A sequence upstream of canonical PDZ-binding motif within CFTR COOH-terminus enhances NHERF1 interaction

Abstract

The development of cystic fibrosis transmembrane conductance regulator (CFTR) targeted therapy for cystic fibrosis has generated interest in maximizing membrane residence of mutant forms of CFTR by manipulating interactions with scaffold proteins, such as sodium/hydrogen exchange regulatory factor-1 (NHERF1). In this study, we explored whether COOH-terminal sequences in CFTR beyond the PDZ-binding motif influence its interaction with NHERF1. NHERF1 displayed minimal self-association in blot overlays (NHERF1, K = 1,382 ± 61.1 nM) at concentrations well above physiological levels, estimated at 240 nM from RNA-sequencing and 260 nM by liquid chromatography tandem mass spectrometry in sweat gland, a key site of CFTR function in vivo. However, NHERF1 oligomerized at considerably lower concentrations (10 nM) in the presence of the last 111 amino acids of CFTR (20 nM) in blot overlays and cross-linking assays and in coimmunoprecipitations using differently tagged versions of NHERF1. Deletion and alanine mutagenesis revealed that a six-amino acid sequence EENKVR and the terminal TRL (PDZ-binding motif) in the COOH-terminus were essential for the enhanced oligomerization of NHERF1. Full-length CFTR stably expressed in Madin-Darby canine kidney epithelial cells fostered NHERF1 oligomerization that was substantially reduced (∼5-fold) on alanine substitution of EEN, KVR, or EENKVR residues or deletion of the TRL motif. Confocal fluorescent microscopy revealed that the EENKVR and TRL sequences contribute to preferential localization of CFTR to the apical membrane. Together, these results indicate that COOH-terminal sequences mediate enhanced NHERF1 interaction and facilitate the localization of CFTR, a property that could be manipulated to stabilize mutant forms of CFTR at the apical surface to maximize the effect of CFTR-targeted therapeutics.

Capitalizing on the heterogeneous effects of CFTR nonsense and frameshift variants to inform therapeutic strategy for cystic fibrosis

“…In contrast, variants located more 3’ including nonsense (E1418X), and two frameshifts (E1418Rfs14X and S1435Gfs14X) that truncate protein at residue 1442 and 1459 respectively generated minimal to moderate amounts of mature truncated protein. The final two variants S1455X and Q1476X, showed no apparent effect on the steady-state amounts of the immature or mature truncated forms of CFTR when compared with WT, as shown previously ( Fig 1C, S2 Fig) [40, 41]. The nonsense and frameshift variants in the 3’region fell into three groups (A-C) based on effects on RNA and protein levels ( Fig 1D ).…”

Integrated Transcriptomic and Proteomic Analysis of Human Eccrine Sweat Glands Identifies Missing and Novel Proteins

“…25 cm ϫ 15 cm skin was collected from the abdominal region of a single donor. Eccrine sweat glands were dissected out from the skin dermis as previously described (12)(13)(14). Approximately 250 eccrine sweat glands were collected and pooled.…”

The chloride anion as a signalling effector

“…To improve the model system, we then applied the differential display method to find differences in the genes expressed by CFDE cells (CF cells) and their CFTR-corrected counterparts, the latter also treated or not with CFTR inhibitors. The use of the CFTR inhibitor NPPB in the CFTR corrected CFDE cells (called CFDE/6Rep-CFTR cells) allowed us to identify functional differences due only to the Cl − transport activity of the CFTR -we selected the differential display spots in which the signal reverted to control values in the presence of NPPB -avoiding possible off-target effects of wild-type CFTR-transfected cells or effects exclusively due to the presence of the CFTR on the plasma membrane, as seems to be the case for regulated upon activation, normally T-expressed, and presumably secreted (RANTES) (Estell et al, 2003) and other PDZ (postsynaptic density protein 95/discs large/zona occludens protein-1 homologous)-dependent genes (Boucherot, Schreiber & Kunzelmann, 2001;Sharma et al, 2016;Watson et al, 2016). In this way, we found and characterized several CFTR-dependent genes, including c-Src (Gonzalez-Guerrico et al, 2002), mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4 (MTND4) (Valdivieso et al, 2007) and CDGSH iron sulfur domain 1 (CISD1) (Taminelli et al, 2008).…”