Endosomal cAMP production broadly impacts the cellular phosphoproteome

Endosomal signaling downstream of G-protein-coupled receptors (GPCRs) has emerged as a novel paradigm with important pharmacological and physiological implications. However, our knowledge of the functional consequences of intracellular signaling is incomplete. To begin to address this gap, we combined an optogenetic approach for site-specific generation of the prototypical second messenger generated by active GPCRs, cyclic AMP (cAMP), with unbiased mass-spectrometry-based analysis of the phosphoproteome. We identified 218 unique, high-confidence sites whose phosphorylation is either increased or decreased in response to cAMP elevation. We next determined that the same amount of cAMP produced from the endosomal membrane led to more robust changes in phosphorylation than the plasma membrane. Remarkably, this was true for the entire repertoire of 218 identified targets and irrespective of their annotated subcellular localizations (endosome, cell surface, nucleus, cytosol). Furthermore, we identified a particularly strong endosome bias for a subset of proteins that are dephosphorylated in response to cAMP. Through bioinformatics analysis, we established these targets as putative substrates for protein phosphatase 2A (PP2A), and we propose compartmentalized activation of PP2A by cAMP-responsive kinases as the likely underlying mechanism. Altogether, our study extends the concept that endosomal signaling is a significant functional contributor to cellular responsiveness to cAMP by establishing a unique role for localized cAMP production in defining categorically distinct phosphoresponses.