Changes in intracellular cAMP reported by a Redistribution® assay using a cAMP-dependent protein kinase-green fluorescent protein chimera

We report on a novel method to monitor changes in intracellular cAMP concentration ([cAMP](i)) within intact living cells using a chimeric fusion of the catalytic subunit of cAMP-dependent protein kinase to green fluorescent protein (PKAcat-GFP). In stably transfected unstimulated fibroblasts, fusion protein fluorescence is highly concentrated in aggregates throughout the cytoplasm and absent in the nucleus. Elevation of [cAMP](i) disperses GFP fluorescence from the cytoplasmic aggregates within minutes. Spot-photobleach measurements show that the rate of exchange of GFP-labeled catalytic subunits at these aggregates increases in proportion to [cAMP](i). For any given stimulus, the response curve for dispersal of GFP fluorescence from aggregates agrees closely with the increase in total [cAMP](i) as measured by standard in vitro methods (SPA). The redistribution of fluorescence is completely reversible: reduction of [cAMP](i) results in return of fluorescence to the cytoplasmic aggregates. Consistent behaviour of PKAcat-GFP is seen in different cell backgrounds. We demonstrate that PKA Redistribution((R)) assays are suitable for measurement of changes in [cAMP](i) brought about by both G(s)- and G(i)-protein-coupled receptor stimulation as well as by inhibition of cAMP phosphodiesterases. (C) 2004 Elsevier Inc. All rights reserved.