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

Peter, Krisztina; Varga, K; Bebök, Zsuzsa; McNicholas-Bevensee, Carmel M.; Schwiebert, Lisa M.; Sorscher, Eric J.; Schwiebert, Erik M.; Collawn, James F.

doi:10.1074/jbc.m209275200

Cited by 30 publications

(43 citation statements)

References 24 publications

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“…In MDCK cells, removing the last three amino acids reduced the half-life of GFP-CFTR in the apical membrane (14). Earlier studies showed that mutating the phenylalanine-based (1,424-1,427) and di-leucine-based (1,430-1,431) motifs reduced endocytosis (17,18). Our functional studies suggest that any alteration in biosynthesis, stability, or endocytosis had a relatively small effect in airway epithelia.…”

Section: Discussionmentioning

confidence: 64%

“…We focused on five parts of this sequence ( (16,17). We generated constructs that deleted combinations of these five regions (Fig.…”

Section: C-terminal Deletion Constructsmentioning

confidence: 99%

“…1 A) interacts with the endocytic adaptor complex AP-2 and mediates entry via clathrin-coated vesicles (3). Mutating this motif reduced CFTR endocytosis and increased cell surface expression (16,17). In addition, a di-leucine-based motif may contribute to endocytosis (18).…”

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confidence: 99%

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Effects of C-terminal deletions on cystic fibrosis transmembrane conductance regulator function in cystic fibrosis airway epithelia

Ostedgaard

Randak

Rokhlina

et al. 2003

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

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To better understand the function of the conserved C terminus of the cystic fibrosis (CF) transmembrane conductance regulator, we studied constructs containing deletions in the C-terminal tail. When expressed in well differentiated CF airway epithelia, each construct localized predominantly to the apical membrane and generated transepithelial Cl ؊ current. The results suggested that neither the C-terminal PSD-95͞Discs-large͞ZO-1 (PDZ)-interacting motif nor other C-terminal sequences were absolutely required for apical expression in airway epithelia. Surprisingly, deleting an acidic cluster near the C terminus reduced both channel opening rate and transepithelial Cl ؊ transport, indicating that it influences channel gating. These results may help explain the relative paucity of CF-associated mutations in the C terminus.

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Section: Discussionmentioning

confidence: 64%

“…We focused on five parts of this sequence ( (16,17). We generated constructs that deleted combinations of these five regions (Fig.…”

Section: C-terminal Deletion Constructsmentioning

confidence: 99%

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Effects of C-terminal deletions on cystic fibrosis transmembrane conductance regulator function in cystic fibrosis airway epithelia

Ostedgaard

Randak

Rokhlina

et al. 2003

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

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“…Transient Transfection of COS-7 Cells-COS-7 cells were transiently transfected using LipofectAMINE PLUS reagent (Invitrogen) according to the manufacturer's protocol. Transfected cells were cultured for 24 -48 h before analysis as described previously (38).…”

Section: Methodsmentioning

confidence: 99%

Efficient Intracellular Processing of the Endogenous Cystic Fibrosis Transmembrane Conductance Regulator in Epithelial Cell Lines

Varga

Jurkuvenaite

Wakefield³

et al. 2004

Journal of Biological Chemistry

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121

116

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The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase A-activated chloride channel that resides on the apical surface of epithelial cells. One unusual feature of this protein is that during biogenesis, ϳ75% of wild type CFTR is degraded by the endoplasmic reticulum (ER)-associated degradative (ERAD) pathway. Examining the biogenesis and structural instability of the molecule has been technically challenging due to the limited amount of CFTR expressed in epithelia. Consequently, investigators have employed heterologous overexpression systems. Based on recent results that epithelial specific factors regulate both CFTR biogenesis and function, we hypothesized that CFTR biogenesis in endogenous CFTR expressing epithelial cells may be more efficient. To test this, we compared CFTR biogenesis in two epithelial cell lines endogenously expressing CFTR (Calu-3 and T84) with two heterologous expression systems (COS-7 and HeLa). Consistent with previous reports, 20 and 35% of the newly synthesized CFTR were converted to maturely glycosylated CFTR in COS-7 and HeLa cells, respectively. In contrast, CFTR maturation was virtually 100% efficient in Calu-3 and T84 cells. Furthermore, inhibition of the proteasome had no effect on CFTR biogenesis in Calu-3 cells, whereas it stabilized the immature form of CFTR in HeLa cells. Quantitative reverse transcriptase-PCR indicated that CFTR message levels are ϳ4-fold lower in Calu-3 than HeLa cells, yet steady-state protein levels are comparable. Our results question the structural instability model of wild type CFTR and indicate that epithelial cells endogenously expressing CFTR efficiently process this protein to post-Golgi compartments.

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“…In either case, CFTR (and presumably also FAK) proteins are likely to play a role in continual turnover involving cycling of subapical membrane vesicles. Also of importance, there is a tyrosine in the carboxyterminus (Y1424) that serves along with I1427 as an internalization cue for endocytosis via clathrin coated pits (Peter et al, 2002). The possibility that tyrosine phosphorylation could regulate the activation and turnover of CFTR opens another possible role for FAK in CFTR containing epithelia.…”

Section: Cftr and Apical Membrane Turnovermentioning

confidence: 99%

CFTR Cl– channel functional regulation by phosphorylation of focal adhesion kinase at tyrosine 407 in osmosensitive ion transporting mitochondria rich cells of euryhaline killifish

Marshall

Watters

Hovdestad

et al. 2009

Journal of Experimental Biology

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SUMMARYCystic fibrosis transmembrane conductance regulator (CFTR) anion channels are the regulated exit pathway in Cl -secretion by teleost mitochondria rich salt secreting (MR) cells of the gill and opercular epithelia of euryhaline teleosts. By confocal light immunocytochemistry, immunogold transmission electron microscopy (TEM), and co-immunoprecipitation, using regular and phospho-antibodies directed against conserved sites, we found that killifish CFTR (kfCFTR) and the tyrosine kinase focal adhesion kinase (FAK) phosphorylated at Y407 (FAK pY407) are colocalized in the apical membrane and in subjacent membrane vesicles of MR cells. We showed previously that basolateral FAK pY407, unlike other FAK phosphorylation sites, is osmosensitive and dephosphorylates during hypotonic shock of epithelial cells (Marshall et al., 2008). In the present study, we found that hypotonic shock and the α 2 -adrenergic agonist clonidine (neither of which affects cAMP levels) rapidly and reversibly inhibit Cl -secretion by isolated opercular membranes, simultaneous with dephosphorylation of FAK pY407, located in the apical membrane. FAK pY407 is rephosphorylated and Cl -secretion rapidly restored by hypertonic shock as well as by forskolin and isoproterenol, which operate via cAMP and protein kinase A. We conclude that hormone mediated, cAMP dependent and osmotically mediated, cAMP independent pathways converge on a mechanism to activate CFTR and Cl -secretion, possibly through tyrosine phosphorylation of CFTR by FAK.

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

Abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that undergoes endocytosis through clathrin-coated pits. Previously, we demonstrated that Y1424A is important for CFTR endocytosis (Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L.

Cited by 30 publications

References 24 publications

Effects of C-terminal deletions on cystic fibrosis transmembrane conductance regulator function in cystic fibrosis airway epithelia

Effects of C-terminal deletions on cystic fibrosis transmembrane conductance regulator function in cystic fibrosis airway epithelia

Efficient Intracellular Processing of the Endogenous Cystic Fibrosis Transmembrane Conductance Regulator in Epithelial Cell Lines

CFTR Cl– channel functional regulation by phosphorylation of focal adhesion kinase at tyrosine 407 in osmosensitive ion transporting mitochondria rich cells of euryhaline killifish

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