ABCMdb

PMID: 20351098

Playford MP, Nurminen E, Pentikainen OT, Milgram SL, Hartwig JH, Stossel TP, Nakamura F
Cystic fibrosis transmembrane conductance regulator interacts with multiple immunoglobulin domains of filamin A.
J Biol Chem. 2010 May 28;285(22):17156-65. Epub 2010 Mar 29., 2010-05-28 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Trp19Cys ABCC7 p.Leu15Pro Point and deletion mutants of IgFLNa and CFTR informed by the models, including disease-causing mutations L15P and W19C, disrupted the binding interaction. Login to comment 21 ABCC7 p.Ser13Phe The CF-causing S13F mutation that disrupts this interaction in vivo leads to a reduced pool of CFTR at apical membrane sites and is prematurely delivered to lysosomes and degraded (11). Login to comment 68 ABCC7 p.Ser13Phe CFTR Peptide Pulldown Assay-Various concentration of FLNa constructs were incubated with increasing concentrations of wild-type or S13F mutant biotin⅐CFTR1-25 peptides FIGURE 1. Login to comment 72 ABCC7 p.Ser13Phe Biotinylated wild-type or S13F mutant CFTR peptides (1.0 ␮M) were incubated with the His-tagged FLNa fragments (0.1 ␮M). Login to comment 97 ABCC7 p.Ser13Phe Biotinylated wild-type or S13F mutant CFTR peptides (1.0 ␮M) were incubated with the GST-tagged FLNa fragments (0.1␮M). Login to comment 102 ABCC7 p.Ser13Phe Structure of Filamin A⅐CFTR Complex 17158 S13F mutant CFTR peptide (Fig. 1B), consistent with previous results (11). Login to comment 113 ABCC7 p.Ser13Phe ABCC7 p.Trp19Cys ABCC7 p.Leu15Pro Amino acid changes reflecting known CF mutations within this peptide (S13F, L15P, or W19C) prevented FLNa binding (Fig. 5C). Login to comment 118 ABCC7 p.Ser13Phe B, His-16-24 and deletion mutant (⌬17,19,21,23 deletion of IgFLNa17, 19, 21, and 23) were pulled down with increasing amounts of the wild-type (WT) or S13F mutant CFTR1-25 peptide. Login to comment 125 ABCC7 p.Ser13Phe ABCC7 p.Trp19Cys ABCC7 p.Leu15Pro Red and blue amino acids indicate residues mutated in CF patients (P5L, S13F, L15P, and W19C). Login to comment 133 ABCC7 p.Ser13Phe In this model, the S13F mutation of CFTR is incapable of forming hydrogen-bonding interactions with the main-chain carbonyl oxygen atom of Val-2472 (Fig. 5A), consistent with in vitro binding experiments (Figs. Login to comment 137 ABCC7 p.Trp19Cys ABCC7 p.Leu15Pro The model also predicted that CF-causing L15P and W19C but not P5L mutants of CFTR perturb the interaction with FLNa. Login to comment 138 ABCC7 p.Trp19Cys ABCC7 p.Leu15Pro Indeed, L15P and W19C synthetic CFTR1-20 peptides did not or barely bound FLNa (Fig. 5C). Login to comment 139 ABCC7 p.Trp19Cys The result also eliminated one of the alignments of CFTR (the second from the bottom in Fig. 4A), as W19C should not affect the binding interaction of this alignment. Login to comment 142 ABCC7 p.Trp19Cys ABCC7 p.Leu15Pro The wild-type, L15P, and W19C peptides were soluble at least at concentration of 1 mg/ml in PBS. Login to comment 153 ABCC7 p.Leu15Pro C, effect of CF-causing point mutations of CFTR on FLNa binding.OnemlofPBSwasaddedto1mgofbiotinylatedCFTR1-20peptides(wild-type,P5L,L15P,andW19C), andthesolutionwascentrifugedat15,000ϫgfor10minatroomtemperature.Allofthepeptidesweresoluble except for P5L mutant peptide (asterisk). Login to comment 169 ABCC7 p.Ser13Phe As anticipated, Myc-FLNa wild-type protein bound to wild-type, but not the S13F mutant CFTR peptide. Login to comment 185 ABCC7 p.Ser13Phe Lysates from these cells were incubated with N-terminal wild type (wt) (1-25) or mutant (1-25 S13F) CFTR peptides. Login to comment 192 ABCC7 p.Ser13Phe FLNa Repeats 9, 12, 17, 19, 21, and 23 Are Necessary for Optimal Surface Expression of CFTR-We previously reported that an interaction with FLNa is important for the expression of CFTR on the cell surface; the surface expression of the CFTR mutant S13F, which is defective in FLNa binding, is dramatically reduced. Login to comment 203 ABCC7 p.Ser13Phe As expected, co-expression of wild-type FLNa had no effect on the surface localization of the FLNa binding-defective CFTR Structure of Filamin A⅐CFTR Complex 17162 mutant S13F. Login to comment 227 ABCC7 p.Ser13Phe ABCC7 p.Trp19Cys ABCC7 p.Leu15Pro Structural Basis for Mutations That Impair the FLNa-CFTR Interaction-Three missense mutations in the FLNa-binding site of CFTR have been reported: S13F, L15P, and W19C. Login to comment 228 ABCC7 p.Ser13Phe The S13F mutation eliminates hydrogen bonding between the main-chain carbonyl oxygen atom of Val or Ala (Val-2472: IgFLNa23 numbering) in IgFLNa repeats 9, 12, 17, 19, 21, and 23, and the bulky Phe residue cannot stack with Val or Ala. Login to comment 229 ABCC7 p.Trp19Cys ABCC7 p.Leu15Pro The model also predicts that CF-causing L15P and W19C mutations in CFTR perturb the interaction with FLNa. Login to comment 230 ABCC7 p.Leu15Pro Mutation of Leu-15 to Pro dramatically changes the structure of the CFTR peptide. Login to comment 232 ABCC7 p.Ser13Phe ABCC7 p.Trp19Cys However, unlike the S13F mutant of CFTR, which is still expressed, albeit reduced ϳ50% compared with wild type, on the cell surface in a mature glycosylated form, the W19C mutant of CFTR remains immature and is retained in the endoplasmic reticulum (ER) (11), suggesting that it causes the disease without disrupting the interaction with FLNa. Login to comment 264 ABCC7 p.Trp19Cys The weak binding affinities determined by the Smith group could explain their inability to demonstrate the critical nature of the W19C mutation in repeat 21. Login to comment