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. 2010 Nov 12;285(46):35825-35.
doi: 10.1074/jbc.M110.131623. Epub 2010 Jul 28.

The cystic fibrosis-causing mutation deltaF508 affects multiple steps in cystic fibrosis transmembrane conductance regulator biogenesis

Patrick H Thibodeau  1 , John M Richardson 3rd, Wei Wang, Linda Millen, Jarod Watson, Juan L Mendoza, Kai Du, Sharon Fischman, Hanoch Senderowitz, Gergely L Lukacs, Kevin Kirk, Philip J Thomas
Affiliations

The cystic fibrosis-causing mutation deltaF508 affects multiple steps in cystic fibrosis transmembrane conductance regulator biogenesis

Patrick H Thibodeau et al. J Biol Chem. .

Abstract

The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ΔF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ΔF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ΔF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.

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Figures

FIGURE 1.
FIGURE 1.
Rescue of ΔF508 CFTR by -3M mutations. The introduction of the -3M mutations (G550E, R553M, R555K) rescues the trafficking defects associated with the ΔF508 mutation and restores near wild type function. A, a schematic of CFTR showing the five distinct domains and the relative locations of the ΔF508 and suppressor mutations is shown. Transmembrane domains are colored blue, and the nucleotide binding domains are yellow. The locations and residue numbers approximating N- and C-terminal domain boundaries are shown for reference. The location of the Phe-508 position is shown as a red circle, and the four RXR motifs are shown as orange squares. The sequence of the -3M combination of suppressor mutations is shown. B, Western blots of CFTR, expressed transiently in HEK-293 cells, show the maturation defects associated with mutation at position 508 and the rescue of these mutants by the inclusion of the -3M mutations. Band B, core glycosylated protein; Band C, complexly glycosylated protein. C, pulse-chase analysis of the CFTR constructs shows an increase in the production of Band C ΔF508 CFTR in the presence of the -3M suppressors. Wild type and ΔF508 CFTR, both with and without the -3M suppressors, are shown. D, functional rescue of the ΔF508 protein accompanies the rescue of CFTR ΔF508 trafficking as measured in whole cell and macropatch techniques of HEK-293T cells expressing the pCMV-CFTR constructs utilized in B. Measurements were made in the presence of PKA and ATP. Holding potential = −80 mV. Data shown are representative of at least three experiments.
FIGURE 2.
FIGURE 2.
Role of R555 in CFTR maturation. A, the -3M second site suppressor mutations are located within the NBD1 domain distal to the Phe-508 locus and do not directly contribute to the surface or structure altered by the deletion of the phenylalanine. Two views of NBD1 are shown rotated ∼90° with respect to one another. The Phe-508 position is shown in red, and the location of the second site suppressor positions are shown in blue. B, analysis of the RXR domains within NBD1 shows high conservation at the 555 position, consistent with its role in the ABC transporter signature motif but much lower conservation at the 516, 518, and 553 positions. C, substitution of the Arg-555 position alters wild type CFTR trafficking. The substitution of R555A, R555G, and R555T resulted in a marked reduction in the formation of band C CFTR, whereas the R555K, as measured by Western blotting of transiently transfected HEK-293 cells displays near wild type CFTR maturation. Data shown are representative of at least four independent experiments.
FIGURE 3.
FIGURE 3.
Influence of the ΔF508 and -3M mutations on NBD1 folding. A and B, expression of the NBD1 protein in E. coli is directly affected by the inclusion of the ΔF508 and the -3M mutations. NBD1 protein was expressed as a fusion with an N-terminal His-Smt3 and assayed by Western blotting after sonication and centrifugation. The soluble protein samples (SOL) are clarified by centrifugation at 40,000 × g relative centrifical force, and the whole cell lysates (WCL) are shown as controls for expression and loading. Representative data are shown. B, soluble production of NBD1 in HEK-293 cells is influenced by the introduction of the ΔF508 and -3M mutations, as measured by β-galactosidase enzymatic activity. Changes in signal intensity reflect changes in soluble NBD production and enzymatic activity. Quantification of the end point β-galactosidase data is presented. Data shown are the mean and S.D. from at least 12 experiments for each mutant.
FIGURE 4.
FIGURE 4.
Structural analysis of CFTR wild type, ΔF508, and suppressed proteins. Limited proteolysis utilizing either trypsin or chymotrypsin was performed to assess the stability of the cytosolic NBD domains. A, a chymotrypsin digestion of CFTR expressed in BHK cells was probed with either L12B4 (residues 385–410) or M3A7 (residues 1373–1382). B, a trypsin digest of CFTR expressed in BHK cells is shown probed with the NBD1 L12B4 and 660 antibodies. The proteolytically stable, putative NBD1 bands are highlighted within dashed lines.
FIGURE 5.
FIGURE 5.
NBD-NBD interactions in CFTR maturation. A, a schematic of ATP-binding sites and the associated binding and dimerization events determined in NBD proteins are shown. Composite sites (Walker A and B) and the ABC transporter signature motif are labeled A, B, and C, respectively. B, mutation of the composite ATP-binding site in NBD1, K464A, adversely affects the trafficking of wild type CFTR. Mutation of the equivalent position in NBD2, K1250A, has minimal effect on CFTR maturation. The NBD-dimer stabilizing mutation, E1371Q, does not dramatically alter the trafficking of the wild type or ΔF508 CFTR proteins when expressed transiently in HEK-293 cells. C, deletion of NBD2 does not dramatically alter the trafficking efficiency of wild type relative to ΔF508 either with or without the -3M mutations. Core (Band B) and complexly glycosylated protein (Band C) are indicated by B* and C* to reflect changes in molecular weight. Data shown are representative of four experiments.
FIGURE 6.
FIGURE 6.
NBD-TMD interactions in CFTR maturation. Mutations in ICL4 at position 1070 were evaluated for effects on the trafficking of wild type, ΔF508, and F508K CFTR, transiently expressed in HEK293 cells. As previously described, the presence of the higher molecular weight band, Band C, is indicative of proper folding and trafficking to the Golgi. A, a model of ICL4-NBD1 interactions was derived from sequence alignments and the Sav1866 crystal structure (2HYD). NBD is shown in green, and ICL4 is shown in blue. The Phe-508 and Arg-1070 residue side chains are shown in red. Two views, rotated by 90 degrees are shown. B, Western blots show the effects of the ICL4 Arg-1070 mutations on the trafficking of wild type, ΔF508, and F508K CFTR. C, the R1070W and -3M suppressor mutations were evaluated for their ability to rescue the ΔF508 mutation either independently or in combination. The inclusion of the -3M and R1070W mutations in combination rescued more ΔF508 CFTR than either suppressor set alone. Cells were cultured at 37 °C and evaluated by Western blotting using the M3A7 antibody. D, trafficking of the F508K missense protein was evaluated with the R1070W mutation. Trafficking of F508K was partially rescued by the R1070W mutation. Data shown are representative of at least four experiments.
FIGURE 7.
FIGURE 7.
A model for CFTR maturation and the influence of suppressor mutations in NBD1 and TMD2. The association of NBD1 with both CFTR TMD components and cellular quality control is in dynamic equilibrium. Decreases in NBD solubility as a result of inefficient folding due to the ΔF508 mutation results in an increase in QC-NBD association. The prolonged NBD-QC interaction ultimately leads to ERAD of CFTR; lower pathway. The -3M suppressor mutations decrease the QC-NBD interaction by stabilizing and/or solubilizing NBD1. The R1070W mutant in TMD2 suppresses ΔF508 by promoting the interactions between NBD1 and ICL4 as required for maturation. By relieving the QC-NBD interactions, the -3M and R1070W mutations promote CFTR maturation; upper pathway. The ER export- competent CFTR structures, full-length, and ΔNBD2 are shown boxed in the upper right.
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