From the Physiologisches Institut, Universität
Zürich Irchel, CH-8057 Zürich, Switzerland, the
¶ Uni-Klinik Regensburg, Department of Dermatology, 93052 Regensburg, Germany, the
Department of Physiology & Pharmacology, University of Queensland, St. Lucia, QLD 4072, Brisbane,
Australia, and the ** Department of Molecular Immunology, Biology III,
University of Freiburg, and Max-Planck-Institut für
Immunobiologie, Freiburg, Germany
Received for publication, December 13, 2000, and in revised form, March 14, 2001
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ABSTRACT |
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Expression of the cystic fibrosis
transmembrane conductance regulator (CFTR) is stringently
controlled by molecular chaperones participating in formation of the
quality control system. It has been shown that about 75% of all CFTR
protein and close to 100% of the
[ Epithelial Cl Recently, several proteins have been identified which bind to B cell
antigen receptors (BCR) (15). These BCR-associated proteins (BAPs) were
identified initially in mouse B cells and have been shown to be
expressed ubiquitously in various types of cells, including CHO cells
(16). BAP31 and other members of this family of proteins are present in
the ER where they exist as integral membrane proteins associated with
membrane immunoglobulin D in B cells (15, 16). It has been suggested
that BAP31 and related proteins play a role in vesicular transport and
control anterograde transport of certain membrane proteins such as
cellubrevin (16, 17). We therefore examined in the present study
whether BAP31 also affects expression of CFTR. We were particularly
interested to see whether expression of [ Cell Culture and Transfection with BAP31 Antisense
Oligonucleotides--
CHO cells expressing either wtCFTR or
[ Western Blotting of CFTR and BAP31--
Cells were lysed in
sample buffer containing 10% SDS and 100 mmol/liter dithiothreitol.
Protein concentration was determined according to a modified
Laury method, and the lysates containing 20 µg of protein were
subjected to SDS-polyacrylamide gel electrophoresis and analyzed by
Western blotting as described previously (21). Proteins were separated
by 7% (CFTR) and 12% (BAP31) SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose membranes (Bio-Rad), and bound antibodies
were detected by enhanced chemiluminescence (Amersham Pharmacia
Biotech) (22). Using the different protocols, antisense transfection
and Western blot analysis were performed three to seven times for each
cell line. A mouse monoclonal antibody (M3A7) was used for detection of
CFTR (kindly provided by Prof. Dr. J. Riordan, Mayo Clinic Scottsdale)
(23). Specific rabbit anti-BAP31 serum has been raised against the GST
fusion protein carrying the C-terminal half of BAP31 (16). Horseradish
peroxidase-conjugated goat anti-mouse antibodies were obtained from
Southern Biotechnology Associates. The goat anti-mouse IgG (H+L)-DTAF
and goat anti-rabbit IgG (H+L)-Texas Red secondary antibody conjugates
were from Jackson Laboratories.
36Cl Patch Clamp Analysis and GFP Fluorescence--
Cell culture
dishes were mounted on the stage of an inverted microscope (IM35,
Zeiss, Oberkochen, Germany) and kept at 37 °C. The bath was
continuously perfused with Ringer solution at a rate of about 20 ml/min. EGFP fluorescence was observed at 480-nm excitation and 520-nm
emission using fluorescence microscopy (Zeiss, Oberkochen, Germany).
Only cells demonstrating EGFP fluorescence were used for patch clamp
analysis. During the experiments, 115 mmol/liter Cl Immunofluorescence--
CHO cells were grown on coverslips,
washed with ice-cold phosphate-buffered saline containing 0.01% azide
and were fixed with 2% paraformaldehyde in phosphate-buffered saline
for 15 min at room temperature and washed three times in
phosphate-buffered saline. Cell were permeabilized with blocking buffer
containing 1% fetal calf serum, 0.01% azide and 0.03% saponin
(Sigma-Aldrich) for 15 min. Cells were incubated with antibodies
diluted in blocking buffer for 15 min at room temperature. Unbound
antibodies were removed by washing with blocking buffer. After
incubation with secondary antibodies, cells were washed three times in
blocking buffer, air dried, and mounted in 10 µl of Fluoromount-G
(Southern Biotechnology Inc.) on a microscope slide. Fluorescence
microscopy was conducted using a LEIKA TCS confocal laser scanning
microscope (Leitz, Germany).
cDNA and in Vitro Transcription of cRNA--
To generate the
cDNA encoding BAP31 missing the C-terminal KKXX motif
(BAP31-KKXX), the BAP31-containing plasmid was digested with
SmaI/BsaAI and re-ligated. The resulting cDNA
was missing the last 26 nucleotides encoding the C-terminal 8 amino
acids. cDNA sequences encoding wtCFTR, [ Double Electrode Voltage Clamp in Oocytes from Xenopus
laevis--
Oocytes were obtained from adult X. laevis
frogs, defolliculated by 1-h treatment with collagenase (type A, Roche
Molecular Biochemicals) and subsequently injected with 10-30 ng of RNA
encoding the individual proteins. Recordings were taken 2-3 days after injection. During two electrode voltage clamp recordings, i/v curves
were obtained every 20 s by voltage clamping the oocyte from Materials and Statistical Analysis--
All used compounds were
of highest available grade of purity. Dimethyl sulfoxide
(Me2SO) was from Merck (Darmstadt, Germany). IBMX and
forskolin were from Sigma. The data are shown as original recordings or
as mean values ± S.E.; n refers to the number of experiments. Statistical analysis was performed according to paired or
unpaired Student's t test. p < 0.05 were
accepted to indicate statistical significance (*).
BAP31 Controls Expression of CFTR and Is Colocalized with CFTR in
CHO Cells--
[
Because Western blot analysis suggested that BAP31 is involved in the
control of synthesis and maturation of CFTR, we performed immunofluorescence stain of both CFTR and BAP31 (Fig.
2). The cells showed a pronounced
staining for BAP31 (Texas red fluorescence) and were also significantly
stained for wtCFTR (DTAF green fluorescence). The overlay (yellow
fluorescence) of both pictures shows co-staining of the two proteins
except for areas close to the plasma membrane (Fig. 2). These data
suggest that both CFTR and BAP31 are colocalized in the ER of CHO cells
and thus support the idea of BAP31 controlling maturation or
trafficking of CFTR.
Inhibition of BAP31 Expression Enhances Cl
We further cotransfected wtCFTR or [ Coexpression of BAP31 and CFTR in Xenopus Oocytes--
We further
examined how coexpression of BAP31 affects activation of wtCFTR in
oocytes of X. laevis. Fig.
6A shows whole cell recordings
obtained from oocytes expressing only wtCFTR or coexpressing wtCFTR
together with BAP31. The corresponding I/V curves are shown in Fig.
6B. The tracings, I/V curves, and the summary (Fig.
6C) of this series of experiments clearly show that
coexpression of BAP31 attenuates significantly the activation of a CFTR
Cl
To further elucidate the impact of BAP expression on CFTR
Cl The present data suggest that the putative integral membrane
protein BAP31 interferes with expression of CFTR in heterologous expression systems. BAP31 was identified initially in the murine myeloma cell line J558L and was shown later to be expressed in various
cell types (15). In the present study, we detected expression of BAP31
in CHO cells and also in various types of human epithelial cells lines
derived from colon, pancreas, kidney collecting duct, and airways (data
not shown). Both BAP29 and BAP31 contain three stretches of hydrophobic
amino acids, suggesting that these proteins are multiple spanning
transmembrane proteins (16). When BAP31 expression was blocked by
antisense oligonucleotides in CHO cells, expression of both wtCFTR or
[ The experiments performed in Xenopus oocytes supply further
evidence for the impact of BAP31 on processing and expression of CFTR.
Interestingly, coexpression of both BAP29 and BAP31 was even more
efficient in inhibiting CFTR expression, compared with the effects of
solely expression of BAP31. Thus, CFTR may interact with BAP31
homodimers or heterodimers of BAP31 and BAP29, which have been reported
previously (16). The present data further stress the importance of the
KKXX motif at the C-terminal end of BAP31, because no
inhibition of CFTR conductance was observed in the absence of this
motif. The KKXX motif is an ER retention signal for residual
proteins of the ER, which cycle between ER and the Golgi (16, 17). It
has also been demonstrated to function as a transport signal (30).
KKXX-carrying proteins bind to COP proteins and are involved
in the retrograde transport from the cis Golgi to the ER
(31, 32). In addition, the KKXX motif may also serve as an
internalization and endocytosis signal (27). The KKXX motif
seems to be crucial for the inhibitory effects of BAP31 on CFTR. As
mentioned above, the KKXX represents an ER retrieval signal
and thus ER overload by overexpressing BAP31 may lead to cell stress,
activation of NF Interestingly, it has been shown recently that virus maturation in the
ER is controlled by an KKXX ER retrieval signal and that
viruses lacking the envelope glycoprotein encoding the KKXX motif have a higher chance for budding at the plasma membrane (36). The
present results suggest that trafficking or maturation of a variety of
transmembrane proteins might be controlled by BAP31, including CFTR.
However, it is unlikely that BAP31 affects expression of all membrane
proteins, because we did not detect an increased expression for the
Phe508] CFTR variant are rapidly degraded
before leaving the endoplasmic reticulum (ER). B cell antigen
receptor-associated proteins (BAPs) are ubiquitously expressed
integral membrane proteins that may control association with the
cytoskeleton, vesicular transport, or retrograde transport from the
cis Golgi to the ER. The present study delivers evidence
for cytosolic co-localization of both BAP31 and CFTR and for the
control of expression of recombinant CFTR in Chinese hamster ovary
(CHO) cells and Xenopus oocytes by BAP31. Antisense
inhibition of BAP31 in various cell types increased expression of both
wild-type CFTR and [
Phe508]CFTR and enabled
cAMP-activated Cl
currents in
[
Phe508]CFTR-expressing CHO cells. Coexpression of
CFTR together with BAP31 attenuated cAMP-activated Cl
currents in Xenopus oocytes. These data therefore
suggest association of BAP31 with CFTR that may control maturation or
trafficking of CFTR and thus expression in the plasma membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
transport is controlled by the cystic
fibrosis transmembrane conductance regulator
(CFTR),1 which is a
Cl
channel and a regulator of other ion channels (1).
Cl
transport and regulation of ion channels is defective
in cystic fibrosis (CF), because of more than 900 different CFTR
mutations. The most common mutation, [
Phe508]CFTR,
leads to a defect in maturation and processing of the CFTR protein and
thus does not allow for expression of sufficient amounts of CFTR in
plasma membranes of epithelial cells and non-epithelial cells that
express recombinant CFTR (2, 3). It has been shown that because of
incomplete folding, [
Phe508]CFTR is not capable of
leaving the ER and thus is not processed to a more mature and
glycosylated form. Therefore, it does not become protease-resistant, is
retained in the ER, and will not undergo complex glycosylation (4). It
is therefore detected as a band of lower molecular weight when analyzed
on an SDS gel (5, 6). Several proteolytic systems, including
proteasomes, contribute to degradation of wild-type CFTR (wtCFTR) and
[
Phe508]CFTR (3, 7). The enzymes and compartments
responsible for degradation of CFTR are part of the ER quality control
system of secretory proteins (8). Binding of CFTR to the ER membrane chaperone calnexin (9) and the cytosolic chaperone Hsp70 or Hsp90 (10,
11) has been demonstrated. Small amounts of [
Phe508]CFTR
that escape proteolysis is trafficked to the cell membrane, where
it functions as cAMP-regulated Cl
channel (12, 13). This
is usually 1% or less of the amount of wtCFTR present in the native
tissue and is probably higher in Xenopus oocytes (2,
14).
Phe508]CFTR can
be influenced by manipulation of the expression of BAP31. The data
presented here demonstrate that BAP31 inhibits both expression of
wtCFTR as well as [
Phe508]CFTR and suggest that BAP31 is
participating in formation of the quality control system.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Phe508]CFTR (kindly provided by Dr. X.-B. Chang and Prof.
J. Riordan, Mayo Clinic Scottsdale, Scottsdale, AZ) were cultured in
-minimum essential medium supplemented with 8% fetal bovine serum,
50 µmol/liter methotrexate, penicillin (100 units/ml) and
streptomycin (100 µg/ml) (Life Technologies, Germany) in a humidified
atmosphere with 5% CO2 (18). Human bronchial epithelial
cells (16HBE14o
) and mouse epithelial-collecting duct cells (M1) were
cultured as described previously (19, 20). About 5 × 106 CHO cells expressing [
Phe508]CFTR were
electroporated in the presence of 12 nmol/liter (40 µg/ml) of a
plasmid encoding the enhanced green fluorescent protein (pEGFP-c1;
CLONTECH, Germany) and about 0.5 µmol/liter (3.5 µg/ml) phosphorothioated (stabilized) oligonucleotides antisense to
the first 20 bases of the BAP31 coding sequence. Control cells were transfected with EGFP and missense oligonucleotides only. Cells were
electroporated (Bio-Rad) at 400 V/500 µF and subsequently kept on ice
for 10 min. Following, cells were resuspended in culture medium and
plated on culture dishes. Alternatively, cells were transfected using 1 µmol/liter antisense DNA and the transfection reagent DOTAP (Roche
Molecular Biochemicals) according to the manufacturer's protocol.
Efflux--
CHO cells grown on
35-mm culture dishes were analyzed for forskolin (10 µmol/liter)
activated 36Cl
efflux. Electroporated CHO
cells were seeded on 35-mm culture dishes and were allowed to grow to
subconfluence for 72 h. After rinsing the culture dishes with
efflux buffer (all mmol/liter) 140 NaCl, 3.3 KH2PO4, 0.83 K2HPO4, 1 CaSO4, 1 MgSO4, 5 HEPES, 10 glucose, pH 7.4, 37 °C) fresh efflux buffer (1 ml) containing 2 µCi/ml
36Cl
was added to each dish. Cultures were
incubated for 2 h at 37 °C and then washed three times for
10 s with efflux buffer. 1 ml of efflux buffer was applied,
removed in 1-min intervals, and replaced by fresh buffer. At 3 min, forskolin (10 µmol/liter) was added to the buffer. Cells were
extracted overnight at 4 °C, remaining
36Cl
was determined, and samples were counted
in scintillation mixture. The percent efflux/min was calculated
according to Ref. 24.
was
replaced by equimolar concentrations of the impermeable anion gluconate
(30Cl
). Patch clamp analysis was performed in the fast
whole cell configuration according to (25). The patch pipettes had an
input resistance of 2-4 megohms and were filled with a solution
containing (mmol/liter) KCl 30, potassium gluconate 95, NaH2PO4 1.2, Na2HPO4
4.8, EGTA 1, CaCl2 0.726, MgCl2 1.034, D-glucose 5, ATP 1. The pH was adjusted to 7.2, the
Ca2+ activity of this solution was 0.1 µmol/liter. The
access conductance was measured continuously and was between 30 and 120 nanosiemens. Currents (voltage clamp) and voltages (current
clamp) were recorded using a patch clamp amplifier (List Medical
Electronic, Darmstadt, Germany) and were displayed continuously on a
pen recorder (Gould Instruments). In regular intervals, membrane
voltages (Vc) were clamped in steps of 10 mV to ± 40 mV. Gm was calculated according to Ohm's law from the
measured I and Vc values (25). CFTR Cl
currents were activated by 3-isobutyl-1-methylxanthine (IBMX; 100 µM) and forskolin (10 µM) (Sigma).
F508]CFTR, BAP31,
BAP31-KKXX, BAP29, and E3/19k were subcloned into
pBluescript SK+ (Stratagene). For in vitro transcription
using either T7 or T3 promoters, plasmids were linearized with
KpnI, BamHI, SalI, and
NotI, respectively. In vitro transcription was
performed using T7 and T3 polymerase, respectively, and a cap analog
(mCAP RNA capping kit, Stratagene).
80
to +40 mV in steps of 20 mV. Conductances were calculated according to
Ohm's law. Oocytes were continuously perfused with amphibian Ringer
containing 96 mM NaCl, 2 mM KCl, 1.8 mM Ca2+, 5 mM HEPES, 1 mM Mg2+ at pH 7.55. CFTR Cl
currents were activated by IBMX (1 mM) and forskolin (10 µM).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Phe508]CFTR does not mature properly and
is therefore excluded from the glycosylation pathway. Thus,
[
Phe508]CFTR appears as a lower molecular weight band on
an SDS gel when compared with wtCFTR. We analyzed expression of wtCFTR
and [
Phe508]CFTR in CHO cells lines stably transfected
with either wtCFTR or [
Phe508]CFTR (4). Mature and fully
glycosylated CFTR of molecular weight of about 180 kDa is detected in
wtCFTR-expressing cells whereas [
Phe508]CFTR appears as a
immature precursor of a lower molecular weight of about 150 kDa (Fig.
1A). CHO cells do also show
expression of BAP31 as indicated by Western blot analysis (Fig.
1A). Both wtCFTR- and [
Phe508]CFTR-expressing
cells were transfected with BAP31 antisense oligonucleotides (+ AS),
which largely reduced expression of BAP31 when compared with control
cells (
AS). Parallel to the reduced expression of BAP31, we detected
an increase in both expression of wtCFTR as well as
[
Phe508]CFTR, suggesting that expression of CFTR is
controlled by BAP31. Experiments were carried out at least in
triplicates but generally 5-7 times. Expression of BAP31 was also
detected in various cultured cell types from pancreas (CFPAC),
collecting duct (M1), colon (HT29 and T84) and
airways (CFDE, 16HBE14o
, 9HTE), although levels of BAP31 expression
varied significantly between the different cell lines. In all theses
cell lines, CFTR is expressed endogenously (data not shown). In mouse
collecting duct cells (M1) and human bronchial epithelial cell lines
(16HBE14o
; HBE), expression of BAP31 was blocked by antisense
treatment. Reduced expression of BAP31 was paralleled by enhanced
expression of CFTR (Fig. 1B). These data suggest that BAP31
also controls expression of endogenous CFTR in airways and collecting
duct of the kidney.
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Fig. 1.
Western blot analysis of the expression of
wtCFTR, [ F508]CFTR and BAP31 in CHO, M1, and
HBE cells. Equal amounts of protein (20 µg) were isolated from
CHO cells, grown in the presence of a BAP31 antisense oligonucleotide
(+AS) or missense oligonucleotide (
AS), and
from M1 and HBE cells growing in either the presence or absence of
BAP31-antisense, and were separated on SDS gels. When grown in the
presence of antisense nucleotides, expression of BAP31 was largely
reduced in CHO cells (A) and the epithelial cell lines M1
and HBE (B). The decrease in BAP31 expression was paralleled
by an increase in CFTR expression in CHO cells, expressing CFTR
exogenously (A), and the epithelial cell lines M1 and HBE,
expressing CFTR endogenously (B). Individual experiments
were carried out 3-7 times.
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Fig. 2.
Co-localization of BAP31 and CFTR in CHO
cells. Immunofluorescence analysis of CFTR and BAB31 in CHO cells
expressing wtCFTR. CFTR and BAP31 were labeled using respective primary
antibodies and goat anti-mouse IgG (H+L)-DTAF (CFTR, green
fluorescence) and goat anti-rabbit IgG (H+L)-Texas Red (BAP31,
red fluorescence) secondary antibody conjugates. The overlay
(yellow fluorescence) of both pictures shows co-staining of
the two proteins except areas around the plasma
membrane.
Conductance and Recovers Cl
Channel Activity in Cells
Expressing [
Phe508]CFTR--
To examine whether BAP31
also affects functional expression of CFTR, we analyzed cells
transfected with BAP31 antisense as well as control cells in a
36Cl
efflux assay. To that end,
electroporated CHO cells were grown on 35 mm-culture dishes and loaded
with 36Cl
. Subsequently,
36Cl
efflux was measured in the absence or
presence of forskolin (10 µmol/liter). As shown in Fig.
3, 36Cl
efflux
was continuously declining in the absence of forskolin (dashed
lines). In wtCFTR-expressing CHO cells, stimulation with forskolin
largely enhanced 36Cl
efflux, indicating
activation of a CFTR Cl
conductance, whereas no
36Cl
efflux was activated in
[
Phe508]CFTR-expressing CHO cells. However, in BAP31
antisense-treated cells expressing [
Phe508]CFTR, forskolin
was able to induce a small but significant
36Cl
efflux, and
36Cl
efflux in wtCFTR-expressing cells was
augmented (solid lines, filled circles). These data suggest
that blocking of BAP31 expression enhances CFTR expression and leads to
a residual Cl
channel activity in
[
Phe508]CFTR-expressing CHO cells.
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Fig. 3.
Cl ion transport measured by
36Cl
efflux in CHO cells expressing wtCFTR or
[
F508]CFTR. Cells were grown in the
presence of BAP31 antisense oligonucleotides (+BAP31-AS) or
missense oligonucleotides (
BAP31-AS). Forskolin (10 µmol/liter) was added after 3 min, and efflux was determined for
another 2 min (solid line). Dashed lines indicate
time course of non-stimulated cells. Asterisk indicates
significant differences between
BAP31-AS- and +BAP31-AS-treated cells
(unpaired Student's t test).
Phe508]CFTR-expressing
CHO cells with a 40-fold molar excess of BAP31 antisense
oligonucleotides and the expression plasmid for green fluorescent
protein (pEGFP-C1). Control cells were transfected with pEGFP-c1 only.
EGFP fluorescence was monitored during subsequent patch clamp
experiments and was used as an indicator for successful transfection.
Only fluorescent-labeled cells were used for patch clamp experiments.
Activation of wtCFTR was studied initially in wtCFTR-expressing cells.
Upon stimulation with IBMX (100 µmol/liter) and forskolin (10 µmol/liter), a large whole cell conductance was activated and the
cell membrane voltage (Vm) was depolarized (Fig.
4, A and B,
upper traces). Partial replacement of extracellular
Cl
by impermeable gluconate (30Cl
) partially blocked
the activated whole cell conductance and further depolarized
Vm, indicating activation of a whole cell Cl
conductance. In the absence of BAP31 antisense,
[
Phe508]CFTR-expressing cells did not show a response to
any of the above maneuvers (Fig. 4, A and B, middle
trace). However, in BAP31 antisense-incubated cells, forskolin and
IBMX were able to activate a residual Cl
conductance as
indicated by changes in whole cell conductance and Vm (Fig. 4,
A and B, lower trace). Fig.
5 summarizes the effects of IBMX and
forskolin and 30Cl
on whole cell conductances and
membrane voltages and clearly indicates a significant cAMP-activated
whole cell Cl
conductance in CHO cells expressing
[
F508]CFTR after blocking expression of BAP31.
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Fig. 4.
Patch clamp analysis of CHO cells expressing
wtCFTR or [ F508]CFTR grown in the presence
of BAP31 antisense oligonucleotide (+BAP31-AS) or
missense oligonucleotide (
BAP31-AS). Recordings
of the whole cell membrane conductance (A) and membrane
voltage (B). Effects of stimulation with forskolin/IBMX (10 µmol/liter and 100 µmol/liter) and partial replacement of
extracellular Cl
by gluconate (30Cl
).
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Fig. 5.
Summary of the patch clamp experiments shown
in Fig. 4. A, whole cell currents were activated by
IBMX and forskolin in CHO cells expressing wtCFTR and CHO cells
expressing [ Phe508]CFTR when grown in the presence of
BAP31 antisense oligonucleotides. B, impact of partial
replacement of extracellular Cl
by gluconate
(30Cl
) on whole cell Cl
conductance
activated by forskolin/IBMX. The whole cell conductance is inhibited by
30Cl
in CHO cells expressing wtCFTR and CHO cells
expressing [
Phe508]CFTR when grown in the presence
of BAP31 antisense oligonucleotides. C, depolarization
of the membrane voltage (Vm) by 30Cl
before and
after stimulation with IBMX/forskolin. Vm is depolarized by
30Cl
after stimulation with forskolin/IBMX of CHO cells
expressing wtCFTR and CHO cells expressing [
Phe508]CFTR
when grown in the presence of BAP31 antisense oligonucleotides. (),
number of experiments; *, significant difference from control (paired
Student's t test).
conductance in Xenopus oocytes. Injection
of a non-translated missense-cRNA did not affect CFTR currents,
suggesting that decrease in CFTR is caused by expression of BAP31
rather than nonspecific effects because of additional cRNA injection
(data not shown).
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Fig. 6.
Impact of BAP31 on the activation of CFTR
Cl conductance in Xenopus oocytes.
A, whole cell currents activated by IBMX and forskolin (1 mmol/liter and 2 µmol/liter) in CFTR expressing oocytes and impact of
the coexpression of BAP31. B, I/V curves for the IBMX- and
forskolin-activated whole cell currents in water-injected control
oocytes and oocytes expressing CFTR (CFTR-BAP31) or
coexpressing CFTR and BAP31 (CFTR+BAP31). C,
summary of the whole cell conductances activated by IBMX and forskolin
in oocytes expressing CFTR or coexpressing CFTR and BAP31. (), number
of experiments; *, significant difference from control (paired
Student's t test); #, significant difference from
CFTR-BAP31 (unpaired Student's t test).
conductance, we coexpressed a BAP31 variant lacking
the last C-terminal 24 nucleotides. This truncated BAP31 version lacks the C-terminal KKEE amino acid motif, which is known as an ER retrieval
sequence (consensus sequence KKXX) and which may also have a
function as a transport or internalization signal (26, 27). The ability
to inhibit CFTR expression in Xenopus oocytes seems to rely
on the presence of the KKXX motif, because
BAP31-KKXX was unable to decrease cAMP-activated CFTR
Cl
conductance (Fig. 7).
Transfection with BAP31 carrying the KKXX may cause an ER
overload and may thus cause stress to the cells (28). To exclude
artificial effects on CFTR Cl
conductance caused by
possible ER stress, we coexpressed CFTR together with the adenovirus
protein E3/19K. E3/19K has been demonstrated to accumulate in ER
membranes and to cause cells stress, thereby activating the
transcription factor NF
B (28). However, ER stress does not seem the
cause for reduced CFTR expression in Xenopus oocytes,
because E3/19K exerted no inhibitory effects on CFTR Cl
conductance (Fig. 7). Finally, BAP29, a protein homologous to BAP31 was
coexpressed together with CFTR and slightly but not significantly
attenuated activation of CFTR upon increase in intracellular cAMP. When
coexpressed together with BAP31, heterodimerization of BAP29 and BAP31
is likely to occur (16). The putative BAP31/BAP29 heterodimer was also
able to inhibit expression of CFTR Cl
conductance, as
shown in Fig. 7. Taken together, these data strongly suggest regulation
of CFTR expression by BAP31, which might be part of the quality control
system.
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Fig. 7.
Summary of whole cell conductances activated
by IBMX/forskolin in wtCFTR-expressing oocytes. Oocytes were
coinjected with cRNA encoding (i) BAP31 lacking the C-terminal
KKXX motif (+BAP31-KKXX), (ii) the ER-localized
transmembrane protein E3/19k, (iii) BAP29, and (iv) BAP29 together with
BAP31. *, significant differences (Student's paired t
test); #, significant difference when compared with whole cell
conductance measured in oocytes expressing CFTR only; (), number of
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Phe508]CFTR seemed to be enhanced. The amount of mature
wtCFTR protein was enhanced when expression of BAP31 was inhibited in
CHO cells, expressing exogenous wtCFTR. Our data show that this effect
is not limited to cells overexpressing CFTR. In airway epithelial and
collecting duct cells, inhibition of BAP31 expression induced enhanced
expression of endogenous CFTR. Expression of mature
[
Phe508]CFTR in CHO cells after BAP31-antisense treatment
was not detected by Western blotting. However, the immature form of the
protein was more abundant in cells treated with BAP31-antisense.
Moreover, functional assays clearly suggest expression of
[
Phe508]CFTR protein in the plasma membranes of
antisense-treated cells. Thus, mutant CFTR protein may be located in
plasma membranes in either an immature unglycosylated form or the small
amounts of mature [
Phe508]CFTR protein cannot be detected
by standard Western blotting. Even longer exposures of the x-ray films
did not show a CFTR protein band of high molecular weight. However, it
is well known from previous studies that only small amounts of CFTR are
required for functional expression in epithelial cells (12, 29).
B, and eventually cell death by apoptosis (28, 33).
In fact, BAP31 probably takes part in the control of programmed cell
death (34, 35). Although we cannot completely rule out a possible
nonspecific effect of BAP31 on CFTR expression, we were able to show
that expression of the protein E3/19k that binds very tightly to the ER
and induces ER overload (28) does not interfere with expression of CFTR in Xenopus oocytes.
-subunit of the Na+/K+-ATPase in BAP31-AS
treated HBE or M1 cells (data not shown). One function of BAP31 could
be that it participates in formation of the quality control mechanism
of membrane protein synthesis, which was postulated for CFTR (37). It
is now well known that biosynthesis of CFTR is strictly controlled by
this mechanism, resulting in degradation of most of the wtCFTR and
basically all of [
Phe508]CFTR (3). Because
[
Phe508]CFTR is by far the most frequent mutation causing
cystic fibrosis, interfering with the expression or function of BAP31
in epithelial cells could be a new way to circumvent the
Cl
channel defect in cystic fibrosis.
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ACKNOWLEDGEMENTS |
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We gratefully thank H. Schauer for expert technical assistance. We thank J. R. Riordan and X.-B. Chang for providing us with the various CHO cell lines and the M3A7-CFTR antibody.
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FOOTNOTES |
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*
This work was supported by DFG Ku756/4-1, Mukoviszidose
e.V., ARC 00/ARCS243, Cystic Fibrosis Australia, and DFG SFB 388.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.
§ These authors contributed equally to the present work.
To whom correspondence should be addressed. Tel.: 61 07 3365 4104; Fax: 61 07 3365 1766; E-mail: kunzelmann@plpk.uq.edu.au.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M011209200
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ABBREVIATIONS |
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The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; ER, endoplasmic reticulum; CHO, chinese hamster ovary cells; GST, glutathione S-transferase; IBMX, 3-isobutyl-1-methylxanthine; EGFP, enhanced green fluorescent protein; BAP, B cell antigen receptor-associated protein; wt, wild-type; HBE, human bronchial epithelial cells; M1, mouse epithelial-collecting duct cells.
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