WS06.1 Stabilization of CFTR at the membrane by EPAC1 activation occurs through interaction with NHERF-1

Abstract

Background CFTR-mediated ion transport is stimulated by increase in subcortical cAMP levels through protein kinase A (PKA) activation thus triggering CFTR phosphorylation and channel opening. However, besides PKA, exchange proteins (EPACs) are also directly activated by cAMP. EPACs are guanine nucleotide exchange factors (GEFs) for Rap family small GTPases and shift to the plasma membrane (PM) in response to cAMP. This suggests that activation of both CFTR and EPACs might be spatially and temporally coincident. Objectives We aimed at elucidating the impact of EPAC1 on CFTR trafficking and PM anchoring. Methods Protein biochemistry and imaging techniques. Results Our results show that in CFBE cells: i. EPAC1 co-localizes and is present in protein complexes involving wt- or F508del-CFTR and this interaction is mediated by NHERF1 but not ezrin. ii. EPAC activation leads to its increased association with CFTR and increased CFTR PM levels. iii. Increase in endogenous cAMP levels increases CFTR membrane levels. iv. EPAC1 knockdown leads to decreased of CFTR PM levels. Conclusion Our results establish that EPAC1 associates to CFTR in membrane complexes with NHERF-1 and enhances CFTR traffic to the PM, making it a novel potential therapeutic target for CF. These data indicate that cAMP signalling regulates CFTR through two distinct but complementary pathways – PKA and EPAC1. Grants from FCT (Portugal) through MCTES EXPL/BIM-MEC/1451/2013 grant and PEst-OE/BIA/UI4046/2011 BioFig centre grant and by 2012 ERS Romain Pauwels Research Award.