No abstract

cAMP/PKA signalling is compartmentalised with tight spatial and temporal control of signal propagation underpinning specificity of response. The cAMP-degrading enzymes, phosphodiesterases (PDEs), localise to specific subcellular domains within which they control local cAMP levels and are key regulators of signal compartmentalisation. Several components of the cAMP/PKA cascade are located to different mitochondrial sub-compartments, suggesting the presence of multiple cAMP/PKA signalling domains within the organelle. The function and regulation of these domains remain largely unknown. Here, we describe a novel cAMP/PKA signalling domain localised at mitochondrial membranes and regulated by PDE2A2. Using pharmacological and genetic approaches combined with real-time FRET imaging and high resolution microscopy, we demonstrate that in rat cardiac myocytes and other cell types mitochondrial PDE2A2 regulates local cAMP levels and PKA-dependent phosphorylation of Drp1. We further demonstrate that inhibition of PDE2A, by enhancing the hormone-dependent cAMP response locally, affects mitochondria dynamics and protects from apoptotic cell death.DOI: http://dx.doi.org/10.7554/eLife.21374.001

Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Here we demonstrate a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. We observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface. Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington’s disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain.

Cyclic AMP (cAMP) activates protein kinase A (PKA) but also the guanine nucleotide exchange factor 'exchange protein directly activated by cAMP' (EPAC1; also known as RAPGEF3). Although phosphorylation by PKA is known to regulate CFTR channel gatingthe protein defective in cystic fibrosis -the contribution of EPAC1 to CFTR regulation remains largely undefined. Here, we demonstrate that in human airway epithelial cells, cAMP signaling through EPAC1 promotes CFTR stabilization at the plasma membrane by attenuating its endocytosis, independently of PKA activation. EPAC1 and CFTR colocalize and interact through protein adaptor NHERF1 (also known as SLC9A3R1). This interaction is promoted by EPAC1 activation, triggering its translocation to the plasma membrane and binding to NHERF1. Our findings identify a new CFTR-interacting protein and demonstrate that cAMP activates CFTR through two different but complementary pathways -the well-known PKA-dependent channel gating pathway and a new mechanism regulating endocytosis that involves EPAC1. The latter might constitute a novel therapeutic target for treatment of cystic fibrosis.