Efficient Endocytosis of the Cystic Fibrosis Transmembrane
Conductance Regulator Requires a Tyrosine-based Signal*
Lawrence S.
Prince
,
Krisztina
Peter,
Sean R.
Hatton,
Lolita
Zaliauskiene,
Laura F.
Cotlin,
J. P.
Clancy§,
Richard B.
Marchase, and
James F.
Collawn¶
From the Department of Cell Biology, the Gregory Fleming James
Cystic Fibrosis Research Center, and the § Department of
Pediatrics, University of Alabama at Birmingham,
Birmingham, Alabama 35294-0005
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ABSTRACT |
We previously demonstrated that the cystic
fibrosis transmembrane conductance regulator (CFTR) is rapidly
endocytosed in epithelial cells (Prince, L. S., Workman, R. B., Jr., and Marchase, R. B. (1994) Proc. Natl. Acad. Sci.
U. S. A. 91, 5192-5196). To determine the structural features
of CFTR required for endocytosis, we prepared chimeric molecules
consisting of the amino-terminal (residues 2-78) and carboxyl-terminal
tail regions (residues 1391-1476) of CFTR, each fused to the
transmembrane and extracellular domains of the transferrin receptor.
Functional analysis of the CFTR-(2-78) and CFTR-(1391-1476) indicated
that both chimeras were rapidly internalized. Deletion of residues
1440-1476 had no effect on chimera internalization. Mutations of
potential internalization signals in both cytoplasmic domains reveal
that only one mutation inhibits internalization, Y1424A. Using a
surface biotinylation reaction, we also examined internalization rates
of wild type and mutant CFTRs expressed in COS-7 cells. We found that
both wild type and A1440X CFTR were rapidly internalized,
whereas the Y1424A CFTR mutant, like the chimeric protein, had ~40%
reduced internalization activity. Deletions in the amino-terminal tail region of CFTR resulted in defective trafficking of CFTR out of the
endoplasmic reticulum to the cell surface, suggesting that an intact
amino terminus is critical for biosynthesis. In summary, our results
suggest that both tail regions of CFTR are sufficient to promote rapid
internalization of a reporter molecule and that tyrosine 1424 is
required for efficient CFTR endocytosis.
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INTRODUCTION |
Cystic fibrosis is caused by mutations in the gene encoding the
cystic fibrosis transmembrane conductance regulator
(CFTR)1 (1), which functions
as a chloride channel on the apical surface of epithelial cells (2, 3).
The most common mutation in CF,
F508, is a temperature-sensitive
mutant that fails to exit the endoplasmic reticulum, presumably because
of a protein folding defect (4).
Previous studies have demonstrated that CFTR is endocytosed (5, 6)
through clathrin-coated vesicles (6, 7), suggesting that CFTR
internalization may provide a mechanism for controlling the
cAMP-stimulated chloride channel activity at the cell surface (6).
Others have suggested that CFTR may play additional roles by regulating
plasma membrane recycling (5, 8) and in clearance of Pseudomonas
aeruginosa from the respiratory tract (9, 10).
The purpose of this study was to determine the structural features of
the CFTR protein required for internalization. Internalization signals
identified to date include tyrosine-based motifs (YXX
or
NPXY, where X is any amino acid and
is a
bulky hydrophobic residue), dileucine motifs, and acidic cluster/casein
kinase II-based motifs (11-15). Initial studies of type III membrane
proteins indicate that the targeting signals occur in the amino- and
carboxyl-terminal cytoplasmic tail regions (16-19).
Our initial studies on the identification of CFTR internalization
signals focused on the two tail regions of the CFTR molecule. Here we
show that both the amino- and carboxyl-terminal cytoplasmic tail
regions of CFTR, residues 2-78 and 1391-1476, are individually sufficient to promote rapid internalization of a reporter molecule, the
transferrin receptor (TR). We also demonstrate both in the context of
chimeric and native proteins that tyrosine 1424 is important for CFTR
endocytosis. Furthermore, we show that the intracellular distribution
of the CFTR-TR chimeras is similar to that of the TR, suggesting that
endocytosis may regulate CFTR activity at the cell surface.
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EXPERIMENTAL PROCEDURES |
Construction of CFTR-TR Chimeras--
The CFTR-TR chimeras were
constructed using the polymerase chain reaction as described previously
(20). A polymerase chain reaction was performed on pKCTR-CFTR cDNA
(also referred to as pGT-CFTR), and unique NheI and
AflII sites were introduced in the 5' and 3' primers,
respectively. For the amino-terminal CFTR tail, the 5' and 3' primers
were 5'-AA-GCT-AGC-CAG-AGG-TCG-CCT-CTG-GAA-AA-3' and
5'-AA-CTT-AAG-GAA-AAAACA-TCG-CCG-AAG-GGC,
respectively. For the carboxyl-terminal CFTR tail, the 5' and 3'
primers were
5'-GCT-AGC-GCA-TTT-GCT-GAT-TGC-ACA-GTA-ATT-3' and
5'-CTT-AAG-TTG-CAC-CTC-TTC-TTCTGT-CTC-CTC-3',
respectively. The polymerase chain reaction-generated fragment was then
subcloned into pBluescript SK+ with a human TR insert
containing these two sites (ATG-ATG-GCT-AGC-CTT-AAG-AGG) encoding a seven-residue cytoplasmic tail with the sequence
Met-Met-Ala-Ser-Leu-Lys-Arg (21). The addition of the two restriction
sites adds three residues, Ala-Ser-Leu, to the tailless TR (
3-59;
Ref. 22), and the amino- and carboxyl-terminal regions of CFTR were
inserted between the Ser and Leu residues (Fig. 1). Mutations were
introduced into the amino- and carboxyl-terminal tail regions using the
Chameleon double-stranded, site-directed mutagenesis kit (Strategene).
The mutations were verified by dideoxynucleotide sequencing (23, 24) of
the BH-RCAS constructs using the Sequenase kit (U.S. Biochemical
Corp.).
Expression of Wild-type TR and CFTR-TR Chimeras--
Human TR
and CFTR-TR chimeras were expressed in chicken embryo fibroblasts
as described previously (25) using the BH-RCAS expression vector
(26, 27).
Internalization Assay--
The rate of transferrin
internalization was determined using the IN/SUR method (28) as
described previously (29).
Construction of CFTR Mutants--
pMT-CFTR (wild type) and
pMT-CFTR-A1440X were kindly provided by Dr. Seng Cheng (Genzyme) (30).
pKCTR-CFTR (wild type) was provided by Dr. Eric Sorscher and the
Gregory James Cystic Fibrosis Research Center Vector Core (University
of Alabama at Birmingham) (31). For mutagenesis of the amino-terminal
region of CFTR, a XmaI-XbaI fragment of
pKCTR-CFTR was subcloned into pSK-Bluescript (Stratagene). For
mutagenesis of the carboxyl-terminal region of CFTR, a
BstXI-SgrAI fragment from pKCTR-CFTR was
subcloned into pSK-Bluescript. CFTR point mutations or deletions in the amino- or carboxyl-terminal tail regions were prepared from the corresponding pSK-Bluescript vectors (containing either the
XmaI-XbaI or BstXI-SgrAI
fragments, respectively) from single-stranded DNA as described
previously (11) by the method of Kunkel (32). Mutants were selected by
restriction mapping or sequencing and then subcloned into the
XmaI-XbaI or BstXI-SgrAI
site of pKCTR-CFTR. The mutations were verified by dideoxynucleotide
sequencing (24) using the Sequenase kit (U.S. Biochemical Corp.)
according to the manufacturer's directions.
Transient Expression of Wild Type and Mutant CFTRs in COS-7
Cells--
Transient expression of wild type or mutant CFTR in
COS-7 cells was performed as described by Cheng et al. (30).
The transfected cells were cultured in Dulbecco's modified Eagle's
medium with 10% fetal calf serum and incubated at 37 °C in
humidified air with 5% CO2 for 48 h.
SPQ Fluorescence Assay of Wild Type and Mutant CFTRs in COS-7
Cells--
CFTR function in individual cells was assayed using the
halide-quenched dye SPQ (30). Briefly, cells were loaded for 10 min
with SPQ (10 mM) by hypotonic shock and then mounted in a specially designed perfusion chamber for fluorescence measurements. Fluorescence (F) of single cells was measured with a Zeiss
inverted microscope, a PTI imaging system, and Hamamatsu camera.
Excitation was at 340 nm, and emission was >410 nm. All functional
studies were at 37 °C. At the beginning of the experiments, cells
were bathed in a quenching buffer (NaI buffer; 130 mM NaI,
5 mM KNO3, 2.5 mM
Ca(NO3)2, 2.5 mM
Mg(NO3)2, 10 mM
D-glucose, 10 mM HEPES), and following
establishment of a stable base line, they were switched to a
halide-free (NO3) dequenching buffer at 200 s. Cells
were stimulated with agonist unless otherwise indicated at 500 s
and then returned to the quenching NaI buffer. Fluorescence was
normalized to the base-line (quenched) value (average fluorescence from
100 to 200 s), with increases presented as percentage of increase F over basal level. Each curve was generated from the mean
values (±S.E.) of either 1) responding cells (defined by increase in dequench slope of >100% following cAMP stimulation (Fig.
8A wt CFTR, and Fig. 8B, wt
CFTR and 1440X CFTR)), or 2) the total
number of screened cells expressing a given construct. The
numbers in parentheses in the keys are
the total cells studied (denominator) and the number of
responding cells (numerator). Statistical analysis was by
2 testing, comparing the number of responding cells in
each condition with the wild-type CFTR (Fig. 8A) or the mock
condition (Fig. 8B). The buffers used in the SPQ assay were
1) NaI buffer, pH 7.3, and 2) NaNO3 buffer (identical to
NaI buffer except that 130 mM NaNO3 replaces NaI).
CFTR Labeling and Internalization--
Cell surface CFTR
biotinylation was performed as described previously (5).
Separation and Isolation of Biotinylated CFTR--
Biotinylated
and nonbiotinylated proteins were separated on an immobilized monomeric
avidin column (Pierce) as described previously (5). The unbound
fraction and biotin eluent fraction were then quantitated as described below.
Immunoprecipitation and Detection of CFTR--
Wild-type or
mutant CFTRs were immunoprecipitated from the pooled fractions (either
unbound fraction or biotin eluent fraction) with either anti-C-terminal
(24-1) or anti-R domain (13-1) monoclonal antibodies generously
supplied by Dr. Seng Cheng (Genzyme). The CFTR immunoprecipitates were
phosphorylated with [
-32P]ATP and
cAMP-dependent protein kinase and then analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography as described
previously (5). Quantitation was performed using a Molecular Dynamics PhosphorImager. The percentage of cell surface CFTR was calculated by
dividing the number of counts/min detected in the CFTR C-band of the
biotin eluent fraction by the total amount of CFTR C-band found in both
the unbound and biotin eluent fractions (5).
Indirect Immunofluorescence--
COS-7 cells expressing
wild-type or mutant CFTRs were trypsinized 12 h after transfection
and plated onto glass coverslips. 48 h later, the coverslips were
fixed in methanol/acetic acid (3:1) for 30 min at
20 °C, rinsed
with 1% BSA in PBS for 10 min at room temperature, and incubated with
either monoclonal antibody 24-1 or 13-1 (diluted 1:50 in PBS/BSA) for
1 h at room temperature. Coverslips were then incubated for 5 min
in 1% BSA in PBS (three times), incubated with Texas Red-conjugated
goat anti-mouse antibody (diluted 1:1000 in PBS, 1% BSA, 5% normal
goat serum) for 1 h at room temperature, and then rinsed for 5 min
in 1% BSA in PBS (3 times). Coverslips were mounted in 1 mg/ml
p-phenylenediamine in a 1:10 mixture of PBS/glycerol and
sealed with nail polish. Slides were analyzed o a Leitz fluorescence
microscope equipped with a Vario Orthomat II camera system and a Texas
Red epifluorescence filter module. Micrographs were prepared on T-Max
400 film processed at ASA 800.
Chicken embryo fibroblasts expressing the CFTR-TR chimeras and
wild-type TR were plated onto glass coverslips and cultured overnight.
The coverslips were then rinsed with PBS and fixed in 2% formaldehyde
in PBS for 15 min at room temperature; rinsed with PBS; quenched with
0.37% glycine, 0.27% NH4Cl in PBS; and permeabilized with
PHS (PBS, 10% horse serum, 0.1% saponin) for 30 min at room
temperature. The coverslips were then incubated with JS8 mouse
anti-chicken TR antibody (diluted in PHS) and with rabbit anti-human TR
(1:500 in PHS) for 30 min at 37 °C and rinsed in PHS at room
temperature. The coverslips were next incubated with Texas Red-labeled
goat anti-mouse IgG1 (1:50 dilution in PHS) and with Oregon
Green-labeled goat anti-rabbit antibody (1:50 in PHS) for 30 min at
37 °C and then rinsed, mounted, and analyzed as described above.
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RESULTS |
CFTR-TR Chimeras Containing the Amino- and Carboxyl-terminal Tails
of CFTR Are Expressed on the Cell Surface and Rapidly
Internalized--
To identify the regions of CFTR important for
endocytosis, we prepared chimeric molecules consisting of the amino
terminus (residues 2-78) and the carboxyl terminus of CFTR (residues
1391-1476), each fused to the transmembrane and extracellular domains
of the human TR (Fig. 1). Both chimeras
along with a wild-type TR and
3-59 TR (a mutant TR that is very
poorly internalized (11)) were expressed in chicken embryo fibroblasts
using BH-RCAS, a replication-competent retroviral vector derived from
the Rous sarcoma virus (26, 27). Cell surface expression of both
chimeras was confirmed using indirect immunofluorescence and
125I-labeled transferrin binding at 4 °C (data not
shown).

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Fig. 1.
Cytoplasmic tail sequences of the TR and
CFTR-TR chimeras. A schematic diagram is shown of wild-type TR
including the native cytoplasmic tail sequence. For each of the CFTR-TR
chimeras, the CFTR amino- and carboxyl-terminal tail sequences replace
the wild-type TR cytoplasmic tail and contain the native transmembrane
(TM) and extracellular domains derived from the TR.
Constructs are referred to throughout by the corresponding names shown
on the left.
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Internalization rates of the CFTR-TR chimeras were monitored using the
IN/SUR method of Wiley and Cunningham (28). Analysis of residue 2-78
CFTR-TR and residue 1391-1476 CFTR-TR indicated that both were
internalized rapidly (ke = 0.126 and 0.061, respectively, similar in fact to the wild-type TR
(ke = 0.090; Fig.
2A). For comparison, the
3-59 TR lacking an internalization signal (11) was internalized
very slowly (ke = 0.009). This suggested that both
cytoplasmic tail regions of CFTR were sufficient to promote TR
endocytosis. To determine if the potential acidic cluster/casein kinase
II region of CFTR (residues 1469-1474) in the carboxyl-terminal tail
was important for endocytosis, we prepared a deletion mutant lacking
this region, CFTR-(1391-1440) (Fig. 1) and compared the
internalization rates of the two chimeras. The results indicate that
CFTR-(1391-1440) and CFTR-(1391-1476) chimeras were internalized with
similar kinetics (ke = 0.063 versus
0.061, respectively; Fig. 2A), suggesting that the
membrane-distal portion of the carboxyl-terminal tail of CFTR was not
required for efficient endocytosis.

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Fig. 2.
Comparisons of the internalization rates of
the CFTR-TR chimeras containing various cytoplasmic tail
mutations. A, chicken embryo fibroblasts expressing
various CFTR-TR chimeras were incubated with prewarmed (37 °C)
125I-labeled transferrin (Tf) for the indicated
times. The amounts of internalized (Internal Tf) and
surface-associated (Surface Tf) radiolabel were determined
as described under "Experimental Procedures." Data are plotted
using the IN/SUR method, in which the slope of the line equals the
endocytic rate constant ke (28). A representative
experiment is shown. B, a summary of the internalization
rates (mean ± S.E.; n = 5 or more) for each of
the CFTR-TR chimeras is shown.
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Next, we analyzed CFTR-TR chimeras that contained point mutations in
potential internalization signals in both cytoplasmic tail regions:
Y38A, L69A, Y1424A, and L1430A (Fig. 1). Analysis of these mutants in
internalization assays indicated that only one mutation, Y1424A,
affected the internalization rate of the chimeras (Fig. 2B;
~40% loss of internalization activity, p < 0.05),
suggesting that this residue might be a part of an internalization signal. Since overexpression of the human wild type transferrin receptor (up to 10-fold over the endogenous receptor) does not affect
internalization of the endogenous receptor in this cell type (not
shown), the endocytic machinery should not be limiting for analysis of
the CFTR chimeras. Therefore, we were unable to determine if the CFTR
chimeras competed for the same cytosolic factors as the endogenous TR receptors.
CFTR-TR Chimeras Co-localize with the Transferrin Receptor--
To
examine the intracellular distribution of the chimeras, we compared
their distribution to that of the endogenous transferrin receptor using
immunofluorescence microscopy. CFTR-(2-78) was localized predominantly
to juxtanuclear structures that largely co-localized with the native
chicken TR receptor (Fig. 3B).
This is similar to co-localization of the human TR expressed in these cells compared with the endogenous receptor (Fig. 3A) except
that the relative surface expression of the wild-type TR appeared to be
much higher (shown in green) than that of CFTR-(2-78)
chimera. Very few vesicles were CFTR-(2-78)- (Fig. 3A) or
CFTR-(1391-1440)- (Fig. 3B) positive only, but there were
vesicles containing only TR (shown in red). The
intracellular distribution of the CFTR-(1391-1440) chimera (Fig.
3C) appeared similar to that of the CFTR-(2-78) chimera but
very different from a "tailless" TR (
3-59 TR) that lacks
intracellular sorting signals and is localized primarily to the cell
surface (Fig. 3D). Comparison of the CFTR-(1391-1440) with
the Y1424A mutant showed little or no difference (not shown). In
nonpermeabilized cells, the wild type and tailless TRs are strongly
positive by immunofluorescence, whereas the two CFTR chimeras are only
weakly positive (not shown). 125I-Labeled transferrin
binding at 4 °C indicated that the relative surface expression of
both CFTR chimeras is approximately 5-10-fold lower than the wild-type
TR (not shown). These results suggest that both chimeras co-localized
with the endogenous TR and therefore were a part of the constitutive
recycling pathway as is the case for the native TR.

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Fig. 3.
Co-localization of CFTR-TR chimeras with the
endogenous TR. Chicken embryo fibroblasts expressing wild-type
human TR (A), CFTR-(2-78) (B), CFTR-(1391-1440)
(C), and 3-59 TR (D), were fixed,
permeabilized, and stained with rabbit anti-human TR antisera followed
by Oregon Green-labeled goat anti-rabbit Ig and with JS8-mouse
anti-chicken TR antibody followed by Texas Red-labeled goat anti-mouse
IgG1. Co-localization of the two proteins is indicated by
yellow fluorescence.
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CFTR and 1440X CFTR Are Rapidly Internalized in COS-7
Cells--
Having established that both cytoplasmic tail regions were
sufficient for endocytosis, we next determined if they were necessary in the context of the CFTR protein. CFTR, unlike the TR, is a type III
membrane protein with 12 membrane-spanning domains. In addition to the
R-domain and the two nucleotide binding domains, both amino- and
carboxyl-terminal tails of CFTR are cytoplasmic in orientation (Fig.
4). Using a cell surface two-step
biotinylation assay to monitor CFTR endocytosis (5) that relies on
biotinylation of the carbohydrate side chains found in extracellular
loop 4 (Fig. 4), we compared the internalization rate of wild-type CFTR to a previously described premature stop mutant, A1440X
(33). First, we confirmed that A1440X expressed in COS-7
cells is maturely glycosylated by monitoring band C formation (Fig.
5A). Next, using the surface
biotinylation assay, we monitored CFTR and A1440X clearance
from the cell surface (Fig.
6A). Interestingly, the A1440X premature stop mutant was internalized faster than
the wild-type CFTR, suggesting, as had been seen for the chimeric protein, that the last 41 residues in CFTR were not necessary for rapid
endocytosis.

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Fig. 4.
Schematic diagram of CFTR and CFTR
mutants. The CFTR mutants used in this study consisted of point
mutations and deletions in the amino-terminal and carboxyl-terminal
cytoplasmic tails. The boxes refer to the wild-type
(WT) amino-terminal tail (residues 1-80), and the
lines represent deleted amino acids from this domain. The
wild-type carboxyl-terminal tail consists of residues 1391-1480.
A1440X contains a stop mutation at residue 1440, and Y1424A
contains an alanine substitution for tyrosine.
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Fig. 5.
Analysis of CFTR and CFTR mutants expressed
in COS-7 cells. CFTR and CFTR mutants expressed in COS-7 cells
were immunoprecipitated from cell lysates 48 h posttransfection.
The immunoprecipitated CFTR or CFTR mutants were then in
vitro phosphorylated with protein kinase A and
[ -32P]ATP and analyzed by SDS-polyacrylamide gel
electrophoresis and autoradiography as described previously (5). The
positions of bands B and C are indicated on the left.
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Fig. 6.
Comparisons of the internalization rates of
CFTR and CFTR mutants. A, internalization of CFTR and
A1440X in COS-7 cells. COS-7 cells transfected with
wild-type CFTR (pMT-CFTR) or A1440X (pMT-1440X) were
analyzed 48 h posttransfection. Cell surface CFTR or CFTR mutants
were biotinylated using a two-step cell surface periodate/LC-hydrazide
biotinylation procedure previously described (5). At zero time, both
steps were conducted at 4 °C to label the entire surface pool of
CFTR. Internalization is monitored by a loss of biotinylation of the
cell surface pool by including a 37 °C incubation period (shown on
the x axis as time in min) between periodate and biotin
LC-hydrazide treatments. Biotinylated and nonbiotinylated proteins were
separated on a monomeric avidin column, and CFTR and A1440X
were in vitro phosphorylated and analyzed by
SDS-polyacrylamide gels and autoradiography to quantitate the amount of
CFTR remaining on the cell surface during the warm-up step. Each time
point represents the mean ± S.E. of 13 experiments for wild-type
CFTR and 6 for the A1440X. B, internalization of
CFTR and Y1424A in COS-7 cells. Cells transfected with wild-type CFTR
(pGT-CFTR) or Y1424A were analyzed as described in A for
percentage CFTR internalized from the cell surface during the warm-up
period. C, percentage of CFTR or Y1424A at the cell surface
under steady-state conditions. Cells transfected with CFTR or Y1424A
were analyzed for total CFTR expression by performing the two-step
biotinylation reaction without a warm-up step. Biotinylated and
nonbiotinylated CFTR or Y1424A was separated on a monovalent avidin
column and quantitated as described for A. The percentage of
CFTR at the cell surface represents the mean ± S.E. of six
experiments. D, wild-type CFTR expression in COS-7 cells
using pMT-CFTR and pGT-CFTR. COS-7 cells transfected with CFTR using
the pMT-CFTR and pGT-CFTR vector were analyzed 48 h
posttransfection. Biotinylated (biotin eluent) and nonbiotinylated
(unbound) CFTR was separated on a monovalent avidin column.
Biotinylated and nonbiotinylated CFTR were then immunoprecipitated from
the two fractions, phosphorylated in vitro with protein
kinase A and [ -32P]ATP, and analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography. The
positions of bands B and C are indicated on the left. The
relative amount of CFTR expressed using the pMT-CFTR vector was always
higher than the pGT-CFTR vector.
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Tyrosine 1424 Is Important for CFTR Endocytosis--
Next, we
tested the only point mutation that affected internalization of the
chimeras, Y1424A. Analysis of this mutation in CFTR revealed that it
was maturely glycosylated (Fig. 5B) but internalized 41%
more slowly than the wild-type CFTR protein (ke = 0.16 versus 0.27 (p < 0.01); Fig.
6B). Since slower internalization would imply that the
steady-state distribution of Y1424A might favor a higher cell surface
distribution pattern, we determined the percentage of CFTR at the cell
surface using surface biotinylation at 4 °C. As predicted, the
percentage of Y1424A at the cell surface was higher than the wild-type
CFTR protein (36.1 versus 23.1% (p < 0.01), respectively; Fig. 6C), supporting the idea that
tyrosine 1424 was important for CFTR endocytosis.
CFTR Expression Levels in COS-7 Cells Affect Internalization
Rates--
In our analysis of wild-type CFTR internalization, we were
surprised to find that the relative internalization rates varied depending on whether the cells were transfected with the pMT-CFTR expression vector (30) or the pGT-CFTR vector (31). The CFTR internalization rate (ke) in COS-7 cells transiently transfected with pMT-CFTR was 0.16 (Fig. 6A), whereas the
CFTR internalization rate in cells transfected with pGT-CFTR was 0.27. Since the promoters were different in the two expression vectors, we
compared the relative expression levels by immunoprecipitating and
in vitro phosphorylating CFTR from cells transfected with the two different expression vectors with [
-32P]ATP
and cAMP-dependent protein kinase and analyzing by
SDS-polyacrylamide gel electrophoresis and autoradiography (5). As is
shown in Fig. 6D, the protein expression levels in
transfected cells using the pMT-CFTR vector were substantially higher,
suggesting that CFTR internalization rates were directly affected by
protein expression levels. A similar phenomenon occurs when the
transferrin receptor is overexpressed in HeLa cells (34), suggesting
that the cellular machinery for removing surface receptors is limiting
and can be saturated. A similar comparison between wild type CFTR (pGT)
and Y1424A (pGT) revealed that the expression levels were similar (Fig.
5B).
Amino-terminal Deletions of CFTR Are Only
Core-glycosylated--
In order to localize the potential
internalization signals in the amino terminus of CFTR, we prepared four
deletion mutants of CFTR:
2-79,
2-59,
35-45, and
60-72.
Expression of each of these mutants in COS-7 cells produced only the B
form of CFTR (Fig. 5C). This protein was sensitive to
endoglycosidase H treatment and could not be biotinylated at the cell
surface (data not shown). To determine the intracellular location of
the transfected CFTR mutants, we examined the cells using indirect
immunofluorescence. As is shown in Fig.
7, the wild-type CFTR protein and
A1440X (Fig. 7, A and B, respectively)
showed a strong juxtanuclear distribution and evidence of surface
staining (a clear outline of the cell borders), whereas all of the
deletion mutants had a reticular staining pattern and little if any
evidence of surface staining. The results suggest, therefore, that all
of the deletion mutants were expressed but failed to exit the
endoplasmic reticulum and reach the cell surface.

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Fig. 7.
Steady state distribution of wild-type CFTR
and amino- and carboxyl-terminal deletion mutants of CFTR in COS-7
cells. COS-7 cells transfected with wild-type CFTR (A),
A1440X (B), 2-79 CFTR (C),
2-59 CFTR (D), 35-45 CFTR (E), and
60-72 (F) were fixed, permeabilized, and subjected to
indirect immunofluorescence using anti-CFTR antibodies. Wild-type CFTR
and A1440X show cell surface staining, whereas the
amino-terminal deletion mutants show a pronounced reticular staining
pattern.
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Finally, as functional proof that the CFTR deletion mutants failed to
reach the cell surface, we tested each of these mutants for CFTR
Cl
channel activity using the SPQ halide efflux assay.
Fig. 8A shows that wild-type
CFTR generated Cl
channel activity after stimulation with
cAMP agonists (20 µM forskolin and 100 µM 3-isobutyl-1-methylxanthine). However, expression of
each of the deletion mutants failed to produce functional CFTR Cl
channels, indicating either that the CFTR deletion
mutants were nonfunctional chloride channels or that they failed to
reach the cell surface or both. The A1440X mutant, unlike
the amino-terminal deletion mutants, had wild-type chloride channel
activity (Fig. 8B). Since the amino-terminal deletion
mutants of CFTR failed to reach the cell surface, we were unable to
determine if this domain is necessary for CFTR endocytosis.

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Fig. 8.
Functional analysis of wild type and deletion
mutants of CFTR using SPQ fluorescence. The change in SPQ
fluorescence is shown for COS-7 cells expressing wild-type CFTR,
2-79 CFTR, 2-59 CFTR, 35-45 CFTR, and 60-72 CFTR
(A) and wild-type CFTR and A1440X (B).
Cells were stimulated with 20 µM forskolin, 50 µM 8-(4-chlorophenylthio)adenosine 3':5'-cyclic
monophosphate, and 100 µM 3-isobutyl-1-methylxanthine (at
arrow, cAMP). The change in SPQ fluorescence for
all of the amino-terminal deletion mutants was not significantly
different from the negative control (mock-transfected cells). Curves
were generated from either 1) the mean ± S.E. of responding cells
(defined by increased rate of dequench following cAMP stimulation of
>100% (wild type CFTR (A), wild type CFTR and
A1440X CFTR (B)) or 2) the total of all screened
cells expressing a given construct. The values in
parentheses are the number of responding cells
(numerator) over the total cells screened
(denominator). A, The wild type CFTR sample was
significantly different from each deletion mutant or mock sample
(p < 0.001 by 2 test for each condition
compared with wild-type CFTR). B, the wild type CFTR and
A1440X CFTR samples were significantly different from the
mock cells (p < 0.001 and 0.025, respectively).
|
|
 |
DISCUSSION |
The conclusions of this study are as follows: 1) the amino and
carboxyl termini of CFTR are each sufficient to promote the endocytosis
of the transferrin receptor; 2) the carboxyl-terminal 40 amino acids of
CFTR are not required for endocytosis; 3) tyrosine 1424 is a critical
part of an internalization signal; and 4) deletions in the
amino-terminal tail region of CFTR result in loss of CFTR surface
expression. Our results clearly indicate that both cytoplasmic tail
domains of CFTR are sufficient to promote internalization of a reporter
molecule, suggesting that these domains are capable of interacting with
the cell's endocytic machinery. Recent results, however, indicate that
both domains interact with other proteins at the cell surface,
suggesting that CFTR internalization and/or function may be regulated
by these interactions (see below).
The only mutation that we identified in our studies that affected CFTR
internalization was found in the carboxyl-terminal tail, Y1424A. This
mutation inhibited endocytosis by approximately 40% in both the native
protein and the chimera. For comparison, a similar mutation in the
tyrosine-based internalization signal of the TR results in an 80% loss
of internalization activity (11). Since the same modification (Tyr
Ala) has a more modest effect on CFTR internalization than TR
internalization, it is tempting to speculate that the CFTR protein
contains more than one internalization signal, such as is the case for
the CD3
-chain (13).
To determine if the tyrosine residue was conserved in other species, we
compared CFTR carboxyl-terminal tail sequences from 10 species (Fig.
9). In all but dogfish, the tyrosine
residue is conserved. In this case, a phenylalanine is found in place of the tyrosine residue. Interestingly, previous studies have demonstrated that phenylalanine can substitute for tyrosine in the TR
internalization signal, YTRF, without a loss of internalization activity (35). In seven of 10 CFTR sequences, the entire motif, YDSI,
is conserved, suggesting that this sequence conforms to the general
pattern of internalization signals, YXX
, and may represent a CFTR internalization signal.

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|
Fig. 9.
Comparison of the amino acid sequences of
various CFTR carboxyl-terminal tails. Amino acid sequence
alignment of C termini of CFTR from human (P13569), monkey (3057116),
rabbit (Q00554), sheep (U20418), bovine (P35071), mouse (M60493), rat
(1901178A), dogfish (P26362), killifish (AF000271), and
Xenopus (U60209) is shown. The tyrosine residue is conserved
among all of the species except the dogfish, which has a phenylalanine
residue. Phenylalanine residues have been shown to substitute for
tyrosine residues and still maintain wild-type internalization activity
for the transferrin receptor (35). All of the sequences except dogfish
conform to the YXX motif common to internalization
signals, where X can be any amino acid and is a
hydrophobic residue.
|
|
Our studies using deletional analysis of the CFTR amino terminus imply
that this region of the molecule is required for proper folding of
CFTR. Several lines of evidence support the idea that the four deletion
mutants failed to reach the cell surface. First, when each mutant was
expressed, only the endoglycoside H-sensitive B form of CFTR was
detected. Second, we were unable to biotinylate CFTR from the surface
of cells expressing any of the mutants. Third, immunofluorescence
studies demonstrated that the protein was being made but was localized
to a reticular staining pattern. Fourth, SPQ analysis of COS-7 cells
demonstrated that cells expressing the wild-type CFTR had chloride
channel activity, whereas the mock-transfected cells and cells
transfected with each of the deletion mutants did not. Clearly, an
intact amino terminus is required for proper CFTR biosynthesis and/or
function, as is suggested by the fact that multiple point mutations in
the amino-terminal tail result in cystic fibrosis (P13569). For
comparison, only one mutation in the carboxyl-terminal tail has been
reported to result in CF, V1397E. This finding and our results on the
deletion mutants are consistent the idea that the amino terminus may
associate with other domains of CFTR for proper CFTR maturation and folding.
Although we were able to show that the amino-terminal tail was
sufficient to promote TR endocytosis, we were unable to show that this
domain was necessary for CFTR endocytosis. Since modification of
Tyr1424 did not completely eliminate CFTR internalization,
the prediction is that another signal exists, but this could be in
either of the two tail regions. Therefore, the role of the amino
terminus in CFTR endocytosis remains unclear. Further complicating this matter, recent results suggest that the amino terminus interacts with
syntaxin 1A (36), and this interaction regulates CFTR activity (37).
Although syntaxin 1A is known to be a component of the vesicle fusion
machinery, disruption of syntaxin 1A/CFTR interaction potentiates
chloride channel activity. Whether the CFTR/syntaxin 1A interaction
regulates trafficking of CFTR or, more directly, modulates the channel
activity of CFTR remains unclear (36). If the amino terminus of CFTR
interacts with syntaxin 1A and/or other domains of CFTR, then the
question is whether this domain would still be available for
interaction with the clathrin and the adaptor molecules at the cell
surface. One interesting possibility is that interaction is regulated
in some manner and that the loss of interaction promotes loss of CFTR
from the cell surface through the latent targeting information found in
the CFTR amino-terminal tail.
The amino terminus may not be the only interacting domain at the cell
surface. Recent studies have also proposed that the last four residues
of the carboxyl-terminal tail of CFTR, Asp-Thr-Arg-Leu, interact with
EBP50 and that this interaction tethers CFTR to the cytoskeleton (38,
39). EBP50, through its PDZ domain, has been proposed to either
regulate the stability of the CFTR protein at the cell surface or
regulate CFTR channel function or perhaps both (38). Although the EBP50
is concentrated at the apical surface in human airway epithelial cells
and associates with CFTR in in vitro binding assays, a
direct linkage has not been established in vivo. Clearly, a
direct comparison of the internalization rates of wild-type CFTR and
A1440X mutant in a polarized epithelial cell line will be
required to clarify the role of the carboxyl terminus in membrane
association and/or endocytosis. Although it might not seem appropriate
for a protein tethered to the cytoskeleton to be endocytosed through
the clathrin-mediated pathway, this is clearly the case for the
2-adrenergic receptor (40), which has recently been
shown to associate with the first PDZ domain of EBP50 (41).
Our results demonstrate that CFTR has sorting signals that allow it to
be rapidly cleared from the cell surface, but what is the physiological
relevance of this? Recent studies indicate that ClC-5, the chloride
channel mutated in Dent's disease, may be involved in acidification of
endosomes (42, 43). Such a role was once proposed for CFTR, but this
was difficult to confirm, especially given the narrow tissue
distribution of CFTR and the widespread phenomenon of endosomal
acidification (44, 45).
If CFTR is not important for endosome acidification, then why would it
be endocytosed? The simplest explanation is that endocytosis may
provide a mechanism for regulating the amount of CFTR at the cell
surface at any one time. If CFTR is tethered to the cytoskeleton (38,
39), then CFTR would be kept out of the recycling pathway. If the
interaction at the cytoskeleton is regulated in any manner, then loss
of association would result in rapid clearance from the cell surface
through interactions with the clathrin-based sorting machinery.
Therefore, the possibility exists that some of the CFTR is in a rapidly
recycling pool and some is tightly associated with the cytoskeleton and
that the distribution of these two pools is somehow regulated. Clearly,
careful structure-function studies of CFTR in polarized epithelial
cells will be required before we will understand the complex
trafficking and regulation of the CFTR molecule.
 |
ACKNOWLEDGEMENTS |
We thank Kevin Kirk, Doug Cyr, Anjaparavanda
Naren, and Erik Schwiebert for critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported by NIDDK, National Institutes of
Health, Grant R29-DK47339 Cystic Fibrosis Foundation Grant COLLAW96PO.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.
Present address: 500 EMRB, University of Iowa, Iowa City, IA 52242.
¶
To whom correspondence should be addressed: Dept. of Cell
Biology, University of Alabama at Birmingham, BHSB 392, UAB Station, Birmingham, AL 35294-0005. Tel.: 205-934-1002; Fax: 205-975-5648; E-mail: jcollawn{at}uab.edu.
The abbreviations used are:
CFTR, cystic
fibrosis transmembrane conductance regulator; , TR, transferrin
receptor; SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium; BSA, bovine serum albumin; PBS, phosphate-buffered saline.
 |
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