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

• Published in: Cellular signalling Vol. 16; no. 8; pp. 907 - 920
• Main Authors: Almholt, Kasper, Tullin, Søren, Skyggebjerg, Ole, Scudder, Kurt, Thastrup, Ole, Terry, Robert
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.08.2004
• Subjects: 3',5'-Cyclic-AMP Phosphodiesterases - antagonists & inhibitors; 3',5'-Cyclic-AMP Phosphodiesterases - metabolism; Animals; C-subunit; cAK; cAMP; cAPK; Cell Nucleus - metabolism; Cells, Cultured; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP - metabolism; Cyclic AMP-Dependent Protein Kinases - metabolism; Cytoplasm - metabolism; Enzyme Activation; GFP; Green Fluorescent Proteins - metabolism; HeLa Cells; Humans; Microscopy, Fluorescence; PKA; Recombinant Fusion Proteins - metabolism; Redistribution; IBMX; PKA; AKAP; ARα2a; dbcAMP; GR; cAMP; cAK; GFP; Redistribution; cAPK; C-subunit; FRET; FRAP
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2004.01.006

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® 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.