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 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.

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.

Rectal epithelial expression of protein kinase A phosphorylation of cystic fibrosis transmembrane conductance regulator

1994 ◽  
Vol 106 (4) ◽  
pp. 890-898 ◽  
Author(s):  
Mrinalini C. Rao ◽  
Grace B. Bissonnette ◽  
Teresa Mahaffey ◽  
William B. Guggino ◽  
Jay L. Goldstein
Keyword(s):  
Cystic Fibrosis ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Kinase A

Dynasore inhibits removal of wild-type and ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) from the plasma membrane

2009 ◽  
Vol 421 (3) ◽  
pp. 377-385 ◽  
Author(s):  
Andrew Young ◽  
Martina Gentzsch ◽  
Cynthia Y. Abban ◽  
Ying Jia ◽  
Patricio I. Meneses ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Small Molecule ◽  
Short Exposure ◽  
Growth Media ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Cystic Fibrosis Transmembrane

Dynasore, a small molecule inhibitor of dynamin, was used to probe the role of dynamin in the endocytosis of wild-type and mutant CFTR (cystic fibrosis transmembrane conductance regulator). Internalization of both wild-type and ‘temperature-corrected’ ΔF508 CFTR was markedly inhibited by a short exposure to dynasore, implicating dynamin as a key element in the endocytic internalization of both wild-type and mutant CFTR. The inhibitory effect of dynasore was readily reversible upon washout of dynasore from the growth media. Corr-4 ({2-(5-chloro-2-methoxy-phenylamino)-4′-methyl-[4,5′]-bithiazolyl-2′-yl}-phenyl-methanonone), a pharmacological corrector of ΔF508 CFTR biosynthesis, caused a marked increase in the cell surface expression of mutant CFTR. Co-incubation of ΔF508 CFTR expressing cells with Corr-4 and dynasore caused a significantly greater level of cell surface CFTR than that observed in the presence of Corr-4 alone. These results argue that inhibiting the endocytic internalization of mutant CFTR provides a novel therapeutic target for augmenting the benefits of small molecule correctors of mutant CFTR biosynthesis.

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