Regulation of CFTR trafficking and membrane anchoring : new insights into cAMP signalling

Cystic Fibrosis (CF) is the most common lethal autosomic recessive disorder among Caucasian population. It is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein, a cAMP-regulated chloride channel expressed at the apical surface of epithelial cells. The most common CF-causing mutation is a deletion of phenylalanine 508 (F508del, present in 85% of CF patients) and leads to CFTR misfolding which is recognized by the endoplasmic reticulum (ER) quality control (ERQC) resulting in ER retention and degradation. Regulation of CFTR at the plasma membrane (PM) is a complex process involving different interaction partners and signalling pathways. Among these, cAMP regulates both channel gating through a protein kinase A (PKA)-dependent process and PM stability through activation of the exchange protein directly activated by cAMP 1 (EPAC1). Here, we aim to provide new insights into three main areas of CFTR regulation namely: 1) CFTR trafficking to the PM; 2) CFTR anchoring at the PM through EPAC1 activation and 3) impact of cAMP levels in the cell homeostasis (using CFTR as a mode). Using interactomics we identified factors specifically involved in the recogniton of ER exit/retention motifs. Extended comparative analysis showed that the CFTR interactome is much more shaped by the folding status of the protein than by its subcellular location. Using protein interaction profiling and bioinformatic analysis, we identified proteins that interact differentially with CFTR under EPAC1 activation. We characterized, for the first time, the actin cytoskeleton dynamics regulators INF2 and CAPZA2 as modulators of CFTR anchoring at the PM. Finally, we dissected the impact of cAMP levels in the stress response and trafficking pathways. We show that PDE2 regulates cAMP levels within the cell, with impact in the EPAC1 pathway, and also ER stress controlling the cellular homeostasisPrograma doutoral BioSys - Sistemas Biológicos, Genômica Funcional & Integrativa (FCT / PD/ 00065 / 2012) da Faculdade de Ciências da Universidade de Lisbo