scholarly journals Cooh-Terminal Truncations Promote Proteasome-Dependent Degradation of Mature Cystic Fibrosis Transmembrane Conductance Regulator from Post-Golgi Compartments

2001 ◽  
Vol 153 (5) ◽  
pp. 957-970 ◽  
Author(s):  
Mohamed Benharouga ◽  
Martin Haardt ◽  
Norbert Kartner ◽  
Gergely L. Lukacs
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cell Surface Biotinylation ◽  
Partially Unfolded ◽  
Cf Transmembrane Conductance Regulator

Impaired biosynthetic processing of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse–chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well.

Ileal smooth muscle dysfunction and remodeling in cystic fibrosis

2012 ◽  
Vol 303 (1) ◽  
pp. G1-G8 ◽  
Author(s):  
P.-A. Risse ◽  
L. Kachmar ◽  
O. S. Matusovsky ◽  
M. Novali ◽  
F. R. Gil ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Smooth Muscle ◽  
Electrical Field Stimulation ◽  
Mouse Strains ◽  
Maximal Velocity ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Velocity Of Shortening ◽  
Cf Transmembrane Conductance Regulator

Patients with cystic fibrosis (CF) often suffer from gastrointestinal cramps and intestinal obstruction. The CF transmembrane conductance regulator (CFTR) channel has been shown to be expressed in vascular and airway smooth muscle (SM). We hypothesized that the absence of CFTR expression alters the gastrointestinal SM function and that these alterations may show strain-related differences in the mouse. The aim of this study was to measure the contractile properties of the ileal SM in two CF mouse models. CFTR−/− and CFTR+/+ mice were studied on BALB/cJ and C57BL/6J backgrounds. Responsiveness of ileal strips to electrical field stimulation (EFS), methacholine (MCh), and isoproterenol was measured. The mass and the cell density of SM layers were measured morphometrically. Finally, the maximal velocity of shortening (Vmax) and the expression of the fast (+)insert myosin isoform were measured in the C57BL/6J ileum. Ileal hyperreactivity was observed in response to EFS and MCh in CFTR−/− compared with CFTR+/+ mice in C57BL/6J background. This latter observation was not reproduced by acute inhibition of CFTR with CFTRinh172. BALB/cJ CFTR−/− mice exhibited a significant increase of SM mass with a lower density of cells compared with CFTR+/+, whereas no difference was observed in the C57BL/6J background. In addition, in this latter strain, ileal strips from CFTR−/− exhibited a significant increase in Vmax compared with control and expressed a greater proportion of the fast (+)insert SM myosin isoform with respect to total myosin. BALB/cJ CFTR−/− ilium had a greater relaxation to isoproterenol than the CFTR+/+ mice when precontracted with EFS, but no difference was observed in response to exogeneous MCh. In vivo, the lack of CFTR expression induces a different SM ileal phenotype in different mouse strains, supporting the importance of modifier genes in determining intestinal SM properties.

scholarly journals Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation

1997 ◽  
Vol 328 (2) ◽  
pp. 353-361 ◽  
Author(s):  
L. Gergely LUKACS ◽  
Gersana SEGAL ◽  
Norbert KARTNER ◽  
Sergio GRINSTEIN ◽  
Fred ZHANG
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Protein Kinase ◽  
Cell Surface ◽  
Protein Phosphorylation ◽  
Chinese Hamster ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Hypertonic Medium ◽  
Cystic Fibrosis Transmembrane

Although the cystic fibrosis transmembrane conductance regulator (CFTR) is primarily implicated in the regulation of plasma-membrane chloride permeability, immunolocalization and functional studies indicate the presence of CFTR in the endosomal compartment. The mechanism of CFTR delivery from the cell surface to endosomes is not understood. To delineate the internalization pathway, both the rate and extent of CFTR accumulation in endosomes were monitored in stably transfected Chinese hamster ovary (CHO) cells. The role of clathrin-dependent endocytosis was assessed in cells exposed to hypertonic medium, potassium depletion or intracellular acid-load. These treatments inhibited clathrin-dependent endocytosis by > 90%, as verified by measurements of 125I-transferrin uptake. Functional association of CFTR with newly formed endosomes was determined by an endosomal pH dissipation protocol [Lukacs, Chang, Kartner, Rotstein, Riordan and Grinstein (1992) J. Biol. Chem. 267, 14568-14572]. As a second approach, endocytosis of CFTR was determined after cell-surface biotinylation with the cleavable sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. Both the biochemical and the functional assays indicated that arresting the formation of clathrin-coated vesicles inhibited the retrieval of the CFTR from the plasma membrane to endosomes. An overall arrest of membrane traffic cannot account for the inhibition of CFTR internalization, since the fluid-phase endocytosis was not effected by the treatments used. Thus the efficient, constitutive internalization of surface CFTR (5% per min) occurs, predominantly by clathrin-dependent endocytosis. Stimulation of protein phosphorylation by cAMP-dependent protein kinase A and by protein kinase C decreased the rate of internalization of cell-surface biotinylated CFTR, and contributed to a substantial diminution of the internal CFTR pool compared with that of unstimulated cells. These results suggest that the rate of CFTR internalization may participate in the determination of the CFTR channel density, and consequently, of the cAMP-stimulated chloride conductance of the plasma membrane.

scholarly journals The human DnaJ homologue (Hdj)-1/heat-shock protein (Hsp) 40 co-chaperone is required for the in vivo stabilization of the cystic fibrosis transmembrane conductance regulator by Hsp70

2002 ◽  
Vol 366 (3) ◽  
pp. 797-806 ◽  
Author(s):  
Carlos M. FARINHA ◽  
Paulo NOGUEIRA ◽  
Filipa MENDES ◽  
Deborah PENQUE ◽  
Margarida D. AMARAL
Keyword(s):  
Cystic Fibrosis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Hsc 70 ◽  
Immature Form ◽  
Cystic Fibrosis Transmembrane

The CFTR (cystic fibrosis transmembrane conductance regulator) gene, defective in cystic fibrosis, codes for a polytopic apical membrane protein functioning as a chloride channel. Wild-type (wt) CFTR matures inefficiently and CFTR with a deletion of Phe-508 (F508del), the most frequent mutation, is substantially retained as a core-glycosylated intermediate in the endoplasmic reticulum (ER), probably due to misfolding that is recognized by the cellular quality control machinery involving molecular chaperones. Here, we overexpressed the heat-shock protein (Hsp) 70 chaperone in vivo and observed no changes in degradation rate of the core-glycosylated form, nor in the efficiency of its conversion into the fully glycosylated form, for either wt- or F508del-CFTR, contrary to previous in vitro studies on the affect of heat-shock cognate (Hsc) 70 on part of the first nucleotide-binding domain of CFTR. Co-transfection of Hsp70 with its co-chaperone human DnaJ homologue (Hdj)-1/Hsp40, however, stabilizes the immature form of wt-CFTR, but not of F508del-CFTR, suggesting that these chaperones act on a wt-specific conformation. As the efficiency of conversion into the fully glycosylated form is not increased under Hsp70/Hdj-1 overexpression, the lack of these two chaperones does not seem to be critical for CFTR maturation and ER retention. The effects of 4-phenylbutyrate and deoxyspergualin, described previously to interfere with Hsp70 binding, were also tested upon CFTR degradation and processing. The sole effect observed was destabilization of F508del-CFTR.

scholarly journals Sorting Nexin 27 (SNX27): A Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking

2018 ◽  
Author(s):  
Mark I. McDermott ◽  
William R. Thelin ◽  
Yun Chen ◽  
Patrick T. Lyons ◽  
Gabrielle Reilly ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Pdz Domain ◽  
Type I ◽  
Apical Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Sorting Nexin ◽  
Cystic Fibrosis Transmembrane

AbstractThe underlying defect in cystic fibrosis is mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel expressed at the apical surface of lung epithelia. In addition to its export and maintenance at the cell surface, CFTR regulation involves repeated cycles of transport through the endosomal trafficking system, including endocytosis and recycling. Many of the known disease mutations cause CFTR intracellular trafficking defects that result in failure of ion channel delivery to the apical plasma membrane. Corrective maneuvers directed at improving transport to the plasma membrane are thwarted by rapid internalization and degradation of the mutant CFTR proteins. The molecular mechanisms involved in these processes are not completely understood but may involve protein-protein interactions with the C-terminal type I PDZ-binding motif of CFTR. Using a proteomic approach, we identify sorting nexin 27 (SNX27) as a novel CFTR binding partner in human airway epithelial Calu-3 cells. SNX27 and CFTR interact directly, with the SNX27 PDZ domain being both necessary and sufficient for this interaction. SNX27 co-localizes with internalized CFTR at sub-apical endosomal sites in polarized Calu-3 cells, and either knockdown of the endogenous SNX27, or over-expression of a dominant-negative SNX27 mutant, resulted in significant decreases in cell surface CFTR levels. CFTR internalization was not affected by SNX27 knockdown, but defects were observed in the recycling arm of CFTR trafficking through the endosomal system. Furthermore, knockdown of SNX27 in Calu-3 cells resulted in significant decreases in CFTR protein levels, consistent with degradation of the internalized pool. These data identify SNX27 as a physiologically significant regulator of CFTR trafficking and homeostasis in epithelial cells.

scholarly journals Impaired Renal HCO3- Excretion in Cystic Fibrosis

2020 ◽  
Vol 31 (8) ◽  
pp. 1711-1727 ◽  
Author(s):  
Peder Berg ◽  
Samuel L. Svendsen ◽  
Mads V. Sorensen ◽  
Casper K. Larsen ◽  
Jesper Frank Andersen ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Metabolic Alkalosis ◽  
Drug Efficacy ◽  
Transmembrane Conductance Regulator ◽  
The Novel ◽  
Transmembrane Conductance ◽  
Collecting Ducts ◽  
Before And After

BackgroundPatients with cystic fibrosis (CF) do not respond with increased urinary HCO3− excretion after stimulation with secretin and often present with metabolic alkalosis.MethodsBy combining RT-PCR, immunohistochemistry, isolated tubule perfusion, in vitro cell studies, and in vivo studies in different mouse models, we elucidated the mechanism of secretin-induced urinary HCO3− excretion. For CF patients and CF mice, we developed a HCO3- drinking test to assess the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in urinary HCO3-excretion and applied it in the patients before and after treatment with the novel CFTR modulator drug, lumacaftor-ivacaftor.Resultsβ-Intercalated cells express basolateral secretin receptors and apical CFTR and pendrin. In vivo application of secretin induced a marked urinary alkalization, an effect absent in mice lacking pendrin or CFTR. In perfused cortical collecting ducts, secretin stimulated pendrin-dependent Cl−/HCO3− exchange. In collecting ducts in CFTR knockout mice, baseline pendrin activity was significantly lower and not responsive to secretin. Notably, patients with CF (F508del/F508del) and CF mice showed a greatly attenuated or absent urinary HCO3−-excreting ability. In patients, treatment with the CFTR modulator drug lumacaftor-ivacaftor increased the renal ability to excrete HCO3−.ConclusionsThese results define the mechanism of secretin-induced urinary HCO3− excretion, explain metabolic alkalosis in patients with CF, and suggest feasibility of an in vivo human CF urine test to validate drug efficacy.

scholarly journals Non-native Conformers of Cystic Fibrosis Transmembrane Conductance Regulator NBD1 Are Recognized by Hsp27 and Conjugated to SUMO-2 for Degradation

2015 ◽  
Vol 291 (4) ◽  
pp. 2004-2017 ◽  
Author(s):  
Xiaoyan Gong ◽  
Annette Ahner ◽  
Ariel Roldan ◽  
Gergely L. Lukacs ◽  
Patrick H. Thibodeau ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Ring Finger ◽  
Transmembrane Conductance Regulator ◽  
Native Conformation ◽  
Transmembrane Conductance ◽  
Sumoylation Site ◽  
Sumo Modification ◽  
Cystic Fibrosis Transmembrane

A newly identified pathway for selective degradation of the common mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del, is initiated by binding of the small heat shock protein, Hsp27. Hsp27 collaborates with Ubc9, the E2 enzyme for protein SUMOylation, to selectively degrade F508del CFTR via the SUMO-targeted ubiquitin E3 ligase, RNF4 (RING finger protein 4) (1). Here, we ask what properties of CFTR are sensed by the Hsp27-Ubc9 pathway by examining the ability of NBD1 (locus of the F508del mutation) to mimic the disposal of full-length (FL) CFTR. Similar to FL CFTR, F508del NBD1 expression was reduced 50–60% by Hsp27; it interacted preferentially with the mutant and was modified primarily by SUMO-2. Mutation of the consensus SUMOylation site, Lys447, obviated Hsp27-mediated F508del NBD1 SUMOylation and degradation. As for FL CFTR and NBD1 in vivo, SUMO modification using purified components in vitro was greater for F508del NBD1 versus WT and for the SUMO-2 paralog. Several findings indicated that Hsp27-Ubc9 targets the SUMOylation of a transitional, non-native conformation of F508del NBD1: (a) its modification decreased as [ATP] increased, reflecting stabilization of the nucleotide-binding domain by ligand binding; (b) a temperature-induced increase in intrinsic fluorescence, which reflects formation of a transitional NBD1 conformation, was followed by its SUMO modification; and (c) introduction of solubilizing or revertant mutations to stabilize F508del NBD1 reduced its SUMO modification. These findings indicate that the Hsp27-Ubc9 pathway recognizes a non-native conformation of mutant NBD1, which leads to its SUMO-2 conjugation and degradation by the ubiquitin-proteasome system.

Rme-1 regulates the recycling of the cystic fibrosis transmembrane conductance regulator

2003 ◽  
Vol 285 (5) ◽  
pp. C1009-C1018 ◽  
Author(s):  
John A. Picciano ◽  
Nadia Ameen ◽  
Barth D. Grant ◽  
Neil A. Bradbury
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Dominant Negative ◽  
Traffic Information ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Endosomal Recycling ◽  
Carboxyl Terminal ◽  
Cystic Fibrosis Transmembrane

Endocytic motifs in the carboxyl terminus of cystic fibrosis transmembrane conductance regulator (CFTR) direct internalization from the plasma membrane by clathrin-mediated endocytosis. However, the fate of such internalized CFTR has remained unknown. Internalized membrane proteins can be either targeted for degradation or recycled back to the plasma membrane. Using cell surface biotinylation and antibody uptake studies, we show that CFTR undergoes constitutive endocytosis and recycling back to the plasma membrane. Expression of dominant negative Rme-1 (a protein that regulates exit from the endosomal recycling compartment) in CFTR-expressing cells results in the expansion of recycling compartments. Transferrin, a marker for the endosomal recycling compartment, and CFTR accumulate in these enlarged recycling endosomes. Such accumulation leads to a loss of cell surface CFTR because it is prevented from being recycled back to the cell surface. In contrast, traffic of the low-density lipoprotein (LDL) is unaffected by the expression of dominant negative Rme-1. In addition, chimeras containing the extracellular domain of the transferrin receptor and the carboxyl terminal tail of CFTR also enter Rme-1-regulated recycling compartments and accumulate in these compartments containing dominant negative Rme-1, suggesting that in addition to endocytic signals, the carboxyl terminal tail of CFTR also contains intracellular traffic information.

Characterization of the internalization pathways for the cystic fibrosis transmembrane conductance regulator

1999 ◽  
Vol 276 (4) ◽  
pp. L659-L668 ◽  
Author(s):  
Neil A. Bradbury ◽  
John A. Clark ◽  
Simon C. Watkins ◽  
Christopher C. Widnell ◽  
H. Skipper Smith ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Apical Membrane ◽  
Airway Epithelial Cells ◽  
Coated Vesicle ◽  
Apical Plasma Membrane ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Coated Pits

Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel give rise to the most common lethal genetic disease of Caucasian populations, CF. Although the function of CFTR is primarily related to the regulation of apical membrane chloride permeability, biochemical, immunocytochemical, and functional studies indicate that CFTR is also present in endosomal and trans Golgi compartments. The molecular pathways by which CFTR is internalized into intracellular compartments are not fully understood. To define the pathways for CFTR internalization, we investigated the association of CFTR with two specialized domains of the plasma membrane, clathrin-coated pits and caveolae. Internalization of CFTR was monitored after cell surface biotinylation and quantitation of cell surface CFTR levels after elution of cell lysates from a monomeric avidin column. Cell surface levels of CFTR were determined after disruption of caveolae or clathrin-coated vesicle formation. Biochemical assays revealed that disrupting the formation of clathrin-coated vesicles inhibited the internalization of CFTR from the plasma membrane, resulting in a threefold increase in the steady-state levels of cell surface CFTR. In contrast, the levels of cell surface CFTR after disruption of caveolae were not different from those in control cells. In addition, although our studies show the presence of caveolin at the apical membrane domain of human airway epithelial cells, we were unable to detect CFTR in purified caveolae. These results suggest that CFTR is constitutively internalized from the apical plasma membrane via clathrin-coated pits and that CFTR is excluded from caveolae.

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