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Am J Physiol Lung Cell Mol Physiol. 2016 Dec 1;311(6):L1170-L1182. doi: 10.1152/ajplung.00363.2016. Epub 2016 Oct 28.

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

Author information

1
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
2
ARIEL Precision Medicine, Pittsburgh, Pennsylvania.
3
Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland.
4
Department of Surgical Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; and.
5
Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland.
6
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; gcutting@jhem.jhmi.edu.

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, Kd = 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 1417EENKVR1422 and the terminal 1478TRL1480 (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.

KEYWORDS:

CFTR; CFTR expression; NHERF; PDZ binding motif; protein-protein interaction

PMID:
27793802
PMCID:
PMC5206395
DOI:
10.1152/ajplung.00363.2016
[Indexed for MEDLINE]
Free PMC Article

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