Cells relay a plethora of extracellular signals to specific cellular responses by using only a few second messengers, such as cAMP. To explain signaling specificity, cAMP-degrading phosphodiesterases (PDEs) have been suggested to confine cAMP to distinct cellular compartments. However, measured rates of fast cAMP diffusion and slow PDE activity render cAMP compartmentalization essentially impossible. Using fluorescence spectroscopy, we show that, contrary to earlier data, cAMP at physiological concentrations is predominantly bound to cAMP binding sites and, thus, immobile. Binding and unbinding results in largely reduced cAMP dynamics, which we term “buffered diffusion.” With a large fraction of cAMP being buffered, PDEs can create nanometer-size domains of low cAMP concentrations. 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). Our study reveals that spatiotemporal cAMP signaling is under precise control of nanometer-size domains shaped by PDEs that gate activation of downstream effectors.
Bibliographical noteFunding Information:
We thank Dr. Frank Schwede (Biolog GmbH, Germany) for discussions regarding the design of 8-FDA-cAMP and all members of the Lohse lab for critical discussions. We are grateful to Dr. Carmine Di Rienzo for discussions and constructive input regarding data analysis. This work was supported by the European Research Council grant TOPAS to M.J.L. and by NIH MIRA (R35-GM126940) to S.S. A.B., P.A., and M.J.L. acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG) (German Research Foundation) through SFB1423, project number 421152132, subproject C03 (P.A. and M.J.L.) and subproject C05 (A.B.), and through SFB688, subproject B08 (M.J.L.).
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- FRET biosensors
- G protein-coupled receptors
- buffered diffusion
- cell signaling
- cyclic AMP
- fluorescence fluctuation spectroscopy
- protein kinase A4
PubMed: MeSH publication types
- Journal Article