(B) Linescan approach used in our experiments. Arrows highlight sites where intracellular esterases cleave both ester bonds. (A) Molecular structure of fluorogenic 8-FDA-cAMP. We therefore set out to address this controversy by developing tools and methods to directly measure and characterize cAMP mobility in intact cells and at physiological levels, and to measure cAMP levels and gradients in real-time and with a spatial accuracy in the nanometer range.ĬAMP dynamics are highly restricted in intact cells. Thus, the important question whether and how intracellular cAMP might be compartmentalized has remained unresolved for decades. Therefore, cAMP should very rapidly equilibrate in a cell ( Rich et al., 2000, Rich et al., 2001, Feinstein et al., 2012, Lohse et al., 2017, Xin et al., 2015), and this would prevent the existence of compartments with different concentrations of cAMP. However, several studies have determined that cAMP is highly diffusible in intact cells ( Bacskai et al., 1993, Nikolaev et al., 2004, Chen et al., 1999, Nikolaev et al., 2006, Richards et al., 2016, Agarwal et al., 2016, Huang and Gillette, 1993) and Table S1), and PDEs have low catalytic rates ( Omori and Kotera, 2007, Conti and Beavo, 2007, Bender and Beavo, 2006). The cAMP-degrading phosphodiesterases (PDEs) have been proposed to play a crucial role in establishing the cAMP gradients that are necessary to create such compartments ( Houslay, 2010, Terrin et al., 2006, Mika et al., 2012, Stangherlin and Zaccolo, 2012). To explain receptor-specific responses observed in experiments and how these responses may differ in different regions of a cell, many researchers have proposed cAMP compartmentalization ( Brunton et al., 1979, Hayes et al., 1980, Buxton and Brunton, 1983). However, it is unclear, how the huge number of receptors that all change global cellular cAMP levels can result in specific cellular responses.

This pathway is central to key physiological functions and, hence, also to many diseases, making it a highly attractive therapeutic target ( Nikolaev et al., 2010, Gold et al., 2013, Zaccolo, 2009, Zaccolo, 2011, Perera and Nikolaev, 2013). Hundreds of cell surface receptors, notably G protein-coupled receptors (GPCRs), signal via the second messenger cyclic adenosine 3’,5’-monophosphate (cAMP) and its effector proteins, in particular protein kinase A (PKA). Our study reveals that spatiotemporal cAMP signaling is under precise control of nanometer-sized domains shaped by PDEs that gate the activation of downstream effectors.

Using FRET-cAMP nanorulers we directly map cAMP gradients at the nanoscale around PDE molecules and the areas of resulting downstream activation of cAMP-dependent protein kinase (PKA). With a large fraction of cAMP being buffered, PDEs can create nanometer-sized domains of low cAMP concentrations. Binding and unbinding results in largely reduced cAMP dynamics which we term ‘ buffered diffusion’. Using fluorescence spectroscopy, we show that – contrary to earlier data – cAMP at physiological concentrations is predominantly bound to cAMP binding sites and, thus, immobile. However, measured rates of fast cAMP-diffusion and slow PDE-activity render cAMP compartmentalization essentially impossible. To explain signaling specificity, cAMP-degrading phosphodiesterases (PDEs) have been suggested to confine cAMP to distinct cellular compartments. Cells relay a plethora of extracellular signals to specific cellular responses using only a few second messengers, such as cAMP.

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