Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells.

We have previously shown (i) that the cystic fibrosis transmembrane regulator (CFTR) locates to lipid raft-like microdomains of epithelial cells upon TNF-α proinflammatory stimulation; and (ii) that TNF-α increases the membrane localization and the channel function of F508del-mutated CFTR. In the present work, we hypothesized that CFTR mutations modify the proteome of lipid rafts in the same proinflammatory conditions. We prepared lipid rafts from HeLa cells transfected with either wild-type or F508del-CFTR and incubated for 10min with 100U/mL of TNF-α. Proteins were extracted, trypsin digested, and peptides analyzed by high resolution MS. Proteins were quantified by a stable isotope labeling with amino acids in cell culture approach. Out of the 22 proteins differentially recruited in lipid rafts after proinflammatory exposure, 17 were increased in F508del cells with respect to wild-type, including two G-protein coupled receptors, three anion transporters, and one cell surface mucin. In both HeLa and bronchial epithelial cells we confirmed that G-protein coupled receptor 5A relocates to lipid rafts along with F508del-CFTR after TNF-α treatment. These results could enlighten the cross-talk between CFTR and TNF-α and its impact on the cell response to proinflammatory challenge.