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Comparative Study
. 2006 Nov 6;175(3):441-51.
doi: 10.1083/jcb.200605050.

PGE(1) stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases

Anna Terrin  1 , Giulietta Di Benedetto, Vanessa Pertegato, York-Fong Cheung, George Baillie, Martin J Lynch, Nicola Elvassore, Anke Prinz, Friedrich W Herberg, Miles D Houslay, Manuela Zaccolo
Affiliations
Comparative Study

PGE(1) stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases

Anna Terrin et al. J Cell Biol. .

Abstract

There is a growing appreciation that the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway is organized to form transduction units that function to deliver specific messages. Such organization results in the local activation of PKA subsets through the generation of confined intracellular gradients of cAMP, but the mechanisms responsible for limiting the diffusion of cAMP largely remain to be clarified. In this study, by performing real-time imaging of cAMP, we show that prostaglandin 1 stimulation generates multiple contiguous, intracellular domains with different cAMP concentration in human embryonic kidney 293 cells. By using pharmacological and genetic manipulation of phosphodiesterases (PDEs), we demonstrate that compartmentalized PDE4B and PDE4D are responsible for selectively modulating the concentration of cAMP in individual subcellular compartments. We propose a model whereby compartmentalized PDEs, rather than representing an enzymatic barrier to cAMP diffusion, act as a sink to drain the second messenger from discrete locations, resulting in multiple and simultaneous domains with different cAMP concentrations irrespective of their distance from the site of cAMP synthesis.

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Figures

Figure 1.
Figure 1.
The PKA-based sensor for cAMP. (A) Schematic representation of the R and C subunits of PKA fused to CFP and YFP, respectively (top), and confocal images showing their localization in HEK293 cells coexpressing the two subunits of the sensor (bottom). (B) Schematic representation of the membrane-targeted version of the PKA-based sensor (top) and distribution of the two subunits in HEK293 cells (bottom) as detected at the confocal microscope. Bars, 10 μm. (C) Apparent activation constants determination for PKA-GFP and mpPKA-GFP.
Figure 2.
Figure 2.
The cAMP response to PGE1 is higher under the plasma membrane than in the bulk cytosol. (A) Wide-field images of a representative HEK293 cell cotransfected with R-CFP (top left) and C-YFP (not depicted) and with mpR-CFP (bottom left) and C-YFP (not depicted). For the same cell, the pseudocolor images of the 480/545-nm emission ratio before (time = 0 s) and after the addition of 10 μM PGE1 (time = 200 s) and 100 μM IBMX (time = 800 s) are shown on the right. Bars, 10 μm. (B) Kinetics of cAMP changes recorded in the cells shown in A. Open circles represent kinetics recorded in the bulk cytosol with PKA-GFP, and closed circles represent kinetics recorded at the plasma membrane with mpPKA-GFP. (C) Summary of all the experiments performed in the same conditions as in A and B. (D) Time to reach half-maximal response (t/2) to 10 μM PGE1. (E) The summary of experiments performed by applying 1 μM PGE1. Error bars indicate SEM. *, 0.01 < P < 0.05; **, 0.005 < P < 0.01; ***, P < 0.005.
Figure 3.
Figure 3.
A unimolecular Epac-based sensor detects different [cAMP] at the plasma membrane and in the bulk cytosol. (A) Schematic representation of the fusion protein constituting the Epac-based cAMP sensors H30 and confocal micrographs showing its distribution in HEK293 cells. (B) Structure and localization of the membrane-targeted version of mpH30. Bars, 10 μm. (C) cAMP dose-response curves measured as the percent FRET changes of H30 (white circles), mpH30 (black circles), and nlsH30 (gray circles). EC50 are 12.5, 20, and 17.5 μM, respectively. (D) Representative kinetics of cAMP changes generated in the cytosol (white circles) and at the plasma membrane (black circles) upon stimulation with 1 μM PGE1 followed by 100 μM IBMX. (E) Summary of the experiments performed as in D. Error bars represent SEM. **, P = 0.002; ***, P = 10−4.
Figure 4.
Figure 4.
Role of PKA in shaping the cAMP gradient between the plasma membrane and the cytosol. (A and B) Representative cAMP kinetic (Romoser et al., 1996) and summary of experiments (B) performed in HEK293 cells cotransfected with PKA and either H30 or mpH30 and challenged with 1 μM PGE1 followed by either total PDE inhibition with 100 μM IBMX or PKA inhibition with 10 μM H89 as indicated. (C and D) Representative kinetics (C) and summary of experiments (D) showing the effect of endogenous PKA inhibition on the cAMP response induced by 10 nM PGE1 at the plasma membrane and bulk cytosol in the absence and presence of the PKA inhibitor H89 (10 μM). In all of the experiments, when the PKA inhibitor was used, cells were preincubated for 10 min with H89, and the inhibitor was present throughout the experiment. Error bars represent SEM. *, P = 0.02; **, P = 0.009; ***, P = 0.0009.
Figure 5.
Figure 5.
Identification of the prevalent PDE affecting the cAMP response to PGE1. Summary of the effect of selective PDE3 (10 μM cilostamide) or PDE4 (10 μM rolipram) inhibition on the cAMP response in the cytosolic (H30) and subplasma membrane (mpH30) compartments upon 1 μM PGE1 stimulation of HEK cells overexpressing untagged PKA. Error bars represent SEM. *, P = 0.01; ***, P < 0.00095.
Figure 6.
Figure 6.
Effect of genetic ablation of selected PDEs on the cAMP gradient between the plasma membrane and bulk cytosol. (A–D) Summary of the effect on the cAMP response generated by 1 μM PGE1 either in the bulk cytosol (open bars) or at the plasma membrane (closed bars) in HEK293 cells expressing either H30 or mpH30 in control cells (A; Romoser et al., 1996) or cells transfected with siRNA oligonucleotides for the selective genetic knockdown of PDE4B (B), PDE4D (C), or both (D). For each experimental group, n ≥ 25. In each panel, the schematics on the right show the observed distribution of the cAMP gradient (in red) and the predicted distribution of endogenous PDEs upon the selective knockdown of specific PDE4 subfamilies. As shown, a lower concentration of cAMP corresponds to a region with higher PDE4 activity. Error bars represent SEM. *, P = 0.04; ***, P = 10−4.
Figure 7.
Figure 7.
Effect of the selective displacement of PDE4 subfamilies on the cAMP gradient. (A and B) Response to 1 μM PGE1 in HEK293 cells expressing the dn variants of PDE4B1 or PDE4B2 (A; Romoser et al., 1996) and expressing the dn variants of PDE4D3 or PDE4D5 (B). For each experimental group, n ≥ 17. Schematics on the right of each panel show the observed cAMP gradient (in red) and the predicted redistribution of endogenous, active PDE4 subfamilies upon overexpression of the cognate, inactive dn proteins (not depicted). Error bars represent SEM. *, P = 0.01; **, P = 0.004.
Figure 8.
Figure 8.
Subcellular localization of PDE4B and PDE4D. Confocal images of HEK293 cells stained with monoclonal antibodies specific for either PDE4B or PDE4D. The control is the secondary antibody alone. Bars, 10 μm.
Figure 9.
Figure 9.
PGE1 stimulation generates a larger cAMP response in the nucleus as compared with the bulk cytosol. (A) Schematic representation of the sensor H30 targeted to the nucleus (nlsH30) and its distribution in HEK293 cells. Bars, 10 μm. (B) Representative kinetics of cAMP changes in response to 1 μM PGE1 as detected in the bulk cytosol in cells transfected with H30 (white circles), at the plasma membrane in cells transfected with mpH30 (black circles), and in the nucleus in cells transfected with nlsH30 (gray circles). (C) Summary of the experiments performed in the aforementioned conditions. (D) Summary of the FRET changes recorded in the bulk cytosol (white bar) and in the nucleus (gray bar) of HEK293 cells cotransfected with either H30 or nlsH30 and siRNA oligonucleotides for PDE4D and stimulated with 1 μM PGE1. Error bars represent SEM. **, P = 0.03; ***, P = 10−4.
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References

    1. Abrahamsen, H., G. Baillie, J. Ngai, T. Vang, K. Nika, A. Ruppelt, T. Mustelin, M. Zaccolo, M. Houslay, and K. Tasken. 2004. TCR- and CD28-mediated recruitment of phosphodiesterase 4 to lipid rafts potentiates TCR signaling. J. Immunol. 173:4847–4858. - PubMed
    1. Bacskai, B.J., B. Hochner, M. Mahaut-Smith, S.R. Adams, B.K. Kaang, E.R. Kandel, and R.Y. Tsien. 1993. Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons. Science. 260:222–226. - PubMed
    1. Baillie, G.S., A. Sood, I. McPhee, I. Gall, S.J. Perry, R.J. Lefkowitz, and M.D. Houslay. 2003. beta-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta-adrenoceptor switching from Gs to Gi. Proc. Natl. Acad. Sci. USA. 100:940–945. - PMC - PubMed
    1. Baillie, G.S., J.D. Scott, and M.D. Houslay. 2005. Compartmentalisation of phosphodiesterases and protein kinase A: opposites attract. FEBS Lett. 579:3264–3270. - PubMed
    1. Barnes, A.P., G. Livera, P. Huang, C. Sun, W.K. O'Neal, M. Conti, M.J. Stutts, and S.L. Milgram. 2005. Phosphodiesterase 4D forms a cAMP diffusion barrier at the apical membrane of the airway epithelium. J. Biol. Chem. 280:7997–8003. - PubMed

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