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

Section: Discussionmentioning

confidence: 99%

“…Previous studies have demonstrated that CFTR is internalized from the cell surface (1-3) through clathrin-coated pits (2,4). Furthermore, CFTR has been shown to interact with PDZ-domain-containing proteins at its COOH terminus (5,6) and syntaxin 1A at its NH 2 terminus (7,8).…”

mentioning

confidence: 99%

Ablation of Internalization Signals in the Carboxyl-terminal Tail of the Cystic Fibrosis Transmembrane Conductance Regulator Enhances Cell Surface Expression

Peter¹,

Varga²,

Bebök³

et al. 2002

“…Functionally active CFTR can be found in purified clathrin-coated vesicles derived from cultured cells and native tissues that constitutively express CFTR (11). Furthermore, studies examining CFTR internalization demonstrate that perturbation of clathrin-coated vesicle formation inhibits the removal of CFTR from the plasma membrane (12,13). More recently, we have shown that in polarized epithelial cells, CFTR is internalized solely through clathrin-coated vesicles and is excluded from other endocytic pathways such as caveolae (12), suggesting that CFTR is specifically recruited to the clathrin-coated vesicle pathway for endocytosis.…”

mentioning

confidence: 95%

“…Thus, immunologically detectable and functionally active CFTR is present in clathrin-coated vesicles isolated from both cultured epithelial cells and intact native tissue (11,29). Moreover, we and others have recently shown that inhibition of clathrin-mediated endocytosis causes a retention of CFTR at the cell surface (12,13). In contrast, disruption of alternate internalization pathways, such as caveolae, has no 1, 3, 5) or GST-CT (lanes 2, 4, 6) was incubated with adaptors isolated from bovine brain.…”

Section: Conformational Models Of the Tyrosine-based Motif In The Carmentioning

confidence: 99%

The Carboxyl Terminus of the Cystic Fibrosis Transmembrane Conductance Regulator Binds to AP-2 Clathrin Adaptors

Weixel

Bradbury

2000

The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid and efficient endocytosis. Since functionally active CFTR is found in purified clathrin-coated vesicles isolated from both cultured epithelial cells and intact epithelial tissues, we investigated the molecular mechanisms whereby CFTR could enter such endocytic clathrin-coated vesicles. In vivo cross-linking and in vitro pull-down assays show that full-length CFTR binds to the endocytic adaptor complex AP-2. Fusion proteins containing the carboxyl terminus of CFTR (amino acids 1404 -1480) were also able to bind AP-2 but did not bind the Golgi-specific adaptor complex AP-1. Substitution of an alanine residue for tyrosine at position 1424 significantly reduced the ability of AP-2 to bind the carboxyl terminus of CFTR; however, mutation to a phenylalanine residue (an amino acid found at position 1424 in dogfish CFTR) did not perturb AP-2 binding. Secondary structure predictions suggest that Tyr 1424 is present in a ␤-turn conformation, a conformation disrupted by alanine but not phenylalanine. Together, these data suggest that the carboxyl terminus of CFTR contains a tyrosine-based internalization signal that interacts with the endocytic adaptor complex AP-2 to facilitate efficient entry of CFTR into clathrin-coated vesicles.

“…Compelling evidence indicates that CFTR enters endosomal compartments through the clathrinmediated endocytic pathway (9 -12). Furthermore, perturbation of clathrin-coated vesicle formation inhibits the removal of CFTR from the plasma membrane (12,13).…”

mentioning

confidence: 99%

μ2 Binding Directs the Cystic Fibrosis Transmembrane Conductance Regulator to the Clathrin-mediated Endocytic Pathway

Weixel

Bradbury

2001

The cystic fibrosis transmembrane conductance regulator (CFTR) contains a conserved tyrosine-based internalization motif, 1424 YDSI, which interacts with the endocytic clathrin adaptor complex, AP-2, and is required for its efficient endocytosis. Although direct interactions between several endocytic sequences and the medium chain and endocytic clathrin adaptor complexes have been shown by protein-protein interaction assays, whether all these interactions occur in vivo or are physiologically important has not always been addressed. Here we show, using both in vitro and in vivo assays, a physiologically relevant interaction between CFTR and the subunit of AP-2. Cross-linking experiments were performed using photoreactive peptides containing the YDSI motif and purified adaptor complexes. CFTR peptides cross-linked a 50-kDa subunit of purified AP-2 complexes, the apparent molecular mass of 2. Furthermore, isolated 2 bound to the sorting motif, YDSI, both in cross-linking experiments and glutathione S-transferase pull-down experiments, confirming that 2 mediates the interaction between CFTR and AP-2 complexes. Inducible overexpression of dominant-negative 2 in HeLa cells results in AP-2 complexes that fail to interact with CFTR. Moreover, internalization of CFTR in mutant cells is greatly reduced compared with wild type HeLa cells. These results indicate that the AP-2 endocytic complex selectively interacts with the conserved tyrosine-based internalization signal in the carboxyl terminus of CFTR, YDSI. Furthermore, this interaction is mediated by the 2 subunit of AP-2 and mutations in 2 that block its interaction with YDSI inhibit the incorporation of CFTR into the clathrin-mediated endocytic pathway.