Mutations in the Amino Terminus of the Cystic Fibrosis Transmembrane Conductance Regulator Enhance Endocytosis

Jurkuvenaite, Asta; Varga, K; Nowotarski, Krzysztof; Kirk, Kevin L.; Sorscher, Eric J.; Li, Yao; Clancy, John; Bebök, Zsuzsa; Collawn, James F.

doi:10.1074/jbc.m508131200

Cited by 35 publications

(54 citation statements)

References 33 publications

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“…Previous studies have shown that increasing CFTR internalization or decreasing endocytic recycling do not necessarily affect the metabolic stability of mature CFTR. For example, the R31L or N287Y mutations may introduce a nonnative internalization motif in CFTR and result in increased plasma membrane internalization (8,9). Additionally, the deletion of the C-terminal PDZ binding motif results in decreased apical surface expression, which reflects less efficient recycling and not a change in endocytosis rates (60).…”

Section: Discussionmentioning

confidence: 99%

“…CFTR mutations are functionally categorized by defects in early biosynthesis and folding (class I, II, and V), impaired chloride channel activity (class III and IV), or destabilization of the mature protein (1)(2)(3). The study of CFTR mutations has provided important insights into the regulation of virtually all aspects of CFTR biology including biosynthesis (4,5), gating (6,7), endocytosis (8,9), and degradation (3,10). Current therapeutic strategies are targeting the basic defects associated with mutant CFTR proteins with the goal of correcting these defects in vivo (11,12).…”

Section: Introductionmentioning

confidence: 99%

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Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR

Thelin¹,

Chen²,

Gentzsch³

et al. 2007

J. Clin. Invest.

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The role of the cystic fibrosis transmembrane conductance regulator (CFTR) as a cAMP-dependent chloride channel on the apical membrane of epithelia is well established. However, the processes by which CFTR is regulated on the cell surface are not clear. Here we report the identification of a protein-protein interaction between CFTR and the cytoskeletal filamin proteins. Using proteomic approaches, we identified filamins as proteins that associate with the extreme CFTR N terminus. Furthermore, we identified a disease-causing missense mutation in CFTR, serine 13 to phenylalanine (S13F), which disrupted this interaction. In cells, filamins tethered plasma membrane CFTR to the underlying actin network. This interaction stabilized CFTR at the cell surface and regulated the plasma membrane dynamics and confinement of the channel. In the absence of filamin binding, CFTR was internalized from the cell surface, where it prematurely accumulated in lysosomes and was ultimately degraded. Our data demonstrate what we believe to be a previously unrecognized role for the CFTR N terminus in the regulation of the plasma membrane stability and metabolic stability of CFTR. In addition, we elucidate the molecular defect associated with the S13F mutation.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR

Thelin¹,

Chen²,

Gentzsch³

et al. 2007

J. Clin. Invest.

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“…Jurkuvenaite et al (42) recently studied R31C and R31L, two CF mutations in the N terminus of CFTR that affect the first AFT (R 29 QR 31 ). Their data demonstrate enhanced internalization from the cell surface, which suggests that some of the reduction in the steady-state levels of band C that we observed with F508del-4RK-CFTR might be explained by enhanced internalization.…”

Section: Rk and G550e Rescue F508del-cftr Channel Gating With Differentmentioning

confidence: 99%

Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms

Roxo-Rosa

Schmidt

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

106

131

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The revertant mutations G550E and 4RK [the simultaneous mutation of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K, and R766K] rescue the cell surface expression and function of F508del-cystic fibrosis (CF) transmembrane conductance regulator (-CFTR), the most common CF mutation. Here, we investigate their mechanism of action by using biochemical and functional assays to examine their effects on F508del and three CF mutations (R560T, A561E, and V562I) located within a conserved region of the first nucleotide-binding domain (NBD1) of CFTR. Like F508del, R560T and A561E disrupt CFTR trafficking. G550E rescued the trafficking defect of A561E but not that of R560T. Of note, the processing and function of V562I were equivalent to that of wild-type (wt)-CFTR, suggesting that V562I is not a disease-causing mutation. Biochemical studies revealed that 4RK generates higher steady-state levels of mature CFTR (band C) for wtand V562I-CFTR than does G550E. Moreover, functional studies showed that the revertants rescue the gating defect of F508del-CFTR with different efficacies. 4RK modestly increased F508del-CFTR activity by prolonging channel openings, whereas G550E restored F508del-CFTR activity to wt levels by altering the duration of channel openings and closings. Thus, our data suggest that the revertants G550E and 4RK might rescue F508del-CFTR by distinct mechanisms. G550E likely alters the conformation of NBD1, whereas 4RK allows F508del-CFTR to escape endoplasmic reticulum retention͞retrieval mediated by AFTs. We propose that AFTs might constitute a checkpoint for endoplasmic reticulum quality control. endoplasmic reticulum quality control ͉ folding ͉ membrane traffic ͉ arginine-framed motifs ͉ trafficking signals

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“…The cells were then transferred to a 378C incubator and cultured for 0 to 12 hours in the presence or absence of 10 mM corr-4a in OPTIMEM (Invitrogen) medium supplemented with 2% (vol/vol) FBS. Cell surface glycoproteins were biotinylated as described previously (23). CFTR was immunoprecipitated with a monoclonal antibody to CFTR (24-1) coupled to protein G agarose, run on 8% SDS-PAGE gels, and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA).…”

Section: Cftr Cell Surface Half-life Experimentsmentioning

confidence: 99%

“…Immunoprecipitation reactions were performed for 2 hours at 48C. Immunoprecipitated proteins were in vitro phosphorylated and visualized by SDS-PAGE followed by autoradiography and phosphorimaging, or by SDS-PAGE followed by Western blotting (23).…”

Section: Immunoprecipitationmentioning

confidence: 99%

Functional Stability of Rescued ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells

Jurkuvenaite

Chen²,

Bartoszewski³

et al. 2010

Am J Respir Cell Mol Biol

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The most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, DF508, results in the production of a misfolded protein that is rapidly degraded. The mutant protein is temperature sensitive, and prior studies indicate that the low-temperature-rescued channel is poorly responsive to physiological stimuli, and is rapidly degraded from the cell surface at 378C. In the present studies, we tested the effect of a recently characterized pharmacological corrector, 2-(5-chloro-2-methoxyphenylamino)-49-methyl-[4,59bithiazolyl-29-yl]-phenyl-methanone (corr-4a), on cell surface stability and function of the low-temperature-rescued DF508 CFTR. We demonstrate that corr-4a significantly enhanced the protein stability of rescued DF508 CFTR for up to 12 hours at 378C (P , 0.05). Using firefly luciferase-based reporters to investigate the mechanisms by which low temperature and corr4a enhance rescue, we found that low-temperature treatment inhibited proteasomal function, whereas corr-4a treatment inhibited the E1-E3 ubiquitination pathway. Ussing chamber studies indicated that corr-4a increased the cAMP-mediated DF508 CFTR response by 61% at 6 hours (P , 0.05), but not at later time points. However, addition of the CFTR channel activator, 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol, significantly augmented cAMPstimulated currents, revealing that the biochemically detectable cell surface DF508 CFTR could be stimulated under the right conditions. Our studies demonstrate that stabilizing rescued DF508 CFTR was not sufficient to obtain maximal DF508 CFTR function in airway epithelial cells. These results strongly support the idea that maximal correction of DF508 CFTR requires a chemical corrector that: (1) promotes folding and exit from the endoplasmic reticulum; (2) enhances surface stability; and (3) improves channel activity.

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Mutations in the Amino Terminus of the Cystic Fibrosis Transmembrane Conductance Regulator Enhance Endocytosis

Cited by 35 publications

References 33 publications

Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR

Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR

Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms

Functional Stability of Rescued ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells

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