Disabled-2 Protein Facilitates Assembly Polypeptide-2-independent Recruitment of Cystic Fibrosis Transmembrane Conductance Regulator to Endocytic Vesicles in Polarized Human Airway Epithelial Cells

Cihil, Kristine M.; Ellinger, P.; Fellows, Abigail; Stolz, Donna B.; Madden, Dean R.; Swiatecka‐Urban, Agnieszka

doi:10.1074/jbc.m112.341875

Cited by 30 publications

(69 citation statements)

References 39 publications

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“…Endocytic and recycling assays were performed as described previously (Cihil et al, 2012;Swiatecka-Urban et al, 2002). For both assays, plasma membrane proteins were first biotinylated at 4°C using membrane-impermeable and cleavable EZ-Link™ Sulfo-NHS-SS-Biotin (Pierce).…”

Section: Endocytic and Recycling Assaysmentioning

confidence: 99%

“…CFTR possesses two transmembrane domains (TMD1 and TMD2); two nucleotide-binding domains (NBD1 and NBD2) that bind to and hydrolyze ATP, regulating the gating of the channel; and a regulatory domain that contains multiple phosphorylation sites (Riordan, 2008). Cl − secretion through CFTR depends on the net balance of channel density at the cell surface and the activity of each individual channel (Cihil et al, 2012). CFTR dysfunction leads to cystic fibrosis, the most common life-threatening recessive disorder in Caucasians.…”

Section: Introductionmentioning

confidence: 99%

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EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1

Lobo

Amaral

Zaccolo

et al. 2016

Journal of Cell Science

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Cyclic AMP (cAMP) activates protein kinase A (PKA) but also the guanine nucleotide exchange factor 'exchange protein directly activated by cAMP' (EPAC1; also known as RAPGEF3). Although phosphorylation by PKA is known to regulate CFTR channel gatingthe protein defective in cystic fibrosis -the contribution of EPAC1 to CFTR regulation remains largely undefined. Here, we demonstrate that in human airway epithelial cells, cAMP signaling through EPAC1 promotes CFTR stabilization at the plasma membrane by attenuating its endocytosis, independently of PKA activation. EPAC1 and CFTR colocalize and interact through protein adaptor NHERF1 (also known as SLC9A3R1). This interaction is promoted by EPAC1 activation, triggering its translocation to the plasma membrane and binding to NHERF1. Our findings identify a new CFTR-interacting protein and demonstrate that cAMP activates CFTR through two different but complementary pathways -the well-known PKA-dependent channel gating pathway and a new mechanism regulating endocytosis that involves EPAC1. The latter might constitute a novel therapeutic target for treatment of cystic fibrosis.

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Section: Endocytic and Recycling Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1

Lobo

Amaral

Zaccolo

et al. 2016

Journal of Cell Science

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“…We have previously studied endocytosis and recycling of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in human airway epithelial cells and HEK293 cells [9][10][11][12][13][14][15] . The biotinylationbased assays described in the manuscript are optimized for examining endocytosis and recycling in epithelial cells cultured under polarizing conditions on semipermeable growth supports.…”

Section: Introductionmentioning

confidence: 99%

“…Endocytic assays are performed as previously described [9][10][11][12][13][14][15] . Cells are cultured on collagen coated semipermeable growth supports 11,14 .…”

Section: Introductionmentioning

confidence: 99%

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The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Cihil

Swiatecka‐Urban

2013

JoVE

Self Cite

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Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior ( i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

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Disabled‐2: A modular scaffold protein with multifaceted functions in signaling

Finkielstein

Capelluto

2016

BioEssays

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Disabled-2 (Dab2) is a multimodular scaffold protein with signaling roles in the domains of cell growth, trafficking, differentiation, and homeostasis. Emerging evidences place Dab2 as a novel modulator of cell-cell interaction; however, its mode of action has remained largely elusive. In this review, we highlight the relevance of Dab2 function in cell signaling and development and provide the most recent and comprehensive analysis of Dab2's action as a mediator of homotypical and heterotypical interactions. Accordingly, Dab-2 controls the extent of platelet aggregation through various motifs within its N-terminus. Dab2 interacts with the cytosolic tail of the integrin receptor blocking inside-out signaling, whereas extracellular Dab2 competes with fibrinogen for integrin α IIb β 3 receptor binding and, thus, modulates outside-in signaling. An additional level of regulation results from Dab2's association with cell surface lipids, an event that defines the extent of cell-cell interactions. As a multifaceted regulator, Dab2 acts as a mediator of endocytosis through its association with the [FY]xNPx[YF] motifs of internalized cell surface receptors, phosphoinositides, and clathrin. Other emerging roles of Dab2 include its participation in developmental mechanisms required for tissue formation and in modulation of immune responses. This review highlights the various novel mechanisms by which Dab2 mediates an array of signaling events with vast physiological consequences.

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Disabled-2 Protein Facilitates Assembly Polypeptide-2-independent Recruitment of Cystic Fibrosis Transmembrane Conductance Regulator to Endocytic Vesicles in Polarized Human Airway Epithelial Cells

Cited by 30 publications

References 39 publications

EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1

EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Disabled‐2: A modular scaffold protein with multifaceted functions in signaling

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