Abstract
The intracellular second messenger cyclic AMP (cAMP) is degraded by phosphodiesterases (PDE). The knowledge of individual families and subtypes of PDEs is considerable, but how the different PDEs collaborate in the cell to control a cAMP signal is still not fully understood. In order to investigate compartmentalized cAMP signaling, we have generated a membrane-targeted variant of the cAMP Bioluminiscence Resonance Energy Transfer (BRET) sensor CAMYEL and have compared intracellular cAMP measurements with it to measurements with the cytosolic BRET sensor CAMYEL in HEK293 cells. With these sensors we observed a slightly higher cAMP response to adenylyl cyclase activation at the plasma membrane compared to the cytosol, which is in accordance with earlier results from Fluorescence Resonance Energy Transfer (FRET) sensors. We have analyzed PDE activity in fractionated lysates from HEK293 cells using selective PDE inhibitors and have identified PDE3 and PDE10A as the major membrane-bound PDEs and PDE4 as the major cytosolic PDE. Inhibition of membrane-bound or cytosolic PDEs can potentiate the cAMP response to adenylyl cyclase activation, but we see no significant difference between the potentiation of the cAMP response at the plasma membrane and in cytosol when membrane-bound and cytosolic PDEs are inhibited. When different levels of stimulation were tested, we found that PDEs 3 and 10 are mainly responsible for cAMP degradation at low intracellular cAMP concentrations, whereas PDE4 is more important for control of cAMP at higher concentrations.
Highlights
The second messenger adenosine 39,59-cyclic monophosphate is involved in a variety of intracellular processes [1]
When the relative responses of the membrane-bound PDE2CAMYEL sensor and the cytosolic CAMYEL sensor for the six different conditions tested in Figure 4 were compared, we found no significant differences between the response measured with PDE2-CAMYEL and that measured with CAMYEL
We have measured intracellular cyclic AMP (cAMP) concentrations in living HEK293 cells with the Bioluminiscence Resonance Energy Transfer (BRET) sensor CAMYEL [17] and a novel membrane-targeted variant that we have made by fusing CAMYEL to the membrane-targeting motif from PDE2
Summary
The second messenger adenosine 39,59-cyclic monophosphate (cAMP) is involved in a variety of intracellular processes [1]. FRET sensors have been used to study cAMP compartmentalization in the much smaller HEK293 cells by targeting FRET sensors to specific subcellular compartments such as plasma membrane, nucleus, or mitochondria [12,13]. Both studies observed a faster cAMP response at the plasma membrane compared to the cytosol after adenylyl cyclase stimulation. In one of these studies [13], but not in the other [12], the maximal cAMP response at the plasma membrane was significantly higher than in the cytosol. The mechanism for achieving cAMP compartmentalization that has most experimental support is that the rates of cAMP degradation differ between compartments due to subcellularly localized PDEs [14,15,16]
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