The Delta F508 mutation shortens the biochemical half-life of plasma membrane CFTR in polarized epithelial cells

Ghanshyam D. Heda1,2, Mridul Tanwani2, and Christopher R. Marino2,3,4

1 Research and 3 Medical Services, Veterans Affairs Medical Center and Departments of 2 Medicine and 4 Physiology and Biophysics, The University of Tennessee Health Sciences Center, Memphis, Tennessee 38163


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although the biosynthetic arrest of the Delta F508 mutant of cystic fibrosis transmembrane conductance regulator (CFTR) can be partially reversed by physical and chemical means, recent evidence suggests that the functional stability of the mutant protein after reaching the cell surface is compromised. To understand the molecular basis for this observation, the current study directly measured the half-life of Delta F508 and wild-type CFTR at the cell surface of transfected LLC-PK1 cells. Plasma membrane CFTR expression over time was characterized biochemically and functionally in these polarized epithelial cells. Surface biotinylation, streptavidin extraction, and quantitative immunoblot analysis determined the biochemical half-life of plasma membrane Delta F508 CFTR to be ~4 h, whereas the plasma membrane half-life of wild-type CFTR exceeded 48 h. This difference in biochemical stability correlated with CFTR-mediated transport function. These findings indicate that the Delta F508 mutation decreases the biochemical stability of CFTR at the cell surface. We conclude that the Delta F508 mutation triggers more rapid internalization of CFTR and/or its preferential sorting to a pathway of rapid degradation.

cystic fibrosis; regulation; membrane protein; endocytosis; chloride channel; cystic fibrosis transmembrane conductance regulator


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MOST CYSTIC FIBROSIS (CF) is caused by a gene mutation that results in the deletion of the phenylalanine at position 508 of CFTR, the cystic fibrosis transmembrane conductance regulator (CFTR) protein (13). The Delta F508 deletion results in a misfolding of the nascent CFTR molecule in the endoplasmic reticulum (ER), leading to its retention in the ER by chaparone proteins and its subsequent ubiquitination and premature degradation by the cytoplasmic proteosome complex (reviewed in Ref. 16). As a consequence, little if any Delta F508 CFTR is processed to its mature, fully glycosylated form or trafficked to the plasma membrane where CFTR normally functions as a cAMP-activated chloride channel. Because the Delta F508 mutant retains some intrinsic chloride transport function (8, 18), interest in approaches that drive Delta F508 CFTR past the ER quality control system and to the cell surface has grown.

Several in vitro studies have shown that surface expression of Delta F508 CFTR can be upregulated by chemical or physical means. Butyrate compounds have been shown to create cAMP-activated chloride currents in cultured Delta F508 cells, presumably by overwhelming the ER quality control system through an increase in gene transcription (4, 28). Glycerol (30) and low temperature (8) stabilize the conformation of newly synthesized Delta F508, allowing some of it to bypass the ER quality control system and reach biochemical maturity. After each of these treatments, an increase in cAMP-activated chloride transport can be detected in the mutant cells. These findings suggest that pharmacological agents that downregulate the ER quality control system might be useful in correcting the chloride transport defect in Delta F508 cells.

For this approach to be clinically effective, the Delta F508 protein must maintain some degree of biochemical stability after reaching the cell surface. Little is known, however, of the fate of the CFTR protein after it reaches the cell surface. Initial work suggested that Delta F508 and wild-type CFTR had similar plasma membrane half-lives (8), but a subsequent in vitro study has shown that cAMP-activated chloride currents in nonpolarized Delta F508 cells are less stable than those in wild-type cells (23). Because plasma membrane expression of the CFTR proteins was not directly examined in that study, the molecular basis for this observation is not known. The functional instability in Delta F508 cells could have been due to more rapid inactivation of Delta F508 channels at the cell surface or to more rapid internalization and/or degradation of the mutant protein. Functional inactivation is supported by data from several studies that show the conduction properties of Delta F508 CFTR differ from those of wild-type CFTR (6, 9, 11). In fact, a recent electrophysiological study has since confirmed that Delta F508 CFTR is more rapidly inactivated than wild-type CFTR in excised membrane patches (31).

Documented differences in vesicle trafficking of Delta F508 and wild-type CFTR (2, 32) suggest a potential role for membrane trafficking in the regulation of plasma membrane CFTR function. However, technical difficulties in getting measurable quantities of the Delta F508 protein to the cell surface have hindered efforts to compare plasma membrane Delta F508 and wild-type CFTR protein expression. The current study overcame that obstacle by the simultaneous treatment of cells with low temperature and sodium butyrate, which act synergistically to markedly upregulate surface CFTR expression (12). With readily detectable levels of Delta F508 CFTR at the cell surface, it became possible to test the hypothesis that the Delta F508 protein is more biochemically unstable than wild-type CFTR. Performed in a polarized epithelial cell line, the experiments presented confirm that the biochemical half-life of plasma membrane Delta F508 CFTR is much shorter than that of wild-type CFTR. These differences in biochemical half-life correlate with transport function, suggesting that rapid internalization and/or degradation of Delta F508 CFTR alone can account for the rapid loss of chloride transport function characteristic of Delta F508 cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cell line. Pig kidney epithelial cells (LLC-PK1), stably transfected with human wild-type or Delta F508-CFTR cDNA were kindly provided by Dr. Seng Cheng (Genzyme, Boston, MA). Cells were grown in low-glucose DMEM supplemented with 10% FBS and 400 µg/ml Genticin (Life Technologies, Grand Island, NY) at 37°C on plastic dishes coated with human placental type IV collagen (Sigma Chemical, St. Louis, MO). For 125I efflux experiments, cells were grown on clear transwell membrane inserts coated with type IV collagen (Corning Costar, Cambridge, MA). For immunocytochemical experiments, cells were grown on collagen-coated glass coverslips.

Upregulation of surface CFTR expression. Transfected LLC-PK1 cells grown to ~70% confluence as described above were treated for 60 h at 27°C in the presence of 5 mM sodium butyrate to upregulate surface CFTR expression (12). Because low temperature partially inhibits cell proliferation, cell overgrowth did not occur during this 60-h treatment, and confluency was rarely achieved.

Biochemical determination of surface CFTR expression over time. After upregulation of surface CFTR expression, cells were washed with butyrate-free media and were incubated at 37°C in media containing 20 µg/ml of cycloheximide, an inhibitor of protein synthesis, for designated time intervals up to 48 h. Cells were then washed with ice-cold PBS, pH 7.4, containing 0.1 mM CaCl2 and 1 mM MgCl2 to inhibit vesicle trafficking, and surface biotinylation was performed as described previously (19). Briefly, the glycosidic moieties of surface membrane proteins were derivatized with sodium periodate and biotinylated using biotin-LC-hydrazide according to company protocol (Pierce, Rockford, IL). The efficiency of surface biotinylation was tested by examining the effect of increasing biotin-LC-hydrazide exposure times on surface CFTR expression. No time-dependent increase in surface CFTR expression occurred with prolonged reagent exposure (data not shown).

After surface biotinylation, cells were lysed with 1% SDS containing 0.2 mM phenylmethylsulfonyl fluoride and 1 mM benzamidine, sonicated to shear DNA molecules, and centrifuged at 10,000 g for 10 min at 4°C to remove cellular debris. The clear supernatants, normalized to 50 µg of total protein, were nutated for 30 min with an excess of streptavidin-coated agarose (SA) beads or uncoated control agarose (CN) beads (Sigma). After incubation, the beads were pelleted, and the supernatants were subjected to 6.5% SDS-PAGE followed by transfer to Hybond-P polyvinylidene difluoride membrane (Amersham, Sunnyvale, CA). The transfer was blocked with 5% nonfat dry milk in 137 mM NaCl, 0.1% Tween 20, and 20 mM Tris · HCl, pH 7.6, and immunoblotted with 1:100 dilution of R3194, an affinity-purified polyclonal anti-CFTR antibody. The specificity of R3194 for CFTR has been characterized previously in transgenic mice (37), in CFTR- and mock-transfected HEK-293 cells (17), and by COOH-terminal CFTR peptide competition experiments (37). CFTR bands were visualized by enhanced chemifluorescence (Amersham) and were quantified using a STORM 860 imaging system with ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

With the use of the above approach, biotinylated surface proteins were irreversibly extracted by the SA bead processing. The efficiency of this extraction step was examined by stripping the immunoblots and reblotting with streptavidin-conjugated horseradish peroxidase followed by Sigma Fast diaminobenzidine color development to confirm the absence of biotinylated proteins in SA bead processed samples (see Fig. 3). Thus the post-SA bead supernatants contain only intracellular (unbiotinylated) proteins, whereas CN bead supernatants contain all cellular proteins (biotinylated and unbiotinylated). Surface CFTR expression was then determined by subtracting the intensity of the CFTR signal after SA bead extraction from the intensity of the total CFTR signal from CN bead processed samples.

Functional determination of surface CFTR expression over time. Chloride secretion was determined by isotopic efflux of 125I from preloaded cells as previously described (33). Briefly, membrane inserts containing LLC-PK1 cells treated with 20 µg/ml of cycloheximide at 37°C were excised, and cells were washed with efflux buffer (140 mM NaCl, 4.7 mM KCl, 1.2 mM CaCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4). The cells were then loaded with 125I by incubation with 3 µCi of 125I-labeled Na (New England Nuclear, Boston, MA) for 1 h at 37°C. Cell monolayers were washed free of excess 125I, and the time-dependent efflux of the isotope into the media was measured after stimulation with the cAMP agonists forskolin (10 µM) and isobutyl methylxanthine (IBMX, 1.5 mM). Cells were then solubilized with 0.1 N NaOH, and 125I radioactivity in the media samples and the final cell lysate was determined by counting gamma emissions (Packard Minaxi gamma counter, series 5000). The rate coefficient of iodide efflux (r) was calculated using the following formula
r=[ln (<IT>R<SUB>1</SUB></IT>)<IT>−</IT>ln (<IT>R<SUB>2</SUB></IT>)]<IT>/</IT>(<IT>t<SUB>1</SUB>−t<SUB>2</SUB></IT>)
where R1 and R2 are the percent of counts remaining in the cell layer at times t1 and t2. Data are presented as means ± SE of replicate experiments. Differential inhibition of 125I efflux by DIDS and diphenylamine-2-carboxylic acid was used to confirm that the cAMP-stimulated efflux from these cells was CFTR mediated.

Immunocytochemical determination of surface CFTR expression. Butyrate/low-temperature-treated cells, grown on collagen-coated coverslips, were fixed for 1 h with 4% paraformaldehyde and then permeabilized with 0.25% saponin in PBS for 10 min. Free aldehyde residues were quenched for 30 min with 50 mM NH4Cl, and cells were blocked with 1% BSA, both prepared in PBS. Cells were then incubated overnight at 4°C with R3194 (polyclonal anti-CFTR) and/or monoclonal antibody 6H (monoclonal against the beta -subunit of rat Na+-K+-ATPase, generously provided by Michael J. Caplan, Yale School of Medicine, New Haven, CT). Anti-CFTR labeling was detected with FITC-conjugated goat anti-rabbit F(ab')2 fragments, and anti-Na+-K+-ATPase labeling was detected with tetramethylrhodamine isothiocyanate-conjugated goat anti-mouse F(ab')2 fragments (Jackson ImmunoResearch Labs, West Grove, PA). For double-label experiments, primary antibodies were applied simultaneously as were the fluorescent-conjugated secondary antibodies. Fluorescent signals were visualized on an Axiophot fluorescent microscope (Zeiss) and digitally stored using Photoshop 4.01 software (Adobe Systems, Mountain View, CA). Photoshop was not used to modify images other than to adjust contrast for improved signal definition. No signal was detected in the absence of primary antibody, indicating that background labeling was low under the experimental conditions employed (data not shown).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because there is no natural cell type that expresses both Delta F508 and wild-type CFTR, transfected cell lines have served as the standard model system for studying differences in the processing and trafficking of CFTR. The most widely used cell lines (CHO, 3T3, C127, and HEK), however, are either unpolarized or of undetermined polarity. Because vesicle trafficking in polarized and unpolarized cells may differ, we sought to examine CFTR surface expression in a transfected mammalian cell line with evidence of cellular polarity. LLC-PK1 cells are transformed epithelial cells from the proximal tubule of pig kidney. Morphological evidence for polarity was obtained by electron microscopy, which demonstrated the appearance of apical tight junctions in monolayers of transfected cells (Fig. 1A). Functional polarity was demonstrated by double-label immunofluorescent microscopy experiments using apical and basolateral membrane markers. As shown in Fig. 1B, Na+-K+-ATPase expression in LLC-PK1 cells is primarily surface and basal in location, consistent with its known distribution along the basolateral membrane of polarized epithelial cells. Although CFTR has both a surface and an intracellular signal in these high-expressing cells, the surface signal is more apically distributed and does not colocalize with that of Na+-K+-ATPase. The prominent intracellular CFTR signal seen in these cells is discussed in greater detail below. These experiments indicate that CFTR-transfected LLC-PK1 cells have both morphological and functional features characteristic of polarized epithelial cells.


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Fig. 1.   Analysis of LLC-PK1 cell polarity. Wild-type LLC-PK1 cells were grown on permeable supports, and their polarity was determined both morphologically and functionally. A: low (left)- and high (right)-magnification electron micrographs showing tight junctions at the apical domain of adjacent cells (arrowheads). Original magnifications were ×12,500 and ×24,000, respectively. Bars, 1 µm. B: double-label immunofluorescence micrographs of cystic fibrosis transmembrane conductance regulator (CFTR; green) and Na+-K+-ATPase (red) distribution in the same LLC-PK1 cells. The distribution of both membrane proteins was captured through the apical, subapical, and basal regions of cells by epifluorescence microscopy. CFTR labeling is seen throughout the cell, but membrane labeling is most prominent in the apical and subapical regions. Na+-K+-ATPase is localized to the plasma membrane at the basal part of the cell. The plasma membrane labeling pattern differs for each.

For direct biochemical study of Delta F508 expression at the plasma membrane, sufficient quantities of the protein had to be driven to the cell surface. To accomplish this, cells were treated at 27°C in the presence of 5 mM sodium butyrate to markedly upregulate surface CFTR expression (12). Previous time course experiments identified 60 h of treatment as optimal for the upregulation of CFTR expression (12). Although wild-type cells did not require butyrate or low temperature treatment for the detection of plasma membrane CFTR, we chose to control our experiments by treating all cells identically. As shown in Fig. 2, mature (band C) Delta F508 CFTR was not detected in LLC-PK1 cells grown at 37°C in the presence or absence of sodium butyrate, nor was it detected by immunoblot analysis in cells grown at 27°C alone. When sodium butyrate and low-temperature treatments were combined, however, there was a marked increase in total Delta F508 CFTR expression that was accompanied by the appearance of the mature, fully glycosylated form (band C). The upregulation of CFTR expression under these experimental conditions was even more pronounced in wild-type LLC-PK1 cells.


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Fig. 2.   Effect of sodium butyrate (SB) and temperature on CFTR protein expression. Delta F508 and wild-type LLC-PK1 cells were grown to ~70% confluence and then were treated for 60 h in the absence (-) or presence (+) of 5 mM sodium butyrate at 37 or at 27°C. After treatment, cells were harvested, lysed, and normalized to 50 µg total protein before SDS-PAGE and immunoblotting with the anti-CFTR antibody R3194. Arrows identify CFTR bands B and C.

Although mature (band C) CFTR has routinely been used as a marker of plasma membrane CFTR expression (5, 8, 30), studies in both native (26, 36) and transfected (21) cells have shown that not all mature CFTR resides at the cell surface. To confirm that Delta F508 CFTR was driven to the surface of LLC-PK1 cells by butyrate and low-temperature treatment, plasma membrane CFTR expression was measured biochemically, functionally, and cytochemically. Surface biotinylation and streptavidin extraction were used to determine how much Delta F508 protein actually reached the cell surface and what molecular form(s) of CFTR were targeted there. As shown in Fig. 3, most of the CFTR in treated Delta F508 and wild-type LLC-PK1 cells did not reach the cell surface. Based on replicate experiments (n = 5), ~35% of CFTR was found to reside at the cell surface (33.9 ± 4.7% for Delta F508 and 35.5 ± 3.4% for wild-type CFTR). These biotinylation experiments also demonstrated that only the mature band C form of CFTR was expressed at the plasma membrane. The immature band B form was unaffected by streptavidin extraction, indicating that it was not biotinylated and therefore not present at the cell surface. Surface expression of Delta F508 CFTR after butyrate and low-temperature treatment was subsequently confirmed cytochemically and functionally (Fig. 4). The relative distribution of CFTR between surface and intracellular compartments, including the prominent intracellular signal, correlated with the above biotinylation data. Functional evidence for surface CFTR expression in both cell lines was obtained by forskolin/IBMX-stimulated 125I efflux assay (Fig. 4).


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Fig. 3.   Plasma membrane expression of CFTR. Delta F508 and wild-type LLC-PK1 cells were grown to ~70% confluence and then were treated with 5 mM sodium butyrate at 27°C for 60 h to upregulate plasma membrane CFTR expression. Cells were then surface biotinylated as described in MATERIALS AND METHODS. Detergent lysates of the biotinylated cells were normalized to total protein (50 µg) and then reacted with streptavidin-conjugated agarose beads (SA) to remove all surface-biotinylated proteins or with plain agarose (control, CN) beads, which remove no protein. Lysate proteins after bead treatment were separated by SDS-PAGE and were transferred to polyvinylidene difluoride membranes. Top: transfer membrane was immunoblotted with R3194 to detect CFTR protein expression. CN lanes depict total CFTR expression, whereas SA lanes show only the unbiotinylated (intracellular) proteins in those samples. Arrows identify CFTR bands B and C. Bottom: the same transfer membrane was stripped of antibodies and reblotted with streptavidin-conjugated alkaline phosphatase followed by diaminobenzidine reaction to detect the presence of biotinylated proteins in each lane. The absence of biotinylated proteins in the SA lanes shows the efficiency of the streptavidin bead extraction step.



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Fig. 4.   Cytochemical and functional detection of plasma membrane CFTR. Expression of Delta F508 (left) and wild-type (right) CFTR at the plasma membrane after butyrate and low-temperature treatment was determined by immunofluorescence microscopy (top) and 125I efflux assay (bottom). Arrows in the immunofluorescence micrographs denote plasma membrane CFTR labeling. Arrows in the 125I efflux graphs note the time when cells were stimulated with forskolin/isobutyl methylxanthine (IBMX). open circle , Control condition (cells grown at 37°C without any butyrate treatment); , 27°C + SB. 125I efflux values represent means ± SE from replicate experiments.

With the use of the surface biotinylation procedure, the half-life of plasma membrane CFTR in Delta F508 and wild-type cells was examined next (Fig. 5). In these experiments, surface CFTR expression was upregulated by pretreatment with sodium butyrate and low temperature. Cells were then transferred to physiological temperature (37°C) and treated for up to 48 h in the presence of cycloheximide. Cells were then surface biotinylated, solubilized, and streptavidin-extracted, with immunoblot analysis being used to quantify CFTR protein expression. The rapid fall in immature (band B) CFTR expression in the Delta F508 cells was due to the inhibition of new protein synthesis by cycloheximide, combined with ER degradation and some conversion to the mature band C form. Although not shown in Fig. 5, band B CFTR was also rapidly degraded in wild-type cells (the signal was undetectable by 4 h, the earliest time point we examined). These findings are consistent with the observations of Ward and Kopito (34), who demonstrated that the immature forms of Delta F508 and wild-type CFTR have similar rates of degradation.


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Fig. 5.   Biochemical half-life of plasma membrane CFTR. Delta F508 and wild-type LLC-PK1 cells were pulsed with butyrate and low temperature and then were chased at 37°C in the presence of 20 µg/ml cycloheximide for the times indicated. At the completion of the chase, cells were transferred to 4°C, and surface proteins were biotinylated, solubilized, and processed through either streptavidin (SA) or control (CN) agarose beads. Postbead lysates, normalized to 50 µg total protein, were immunoblotted with R3194 anti-CFTR antibody. Arrows identify CFTR bands B and C.

In Delta F508 cells, total band C CFTR expression was nearly eliminated by 6 h. In contrast, total band C CFTR expression in the wild-type cells persisted for 48 h. Although most of the band C CFTR was intracellular, the persistence of a streptavidin-extractable pool for as long as 48 h provides biochemical evidence for the presence of plasma membrane CFTR expression in wild-type cells for at least this length of time. Because the relative distribution of CFTR between intracellular and plasma membrane compartments did not change appreciably over time (data not shown), the plasma membrane half-life of each protein was estimated by quantifying the rate of degradation of total band C CFTR. From replicate experiments (n = 7), we calculated the biochemical half-life of plasma membrane CFTR in Delta F508 cells to be ~4 h, whereas the biochemical half-life of plasma membrane CFTR in wild-type cells exceeded 48 h.

To rule out the possibility that the relatively long plasma membrane half-life of wild-type CFTR was an artifact of overexpression, these biochemical and functional studies were repeated under conditions of comparable total CFTR expression in the two cells lines. Based on data from Fig. 2, wild-type cells treated with sodium butyrate at 37°C had CFTR expression levels that were comparable to those found in Delta F508 cells treated with sodium butyrate at 27°C. Thus cells were pretreated in this manner and then processed and analyzed as described previously. Under these conditions of comparable total CFTR expression, the biochemical half-life of band C CFTR in wild-type cells continued to exceed 24 h. Surface biotinylation experiments also confirmed the presence of plasma membrane CFTR for at least this length of time (Fig. 6). This contrasts with the near absence of any detectable Delta F508 protein after 6 h of chase. Although we were able to detect some functional Delta F508 CFTR at these early time points by the qualitative 125I efflux assay, no functional CFTR expression was detected in Delta F508 cells after 24 h of chase, which contrasts sharply with the persistence of functional CFTR in the wild-type cells at this time. Similar kinetic studies were performed in transfected (unpolarized) C127 cells, where Delta F508 expression exceeds that of wild-type CFTR after butyrate and low-temperature treatment, and similar half-life values for Delta F508 and wild-type CFTR were obtained (data not shown). Thus several lines of evidence indicate that the difference in plasma membrane expression of Delta F508 and wild-type CFTR reflects the biology of these two protein species and not simply overexpression.


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Fig. 6.   Half-life of plasma membrane CFTR under conditions of comparable levels of CFTR expression. To confirm that the long plasma membrane half-life of wild-type CFTR was not an artifact of overexpression, an experiment was performed under conditions of comparable total CFTR expression in both cell types. Based on data in Fig. 2, Delta F508 cells treated with 5 mM sodium butyrate at 27°C were compared with wild-type cells treated with 5 mM sodium butyrate at 37°C. After treatment, all cells were transferred to 37°C and were incubated in the presence of 20 µg/ml cycloheximide for the times shown. Top: biochemical half-life of Delta F508 and wild-type CFTR after surface biotinylation, SA or CN bead extraction, and immunoblotting with R3194. Bottom: 125I efflux from forskolin/IBMX-stimulated cells at baseline () and after 24 h of chase at 37°C (). open circle , Control condition (cells grown at 37°C without any butyrate treatment or chase period). The biochemical and functional stability of wild-type CFTR continues to exceed 24 h, even when relative overexpression is corrected.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Until the long-term goals of gene therapy are realized, strategies designed to upregulate plasma membrane expression of Delta F508 CFTR remain an important potential therapeutic option in CF (16, 29). Much emphasis has been placed on the biogenesis of Delta F508 CFTR, with the goal being to overcome its biosynthetic arrest and thereby increase the delivery of functional Delta F508 CFTR channels to the cell surface. Effective correction of the CF defect, however, will require stable expression of Delta F508 after reaching the plasma membrane, and one study has shown that the functional half-life of plasma membrane Delta F508 in intact cells is less than that of wild-type CFTR (23). The current study sought to test the hypothesis that the short functional half-life in Delta F508 cells results from more rapid removal of CFTR channels from the cell surface.

To study the biochemical stability of plasma membrane Delta F508 CFTR, sufficient quantities of the mutant protein needed to be driven to the cell surface. The current study took advantage of combination therapy with sodium butyrate and low temperature to increase surface Delta F508 protein expression to levels sufficient for the direct measurement of CFTR protein half-life by a quantitative immunoblot technique (12). Thus, in all experiments, cells were pretreated with sodium butyrate and low temperature to upregulate CFTR protein expression and then were followed at 37°C to examine the plasma membrane half-life of each CFTR species. Because cell polarity can affect membrane trafficking, all experiments were performed in a polarized epithelial cell line (LLC-PK1).

After butyrate and low-temperature treatment, most of the CFTR in these cells was intracellular. Surface biotinylation experiments demonstrated that only the mature, fully glycosylated (band C) form of CFTR reached the cell surface. No evidence for the direct targeting of immature (electrophoretic bands A and B) CFTR to the plasma membrane was found. Quantitative analysis of CFTR distribution in butyrate/low-temperature-treated LLC-PK1 cells demonstrated that ~65% of band C CFTR remained intracellular. This was true for both Delta F508 and wild-type CFTR and is similar to observations made in other cell systems (26, 36). Although changes in band C CFTR expression correlated with changes in plasma membrane CFTR expression in transfected LLC-PK1 cells, this may not be true in all cell types. Thus surface protein labeling techniques remain the most accurate means of measuring plasma membrane CFTR expression.

The biochemical finding of a prominent intracellular compartment of CFTR was confirmed immunocytochemically. After butyrate/low-temperature treatment, both Delta F508 and wild-type cells labeled similarly, with modest surface but prominent intracellular signals. The identity of the intracellular signal in each cell type remains unknown. In the wild-type cells, surface biotinylation demonstrated that most of the unbiotinylated CFTR is of the band C form and therefore is presumably in a post-ER compartment (Golgi, endosomes, transport vesicles, etc.). In Delta F508 cells, where most of the unbiotinylated CFTR is of the band B form, a large ER component to the intracellular signal would be expected. Identification of the sources of intracellular CFTR labeling in these cells will, however, require much additional study.

The kinetics of Delta F508 and wild-type CFTR degradation over time was examined at physiological temperature and under conditions of protein synthesis inhibition. The rate of CFTR degradation was quantified biochemically and confirmed by functional assay. Replicate experiments indicate that the biochemical half-life of plasma membrane Delta F508 CFTR is ~4 h, whereas the biochemical half-life of plasma membrane wild-type CFTR exceeds 48 h. These values correlate with changes in 125I efflux and are remarkably similar to the functional data generated by Lukacs et al. (23) in nonpolarized C127 cells. This time-dependent correlation between the rate of plasma membrane CFTR degradation and the loss of cAMP-stimulated 125I efflux indicates that the functional instability in Delta F508 cells can be theoretically attributed to more rapid degradation of plasma membrane Delta F508 CFTR. The data, however, do not exclude a contributory role for channel inactivation in this process.

These findings establish that the Delta F508 mutation has a negative effect on the biochemical stability of CFTR at the cell surface. This biochemical instability must be due to more rapid internalization of mutant protein and/or its selective targeting for rapid degradation. CFTR is endocytosed in clathrin-coated vesicles (1, 22), and the molecular signal for CFTR internalization may reside in its cytoplasmic tail (10, 24, 25). The Delta F508 mutation, however, is in the first nucleotide-binding domain and is some distance from the cytoplasmic end of the molecule. Thus CFTR must have another internalization signal, or the Delta F508 mutation must have an indirect effect on the COOH-terminal signal(s). Based on our understanding of CFTR folding during biogenesis, it is attractive to hypothesize that the Delta F508 mutation affects CFTR folding in such a manner that the COOH-terminal internalization signal is altered. The fundamental question is what is the signal? COOH-terminal tyrosine-based sequences have been implicated in the positioning of membrane proteins in coated pits (7, 20), and one recent study has implicated phosphorylation of tyrosine-1424 in the regulation of CFTR endocytosis (25). On the other hand, the rate of internalization of Ste6, a yeast homolog of CFTR, and Ste3p, another yeast membrane protein, are regulated by ubiquitination (14, 27). Because ubiquitination is the major signal for the degradation of both wild-type and Delta F508 CFTR during biogenesis (35), it is provocative to speculate that it is also involved in plasma membrane CFTR degradation. During CFTR biosynthesis, ubiquitination targets the immature forms of both Delta F508 and wild-type CFTR to rapid proteosome degradation with similar kinetics (34). A pool of wild-type CFTR, however, escapes this fate, becomes fully glycosylated, and reaches the plasma membrane. If ubiquitination was also the signal for plasma membrane CFTR degradation, one would have to postulate that the wild-type and mutant proteins were differentially ubiquitinated at the cell surface to account for their different rates of degradation. Because the yeast data suggest that the proteasome complex is not the final target of ubiquitinated plasma membrane Ste6 (15), one must also consider the possibility that ubiquitination of plasma membrane CFTR serves a different role (e.g., signaling for internalization and/or sorting rather than proteosome degradation). Although provocative, a role for ubiquitination in the downregulation of surface CFTR expression remains highly speculative at this time.

Alternatively, mutant and wild-type CFTR may have similar rates of internalization but are differentially sorted in the endocytic compartment, with Delta F508 being targeted for rapid lysosomal degradation while wild-type CFTR is recycled back to the plasma membrane. In this case, the Delta F508 mutation might be affecting the interaction of CFTR with specific GTP-binding (Rab) proteins that regulate vesicle trafficking (reviewed in Ref. 3). Rab4 has been most closely associated with sorting endosomes, so an understanding of its interactions with Delta F508 and wild-type CFTR would be of particular interest. Last, one must consider the effect of phosphorylation on CFTR trafficking, particularly in light of data showing that the rate of endocytosis differs in Delta F508 and wild-type cells after cAMP activation (2). Although the current study did not examine the biochemical stability of plasma membrane CFTR under stimulatory conditions, the basal phosphorylation state of these two CFTR species may differ, and this, in turn, could expose different internalization signals.

In conclusion, the Delta F508 mutation dramatically reduces the residence time of CFTR at the cell surface. This must be due to an effect of the mutation on the rate of CFTR internalization and/or its intracellular targeting. Although little is known about this aspect of CFTR biology, it has clear implications for any therapeutic strategies that rely on delivering more Delta F508 protein to the cell surface.


    ACKNOWLEDGEMENTS

We thank Dr. Raymond A. Frizzell for helpful discussions and acknowledge the technical assistance of Virginia Jeanes.


    FOOTNOTES

This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (C. R. Marino) and by the Cystic Fibrosis Foundation (C. R. Marino).

Address for reprint requests and other correspondence: C. R. Marino, Medical Service (111), VA Medical Center, 1030 Jefferson Ave., Memphis, TN 38104 (E-mail: cmarino{at}utmem.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 May 2000; accepted in final form 9 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 280(1):C166-C174