A Framework to Map cAMP Signalling Nanodomains: Phosphodiesterase-Centred Phosphoproteome-Interactome Networks...

Intracellular signalling is commonly represented as linear pathways that connect receptors to downstream effectors. While such models have been instrumental in defining signalling cascades, they fail to capture the spatial organisation that underlies signalling specificity in living cells. The cyclic adenosine monophosphate (cAMP) pathway provides a well-established example of this principle, where signalling is compartmentalised into nanometre-scale domains that generate highly localised and functionally distinct responses. However, a comprehensive framework for defining the molecular composition, spatial organisation, and functional outputs of these signalling domains and how they adapt to perturbations remains lacking. Here, we discuss how integrative proteomics approaches can be used to reconstruct the subcellular cartography of compartmentalised signalling networks. By combining isoform-specific interactomics, quantitative phosphoproteomics, network analysis, and spatial annotation, individual signalling platforms can be mapped within their native intracellular context. We introduce phosphoproteome-interactome networks (pPINs), a systems-level framework that integrates molecular interactions, subcellular localisation, and phosphorylation responses to define phosphodiesterase (PDE)-centred signalling platforms and their associated signalling outputs. Using PDE3A isoforms as an example, we illustrate how pPINs uncover multiple spatially distinct cAMP signalling nanodomains in cardiac myocytes and reveal previously unrecognised biology, including a nuclear PDE3A2/SMAD4/HDAC-1 platform that locally constrains PKA activity and suppresses prohypertrophic gene expression. More broadly, pPINs provide a conceptual and computational framework for resolving the spatial architecture of intracellular signalling networks and establish a foundation for precision therapeutic strategies targeting discrete signalling microenvironments.