Epac Based Sensors

The first unimolecular FRET probes for cAMP exploited Epac as the sensor for the cyclic nucleotide (DiPilato et al. 2004; Ponsioen et al. 2004). One of these indicators was generated by fusing the amino terminus of the Epac 1 protein to CFP and the C-terminus to YFP (Ponsioen et al. 2004). Such sensors localize to the cytosol and to membranes, in particular to the nuclear envelope and to perinuclear compartments (Ponsioen et al. 2004). To generate a cytosolic variant, the DEP domain (amino acids 1-148) was deleted. Indeed, this chimera locates almost exclusively in the cytosol. Additional mutations (T781A, F782A) were introduced to generate the sensor CFP-Epac(5DEP-CD)-YFP (also termed H30), which is catalytically inactive (Ponsioen et al. 2004) (Fig. 2a,b).

The lower sensitivity to cAMP of these Epac-based sensors (20 |M, Table 1) as compared to the PKA-based probes limits their application to systems characterized by large changes in cAMP concentration.

Further modifications to the Epac-based indicators have been introduced to target the sensor to specific subcellular compartments. In particular, a short polypeptide called mp and corresponding to the N-terminal targeting signal from the Lyn kinase (Resh 1999) was fused to the N-terminal end of H30. The mp sequence is post-translationally myristoylated and palmitoylated, therefore targeting the sensor to the

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Fig. 2 Epac 1-based sensor. (a) Schematic representation of the mechanism of action of the Epac 1-based sensor H30. (b) Schematic structure of H30 (left panel) and cytosolic distribution of the probe (right panel). (c) Schematic structure of mpH30 (left panel) and localization of the probe at the plasma membrane (rightpanel). (d) Schematic structure of nlsH30 (leftpanel) and localization of the probe in the nucleus (right panel) shown as image of the fluorescence emission (left) and its overlay with the transmitted light image of the same cells (right). (e) Dose-response curves showing FRET changes at different cAMP concentration for the three Epac 1-based sensors. For these measurements, HeLa cells expressing each cAMP sensor were injected with known concentrations of cAMP via a patch pipette and FRET changes recorded (Terrin et al. 2006). (f) Kinetics of FRET changes recorded with the three different sensors upon addition, via a patch pipette, of 100 |M cAMP. Each curve represents the average of five independent measurements

Fig. 2 Epac 1-based sensor. (a) Schematic representation of the mechanism of action of the Epac 1-based sensor H30. (b) Schematic structure of H30 (left panel) and cytosolic distribution of the probe (right panel). (c) Schematic structure of mpH30 (left panel) and localization of the probe at the plasma membrane (rightpanel). (d) Schematic structure of nlsH30 (leftpanel) and localization of the probe in the nucleus (right panel) shown as image of the fluorescence emission (left) and its overlay with the transmitted light image of the same cells (right). (e) Dose-response curves showing FRET changes at different cAMP concentration for the three Epac 1-based sensors. For these measurements, HeLa cells expressing each cAMP sensor were injected with known concentrations of cAMP via a patch pipette and FRET changes recorded (Terrin et al. 2006). (f) Kinetics of FRET changes recorded with the three different sensors upon addition, via a patch pipette, of 100 |M cAMP. Each curve represents the average of five independent measurements plasma membrane (Fig. 2c). Another variant of H30 was generated by fusing a nuclear localization sequence (nls) to the C-terminus of the sensor, which effectively targets the cAMP indicator to the nuclear compartment (Fig. 2d). These differently targeted probes have proved to be very useful tools to study compartmentalized cAMP signaling. The combined use of the differently targeted H30 probes allowed the dissection of the cAMP response in the different subcellular compartments in the model cell line HEK293 (Terrin et al. 2006). This study shows that prostaglandin 1 (PGE1) stimulation of HEK293 cells generates multiple contiguous intracellular domains with different cAMP concentration. In particular, PGE1 stimulation generates a larger pool of cAMP in the sub-plasma membrane compartment as compared to the bulk cytosol. In addition, this study shows that in a subcellular compartment, such as the nucleus, located deep inside the cell and far away from the site of synthesis of the second messenger, the concentration of cAMP can be higher as compared to the surrounding cytosol. This is an important observation because it confirms that mechanisms are in place that allow a subset of PKA that are anchored at a distance from the plasma membrane to be selectively activated without the concomitant activation of other PKA subsets that may be located closer to the site of cAMP synthesis. What is the mechanism responsible for such com-partmentalization of cAMP? Pharmacological inhibition of PDEs associated with genetic manipulation of these enzymes by using small RNA interfering and overexpression of dominant negative constructs of selected PDEs demonstrate that compartmentalized PDE4B and PDE4D are responsible for selectively modulating the concentration of cAMP in individual subcellular compartments in HEK293 cells (Terrin et al. 2006).

The obvious advantage of using the same sensor targeted to different subcel-lular compartments is that, in principle, the readings of the different probes are directly comparable. However, careful characterization of the individual sensors is necessary as even minor changes in the sequence of a probe may affect its performance. For example, in the case of the H30, mpH30 and nlsH30 indicators, although the introduction of targeting sequences did not significantly affect the sensitivity of each probe variant to cAMP (Fig. 2e and Table 1), the kinetics of FRET changes resulted in being significantly faster for the nlsH30 sensor as compared to the H30 and mpH30 variants (Fig. 2f), therefore hindering reliable conclusions on the different kinetics of cAMP changes in the individual compartments.

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