A key feature of signal transduction is the convergence of a very large variety of extracelluar stimuli onto an extremely limited number of intracellular second messengers. By transducing inside the cell the highly variable and complex extracellular information, second messengers can tightly and specifically modulate a disparate variety of intracellular functions and a signalling code based simply on messenger concentration grading appears insufficient to ensure the necessary specificity and diversity to signalling. In the last decade much attention has thus been focused on the spatial and temporal dynamics of second messengers and it has become clear that specificity of response is attained through a versatile use of speed, amplitude and spatio-temporal patterning of signalling molecules (Zaccolo et al. 2002). In particular, it has become clear that targeting and compartmentation of signalling enzymes and effectors close to their activators and targets is crucial to ensure tight regulation and specificity of action of signalling cascades.
The cAMP/PKA signalling pathway modulates a large variety of cellular functions, as diverse as growth, movement, metabolism and synaptic plasticity. cAMP signalling relies on the organisation of macromolecular complexes in the generation of which a central role is played by A kinase anchoring proteins (AKAPs). AKAPs can act as multiscaffolding proteins that are targeted to specific subcellular locations and can bind simultaneously the main effector of cAMP, PKA and several other signalling molecules (such as plasma membrane receptors, phosphatases, PKC, etc.). The organization of the cAMP/PKA pathway in such signalling domains is thought to be crucial for achieving specificity of response (Edwards & Scott, 2000) and although the concept of cAMP/PKA compartmentalized signal transduction is largely accepted, the detailed description of how this is operated is still missing and the information on the spatio-temporal dynamics of cAMP is extremely poor. This is mainly due to the lack of methodologies for the analysis of cAMP changes in live cells. We recently generated a sensor for cAMP by genetically linking the R and C subunits of PKA each to a different mutant of GFP (Zaccolo et al. 2000). These mutants (i.e. EYFP and ECFP) show spectral characteristics that make them a suitable pair for fluorescence resonance energy transfer (FRET). FRET is a quantum-physical phenomenon whereby the energy of the excited state of a donor fluorophore (ECFP) is transferred to an acceptor fluorophore (EYFP) that lies in its close proximity (< 10-10 m). Cells transfected with this probe can be analysed at the fluorescence microscope by exciting ECFP only and changes in FRET are conveniently measured as Δ(emission cyan/emission yellow). This methodology allows fast and reproducible monitoring of [cAMP]i changes in single cells with a high spatial (< 10-8 m) and time (< 10-1 s) resolution. By using this sensor we could gain an important insight into the spatio-temporal dynamics of β-adrenergic signalling in heart cells (Zaccolo & Pozzan, 2002). In particular we could show that: (i) β-adrenergic stimulation generates in cardiac myocytes multiple local microdomains of high cAMP in correspondence of the Z band in the sarcomere; (ii) the restricted pools of cAMP show a range of action as small as ~1 µm and free diffusion of the second messenger is limited by the activity of phosphodiesterases; (iii) the steep gradients of cAMP specifically activate a subset of protein kinases A anchored in proximity of the Z band.
M.Z. is supported by Telethon Italy.