| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 51, 49952-49957, December 20, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
§¶,§¶,§,
,§,§, and§
From the Departments of Cell Biology,
Physiology and Biophysics, and ** Medicine and the
§ Gregory Fleming James Cystic Fibrosis Research Center,
University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
Received for publication, September 10, 2002
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
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. F., Clancy,
J. P., Marchase, R. B., and Collawn, J. F. (1999)
J. Biol. Chem. 274, 3602-3609). Here we show that a
second substitution in the carboxyl-terminal tail of CFTR, I1427A, on
Y1424A background more than doubles CFTR surface expression as
monitored by surface biotinylation. Internalization assays indicate
that enhanced surface expression of Y1424A,I1427A CFTR is caused by a
76% inhibition of endocytosis. Patch clamp recording of chloride
channel activity revealed that there was a corresponding increase in
chloride channel activity of Y1424A,I1427A CFTR, consistent with the
elevated surface expression, and no change in CFTR channel properties.
Y14124A showed an intermediate phenotype compared with the double
mutation, both in terms of surface expression and chloride channel
activity. Metabolic pulse-chase experiments demonstrated that the two
mutations did not affect maturation efficiency or protein half-life.
Taken together, our data show that there is an internalization signal
in the COOH terminus of CFTR that consists of
Tyr1424-X-X-Ile1427
where both the tyrosine and the isoleucine are essential residues. This
signal regulates CFTR surface expression but not CFTR
biogenesis, degradation, or chloride channel function.
The cystic fibrosis transmembrane conductance regulator
(CFTR)1 is a cAMP-activated
chloride channel that resides at the apical surface of epithelial
cells. 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
NH2 terminus (7, 8). How these interactions affect cell
surface expression is not clear, but they imply that CFTR may exist in
at least two cell surface pools, one tethered to the actin cytoskeleton
and one associated with the endocytic pathway. Subcellular localization studies reveal that CFTR is found in the endosomes in epithelial cells
(9), supporting the view that CFTR enters the endocytic pathway.
Whether CFTR is constitutively recycled is not known.
In previous studies, our laboratory demonstrated that a key feature of
CFTR endocytosis was the presence of a tyrosine residue at position
1424 in the COOH-terminal tail of CFTR. Because tyrosine-based signals
have been proposed to consist of the motif YXX Construction of CFTR Mutants--
CFTR (wild-type) was provided
by the Gregory James Cystic Fibrosis Center Vector Core and Dr. Jeong
Hong. The construction of the Y1424A mutant was described previously
(3). For construction of the Y1424A,I1427A mutant, a
BstXI-SgrAI fragment that coded for the
COOH-terminal tail region of Y1424A CFTR was subcloned into
pSK-Bluescript (Stratagene). A second-site mutation was prepared from
the corresponding pSK-Bluescript vector containing the
BstXI-SgrAI fragment from single-stranded DNA as
described previously (11) by the method of Kunkel (12). Mutants were
selected by sequencing and then subcloned into the
BstXI-SgrAI site of pGT-1-CFTR. The mutations
were verified by dideoxynucleotide sequencing (13) using the Sequenase
kit (U. S. Biochemical Corp.) according to the manufacturer's directions.
Cell Culture and Transient Transfection of COS-7
Cells--
COS-7 cells were cultured as described previously (3) and
transiently transfected using LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's directions. The cells were incubated at 37 °C in a humidified incubator for 24-48 h before analysis.
Immunoprecipitation of CFTR Protein--
One 100-mm dish of
transfected COS-7 cells was used 48 h post-transfection. CFTR was
immunoprecipitated using a polyclonal antibody to nucleotide binding
domain 1 (a generous gift from Dr. David Bedwell at the
University of Alabama at Birmingham) and phosphorylated with
[ Biotinylation of Surface CFTR--
Cell-surface biotinylation of
glycoproteins and detection of CFTR were performed as described
previously (1) with the following modifications. After biotinylation
and lysis, samples were divided into two equal samples and
immunoprecipitated with anti-CFTR nucleotide binding domain 1 antibody and protein A-agarose. One of the immunoprecipitated samples
was then eluted from the beads using Laemmli sample buffer (without
bromphenol blue), diluted in RIPA buffer (150 mM NaCl, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM
Tris-HCl, pH 8.0) 10-fold, and the biotinylated fraction was captured
with avidin-Sepharose beads (Pierce). Both total CFTR and biotinylated CFTR were then in vitro phosphorylated using
[ Metabolic Pulse-Chase Assays--
One day post-transfection,
COS-7 cells (one 35-mm dish/time point) were rinsed three times and
incubated in methionine-free Dulbecco's modified Eagle's medium for
1 h and then pulsed in the same media containing 200 µCi/ml trans-[35S]methionine (ICN
Biomedicals). Pulse-labeled cells were chased for 0, 4, 14, 18, or
24 h in complete media. At each time point, the cells were placed
on ice and rinsed with cold phosphate-buffered saline, lysed in RIPA
buffer, and incubated for an additional 30 min on ice. CFTR was
immunoprecipitated from the post-nuclear supernatants and analyzed by
SDS-PAGE and autoradiography (PhosphorImager, Amersham
Biosciences). Calculation of the protein half-lives was performed as described by Straley et al. (1998) (14).
Whole Cell Patch Clamp Assays--
Individual dishes of
transfected COS-7 cells were used in electrophysiological recordings as
described previously (15). One modification is that PClamp 8.0 software
was used in this study. COS-7 cells were transiently transfected with
each of the CFTR constructs along with pGL-1 (pGreen Lantern-1, a green
fluorescence protein (GFP) plasmid). Under these conditions, >90% GFP
and CFTR co-transfectants respond to cyclic AMP mixture (250 µM 8-Br-cAMP and chlorophenyl thio-cAMP plus 2 µM forskolin) treatment with an increase in whole cell
Cl Single Channel Patch Clamp--
Assays of single channel
recordings were obtained from membrane patches in both the
cell-attached and inside-out configurations. Recording pipettes were
constructed from borosilicate glass capillaries (Warner Instrument
Corporation, Hamden, CT) using a Narishige PC-10 microelectrode puller
(Narishige Scientific Instrument Laboratory, Tokyo, Japan) and were
fire-polished with a Narishige microcentrifuge. The pipettes
were partially filled with standard pipette solution and had tip
resistances of 5-10 megohms. Experiments were performed at room
temperature (20-22 °C). Currents were recorded at 50-60 mV
(negative to pipette potential) using an Axopatch 200B patch clamp
amplifier (Axon Instruments, Union City, CA) low pass-filtered at 1000 Hz (LPF-8, Warner Instruments), sampled every 100 µs with a Digidata
1321A interface (Axon Instruments), and stored onto the computer hard
disk using PClamp 8 software (Axon Instruments). A brief protocol of
stepping the holding potential from Endocytosis Assays--
Internalization assays were performed as
described previously (3) that included the modifications of the surface
biotinylation assay described above.
Mutations in the Carboxyl-terminal Tail of CFTR Increase Surface
Expression--
Our hypothesis in these experiments is that if both
tyrosine 1424 and isoleucine 1427 are important for CFTR
internalization, complete disruption of these residues should increase
CFTR surface expression. Because little is known concerning the nature
of CFTR endocytosis and recycling or how these processes affect CFTR
function, we constructed a double substitution COOH-terminal mutant in
which both tyrosine 1424 and the isoleucine 1427 were changed to alanine.
First, we determined the effects of these substitutions on CFTR surface
expression by comparing the percentage of wild-type and mutant CFTR at
the cell surface using a surface biotinylation assay. COS-7 cells
expressing wild-type, Y1424A, and Y1424A,I1427A CFTR were
surface-biotinylated and lysed in RIPA buffer (see "Materials and
Methods"). Total CFTR was measured following immunoprecipitation from
50% of the lysate detected by in vitro phosphorylation
([ Mutations at Tyr1424 and Ile1427 Do Not
Alter CFTR Maturation Efficiency or Protein Half-life--
To test the
effects of these mutations on maturation efficiency and protein
half-life, we performed metabolic pulse-chase experiments on COS-7
cells expressing wild-type, Y1424A, and Y1424A,I1427A CFTR. CFTR
half-lives were measured 24 h post-transfection. The results in
Fig. 2 show that the half-lives for
wild-type (Wt), Y1424A, and Y1424A,I1427A CFTR were
10.3 ± 2.3, 11.3 ± 2.6, and 11.3 ± 1.5 h
(mean ± S.D.). This finding indicated that the elevated surface
expression of the mutants was not attributed to enhanced protein
half-life.
In the same series of experiments, we also compared the amount of
immaturely glycosylated CFTR (Band B) at 0 time with the amount of maturely glycosylated CFTR (Band C) at 4 h
(top panel). The average maturation efficiency for wild-type
(Wt), Y1424A, and Y1424A,I1427A CFTR were 32, 31, and 31%,
respectively (bottom right panel). This finding demonstrated
that elevated surface expression of Y1424A,I1427A CFTR was not because
of alterations in maturation efficiency.
Tyrosine 1424 and Isoleucine 1427 Are Necessary for CFTR
Endocytosis--
To test whether elevated surface expression was
attributed to alterations in the internalization rate of CFTR, we
performed internalization assays on COS-7 cells expressing wild-type,
Y1424A, and Y1424A,I1427A CFTR. Using a warm-up period between
periodate and the biotin LC-hydrazide treatments (0 or 2.5 min),
we monitored the loss of the surface pool of CFTR (see "Materials and
Methods"). During this warm-up period, previously oxidized
carbohydrate residues are internalized and therefore do not react with
the membrane-impermeant biotin LC hydrazide (1). A representative
internalization assay for each of the constructs is shown in Fig.
3, top panel. A summary of
eight assays is shown in the lower panel. For wild-type
CFTR, 34% of the surface pool was internalized in 2.5 min. For Y1424A and Y1424A,I1427A CFTR, internalization dropped to 21 and 8%, respectively, during the same time period. These results demonstrate that CFTR endocytosis is inhibited by 76% when these two residues are
modified.
The Y1424A and Y1424A,I1427A CFTR Have Normal Chloride Channel
Properties--
Because the biochemical data suggested that a specific
motif in the CFTR COOH terminus dramatically affected endocytosis and because point mutations in the NH2 terminus lead to both
disruption of binding to docking machinery and changes in CFTR ion
channel function, we tested whether the mutation of Tyr1424
and Ile1427 affected chloride channel function. Whole cell
patch clamp recordings were performed to assess the total population of
CFTR Cl
Single channel biophysical properties of wild-type, Y1424A, and
Y1424A,I1427A CFTR were also assessed. Before the recording of CFTR
Cl The data presented here demonstrate that two key residues in the
COOH-terminal tail dramatically regulate the steady-state distribution
of CFTR between the cell surface and intracellular sites. This is the
first demonstration of a CFTR mutant whose activity is actually
enhanced relative to wild-type CFTR. We established this observation
using both surface biotinylation and patch clamp measurements.
In examining the mechanism for the elevated surface expression of CFTR,
we first showed that total expression levels of wild-type, Y1424A, and
Y1424A,I1427A were the same. We next demonstrated that maturation
efficiency and protein half-life were unaffected, suggesting that a
primary alteration caused by these substitutions involved changes in
distribution between the intracellular and cell surface compartments.
This alteration could result from decreased internalization or
increased recycling or both. Moreover, we showed that Y1424A,I1427A
CFTR was internalized much more slowly than the native protein (76%
inhibition at 2.5 min) with an internalization rate of ~2%/min.
Several observations suggest that the only internalization signal in
CFTR is the
Tyr1424-X-X-Ile1427 motif
in the COOH-terminal tail. First, the ablation of the only endocytosis
signal in the transferrin receptor YTRF resulted in a similar loss of
internalization activity (11). Furthermore, the rate of endocytosis of
the 20YTRF23 The signal identified here, YXXI, appears to function only
as an internalization signal and not a "down-regulation" signal for
conferring CFTR degradation. If YXXI was important to
mediate CFTR degradation, metabolic pulse-chase experiments would have revealed an extended half-life when the signal was inactivated. Our
studies indicate that CFTR lacking YXXI is stabilized at the cell surface because endocytosis of this mutant is severely
compromised. This also suggests that CFTR participates in the
membrane-recycling pathway. This idea is consistent with previously
reported immunolocalization studies that have shown that CFTR
co-localizes with rab4, a component of recycling endosomes (9). The
reasons why CFTR would be part of this pathway are unclear, but it may
be to regulate the amount of functional chloride channels at the cell
surface in the same manner as aquaporins and glucose transporters are
regulated (20-25).
The specific residues identified by these studies, YXXI,
that are important for CFTR endocytosis are conserved in the ten COOH-terminal tail sequences spanning from Xenopus to human
(3). The tyrosine residue is conserved among all species with the
exception of the dogfish, which has a phenylalanine residue. The
isoleucine residue is conserved in 7 of 10 sequences with a very
conservative leucine residue substitution in the other three,
indicating that this motif, YXX(I/L), is highly
conserved in the sequences identified to date. Both FXXL
(dogfish) and YXX(I/L) conform to the YXX The identification of the YXXI signal is also consistent
with recent studies that a region that includes this sequence interacts with the endocytic clathrin adaptor complex AP-2 using plasmon resonance analysis (19). Together, their study (19) and ours support
the view that CFTR endocytosis occurs through clathrin-coated pits. Our
study shows that two residues in the COOH-terminal tail, tyrosine 1424 and isoleucine 1427, regulate the steady-state distribution of CFTR
between the plasma membrane and intracellular sites. This raises the
important and testable hypotheses that the Y1424,I1427 signal controls
CFTR entry into clathrin-coated pit regions at the apical membrane and
that ablation of this signal abrogates one type of microdomain
targeting in polarized epithelial cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
where is a large hydrophobic residue and X is any residue (10), we
tested the hypotheses that the isoleucine residue at position 1427 is
important for CFTR endocytosis and that ablation of this putative
signal YXXI would increase the steady-state surface
expression of CFTR. To this end, we performed an integrated series of
biochemical and electrophysiological assays designed to study
maturation efficiency, trafficking, and Cl
channel
function of the wild-type and two COOH-terminal mutant CFTR proteins.
We find that the substitution of Tyr1424 and
Ile1427 with alanine residues resulted in a 2-fold increase
in surface expression, whereas the single Y1424A mutation shows an
intermediate phenotype. CFTR internalization assays revealed that the
elevated surface expression was attributed to a dramatic decrease in
endocytosis, suggesting that these residues are necessary for CFTR
internalization. Because the chloride channel activity and relative
surface expression of Y1424A and I1427A CFTR are elevated to a similar
extent, we propose that these substitutions affect protein trafficking
but not CFTR chloride channel function. To our knowledge, this is the
first CFTR mutant that has enhanced rather than diminished activity at
the cell surface because of attenuation of internalization.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
32P]ATP (PerkinElmer Life Sciences) and
cAMP-dependent protein kinase (Promega). Labeled CFTR was
analyzed by SDS-PAGE, autoradiography, and phosphorimaging analysis as
described previously (3).
32P]ATP (PerkinElmer Life Sciences) and
cAMP-dependent protein kinase (Promega) as described
previously (3).
conductance. Background levels of cyclic AMP-activated
Cl conductance were monitored in non-transfected cells in
the same dish that lack GFP fluorescence, in mock-transfected cells,
and in parental cells. In these whole-cell recordings, the bath
(extracellular) solution contained 145 mM Tris-Cl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 60 mM sucrose, and 5 mM
HEPES, pH 7.45. The pipette (intracellular) solution contained 145 mM Tris-Cl, 5 mM HEPES, 100 nM
CaCl2 and MgCl2 (chelated with 2 mM
EGTA), and 5 mM Mg2+-ATP, pH 7.45. These
solutions were designed to study the only current flowing through
Cl channels because Cl is the only permeant
ion in solution, to clamp intracellular Ca2+ at ~100
nM, and to prevent swelling-activated Cl
currents with added sucrose in the bath solution.
100 to +100 mV and back to 100
mV served to inactivate a contaminating voltage-dependent
Cl channel (probably ClC-2) that was
hyperpolarization-activated but inactivated permanently by a +100-mV
pulse. The pipette solution contained (in mmol/liter): 150 NaCl, 1 MgCl2, 1 CaCl2, 5 HEPES, pH 7.4. The bath
solution contained (in mmol/liter): 150 NaCl, 1 MgCl2, 5 EGTA, 5 HEPES, pH 7.4.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and protein kinase A) and analyzed by
SDS-PAGE and autoradiography (Fig. 1,
top panel, total CFTR). CFTR was also immunoprecipitated from the other half of the lysate. This fraction was then eluted from
the protein-A beads, reprecipitated using monomeric avidin-Sepharose (to separate biotinylated CFTR), and detected as described above for
the total CFTR (Fig. 1, top panel). The percentage CFTR at the cell surface was markedly increased for Y1424A,I1427A CFTR compared
with both wild-type (108% increase, n = 10, p < 0.001) and Y1424A CFTR (59% increase,
n = 10, p < 0.001) (Fig. 1,
bottom panel). The surface biotinylation data indicated that
modification of residues Tyr1424 and Ile1427
increased the steady-state surface expression of CFTR. The potential mechanisms that could account for these differences include changes in
1) maturation efficiency, 2) protein half-life, or 3) internalization and/or recycling rates.
View larger version (25K):
[in a new window]
Fig. 1.
Surface expression levels of wild-type and
mutant CFTR in COS-7 cells. The levels of expression of
wild-type (Wt), Y1424A, and Y1424A,I1427A CFTR were analyzed
in COS-7 cells 48 h after transfection. Cells were lysed in RIPA
buffer, and CFTR was immunoprecipitated using an anti-nucleotide
binding domain 1 polyclonal antibody. Total, total
CFTR from 50% of the lysate. Biotinylated CFTR was eluted from the
antibody-protein A beads and reprecipitated using avidin-Sepharose
beads. Biotinylated, CFTR from 50% of the lysate. Mock
transfected cells were used as a negative control (lanes 1 and 5). Immunoprecipitated (Total) and
reprecipitated (Biotinylated) CFTR were in vitro
phosphorylated with protein kinase A and [ -32P]ATP and
analyzed by SDS-PAGE and autoradiography. A representative gel of 10 is
shown (top panel). The relative amounts of wild-type
(lanes 2 and 6), Y1424A (lanes 3 and
7), and Y1424A,I1427A CFTR (lanes 4 and
8) are shown. The averages ± S.E. were calculated from
the phosphorimaging analysis from 10 independent experiments. *,
p < 0.02; +, p < 0.001 (compared with
wild-type CFTR (lower panel)).
View larger version (34K):
[in a new window]
Fig. 2.
Point mutations in the CFTR COOH terminus do
not affect protein stability or maturation efficiency. The protein
turnover and processing of CFTR and CFTR mutants were monitored in
COS-7 cells 24 h after transfection in metabolic pulse-chase
experiments. After a 1-h pulse and the indicated chase time periods,
the cells were lysed in RIPA buffer and CFTR or CFTR mutants were
immunoprecipitated and analyzed as described under "Materials and
Methods." Mock (M) transfected cells were used as a
negative control. The top panel shows a representative gel.
Bands B and C of CFTR indicated on the
left. The average half-lives and maturation efficiencies
from four independent experiments shown below demonstrate that the
half-lives (left panel) and maturation efficiencies
(right panel) are not affected by these two CFTR
substitutions.
View larger version (31K):
[in a new window]
Fig. 3.
Comparisons of the internalization
rates of CFTR and CFTR mutants. COS-7 cells transfected with
wild-type, Y1424A, or Y1424A,I1427A CFTR were analyzed 48-h
post-transfection. Wild-type or mutant CFTR was biotinylated using a
two-step surface periodate/LC-hydrazide biotinylation procedure. At
zero time, both steps were conducted at 4 °C to label the entire
pool of CFTR. Internalization was monitored by a loss of biotinylated
of the cell surface pool by including a 37 °C incubation period
(Time (min)) between periodate and biotin
LC-hydrazide treatments. Biotinylated CFTR and total CFTR were detected
as shown in Fig. 1. The percentage of wild-type, Y1424A, and
Y1424A,I1427A CFTR internalized after 2.5 min was 34, 18, and 8 respectively. 1 of 8 representative experiments is shown. In the
bottom panel, the percentage of CFTR internalized at
each time point was calculated based on phosphorimaging analysis
(averages from eight experiments: *, p < 0.05 compared
with Wt; +, p < 0.001 compared with Wt).
channels in the plasma membrane of transfected
COS-7 cells. Tris-Cl-containing solutions were used in bath
(extracellular) and pipette (intracellular) solutions so that
Cl was the only major permeant ionic species in the
recordings. GFP was also expressed together with the CFTR-bearing
vectors to detect cells that were successfully transfected prior to
recording. Cells that did not express GFP served as internal controls.
Three sets of transiently transfected COS-7 cells were examined in
parallel with the above biotinylation experiments (Table
I). In agreement with the surface
biotinylation assays, CFTR whole cell Cl currents in
Y1424A CFTR and Y1424A,I1427A CFTR-transfected cells were elevated
compared with wild-type CFTR-expressing cells (Table I), suggesting
that the elevated Cl channel activity was the result of
the elevated surface expression of CFTR. Typical whole cell current
traces after stimulation with cAMP agonist mixture for wild-type and
mutant CFTR are shown in Fig.
4A. Fig. 4B shows
wild-type CFTR Cl current-voltage relationships
demonstrating insensitivity of the currents to DIDS (100 µM) and inhibition of the currents by glibenclamide (100 µM). These pharmacological properties are consistent with
wild-type CFTR (16). The time and voltage independence of the currents
and the linear I-V relationship are also consistent with CFTR chloride
channel activity. Fig. 4, C and D, show the Y1424A and Y1424A,I1427A Cl current-voltage
relationships, respectively, and indicate that although the
sensitivities to DIDS and glibenclamide remain similar to wild-type
(Fig. 4B), the total current is elevated in the single and
double mutants. Whereas the representative I-V plots show a
variability in sensitivity to glibenclamide, inhibition with this
Cl channel-blocking drug was only partial ranging from 50 to 90% for both wild-type and mutant currents.
Summary of whole cell patch clamp recordings for wild-type CFTR and for
CFTR mutants shows elevated activity in the mutants relative to wild
type
View larger version (23K):
[in a new window]
Fig. 4.
Chloride channel activity of the CFTR
COOH-terminal mutants is normal but expression of the mutants is
elevated. Table I shows the complete summary of the whole-cell
patch clamp data. Panel A showed typical whole-cell current
records. Typical whole-cell I-V plots for wild-type CFTR (panel
B), Y1424A (panel C), and Y1424A,I1427A (panel
D) showing cyclic AMP-stimulated chloride currents in the absence
of blockers (squares), presence of DIDS (upward
triangles), and presence of glibenclamide (inverted
triangles). A non-green cell showing background cyclic
AMP-stimulated chloride currents is also shown in each plot
(circles). A linear I-V relationship and time- and
voltage-independent kinetics are hallmarks of CFTR channels and were
similar in nature between wild type (WT) and the mutants. Panel
E shows representative single channel current traces for WT and
the mutants. Although these segments of recordings show more channels
and more "wave-like" cooperative gating in the mutants
versus the wild type, N or number of channels per
patch could not be calculated because a quiet 0-channel base line was
never reserved in patches that contained CFTR channels.
channel properties under cAMP-stimulated conditions
was undertaken, voltage steps between 100 and +100 mV were necessary
to inactivate a pseudo-channel with similar Cl
conductance as CFTR. The properties of this channel were not inconsistent with ClC-2, known to be expressed in COS-7 cells (17).
Representative recordings of wild-type, Y1424A, and Y1424A,I1427A CFTR
at 50-60 mV (negative to pipette potential) are shown in Fig.
4E. Single channel conductance for all three constructs was 7-8 picoSiemens for stretches of the recordings where a subset of the
channels could be analyzed. Biophysical analysis of single channel
kinetics was not possible, because each patch obtained from a
positively transfected cell had at least 10 channels. We could never
obtain patches with a single channel. Furthermore, a base line without
channel openings was not observed. Nevertheless, the whole cell and
single channel recordings together show that the difference in
Cl channel activity is attributed to elevated surface
expression without a significant change in CFTR chloride channel
properties among wild-type, Y1424A, and Y1424A,I1427A CFTR.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20ARTA23 mutant was the same as a
transferrin receptor containing only a 4-amino acid cytoplasmic tail,
indicating that this motif and more specifically these two residues
were the only residues in the 61-amino acid cytoplasmic tail of the
transferrin receptor that were necessary for endocytosis. Second, the
internalization rate of Y1424A,I1427A CFTR is comparable with the rate
of bulk flow lipid uptake via the endocytic pathway (~2%/min.) (18), suggesting that the residual internalization activity observed in these
studies reflects nonspecific uptake through clathrin-coated pits.
Considering that clathrin-coated pits constitute ~2% of the cell
surface (18), our findings suggest that the double mutant has
completely lost the ability to concentrate in these surface domains.
This result has particular significance given the increasing evidence
that CFTR enters the endocytic pathway via clathrin-coated pits (2, 4,
19).
motif common to internalization signals, where X is any
amino acid and is a hydrophobic residue (10).
| |
ACKNOWLEDGEMENT |
|---|
We thank the Gregory Fleming James Cystic Fibrosis Research Center for their support of this research.
| |
FOOTNOTES |
|---|
* This work was supported in part by a fellowship from the Research Development Program of the Cystic Fibrosis Foundation (CFF) (to K. P. and K. V.), Grants COLLAWOOGO from the CFF and DK 60065 from the National Institutes of Health (to J. F. C.), Grant DK 54367 from the National Institutes of Health (to E. M. S.), and a grant from the Research Development Program of the CFF and the National Institutes of Health (to E. J. S.).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.
¶ Both authors contributed equally to this work.
To whom correspondence should be addressed: Gregory Fleming
James Cystic Fibrosis Research Center, University of Alabama, MCLM 350, 1918 University Blvd., Birmingham, AL 35294-0005. Tel.: 205-934-1002;
E-mail: jcollawn@uab.edu.
Published, JBC Papers in Press, October 9, 2002, DOI 10.1074/jbc.M209275200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; RIPA, radioimmune precipitation assay buffer; GFP, green fluorescent protein; DIDS, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. |
Prince, L. S.,
Workman, R. B., Jr.,
and Marchase, R. B.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
5192-5196 |
| 2. | Lukacs, G. L., Segal, G., Kartner, N., Grinstein, S., and Zhang, F. (1997) Biochem. J. 328, 353-361 |
| 3. |
Prince, L. S.,
Peter, K.,
Hatton, S. R.,
Zaliauskiene, L.,
Cotlin, L. F.,
Clancy, J. P.,
Marchase, R. B.,
and Collawn, J. F.
(1999)
J. Biol. Chem.
274,
3602-3609 |
| 4. |
Bradbury, N. A.,
Cohn, J. A.,
Venglarik, C. J.,
and Bridges, R. J.
(1994)
J. Biol. Chem.
269,
8296-8302 |
| 5. | Wang, S., Raab, R. W., Schatz, P. J., Guggino, W. B., and Li, M. (1998) FEBS Lett. 427, 103-108[CrossRef][Medline] [Order article via Infotrieve] |
| 6. |
Short, D. B.,
Trotter, K. W.,
Reczek, D.,
Kreda, S. M.,
Bretscher, A.,
Boucher, R. C.,
Stutts, M. J.,
and Milgram, S. L.
(1998)
J. Biol. Chem.
273,
19797-19801 |
| 7. | Naren, A. P., Nelson, D. J., Xie, W., Jovov, B., Pevsner, J., Bennett, M. K., Benos, D. J., Quick, M. W., and Kirk, K. L. (1997) Nature 390, 302-305[CrossRef][Medline] [Order article via Infotrieve] |
| 8. |
Naren, A. P.,
Quick, M. W.,
Collawn, J. F.,
Nelson, D. J.,
and Kirk, K. L.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
10972-10977 |
| 9. |
Webster, P.,
Vanacore, L.,
Nairn, A. C.,
and Marino, C. R.
(1994)
Am. J. Physiol.
267,
C340-C348 |
| 10. | Trowbridge, I. S., Collawn, J. F., and Hopkins, C. R. (1993) Ann. Rev. Cell Biol. 9, 129-161[CrossRef] |
| 11. | Collawn, J. F., Stangel, M., Kuhn, L. A., Esekogwu, V., Jing, S., Trowbridge, I. S., and Tainer, J. A. (1990) Cell 63, 1061-1072[CrossRef][Medline] [Order article via Infotrieve] |
| 12. |
Kunkel, T. A.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
488-492 |
| 13. | Tabor, S., and Richardson, C. C. (1987) Biochemistry 84, 4767-4771 |
| 14. |
Straley, K. S.,
Daugherty, B. L.,
Aeder, S. E.,
Hockenson, A. L.,
Kim, K.,
and Green, S. A.
(1998)
Mol. Biol. Cell
9,
1683-1694 |
| 15. |
Moyer, B. D.,
Loffing, J.,
Schwiebert, E. M.,
Loffing-Cueni, D.,
Halpin, P. A.,
Karlson, K. H.,
Ismailov, I. I.,
Guggino, W. B.,
Langford, G. M.,
and Stanton, B. A.
(1998)
J. Biol. Chem.
273,
21759-21768 |
| 16. |
Schwiebert, E. M.,
Morales, M. M.,
Devidas, S.,
Egan, M. E.,
and Guggino, W. B.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2674-2679 |
| 17. | Thiemann, A., Grunder, S., Pusch, M., and Jentsch, T. J. (1992) Nature 356, 57-60[CrossRef][Medline] [Order article via Infotrieve] |
| 18. |
Mukherjee, S.,
Ghosh, R. N.,
and Maxfield, F. R.
(1997)
Physiol. Rev.
77,
759-803 |
| 19. |
Weixel, K. M.,
and Bradbury, N. A.
(2001)
J. Biol. Chem.
276,
46251-46259 |
| 20. | Brown, D., Katsura, T., Kawashima, M., Verkman, A. S., and Sabolic, I. (1995) Histochem. Cell Biol. 104, 1-9[CrossRef][Medline] [Order article via Infotrieve] |
| 21. |
Katsura, T.,
Verbavatz, J. M.,
Farinas, J., Ma, T.,
Ausiello, D. A.,
Verkman, A. S.,
and Brown, D.
(1995)
Proc. Natl. Acad. Sci. U.S.A.
92,
7212-7216 |
| 22. |
Nielsen, S.,
Chou, C. L.,
Marples, D.,
Christensen, E. I.,
Kishore, B. K.,
and Knepper, M. A.
(1995)
Proc. Natl. Acad. Sci. U.S.A.
92,
1013-1017 |
| 23. |
Jhun, B. H.,
Rampal, A. L.,
Liu, H.,
Lachaal, M.,
and Jung, C. Y.
(1992)
J. Biol. Chem.
267,
17710-17715 |
| 24. |
Holman, G. D., Lo,
Leggio, L.,
and Cushman, S. W.
(1994)
J. Biol. Chem.
269,
17516-17524 |
| 25. |
Pessin, J. E.,
Thurmond, D. C.,
Elmendorf, J. S.,
Coker, K. J.,
and Okada, S.
(1999)
J. Biol. Chem.
274,
2593-2596 |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  R. F. Goldstein, A. Niraj, T. P. Sanderson, L. S. Wilson, A. Rab, H. Kim, Z. Bebok, and J. F. Collawn VCP/p97 AAA-ATPase Does Not Interact with the Endogenous Wild-Type Cystic Fibrosis Transmembrane Conductance Regulator Am. J. Respir. Cell Mol. Biol., June 1, 2007; 36(6): 706 - 714. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Broughman, L. Sun, S. Umar, J. Scott, J. H. Sellin, and A. P. Morris Chronic PKC-beta activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane current generation Am J Physiol Gastrointest Liver Physiol, August 1, 2006; 291(2): G318 - G330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Malik, S. R. Price, W. E. Mitch, Q. Yue, and D. C. Eaton Regulation of epithelial sodium channels by the ubiquitin-proteasome proteolytic pathway Am J Physiol Renal Physiol, June 1, 2006; 290(6): F1285 - F1294. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Jurkuvenaite, K. Varga, K. Nowotarski, K. L. Kirk, E. J. Sorscher, Y. Li, J. P. Clancy, Z. Bebok, and J. F. Collawn Mutations in the Amino Terminus of the Cystic Fibrosis Transmembrane Conductance Regulator Enhance Endocytosis J. Biol. Chem., February 10, 2006; 281(6): 3329 - 3334. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Bebok, J. F. Collawn, J. Wakefield, W. Parker, Y. Li, K. Varga, E. J. Sorscher, and J. P. Clancy Failure of cAMP agonists to activate rescued {Delta}F508 CFTR in CFBE41o- airway epithelial monolayers J. Physiol., December 1, 2005; 569(2): 601 - 615. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Rene, M. Taulan, F. Iral, J. Doudement, A. L'Honore, C. Gerbon, J. Demaille, M. Claustres, and M.-C. Romey Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway Nucleic Acids Res., September 16, 2005; 33(16): 5271 - 5290. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Varga, A. Jurkuvenaite, J. Wakefield, J. S. Hong, J. S. Guimbellot, C. J. Venglarik, A. Niraj, M. Mazur, E. J. Sorscher, J. F. Collawn, et al. Efficient Intracellular Processing of the Endogenous Cystic Fibrosis Transmembrane Conductance Regulator in Epithelial Cell Lines J. Biol. Chem., May 21, 2004; 279(21): 22578 - 22584. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-M. Zhang, X.-T. Wang, H. Yue, S. W. Leung, P. H. Thibodeau, P. J. Thomas, and S. E. Guggino Organic Solutes Rescue the Functional Defect in {Delta}F508 Cystic Fibrosis Transmembrane Conductance Regulator J. Biol. Chem., December 19, 2003; 278(51): 51232 - 51242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. S. Ostedgaard, C. Randak, T. Rokhlina, P. Karp, D. Vermeer, K. J. Ashbourne Excoffon, and M. J. Welsh Effects of C-terminal deletions on cystic fibrosis transmembrane conductance regulator function in cystic fibrosis airway epithelia PNAS, February 18, 2003; 100(4): 1937 - 1942. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
