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. 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

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Free PMC article

A Sequence Upstream of Canonical PDZ-binding Motif Within CFTR COOH-terminus Enhances NHERF1 Interaction

Neeraj Sharma et al. Am J Physiol Lung Cell Mol Physiol. .
Free PMC article

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.

Figures

Fig. 1.
Fig. 1.
Expression of NHERF1 and CFTR in human eccrine sweat gland. A: immunohistochemistry of a human skin showing apical membrane immunoreactivity of NHERF1 in the reabsorptive ducts of the eccrine sweat glands (original magnification ×20). B: immunohistochemistry of a human lung tissue showing apical membrane immunoreactivity of NHERF1 in the bronchiolar epithelial cells (original magnification ×20). Arrow indicates NHERF1 staining. Inset shows magnification ×40. C: representative MS/MS spectrum for NHERF1 protein in eccrine sweat gland dissected out from human skin. D: heat map of gene expression of 37 genes (rows) in 6 tissues (columns) based on RNA-Seq data. *CFTR and NHERF1 (SLC9A3R1). RNA-Seq data were obtained from The Human Protein Atlas, from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database for the pancreas (accession numbers: ERR315466, ERR315429, ERR315436), skin (ERR315372, ERR315376, ERR315339), lung (ERR315424, ERR315444, ERR315487), colon (ERR315348, ERR315403, ERR315462), and kidney (ERR315443, ERR315494, ERR315468). Eccrine sweat gland RNA-Seq data were obtained in house. Color scale represents log10 transformed fragments per kilobase of transcript per million mapped reads (FPKM) + 1. Darker colors indicate higher expression.
Fig. 2.
Fig. 2.
CFTR COOH-terminus enhances formation of NHERF1 oligomers at nanomolar concentrations. A: binding of HA-tagged NHERF1 with full-length His-tagged NHERF1, PDZ1 (a.a. 11–101), and PDZ2 (a.a. 152–358) detected with anti-HA antibody (1:20,000). His-tagged NHERF1, PDZ1, and PDZ2 (500 ng each) were run on SDS-PAGE and transferred to the polyvinylidene difluoride membrane, and blot was overlaid with increasing concentrations of HA-tagged NHERF1 (1 nM, 10 nM, 100 nM, 500 nM, 1 μM, 1.5 μM, 2.0 μM, 2.5 μM, and 3 μM). Values are means ± SE (n = 3). B: effect of C111 on the binding of HA-NHERF1 with His-tagged NHERF1, PDZ1, and PDZ2. C111 refers to last 111 amino acids (a.a. 1370–1480) of human CFTR. In simultaneous overlay, HA-NHERF1 (10 nM) + C111 (20 nM) were overlaid at the same time onto the immobilized His-tagged NHERF1, PDZ1, and PDZ2. In sequential overlay, incubation with C111 (20 nM) was followed by HA-NHERF1 (10 nM) with a PBST wash in between (C111>>>>HA-NHERF1) or vice versa (HA-NHERF1>>>>C111). Incubation time was 2 h for both simultaneous and sequential overlays. Values are means ± SE (n = 5). ***P < 0.0001, one-way ANOVA (comparison with NHERF1 oligomerization by itself). C: coimmunoprecipitation of His-NHERF1 by HA-NHERF1 (10 nM each) in the absence or presence of C111 (20 nM). Graph illustrates fold increase in His-NHERF1 binding to HA-NHERF1 by C111. Values are means ± SE (n = 3). ***P < 0.001, one-way ANOVA. D: analysis of HA-NHERF1 oligomers in the presence of C111 or C111 mutants (C26, C111ΔTRL, and alanine substitution of 1417EENKVR1422) using a cross-linker N-hydroxysuccinimide ester (BS3). The reaction was quenched with ethanolamine (100 nM) at the time indicated. The 0-min sample had ethanolamine added before cross-linker. Oligomers of NHERF1 were detected by immunoblotting with anti-HA antibody.
Fig. 3.
Fig. 3.
Identification of a six-amino acid sequence in the CFTR COOH-terminus involved in enhancing NHERF1 oligomers. A: diagram of C111 (a.a. 1370–1480) deletions showing regions 1404–1425 and 1477–1480 (shaded) as essential for enhancing NHERF1 oligomers. Blots are the overlays of 10 nM HA-NHERF1 and 20 nM of each CFTR COOH-terminal deletion fragments C111-DelTRL (deletion a.a. 1478–1480), C77 (a.a. 1404–1480), C55 (a.a. 1426–1480), C26 (a.a. 1455–1480), and Del2 (a.a. 1370–1394, 1404-24, and 1477–1480) onto the immobilized His tagged NHERF1, PDZ1, and PDZ2 (500 ng each). Binding of HA-NHERF1 with His-NHERF1 and PDZ fragments was detected using anti-HA antibody (1:20,000). The graph illustrates the fraction of NHERF1 binding to the corresponding His tagged constructs in the presence of C111 deletion mutants. Values are means ± S.E. (n = 5). ***P < 0.0001, one-way ANOVA, as in Fig. 2B. B: diagram of C111 constructs with various alanine substitutions (white regions with A) used to identify a.a. 1417–1422 as essential for NHERF1 oligomers. Blots are the overlay assays as described above using the CFTR COOH-terminus (C111) with the following residues mutated to alanine: IE-QF (1404IEAMLECQQF1413), 1414LVI1416, 1417EEN1419, 1420KVR1422, and 1423QYD1425. The graph illustrates quantitation of the blots as described above. Values are means ± SE (n = 5). ***P < 0.0001. C: diagram of C111 constructs with single alanine amino acid substitutions at the corresponding position, as indicated by the labels on the figure. Graph represents the quantitation of the blot overlay assays as described above. Values are means ± SE (n = 3). ***P < 0.0001.
Fig. 4.
Fig. 4.
Formation of NHERF1 oligomers by 1417EENKVR1422 sequence in CFTR COOH-terminus is not independent of terminal PDZ-binding motif. A: effect of HA-NHERF1 binding to His-tagged NHERF1, PDZ1, and PDZ2 by preincubation with increasing concentration of C111. His-tagged NHERF1, PDZ1, and PDZ2 on the membrane were overlaid with C111 (20 nM) for 2 h. After PBST wash, the membrane was overlaid with HA-NHERF1 (10 nM) preincubated with C111 at 1 nM, 10 nM, 100 nM, or 200 nM for 2 h. Values are means ± SE (n = 3). B: effect of HA-NHERF1 binding to the His-tagged NHERF1, PDZ1, and PDZ2 by preincubation with C111 mutants. His-tagged NHERF1, PDZ1, and PDZ2 on the membrane were overlaid with C111 (20 nM) for 2 h. After PBST wash, the membrane was overlaid with HA-NHERF1 (10 nM) preincubated with C111, C111-Del2, C111-EENKVR-alanine mutant, and C111-DelTRL (100 nM each) for 2 h. Values are means ± SE (n = 3). ***P < 0.0001, one-way ANOVA.
Fig. 5.
Fig. 5.
1417EENKVR142 and PDZ-binding motif in full-length CFTR foster NHERF1 interaction in polarized epithelial cell line. Coimmunoprecipitation of myc-NHERF1 by HA-NHERF1 from the protein lysate of MDCK II-FRT cells stably expressing full-length GFP-CFTR-wild type, GFP-CFTR alanine mutants (1417EENKVR1422, 1417EEN1419, 1420KVR1422), and GFP-CFTR-DelTRL. Both myc-NHERF1 and HA-NHERF1 were transiently cotransfected. Graph illustrates fold increase in myc-NHERF1 binding to HA-NHERF1 in CFTR expressing MDCK cells compared with MDCK parentals. Values are means ± SE (n = 3). ***P < 0.0001, one-way ANOVA.
Fig. 6.
Fig. 6.
1417EENKVR1422 and PDZ-binding motif are required for apical localization of full-length CFTR in polarized MDCK II cells. Confocal images (×63 magnification) of immunostained MDCK-FRT cells imaged in the xz-plane (scanned apical to basal membrane) are shown. First column shows MDCK FRT cells transiently expressing full-length GFP wild-type CFTR as control. Second, third, and fourth columns show the effect of coexpression of myc-tagged C111, C111-EENKVR-alanine mutant, and C111-DelTRL, respectively, on the localization of full-length GFP wild-type CFTR. Expression of the myc-tagged C111 constructs is shown in red detected by Alexa Fluor 647, and GFP-tagged WT CFTR in green detected by Alexa Fluor 488. All cells were counterstained with DAPI (blue) to detect the nuclei. Scale bar, 5 μm.

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