Changes in intracellular cAMP reported by a Redistribution assay using a cAMP-dependent protein kinase-green fluorescent protein chimera
Research output: Contribution to journal › Journal article › Research › peer-review
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 Gs- and Gi-protein-coupled receptor stimulation as well as by inhibition of cAMP phosphodiesterases.
Original language | English |
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Journal | Cellular Signalling |
Volume | 16 |
Issue number | 8 |
Pages (from-to) | 907-20 |
Number of pages | 14 |
ISSN | 0898-6568 |
DOIs | |
Publication status | Published - 2004 |
Externally published | Yes |
- 3',5'-Cyclic-AMP Phosphodiesterases, Animals, CHO Cells, Cell Nucleus, Cells, Cultured, Cricetinae, Cricetulus, Cyclic AMP, Cyclic AMP-Dependent Protein Kinases, Cytoplasm, Enzyme Activation, Green Fluorescent Proteins, HeLa Cells, Humans, Microscopy, Fluorescence, Recombinant Fusion Proteins
Research areas
ID: 43349020