Abstract
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.
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Cellular Signalling |
| Vol/bind | 16 |
| Udgave nummer | 8 |
| Sider (fra-til) | 907-20 |
| Antal sider | 14 |
| ISSN | 0898-6568 |
| DOI | |
| Status | Udgivet - 2004 |
| Udgivet eksternt | Ja |