G551D‐CFTR needs more bound actin than wild‐type CFTR to maintain its presence in plasma membranes

Abstract

Cystic Fibrosis is due to mutations in the CFTR gene. The missense mutation G551D (approx. 5% of cases) encodes a CFTR chloride channel with normal cell surface expression but with an altered chloride channel activity, leading to a severe phenotype. Our aim was to identify specific interacting proteins of G551D-CFTR which could explain the channel defect. Wild-type CFTR (Wt-CFTR) was co-immunoprecipitated from stably transfected HeLa cells and resolved by 2D gel electrophoresis. Among the detected spots, one was expressed at a high level. Mass Spectrometry revealed that it corresponded to actin which is known to be involved in the CFTR's channel function. To assess whether actin could be involved in the altered G551D-CFTR function, its basal expression was studied. Because actin expression was the same in wt- and in G551D-CFTR expressing cells, its interaction with both wt- and G551D-CFTR was studied by co-immunoprecipitation, and we found that a higher amount of actin was bound onto G551D-CFTR than onto Wt-CFTR. The role of actin upon wt- and G551D-CFTR function was further studied by patch-clamp experiments after cytochalasin D treatment of the cells. We found a decrease of the very weak currents in G551D-CFTR expressing cells. Because a higher amount of actin is bound onto G551D-CFTR than onto Wt-CFTR, it is likely to be not involved in the mutated CFTR's defect. Nevertheless, because actin is necessary to maintain the very weak global currents observed in G551D-CFTR expressing HeLa cells, we conclude that more actin is necessary to maintain G551D-CFTR in the plasma membrane than for Wt-CFTR.

A Proteomic Variant Approach (ProVarA) for Personalized Medicine of Inherited and Somatic Disease

“…Two previous studies used 2D-electrophoresis to identify G551D-CFTR interacting proteins [55, 56]. These studies noted that calu menin (CALU) and actin are recovered with increased affinity for the G551D variant relative to WT-CFTR [55, 56]. While our PIP analysis failed to identify actin as a differentially bound protein, we did observe an increased affinity of CALU for G551D-CFTR (Table S6).…”

“…While 88% of proteins were recovered with both WT-and G551D-CFTR, 325 proteins exhibited a statistically significant difference in affinity (Figure 1C; Table S6), a value that is consistent with the low pairwise Jaccard similarity index with WT-CFTR, but surprisingly large given the ability of both variants to navigate the cellular compartments associated with protein biogenesis and trafficking. Two previous studies used 2D-electrophoresis to identify G551D-CFTR interacting proteins [55, 56]. These studies noted that calu menin (CALU) and actin are recovered with increased affinity for the G551D variant relative to WT-CFTR [55, 56].…”

“…The data presented herein reveals that the PIP of the G551D-CFTR polypeptide is vastly different from that of its WT counterpart, which could account for the differential behavior of these variants at the PM. Furthermore, Trouve et al , showed that the G551D variant exhibits increased binding to actin relative to the affinity displayed by WT-CFTR and this actin binding is required to maintain the weak basal chloride channel activity exhibited by the G551D variant [56], suggesting that this actin binding is required to maintain the variant in the PM. The G551D PIP reveals reduced binding to actin related proteins suggesting that the G551D variant would not be maintained at the PM, but rather localized within endocytic recycling vesicles.…”

Revisiting CFTR Interactions: Old Partners and New Players

“…Class II mutants exhibited, as before, an increased interaction with the degradation machinery and proteostasis network components whereas G551D-CFTR, despite its ability to traffic to the PM, suffered from an overall reduction in binding affinity compared to WT-CFTR, suggesting a lack of key interactions that could explain its channel gating defect. Previously, it was shown that G551D-CFTR anchoring at the PM needed more bound actin compared with WT-CFTR [157].…”

<p>Genomics and Transcriptomics: The Powerful Technologies in Precision Medicine</p>

“… 73 – 75 More than 2000 mutations have been identified in the CFTR gene which the most notable of them are: p.Phe508del or c.1521_1523delCTT, G542X, and G551D. 76 – 78 Each one of these mutations (based on the defect that occurs in the CFTR protein) leads to different forms of molecular pathways in the CF disease. Note that this difference may not be easily visible in clinical symptoms of patients.…”