A Mutation in the Cystic Fibrosis Transmembrane Conductance
Regulator Generates a Novel Internalization Sequence and Enhances
Endocytic Rates*
Mark R.
Silvis,
John A.
Picciano,
Carol
Bertrand,
Kelly
Weixel
,
Robert J.
Bridges, and
Neil A.
Bradbury§
From the Cystic Fibrosis Research Center, Department of Cell
Biology and Physiology and the
Department of Medicine,
University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
15261
Received for publication, December 17, 2002, and in revised form, January 14, 2003
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ABSTRACT |
Cystic fibrosis is a common lethal genetic
disease among Caucasians. The cystic fibrosis gene encodes a cyclic
adenosine monophosphate-activated chloride channel (cystic fibrosis
transmembrane conductance regulator (CFTR)) that mediates
electrolyte transport across the luminal surfaces of a variety of
epithelial cells. Mutations in CFTR fall into two broad categories;
those that affect protein biosynthesis/stability and traffic to the
cell surface and those that cause altered channel kinetics in proteins
that reach the cell surface. Here we report a novel mechanism by which
mutations in CFTR give rise to disease. N287Y, a mutation within
an intracellular loop of CFTR, increases channel endocytosis from the
cell surface without affecting either biosynthesis or channel gating.
The sole consequence of this novel mutation is to generate a novel
tyrosine-based endocytic sequence within an intracellular loop in CFTR
leading to increased removal from the cell surface and a reduction in
the steady-state level of CFTR at the cell surface.
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INTRODUCTION |
Disruption of intracellular trafficking events has been recognized
as the underlying molecular basis for a growing number of human genetic
diseases including diabetes mellitus, familial hypercholesterolemia,
Hermansky-Pudlak syndrome,
1-antitrypsin activity, and
cystic fibrosis (CF)1 (1).
Cystic fibrosis, a common lethal autosomal recessive genetic disease,
results from mutations in the CF transmembrane conductance regulator
(CFTR) and affects ~1 in 2,000 live births in Caucasians (2-4).
CFTR, a member of the ATP-binding cassette transporter family of
proteins (3), functions as a cAMP-activated anion channel and channel
regulator that resides at the apical plasma membrane of polarized
epithelia where it regulates epithelial secretions in the respiratory
and gastrointestinal tracts (5-7). Cystic fibrosis is characterized by
high sweat chloride levels, pulmonary disease, and pancreatic
insufficiency, although other organs including kidney, liver, and
tissues of the reproductive tract are also affected (2, 4). The most
common disease-causing mutation in CFTR is a 3-base pair deletion,
resulting in the loss of a phenylalanine residue at position 508 (
F508) (3). This mutation accounts for ~70% of all mutant alleles
and generates a protein that is unable to fold appropriately. Misfolded
F508 CFTR is retained within the endoplasmic reticulum (ER) and
eventually degraded by the proteasome. Although the molecular basis for
disease in patients bearing the
F508 mutation has been extensively
studied, little is known about the molecular bases for disease in the
more than 1000 different mutations that have been detected in CF
patients (Cystic Fibrosis Genetic Analysis Consortium,
www.genet.sickkids.on.ca/CFTR).
In addition to its localization within the apical plasma membrane,
morphological, biochemical, and functional data indicate that CFTR is
also localized to endosomal and recycling compartments (8-11). The
distribution of CFTR between endosomes and the plasma membrane appears
to be in a dynamic equilibrium. The rapid efficient endocytosis of CFTR
is mediated by clathrin-coated pits in both polarized and non-polarized
cells, and CFTR can be detected in isolated clathrin-coated vesicles
(9). Efficient internalization of integral membrane proteins relies on
the presence of short peptide sequences within their cytoplasmic
domains. Tyrosine- and dileucine/leucine-isoleucine-based endocytic
signals have been the most extensively studied endocytic signals
identified in type I and type II membrane proteins (12, 13). Studies utilizing full-length (14) and chimeric proteins fused to either the
transferrin receptor (15) or the interleukin 2 receptor (16) have
identified a tyrosine-based motif (1424YDSI) in the
carboxyl tail of CFTR. Moreover, cross-linking and in vitro
pull-down assays demonstrated that Tyr1424 associates with
the µ subunit of the AP-2 component of the clathrin endocytic
machinery (17). Recently an individual with mutation N287Y (991A
T)
was identified based on a diagnosis of elevated sweat electrolytes
(18). Since tyrosine-based signals are important in endocytic
targeting, we hypothesized that the N287Y mutation generated a novel
additional internalization signal in CFTR (Fig. 1a), leading
to reduced cell surface expression of CFTR as a result of increased
endocytic activity. Using site-directed mutagenesis in conjunction with
morphological, biochemical, and functional assays, we demonstrate that
N287Y CFTR generates a novel endocytic sequence enhancing the endocytic
rate of CFTR compared with wild type. Such increased endocytosis leads
to a reduced steady-state level of CFTR at the plasma membrane, likely
accounting for the CF phenotype observed in patients bearing this
mutation. Moreover, since neither biosynthesis nor single channel
kinetics are appreciably altered in this mutation, the sole molecular
basis for this disease-causing mutation appears to be altered endocytic
rates. These studies reveal not only a novel class of CFTR mutations
but also a new paradigm in the localization of endocytic sequences in
polytopic proteins.
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EXPERIMENTAL PROCEDURES |
Construction of CFTR Mutants--
Full-length human CFTR
cDNA from pBQ4.7 was subcloned into the pcDNA5/FRT vector
(Invitrogen). An NheI site was introduced (QuikChange
Site-Directed Mutagenesis kit, Stratagene) upstream of the CFTR coding
region, and CFTR was subcloned into pcDNA5/FRT using restriction
site NheI and a pre-existing carboxyl-terminal XhoI site into the multiple cloning site within the
pcDNA5/FRT vector. The N287Y mutation was introduced into the pFRT
CFTR wild-type vector using the QuikChange Site-Directed mutagenesis
kit (Stratagene). The sequences of the pcDNA5/FRT-CFTR wild type
and N287Y were verified prior to use for expression.
Cell Lines and Transfections--
293 and CHO
Flp-InTM (Invitrogen) isogenic cell lines expressing
wild-type,
F508, and N287Y CFTR from the same genomic locus were generated according to the manufacturer's instructions.
Confirmation of expression was determined by immunofluorescence
microscopy, immunoblot, and immunoprecipitation. All cells were grown
at 37 °C in 5% CO2 under standard conditions. Confluent
monolayers of Madin-Darby canine kidney cells (type II) grown on
permeable filter supports were transiently transfected with either
wild-type or N287Y CFTR using calcium phosphate. Seventy-two hours
following transfection cells were processed for domain-selective biotinylation.
Metabolic Labeling and Immunoprecipitation--
Following
incubation in methionine- and cysteine-free
minimum Eagle's
medium for 30 min at 37 °C, 293 cells were pulsed in the same
medium containing 100 µCi/ml [35S]methionine and
[35S]cysteine (>1000 Ci/mmol, Amersham Biosciences) for
30 min at 37 °C. For chasing, the labeling medium was replaced with
complete
minimum Eagle's medium supplemented with 7% serum and 1 mM each of cold methionine and cysteine. Metabolically
labeled CFTR was isolated by immunoprecipitation according to standard
protocols (9) using an antibody directed against a region in the
carboxyl-terminal domain of CFTR (clone M3A7, Upstate Biotechnology,
Lake Placid, NY).
Cell Surface Biotinylation--
Cell surface biotinylation was
performed as described previously (19). Cell surface proteins were
biotinylated in the presence of 1 mg/ml
sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithioproprionate (EZ-Link
sulfo-NHS-SS-biotin, Pierce) in buffer (10 mM HEPES (pH 8.0), 3 mM KCl, 1 mM MgCl2, 0.5 mM CaCl2) at 4 °C. Cells were washed in
ice-cold phosphate-buffered saline supplemented with bovine serum
albumin (0.1%) and lysed. Biotinylated CFTR was isolated by
precipitation on streptavidin-Sepharose (Sigma). Precipitates were
resolved by SDS-PAGE, transferred to nitrocellulose, and probed with a
monoclonal antibody (M3A7) against CFTR. Biotinylated CFTR was
visualized by enhanced chemiluminescence and quantified by
densitometric analysis. To quantify cell surface insertion of CFTR,
cells metabolically labeled using a pulse-chase protocol were
biotinylated during the subsequent 1-h chase period. Cells were washed
with ice-cold phosphate-buffered saline supplemented with 0.1% bovine
serum albumin, 1 mM MgCl2, and 0.1 mM CaCl2 and lysed. Biotinylated CFTR was
isolated by immunoprecipitation with M3A7 anti-CFTR antibodies and then
on streptavidin-Sepharose (Sigma). Biotinylated CFTR was visualized
with fluorography, and radioactivity was measured by phosphorimage analysis.
Patch Clamp Analysis--
Whole cell and cell-attached patch
clamp studies were performed on CHO Flp-In cells expressing wt CFTR or
N287Y CFTR. The patch pipette solution for whole cell studies contained
100 mM L-aspartic acid, 100 mM CsOH, 40 mM CsCl, 1 mM NaCl, 1 mM EGTA and
10 mM TES (pH 7.2). One millimolar Mg-ATP and 50 µM Mg-GTP were added to the pipette solution before each
experiment. The bath solution contained 140 mM NaCl, 4 mM CsCl, 1 mM CaCl2, 5 mM glucose, 30 mM mannitol, and 10 mM TES (pH 7.4). Mannitol was used to avoid the stimulation
of swelling-activated chloride channels. The mannitol was eliminated
from the bath solution for the cell-attached patch clamp studies, and
this same solution was used in the patch pipette. All experiments were
performed at 37 °C. Cells were stimulated with 10 µM
forskolin and 100 µM cpt-cAMP added to the bath
perfusate. Current-voltage curves were constructed using voltage pulses
of 250-ms duration over a range of
70 to +70 mV at 10-mV increments. Data acquisition and analysis were performed using pClamp software (version 8.0, Axon Instruments). Values for Po
were obtained from amplitude histograms of multichannel patches.
Channel records of at least 3-min duration were evaluated. The maximum
current amplitude was noted and divided by the single channel amplitude to obtain an estimate of N for each cell.
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RESULTS |
ER Export and Polarity of CFTR Distribution Is Preserved in N287Y
CFTR--
The cellular distribution of N287Y CFTR was initially
examined since intracellular retention of mutant CFTRs by the ER
quality control and subsequent failure to mature to a
complex-glycosylated form is the most prevalent form of CF. In contrast
to
F508 CFTR, which displayed an ER-like perinuclear distribution
pattern (Fig. 1c), both
wild-type and N287Y CFTR were seen at the cell surface (Fig. 1,
b and d). Similar results were obtained for N287Y
CFTR stably expressed in CHO cells (data not shown). These results suggest that intracellular retention is unlikely to account for the
clinical phenotype associated with N287Y CFTR. To determine whether the
N287Y mutation altered the polarization of CFTR in epithelial cells, we
transiently expressed wild-type and N287Y CFTR in Madin-Darby canine
kidney cells. Confocal immunofluorescence microscopy demonstrated that
wild-type CFTR was polarized to the apical plasma membrane (Fig.
2a) with little or no staining
of the basal membrane (Fig. 2e). The ratio of wild-type CFTR
in the apical membrane versus the basolateral membrane was
10.6 ± 2.9 as determined by domain-selective cell surface
biotinylation (Fig. 2g). Similarly N287Y CFTR was also
polarized to the apical plasma membrane (Fig. 2b). The ratio
of N287Y CFTR in the apical membrane relative to the basolateral
membrane was 8.1 ± 3.1 as determined by domain-selective cell
surface biotinylation. Thus, abnormalities in the polarized
distribution of N287Y CFTR compared with wild-type CFTR cannot account
for the disease phenotype associated with this mutation.

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Fig. 1.
Schematic representation of CFTR and
immunofluorescence localization of F508,
wild-type, and N287Y CFTR. a, CFTR was drawn according
to the model predicted in Ref. 3. The shaded circle shows
the N287Y mutated residue within the second intracellular loop
(CL-2). The mutation is designated as the original
amino acid residue (first letter), its location
(number), and the amino acid to which it was changed
(second letter). NBF, nucleotide binding fold;
R, regulatory domain. b-d, Flp 293 cells were
stably transfected with wild-type (b), F508
(c), or N287Y (d) CFTR or mutant CFTR. CFTR was
visualized by immunostaining with a monoclonal antibodies against the
CFTR (M3A7 and L12B4, Upstate Biotechnology) and Alexa-488
anti-mouse secondary antibody (Molecular Probes, Eugene, OR).
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Fig. 2.
Polarized distribution of wild-type and
N287Y CFTR in Madin-Darby canine kidney cells. Confocal
micrograph of Madin-Darby canine kidney II cells transiently expressing
wild-type and N287Y CFTR. CFTR fluorescence is green (M3A7
and L12B4, Upstate Biotechnology), and ZO-1 (Zymed
Laboratories Inc., San Francisco, CA), a protein in tight
junctions that separates apical and basolateral membrane domains, is
red. Images show confocal sections taken at the plane of the
apical membrane (a and b), plane of the tight
junction (c and d), and plane of the basal
membrane (e and f) for wild-type (a,
c, and e) and N287Y (b, d,
and f) CFTR. g, immunoblot of cells expressing
wild-type and N287Y CFTR following domain-selective cell surface
biotinylation. Mature, fully glycosylated CFTR band C is ~190
kDa. Ap, apical membrane; Bl, basolateral
membrane.
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N287Y CFTR Shows Altered Cellular Distribution--
Quantitative
immunoblot analysis of whole cell lysates from isogenic wild-type and
N287Y CFTR-expressing cells revealed that steady-state levels of
protein expression were identical in each cell line (Fig.
3, a and b).
Moreover the ratio of fully glycosylated mature band C CFTR to immature
core-glycosylated band B CFTR was not altered by the N287Y mutation
compared with wild-type CFTR (7.2 ± 0.5 and 6.9 ± 0.5, mean ± S.E., n = 4 for wild-type and N287Y CFTR,
respectively). In addition, the complex- and core-glycosylated forms of
wild-type and N287Y CFTR could be distinguished by their sensitivity to
endoglycosidases, which was not different between the two cell lines
(Fig. 3c). To evaluate the amount of CFTR present at the
plasma membrane, cell surface biotinylation experiments were performed.
In both wild-type and N287Y CFTR-expressing cell lines, biotinylated
CFTR was detected as a single band of ~170 kDa, consistent with the
presence of mature fully glycosylated CFTR at the cell surface.
However, densitometric analysis revealed that the level of biotinylated
N287Y CFTR was only ~50% of that for wild-type CFTR (Fig. 3,
a and b). The detection of the mature complex-glycosylated form of wild-type and N287Y CFTR in immunoblots and at the cell surface demonstrates that biosynthesis and
intracellular transport occurred.

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Fig. 3.
Steady-state levels and cell surface
expression of wild-type and N287Y CFTR. a, expression
level of total and cell surface CFTR. Equal amounts of metabolically
labeled 293 cells expressing wild-type or N287Y CFTR were subject to
immunoprecipitation using the M3A7 anti-CFTR antibody followed by
phosphorimage analysis (top). Plasma membrane proteins were
biotinylated with 1 mg/ml sulfo-NHS-SS-biotin for 60 min at 4 °C and
isolated on streptavidin-Sepharose, and the precipitate was subjected
to immunoblot analysis using M3A7 anti-CFTR antibodies
(bottom). The sulfo-NHS-SS-biotin remains
membrane-impermeant during the labeling as shown by the lack of
biotinylated core-glycosylated forms. Complex- and core-glycosylated
forms are indicated by black and white arrows,
respectively. b, the expression level of wt and N287Y CFTR
at the cell surface (biotinylated) and post-ER compartments
(complex-glycosylated). Data represent means ± S.E.
(n = 3) and were normalized by expressing the data as a
percentage of wild type, designated as 100%. c, glycosidase
sensitivity of wild-type and N287Y CFTR. Cell lysates were incubated in
the presence or absence of endoglycosidase H (15 milliunits of
endo H) or peptide-N-glycosidase F (250 milliunits of PNGase F) for 3 h at 37 °C. Proteins
were resolved by SDS-PAGE and probed with M3A7 anti-CFTR antibodies.
Complex-, core-, and deglycosylated CFTR are indicated by
black, white, and gray
arrowheads, respectively.
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Biosynthesis of N287Y CFTR Is Not Impaired--
To better evaluate
the efficiency of N287Y CFTR biosynthesis, pulse-chase experiments were
performed (Fig. 4, a and
b). Immediately following the pulse, the entire
immunoprecipitated label was associated with a broad band migrating at
~140 kDa. This band was identified as ER-associated core-glycosylated
CFTR based on the following criteria. First, its electrophoretic
mobility corresponds to that previously observed (for band B) in other
CFTR-expressing cell lines (20, 21). Second, it was immunoprecipitated
with several monoclonal antibodies against CFTR but not with an
unrelated control antibody (data not shown). Third, it was detected
only in 293 cells stably transfected with CFTR cDNA and not in
"parental" 293 FlpTM cells (21). Band B CFTR intensity
decreased during the chase, concomitant with the appearance of a slower
migrating species (~160-180 kDa; Fig. 4, a and
b) corresponding to mature fully glycosylated CFTR (band C).
Following a 30-min pulse, labeling of band B decreased rapidly over 60 min with mature band C CFTR first becoming detectable at 30 min of
chase and reaching a plateau by 120 min. Labeling of band C CFTR did
not show any evidence of decline during chase periods up to 480 min,
consistent with observations that band C CFTR is considerably more
stable than band B CFTR. As reported previously, the conversion of the
wild-type band B CFTR to band C was not very efficient, reaching a
maximum of ~ 40% after ~2 h of chase (with the remaining 60%
of synthesized band B being degraded). The apparently normal
biosynthesis of mature fully glycosylated N287Y CFTR suggests that
targeting to the plasma membrane is largely intact. Wild-type and N287Y
CFTR were pulse-labeled with [35S]methionine, and those
molecules that arrived at the cell surface were biotinylated throughout
the subsequent chase. Biotinylated CFTR was affinity-isolated and
visualized by fluorography (Fig. 4, c and d). The
cell surface targeting efficiency of N287Y CFTR was 91 ± 4%
(mean ± S.E., n = 3) of wild-type CFTR,
suggesting that biosynthesis and plasma membrane delivery of N287Y CFTR
is largely uncompromised. The turnover of complex-glycosylated
wild-type and N287Y CFTR was assessed by pulse-chase labeling. As
previously shown (21, 22), the t1/2 of stably expressed complex-glycosylated wild-type CFTR was ~12-14 h (Fig. 4e). The stability of complex-glycosylated N287Y CFTR stably
expressed in 293 cells was not significantly different from that
observed for wild-type CFTR.

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Fig. 4.
Biosynthetic maturation, cell surface
targeting, and stability of wild-type and N287Y CFTR.
a, cells were labeled metabolically with 100 µCi of
[35S]methionine for 30 min. After the pulse, the
[35S]methionine-containing medium was replaced by
complete medium supplemented with 1 mM methionine and
chased for the times indicated above each film image.
Clarified cell lysates were subject to immunoprecipitation with CFTR
antibodies and analyzed by SDS-PAGE fluorography. Shown is a fluorogram
from a typical experiment. The arrowheads indicate the
mobilities of the immature core-glycosylated (open
arrowhead) and the mature complex-glycosylated (filled
arrowhead) forms of CFTR. b, integrated intensities of
the immature (open circles) or mature (closed
circles) CFTR were obtained by phosphorimage analysis and
represent mean ± S.E. (n = 4).
c, targeting efficiency of newly synthesized CFTR to the
cell surface. Following pulse labeling of wt or N287Y CFTR for 25 min,
plasma membrane insertion of the ion channels was determined by
biotinylation during a 1-h chase at 37 °C using freshly
dissolved sulfo-NHS-SS-biotin (1 mg/ml) every 15 min. Biotinylated CFTR
was precipitated with the M3A7 antibody and then isolated on
streptavidin-agarose. Labeled CFTR was visualized by fluorography and
quantified by phosphorimage analysis. d, integrated
densities of wt and N287Y CFTR were obtained by phosphorimage analysis
and represent mean ± S.E. (n = 3).
e, cells were pulsed with
[35S]methionine/cysteine for 30 min as described under
"Experimental Procedures" and then chased for the indicated
times. Inset, the data were fit with a single exponential
term by linear least-squares regression analysis.
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N287Y CFTR Displays Abnormal Endocytic Trafficking--
Since
biosynthesis and membrane insertion of N287Y CFTR was uncompromised, we
hypothesized that the reduction in cell surface N287Y CFTR compared
with the wild type was likely due to an increase in endocytic retrieval
from the plasma membrane. Endocytic internalization was determined by
detecting the extent to which cell surface biotinylated CFTR was
endocytosed and refractory to subsequent removal of remaining cell
surface biotin following exposure to membrane impermeant thiol-reducing
agents (19). Direct evidence for enhanced endocytosis of N287Y CFTR was
obtained by determining the amount of thiol-resistant biotinylated CFTR
with respect to time. The rapid increase in thiol-resistant
biotinylated wild-type and N287Y CFTR indicates that both constructs
are internalized with high efficiency (Fig. 5). Approximately 32% of wild-type CFTR
was endocytosed during the first 5 min of incubation, showing an
internalization rate of 6.6 ± 0.5%/min (n = 4),
a rate similar to that reported previously (15-17). In contrast, the
internalization rate of N287Y CFTR was nearly 2-fold faster (12.4 ± 0.4%/min, n = 4), implying that the tyrosine
substitution enhanced the endocytic activity of CFTR.

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Fig. 5.
Internalization efficiency of CFTR is
increased by the N287Y mutation. The rate of removal of CFTR from
the cell surface was monitored as an increase in biotinylated CFTR
resistant to thiol cleavage of the biotin moiety as described under
"Experimental Procedures." Cells stably expressing wild-type
(filled circle) or N287Y (open circle) CFTR were
subjected to cell surface biotinylation at 4 °C, washed, and left to
internalize CFTR at 37 °C. Biotin remaining at the cell surface was
removed using a membrane-impermeant reducing agent. Cells were lysed,
biotinylated proteins were isolated on streptavidin beads, and CFTR was
detected by immunoblot analysis. Results are means ± S.E. for
four separate experiments.
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Channel Density, but Not Single Channel Properties, Is Altered in
N287Y CFTR-expressing Cells--
We have documented abnormalities in
the endocytic, but not biosynthetic, traffic of N287Y CFTR that result
in a decrease in biotinylatable CFTR at the cell surface. We propose
that this reduction is sufficient to lead to the mild clinical
phenotype observed in this patient. However, it is formally possible
that there are also alterations in the biophysical fingerprint of N287Y CFTR compared with wild type. To confirm that N287Y CFTR is functional at the plasma membrane, electrophysiological recordings were performed in the whole cell and cell-attached patch configurations on CHO cells
stably expressing either wild-type or N287Y CFTR. Whole cell
measurements were used to obtain an estimate of channel density. Expression of both wild-type and N287Y CFTR conferred cAMP-stimulated whole cell currents in CHO cells (Fig. 6)
with no change in base-line current in the absence of cAMP. The
cAMP-stimulated whole cell conductance was 2.6-fold higher in wt
CFTR-expressing cells compared with N287Y CFTR-expressing cells.
Cell-attached patch studies revealed the single channel properties of
the N287Y CFTR were nearly identical to that of wt CFTR. The single
channel conductance's were 9.7 ± 0.35 versus 9.8 ± 0.39 picosiemens and open probabilities were 0.48 ± 0.04 versus 0.46 ± 0.05 for N287Y CFTR and wt CFTR, respectively. These results strongly support the conclusion that the
lower whole cell conductance of the N287Y CFTR-expressing cells is the
result of a lower density of functional channels in the plasma
membrane.

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Fig. 6.
Single channel kinetics are unaltered by the
N287Y mutation. Shown are the whole cell current-voltage
curves and single channel current traces from wt CFTR and N287Y
CFTR-expressing CHO cells. a, whole cell current-voltage
curves were obtained from n = 8 cells in each group
before and after stimulation with 10 µM forskolin plus
100 µM cpt-cAMP. Plotted are the steady-state currents
after stimulation minus the base-line currents before stimulation.
b and c, base-line currents prior to cAMP
stimulation were identical for wt and N287Y CFTR-expressing cells.
Shown are the current traces of single channel activity from
cell-attached patches of wt and N287Y CFTR-expressing cells stimulated
with forskolin and cpt-cAMP at a holding potential of 80 mV. The
dashed line indicates the zero current level. Each patch had
a total of five active channels. Results are representative of five
similar experiments.
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 |
DISCUSSION |
We have demonstrated that the N287Y mutation in CFTR causes
clinical disease by dramatically increasing the rate at which CFTR is
sequestered from the plasma membrane without altering its channel
properties. Although there are several classes of CF-causing
mutations, they nonetheless can be broadly grouped into two main
categories (23, 24). The first group includes those mutants that affect
channel density at the cell surface either by affecting protein
synthesis (class I); by reducing protein folding, processing, and
export from the endoplasmic reticulum to the plasma membrane (class
II); or by altering stability and abundance of mRNA or protein
(class V). Disease-associated mutations within the second group
generate CFTR molecules that reach the cell surface but fail to
appropriately transport chloride ions either because of a defect in
regulation by ATP and protein kinase A (class III) or channel
conductance (class IV). Several mutations, including the
F508
mutation, display disruption in both protein traffic and ion channel
activation/conductance. Recently Lukacs and colleagues (22) have
suggested a class VI mutation that would include mutations in CFTR that
alter protein stability within the plasma membrane by increasing
degradation rates. Several point mutations have been described in the
amino-terminal cytoplasmic loops of CFTR in CF patients (25, 26). Based
upon these studies and others (27), the intracellular loops of CFTR are
emerging as significant contributors to CFTR trafficking and function. Approximately half of the disease-causing point mutations identified in
the second intracellular loop give rise to processing mutants; the rest
appear to process appropriately but have significantly reduced open probabilities.
Here we report a novel class of CFTR mutation. In contrast to
previously reported mutations in CFTR, the biosynthesis, exocytic insertion into the plasma membrane, overall protein stability, and
single channel kinetics are unaltered; only endocytic kinetics are
changed. The N287Y mutation, a mutation in the second intracellular loop, results in a channel that is biosynthetically and biophysically normal but has greater endocytosis kinetics compared with wild-type CFTR. This increased endocytic rate results in a significant reduction (~50%) in steady-state CFTR levels in the plasma membrane as
determined by cell surface biotinylation and whole cell patch clamp
analysis. Similarly targeting of CFTR to the apical plasma membrane
domain of polarized epithelial cells is unaffected by the N287Y
mutation. In contrast to other mutations in the second intracellular
loop, the N287Y mutation has no functional consequence on the gating kinetics of CFTR. Thus open probability and single channel conductance are essentially wild type. The only physiological consequence of the
N287Y mutation is therefore to produce a more rapidly endocytosed protein, reducing steady-state levels in the plasma membrane. The
genotype of the initial patient reported to have the N287Y mutation was
F508/N287Y (18). Since little or no cell surface CFTR is produced by
the
F508 allele, the only cell surface CFTR protein is produced by
the N287Y allele. This would lead to a 75% reduction in the amount of
CFTR at the cell surface in this patient compared with an individual
homozygous for wild-type CFTR.
The most parsimonious interpretation of our data is that the N287Y
mutation generates a novel tyrosine-based endocytic motif. Yet the
mutant tyrosine-containing sequence (284MIENLRQT
284MIEYLRQT) does not conform to the
canonical NPXY or YXX
sequences where
X is a variable amino acid and
is a bulky hydrophobic. However, non-canonical tyrosine-based endocytic motifs have recently been described in ionotropic receptors (28) that result in
AP-2-mediated targeting to the clathrin-dependent endocytic
pathway. Thus it is possible that the N287Y mutation results in the
generation of a sequence that displays modest affinity for the AP-2
clathrin adaptor complex. Although we have focused upon the role of the N287Y mutation in mediating enhanced endocytosis of CFTR, it is still a
formal possibility that the N287Y mutation could also reduce the
endocytic recycling rate. However, it still remains to be determined
whether CFTR does indeed recycle or whether endocytosed CFTR is
degraded. It is of interest to note that the N287Y mutation is a gain
of function mutation and appears to generate an endocytic signal that
is present within the body of the protein rather than at the termini of
the protein, a localization not previously identified in polytopic
membrane proteins. The first identification of an endocytic targeting
sequence and a disease-causing mutation within that sequence was the
NPVY sequence identified in the cytoplasmic tail of the low density
lipoprotein receptor; mutations in this sequence result
in an inhibition of endocytic internalization of the low density
lipoprotein receptor (29) with concomitant increases in plasma low
density lipoprotein. In contrast, our studies report a mutation that
generates an additional endocytic signal resulting in increased rates
of endocytosis and loss of CFTR from the cell surface. In the broader
context of molecular mechanisms underlying the pathology of human
genetic diseases, the significance of our observations lies in the
recognition that mutations can reduce the expression level of a
membrane protein not only by impairing its biosynthesis or stability
(or in the case of ion channels their biophysical fingerprint) but also
by accelerating endocytic retrieval from the plasma membrane.
 |
ACKNOWLEDGEMENTS |
We thank S. W. Watkins for help with
immunofluorescence imaging and analysis and R. A. Frizzell for
critical review of the manuscript.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DK57583 (to N. A. B.) and P50DK56490 (Project 1 to R. J. B. and Project 2 to N. A. B.).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.
§
To whom correspondence should be addressed: Dept. of Cell Biology
and Physiology, University of Pittsburgh School of Medicine, S306 BST
South, 3500 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-2845; Fax:
412-648-2844; E-mail: nabrad@pitt.edu.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212843200
 |
ABBREVIATIONS |
The abbreviations used are:
CF, cystic fibrosis;
CFTR, CF transmembrane conductance regulator;
ER, endoplasmic
reticulum;
CHO, Chinese hamster ovary;
wt, wild-type;
TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic
acid;
cpt-cAMP, chlorophenylthio-cAMP;
sulfo-NHS-SS-biotin, sulfosuccinimidyl-2-(biotin-
amido)ethyl-1,3-dithioproprionate.
 |
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