added at the end of test period.