title: 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, 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 sequence1417EENKVR1422and the terminal1478TRL1480(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.
authors: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors: Neeraj
authors: N
authors: Sharma
authors: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.|ARIEL Precision Medicine, Pittsburgh, Pennsylvania.
authors: Jessica
authors: J
authors: LaRusch
authors: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors: Patrick R
authors: PR
authors: Sosnay
authors: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors: Laura B
authors: LB
authors: Gottschalk
authors: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors: Andrea P
authors: AP
authors: Lopez
authors: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors: Matthew J
authors: MJ
authors: Pellicore
authors: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors: Taylor
authors: T
authors: Evans
authors: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors: Emily
authors: E
authors: Davis
authors: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors: Melis
authors: M
authors: Atalar
authors: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors: Chan-Hyun
authors: CH
authors: Na
authors: Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors: Gedge D
authors: GD
authors: Rosson
authors: Department of Surgical Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; and.
authors: Deborah
authors: D
authors: Belchis
authors: Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland.
authors: Michal
authors: M
authors: Milewski
authors: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors: Akhilesh
authors: A
authors: Pandey
authors: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; gcutting@jhem.jhmi.edu.
authors: Garry R
authors: GR
authors: Cutting
mesh_terms: D000328:Adult; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016764:Cell Polarity; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism*; D004285:Dogs; D004439:Eccrine Glands / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D054731:PDZ Domains; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D040901:Proteomics; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: CFTR; CFTR expression; NHERF; PDZ binding motif; protein-protein interaction