Deletion of a phenylalanine residue at position 508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the principal cause of cystic fibrosis (CF). Altered interactions of F508del CFTR with endoplasmic reticulum quality control proteins promote its proteasomal degradation. However, it is believed that crucial CFTR-interacting proteins (CIPs) important for trafficking and function of CFTR remain unknown. In this study, the NBD1 of CFTR was used as 'bait' in affinity chromatography to capture novel interacting proteins. Analysis of the recovered proteins by high resolution two-dimensional electrophoresis followed by mass spectrometry (Maldi-Toff) identified annexin V as binding partner of CFTR-NBD1.

Annexin V belongs to a class of calcium-dependent phospholipids binding proteins, some of which have been implicated in membrane-related events along exocytosis and endocytosis pathways. Annexin V is a protein kinase C (PKC) inhibitor protein, which binds acidic phospholipids. This protein is overexpressed in CF epithelial cells and it was recently shown that annexin V is necessary for CFTR membrane expression and function. We report here that annexin V has a stimulatory effect on CFTR-mediated Cl- currents in HEK cells taking part in the traffic regulation. Since annexin proteins are well known to control protein trafficking, we examined whether it contributed to CFTR trafficking. When patch clamp experiments were preformed by overexpressing the CFTR mutant Y1424A/I1427A, that disrupts the internalization signal, the effect of annexin V overexpression was no longer observed. This suggests a contribution of annexin V on CFTR endocytosis.

Work supported by EU grant FP6-LSH-2005-037365 (TargetScreen2). SFB699 A6/A7