1 Department of Physiology, University of Saarland, D-66421 Homburg,
Germany
2 Department of Clinical Pharmacology, University of Bern, Murtenstrasse 35,
3010 Bern, Switzerland
3 Department of Geriatrics, Geneva University Hospital, CH-1211 Geneva 14,
Switzerland
* Author for correspondence (e-mail: phlmery{at}uniklinik-saarland.de)
Accepted 14 June 2002
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Summary |
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Key words: TRPC proteins, PDZ domain, EBP50, Membrane localization
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Introduction |
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Over the last years, the cDNAs encoding a large number of ion channels have
been cloned and, in parallel, mutations that affect channel properties have
been identified. It has become obvious that in a number of `channelopathies',
the defect is associated with a failure of the intracellular sorting machinery
to deliver an otherwise functional channel to its correct destination. This is
true for the CFTR mutation F508, which occurs in nearly 70% of cystic
fibrosis patients among Caucasians (Cheng
et al., 1990
; Pasyk and
Foskett, 1997
), as well as for HERG potassium channel mutations
that cause the cardiac `long Q-T' syndrome
(Zhou et al., 1998
). Other
diseases (such as Liddle's syndrome) are caused by a gain of channel function
due to an increased stability of the channel at the plasma membrane
(Rotin et al., 2001
). These
findings have prompted a number of groups to look for specific signals within
the amino acid sequence of channel proteins that could regulate their
intracellular transport and surface localization. By using domain swap or
deletion experiments as well as site-directed mutagenesis, several types of
motifs have been identified including the PDZ-binding domains. The latter
correspond to short consensus sequences (S/T-X-L/V/I/M or F/Y-X-F/Y/A, where X
is any amino acid) located at the C-terminus of numerous receptors, ion
channels and transporters (Songyang et
al., 1997
). PDZ-binding domains specifically interact with modular
80-90 amino acid sequences named PDZ domains after PDS-95, Dlg-A and ZO-1,
three proteins in which these motifs were first described.
A clear demonstration for a role of a PDZ domain protein in channel
localization has emerged from analysis of Drosophila
phototransduction. Vision in Drosophila uses the fastest known
PLC-dependent signaling cascade, taking just a few milliseconds to go from
activation of rhodopsin to the generation of a receptor potential and less
than 100 milliseconds to terminate the response (for a review, see
Montell, 1999). An important
strategy used by Drosophila photoreceptors to both increase vision
speed and allow a fine tuning of the light response, is the organization of
the signaling components into a macromolecular complex (or transducisome) via
the adaptor protein INAD (inactivation no afterpotential D). INAD
(Shieh and Niemeyer, 1995
)
consists primarily of five PDZ domains and can bind directly to different
proteins (Montell, 1999
)
including protein kinase C (PKC), phospholipase C (PLC), the light-activated
cation channel TRP and the actin-binding protein NINAC. The transducisomes
localize to the rhabdomeres, a subcellular compartment consisting of 60,000
microvilli and containing the 108 molecules of rhodopsin found in
Drosophila photoreceptors. In inaD null mutants,
photoreceptors have profound signaling defects
(Chevesich et al., 1997
;
Tsunoda et al., 1997
). TRP,
PLC and PKC no longer localize to the rhabdomeres, but instead are randomly
distributed throughout either the plasma membrane (in the case of TRP) or the
cytoplasm (PLC and PKC). Recent studies have shown that the INAD-TRP
interaction is not required for targeting but rather for anchoring of the
preassembled transducisomes in the rhabdomere
(Li and Montell, 2000
;
Tsunoda et al., 2001
).
While it is accepted that members of the TRPC subfamily of
Drosophila TRP homologs form channels that are activated in a
PLC-dependent manner (Clapham et al.,
2001), the mechanisms that control their assembly and cell surface
expression are largely unknown. It has recently been suggested that the PDZ
domain protein NHERF (Na+/H+ exchanger regulatory
factor) (Weinman et al., 1995
)
and its human ortholog EBP50 [ezrin/radixin/moesin (ERM)-binding
phosphoprotein 50] (Reczek et al.,
1997
) may be functional analogs of INAD in mammalian cells.
Indeed, the PDZ domains of NHERF have been shown to bind to several members of
the phospholipase Cß family (Reczek
and Bretscher, 2001
; Tang et
al., 2000
) and to the mammalian TRP homologs TRPC4 and TRPC5
(Tang et al., 2000
). In
addition, NHERF interacts with the actin-binding proteins of the ERM family
(Reczek et al., 1997
) via its
C-terminal 30 amino acids and may link the PLC-ß and TRPC4/5 to the
cytoskeleton. In this study, we demonstrate that the PDZ-binding domain of
TRPC4 controls its localization and surface expression in transfected human
embryonic kidney (HEK) 293 cells. Our results also suggest that the
interaction between EBP50 and the membrane-cytoskeletal adaptors of the ERM
family may play an active role in the cell surface delivery of TRPC4 by
facilitating the translocation of TRPC4-bearing vesicles from the cortical
actin layer to the plasma membrane.
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Materials and Methods |
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Plasmid constructions
PolyA+ mRNA was purified from HEK293 cells using the Fast Track
2.0 kit (InVitrogen, Groningen, The Netherlands). First strand cDNA was
synthesized by using the AMV reverse transcriptase (InVitrogen) and oligo-dT
as primers. The entire coding region of the EBP50 cDNA was then amplified by
PCR. The resulting product was gel purified, subcloned into the pGEM-T vector
(Promega) and sequenced (MWG biotech). The cDNA sequences encoding residues
10-101 and 144-247 of EBP50 were amplified by PCR using mutant primers
containing appropriated restriction sites. The resulting products, encoding
either the PDZ1 or the PDZ2 domain of EBP50, were then subcloned into the
pGEX-5X-1 vector (Amersham Pharmacia biotech) to produce
glutathione-S-transferase (GST)-PDZ fusion proteins.
The cDNA encoding YFP-EBP50 was generated as follows: the coding region of
EBP50 was amplified by PCR. A BglII site was introduced at the
3'end, after the stop codon, to facilitate subsequent cloning. The start
codon (ATG) was replaced by the GCT nucleotides to create a NheI site
(5'-GCTAGC-3'). The region corresponding to nucleotides 1879-2621
of the pIRES-EYFP vector (Clontech), encoding the enhanced yellow fluorescent
protein (EYFP), was amplified by PCR. In the antisense primer, the stop codon
(TAA) was replaced by a TCT codon. The latter was included into a
Xba1 site (5'-TCTAGA-3') to allow the fusion in frame
with EBP50. The resulting products were gel purified and ligated into pGEM-T
[leading to the pGEM/EBP50(Met) and pGEM/EYFP(
stop) constructs].
All recombinant sequences were determined to be free of PCR errors by
nucleotide sequence analysis. The NheI-SacI fragment was
excised from pGEM/EBP50(
Met) and subcloned into the
XbaI-SacI sites of pGEM/EYFP(
stop). The resulting
construct was then digested with BamHI and NotI and inserted
into pcDNA3(+) (InVitrogen). The EBP50 mutant lacking the
ERM-binding domain was generated as follows: the region corresponding to
nucleotides 596-1193 of the EBP50 cDNA was amplified by PCR using pGEM/EBP50
as template. Nucleotides 1191-1193 (which encodes A328) were
replaced by the TAG stop codon using a sequence-mutated antisense primer. The
PCR product was subcloned into the pGEM-T vector. Following sequence analysis,
the insert was excised from pGEM using EcoRI and NotI and
ligated into pcDNA3/YFP-EBP50. The resulting construct encodes a
YFP-EBP50 fusion protein lacking the 31 EBP50 C-terminal amino acids and
referred to as YFP-
ERM. Nucleotides are numbered according to the
sequence deposited in the GenBank under the accession number AF015926.
The cDNA encoding TRPC4 (accession number AAF22928) was isolated as
previously described and subcloned into the pGEM-T vector. The 5'
untranslated region was removed and the construct was epitope-tagged at the
N-terminus with the myc peptide MEQKLISEEDLLR. The first methionine codon was
included within the sequence characteristic for translation initiation
5'-GCCGCCATGG-3' as specified previously
(Kozak, 1991). The TRPC4
mutant lacking the last three C-terminal amino acids (called myc-
TRL)
was generated by PCR-based mutagenesis. The region corresponding to
nucleotides 2644-2907 of the TRPC4 cDNA was amplified and the nucleotides
2905-2907 encoding T890 were replaced by a premature stop codon in
the antisense primer. The resulting product was inserted into pGEM-T.
Sequencing was performed to confirm the deletion and to verify that base
misincorporation did not occur during DNA amplification. The PCR fragment was
then excised from pGEM using XhoI and NotI and ligated into
the similarly cut pGEM/myc-TRPC4. The cDNAs encoding myc-TRPC4 and
myc-
TRL were then subcloned into the NotI site of
pcDNA3(+) (InVitrogen). All PCR primers sequences are available
upon request.
Cell culture and transfection
HEK293 cells and JEG-3 cells were cultured in DMEM-F12 medium supplemented
with 10% heat-inactivated fetal calf serum, 50 U/ml penicillin and 50 µg/ml
streptomycin. Transfections were carried out by the calcium phosphate
co-precipitation method as previously described
(Mery et al., 2001).
GST-pulldown assays
GST-PDZ1, GST-PDZ2 as well as the GST alone were expressed in BL21 E.
coli strain. Production of the fusion proteins was initiated by adding
0.1 mM isopropyl-ß-thio-D-galactopyranoside (IPTG) to the bacterial
cultures grown to an A600 of 0.6. After 90 minutes of induction at
37°C, bacteria were harvested and resuspended in icecold PBS containing 1%
Triton X-100, 1 mM EDTA and a cocktail of protease inhibitors (Boehringer
Mannheim). Bacteria were lysed by three cycles of rapid freeze/thawing
followed by 12 passages through a 23-gauge needle. The insoluble material was
removed by centrifugation for 5 minutes at 10,000 g and 4°C and
the supernatant was incubated with glutathione-sepharose beads for 1 hour at
room temperature. After four washes with the lysis buffer, an aliquot of beads
was removed: bound proteins were released by incubating the beads in 2x
Laemmli buffer (125 mM Tris-HCl, 20% glycerol, 6% SDS, 10%
ß-mercaptoethanol, pH 6.8) for 15 minutes at 37°C. Eluates were
analyzed on 12% SDS-PAGE and then stained with Coomassie blue. HEK293 cells
expressing either myc-TRPC4 or myc-TRL as well as the mock-transfected
cells were lysed 48 hours after transfection in PBS containing 1% Triton
X-100, 1 mM EDTA and a cocktail of protease inhibitors (Boehringer Mannheim).
Protein concentrations were determined with the bicinchonic acid (BCA)
procedure (Sigma) using bovine serum albumin as standard. Samples (200 µg
of triton-solubilized proteins) were incubated for 3 hours at 4°C with
glutathione-sepharose beads, charged with either GST, GST-PDZ1 or GST-PDZ2.
The beads were then harvested by centrifugation and washed four times with the
lysis buffer at 4°C. Bound proteins were eluted with 50 µl of 2x
Laemmli buffer and analyzed, together with aliquots of the total cell
extracts, by SDS-PAGE and immunoblot.
Co-immunoprecipitation
48 hours after transfection, HEK293 cells were rinsed twice with PBS pH 7.4
and then collected from plates without trypsinisation, by incubation in the
same buffer supplemented with 2 mM EDTA. The harvested cells were centrifuged
at 1000 g and 4°C for 5 minutes and resuspended in ice-cold lysis
buffer (PBS pH 7.4 containing 1% triton, 1 mM EDTA and a cocktail of protease
inhibitors). The lysates were rotated at 4°C for 30 minutes to further
solubilize the proteins and cellular debris were removed by centrifugation for
5 minutes at 1000 g and 4°C. Samples (300 µg of proteins) were
mixed with anti-YFP antibodies (2 µg) and rotated for 90 minutes at
4°C. Antigen-antibody complexes were then precipitated by adding 50 µl
of protein G-agarose beads (Roche diagnostics) pre-equilibrated in the lysis
buffer. After rocking the reaction mixtures for 16 hours at 4°C, the beads
were pelleted and washed four times with the lysis buffer. The
immunoprecipitates were eluted by boiling the samples in 50 µl 2x
Laemmli buffer for 5 minutes and then fractionated by SDS-PAGE.
Cell surface biotinylation
Dishes with confluent transfected HEK293 cells were placed on ice and
washed twice with ice-cold PBS pH 8 containing 1 mM MgCl2 and 0.5
mM CaCl2 (PBSB). Cells were then incubated for 30 minutes at
4°C with NHS-LC-biotin (final concentration 0.5 mg/ml), freshly diluted in
PBSB. Biotinylation was terminated by rinsing the dishes twice with PBSB
containing 0.1% bovine serum albumin (to quench the unbound NHS-LC-biotin) and
once with PBS pH 7.4 without Ca2+/Mg2+. Cells were
collected from plates without trypsinisation and lysed as described above.
Following measurement of the protein content of the cell extract, samples (900
µg of protein) were added to 200 µl of avidin-agarose beads,
pre-equilibrated in the lysis buffer and rotated for 3 hours at 4°C. The
biotin-avidin agarose complexes were then harvested by centrifugation and
washed four times with the lysis buffer supplemented with 0.25 M NaCl (final
concentration 0.4 M NaCl). The beads were then resuspended in 150 µl of
2x Laemmli buffer and incubated at 37°C for 15 minutes prior to
SDS-PAGE. Experiments were carried out to rule out that: (1) cytosolic
proteins in damaged cells are biotinylated and thus contribute to the pool
assumed to represent surface molecules; and (2) intracellular myctagged
proteins can bind to avidin-agarose beads in a biotin-independent way. As
shown in Fig. 5C, YFP was found
in the lysate from the biotinylated HEK293 monolayers but not in the fraction
recovered from the avidin beads. Similarly, myc-tagged channels were not
detected in affinity precipitates from myc-TRPC4- or myc-TRL-expressing
cells not exposed to NHS-LC-biotin.
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Preparation of membrane fractions
Transfected HEK293 cells were grown to confluence, washed with PBS pH 7.4
and then collected from plates without trypsinisation, by incubation in PBS
containing 2 mM EDTA for 2 minutes. The cells were centrifuged at 700
g for 5 minutes, resuspended in ice-cold lysis buffer (50 mM Tris-HCl
buffer pH 7.5 supplemented with 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA
and protease inhibitors) and rotated at 4°C for 30 minutes. The cells were
subjected to three freezethaw cycles and then sheared 10 times with 23-gauge
needles. Normal osmolarity was restored by adding 150 mM NaCl. Postnuclear
supernatants, prepared by spinning the cell homogenate at 100 g and
4°C for 10 minutes, were then centrifuged for 1 hour at 100,000 g
and 4°C in a T160 rotor (Beckman Coulter), resulting in a clear
cytoplasmic fraction and a membrane pellet. The latter was washed once with
the lysis buffer supplemented with 150 mM NaCl and then solubilized in PBS
containing 1% triton, 0.2% SDS and a cocktail of protease inhibitors at
4°C. The insoluble material was removed by centrifugation at 10,000
g for 5 minutes at 4°C. Protein concentrations were determined
with the bicinchonic acid procedure (Sigma) using bovine serum albumin as
standard. Isolated crude membranes were subjected to SDS-PAGE.
SDS-PAGE and immunoblots
Proteins were fractionated on a SDS-PAGE polyacrylamide gel and
electrophoretically transferred to Immobilon-P membranes (Millipore Corp.) in
25 mM Tris-HCl, 0.19 M glycine and 20% ethanol. The polyvinylidene difluoride
membranes were blocked overnight at 4°C in PBS containing 5% nonfat dry
milk and 0.05% Tween-20, rinsed twice with water and then incubated for 2
hours at room temperature with the primary antibody (anti-myc or anti-YFP)
diluted 1:1000 in PBS supplemented with 0.05% Tween-20 and 2.5% nonfat milk.
After three washes with PBS/0.05% Tween-20, the membranes were allowed to
react for 1 hour at room temperature with a peroxidase-conjugated goat
anti-mouse IgG antibody, diluted 1:10,000 in PBS/0.05% Tween-20. The blots
were washed three times with PBS/0.05% Tween-20 and the immune complexes were
visualized by chemiluminescence. When required, bands were quantified with a
LKB Ultrascan XL laser densitometer and the ImageQuant software.
Indirect immunofluorescent microscopy
HEK293 and JEG-3 cells were seeded on polyornithine-coated coverslips at
low density. Twenty-four hours after the plating, HEK293 cells were
co-transfected with 0.9 µg of the vector encoding either myc-TRPC4 or the
TRL mutant and 0.3 µg of the plasmid encoding YFP-EBP50. JEG-3 cells
were transfected with 0.5 µg of the vector encoding either the YFP alone,
YFP-EBP50 or YFP-
ERM. Two days after the transfection, cells were
rinsed twice with PBS pH 7.4 containing 1 mM CaCl2 and 0.5 mM
MgCl2 (PBS C/M), fixed in 4% paraformaldehyde for 15 minutes and
then permeabilized using 0.4% triton for 3 minutes. Blocking of nonspecific
binding sites was performed by incubating the monolayers with PBS-C/M
containing 0.2% gelatin and 2% normal goat serum (Sigma) for 30 minutes.
Transfected cells were stained for 1 hour with the primary antibody at the
indicated dilutions [anti-CD4 (1:200), anti-myc (1:300), anti-NHERF/EBP50
(1:200), antiezrin (1:200), anti-ß-tubulin (1:200), anti-vimentin
(1:100), rhodamine-phalloidin (1:200)], washed three times with the blocking
solution and then incubated for 1 hour with a 1:300 dilution of the
appropriate secondary antibody [Alexa 594-conjugated anti-mouse IgG or Alexa
488-conjugated anti-rabbit IgG (Molecular probes)]. All antibody dilutions
were prepared in PBS-C/M supplemented with 0.2% gelatin and 2% normal goat
serum and the incubations were carried out at room temperature. After
extensive washing with PBS-C/M, coverslips were mounted using Prolong antiface
reagent (Molecular Probes) and viewed with an upright Olympus BX50
fluorescence microscope equipped with a 75W Xe arc lamp and with standard FITC
and Texas Red filter sets (AHF Analysentechnik, Germany). Images were
collected through an Olympus 100x oil immersion objective (UPlanApo, NA
1.35) with a CCD camera (Sony) and digitized with microslicer imaging software
(Bernd Lindemann, Homburg). For some experiments, adherent cells were
pre-extracted in buffer A (140 mM KCl, 10 mM NaCl, 2 mM MgCl2, 10
mM Hepes, 0.1 mM CaCl2, 1.1 mM EGTA pH 7.2) containing 10 µg/ml
digitonin for 5 minutes on ice before fixation with 4% PFA in buffer A. The
fixed cells were washed twice with buffer A, twice with PBS C/M and then
processed as previously described.
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Results |
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Addition of a YFP tag at the N-terminus of EBP50 facilitates its
detection without affecting its subcellular localization
To be able to study the localization of the scaffold protein in both living
and fixed cells, we generated a cDNA construct encoding EBP50 N-terminally
fused to the yellow fluorescent protein (YFP). In JEG-3 cells, which derive
from placental syncytiotrophoblasts, EBP50 is particularly abundant and
specifically associates with the numerous microvilli of the apical surface. To
rule out the possibility that addition of the YFP tag affects EBP50
localization or trafficking, the fusion protein was expressed in JEG-3 cells
and its subcellular distribution was analyzed by indirect immunofluorescence
microscopy. As shown in Fig. 2,
the staining pattern of YFP-EBP50 (Fig.
2C) was very similar to that seen for ezrin
(Fig. 2A) or endogenous EBP50
(Fig. 2B). In contrast, the YFP
(Fig. 2D) was mainly found in
the cytosol and the nucleus of transfected cells.
|
Deletion of the TRL motif prevents the association of TRPC4 with
YFP-EBP50 in HEK293 cells
To determine whether YFP-EBP50 can bind to TRPC4 in a cellular context,
co-immunoprecipitation experiments were performed. YFP-EBP50 or YFP were
co-expressed with either myc-TRPC4 or myc-TRL in HEK293 cells. As shown
in Fig. 3 (lower panel),
myc-TRPC4 can be co-immunoprecipated with YFP-EBP50 but not with the YFP
alone. Removal of the TRL motif completely abolished the association with the
scaffold protein, indicating that formation of the TRPC4/YFP-EBP50 complex in
HEK293 cells requires the PDZ-binding cassette of TRPC5. Note that expression
of YFP-EBP50 in HEK293 cells allowed immunological detection of two bands at
65-70 kDa with antibodies to YFP (Fig.
3, top panel). Treatment of the cell lysate with calf intestinal
phosphatase resulted in the collapse of the 70 kDa band into the 65 kDa
species (data not shown), indicating that the heterogeneity was due to
phosphorylation. Thus, YFP-EBP50, similar to endogenous EBP50, is
constitutively phosphorylated in HEK293 cells, possibly by the G
protein-coupled receptor kinase 6A (GRK6A)
(Hall et al., 1999
).
|
Deletion of the TRL motif dramatically alters the subcellular
distribution of TRPC4 in HEK293 cells
Indirect immunofluorescence microscopy was then used to analyze the
distribution of myc-TRPC4 and myc-TRL in HEK293 cells co-expressing
YFP-EBP50. The localization of the myc-tagged molecules was compared with that
of CD4, a transmembrane protein that does not interact with EBP50. In
agreement with the idea that the N-terminal domain of TRP proteins is oriented
towards the cytoplasm, myc-TRPC4 and myc-
TRL were detected in cells
permeabilized with either Triton X-100
(Fig. 4J,L) or digitonin
(Fig. 4P,R) but not in intact
cells (Fig. 4D,F). As shown in
Fig. 4B, non permeabilized
CD4-expressing cells were heavily stained with an antibody directed against
the extracellular domain of the receptor, indicating that CD4 was highly
expressed on the surface membrane. In triton-permeabilized cells, the staining
patterns of CD4 (Fig. 4H) and
myc-TRPC4 (Fig. 4J) looked very
similar. Both proteins were found to be evenly distributed at the plasma
membrane and to localize to a juxtanuclear compartment, most probably
representing the Golgi apparatus. In contrast, the mutant lacking the TRL
motif accumulated into cell outgrowths and exhibited a clustered appearance
(Fig. 4L). The latter was very
similar to the patchy staining that has been described for murine TRPC4
C-terminally fused to the GFP and expressed in HEK293 cells
(Schaefer et al., 2000
). These
findings suggest that EBP50 or another PDZ protein with similar specificity
[such as the NHE3-kinase A regulatory protein (E3KARP)
(Yun et al., 1997
), the
CFTR-associated protein 70 (CAP70) (Wang
et al., 2000
), or the CFTR-associated ligand (CAL)
(Cheng et al., 2001
)] controls
TRPC4 localization in HEK293 cells.
|
Expression of YFP-EBP50 in HEK293 resulted in a diffuse fluorescence signal
throughout the cell that was not significantly influenced by co-expression of
either CD4 (Fig. 4A,G),
myc-TRPC4 (Fig. 4C,I) or
myc-TRL (Fig. 4E,K). In
contrast to the transmembrane proteins
(Fig. 4N,P,R), YFP-EBP50 was
almost completely extracted by a pretreatment of the cells with a low
concentration of digitonin prior to fixation
(Fig. 4M,O,Q).
Deletion of the TRL motif at the C-terminus of TRPC4 decreases the
fraction of channel associated with the plasma membrane
Whereas immunostaining clearly revealed differences in the subcellular
distribution of the wild-type and truncated TRPC4 proteins, it did not allow
quantification of channel expression on the cell surface. Thus, to compare the
plasma membrane association of myc-TRPC4 and myc-TRL in HEK293 cells
(co-expressing YFP-EBP50), we performed cell surface biotinylation
experiments. Equal amounts of transfected cells were treated with
NHS-LC-biotin (at 4°C to prevent internalization of the biotin derivative)
before being solubilized in Triton X-100. A portion of the resulting extract
was retained as the total fraction. The remaining lysate was incubated with
immobilized avidin to recover the biotinylated proteins. Serial dilutions of
the total and surface fractions were resolved by SDS-PAGE, transferred to PVDF
membrane and probed with the anti-myc antibody. Immunoreactive bands were
visualized by ECL and quantified with a densitometer. Optical densities were
then plotted against the respective amounts of total or biotinylated protein
loaded. As shown in Fig. 5A,
the intensity of the measured ECL signal was linear with respect to protein
concentrations between 5 and 30 µg and volumes of avidinprecipitate between
10 and 40 µl. The slopes of the linear correlations were determined as
described in Materials and Methods. The relative contents of myc-TRPC4 and
myc-
TRL in the surface fraction and in the corresponding total extract,
referred to as RS and RT, respectively, were then
calculated by dividing the slope obtained from the myc-TRPC4-expressing cells
by the slope derived from the myc-
TRL-expressing cells. Finally,
RS/RT represents the relative content of myc-TRPC4 and
myc-
TRL in the surface fraction corrected for any differences in the
level of channel expression in the total extracts. Control experiments were
performed to confirm that the avidin-bound myc-TRPC4 and myc-
TRL were
derived only from the biotinylated pool of the surface molecules (see
Materials and Methods and Fig.
5C).
As shown in Fig. 5B,
expression on the cell surface was higher (2.35-fold, P<0.01,
one sample t-test) for myc-TRPC4 than for myc-
TRL in HEK293
cells co-expressing YFP-EBP50. Thus, deletion of the TRL motif not only
changed the subcellular distribution of TRPC4 but also decreased its cell
surface expression. These results may indicate that the anterograde
trafficking of myc-
TRL to the plasma membrane is less efficient. They
may also reflect a reduction in the residence time of the mutant at the cell
surface. Indeed, EBP50 that interacts with the actin-binding protein of the
ERM family, has been suggested to either prevent the internalization or
facilitate the recycling to the plasma membrane of its target molecules. To
determine whether the EBP50-ERM interaction influences the cell surface
expression of TRPC4, a truncated form of EBP50 that lacks the ERM-binding
domain was generated and fused to the YFP. The capability of the mutant
(referred to as YFP-
ERM) to interact with ezrin and myc-TRPC4 was
analyzed first.
The YFP-EBP50 mutant lacking the ERM-binding site does not localize
to the ezrin-enriched microvilli in JEG-3 cells but still associates with
TRPC4 in HEK293 cells
As previously described, when expressed in JEG-3 cells, YFP-EBP50
(Fig. 6A) preferentially
localized to the ezrin-enriched microvilli
(Fig. 6A,B) of the apical
surface. In contrast, YFP-ERM was found to be mainly cytosolic
(Fig. 6C), the residual apical
staining probably reflecting the association of the truncated mutant with
endogenous EBP50 through PDZ-PDZ interactions
(Fouassier et al., 2000
). This
redistribution was not due to an alteration of the morphology of the
ezrin-rich structures in cells expressing YFP-
ERM
(Fig. 6D) and strongly suggests
that the interaction with ezrin determines EBP50 localization in JEG-3
cells.
|
In HEK293 cells, YFP-EBP50 and YFP-ERM were expressed at similar
levels and deletion of the 31 C-terminal amino acids of EBP50 did not impair
its interaction with myc-TRPC4 (Fig.
7A). The subcellular distributions of the YFP-tagged proteins in
myc-TRPC4- and myc-
TRL-expressing cells were compared upon cell
fractionation. As shown in Fig.
7B (left panel), the partition of YFP-EBP50 between cytosolic and
crude membrane fractions was not influenced by the co-expression of either
myc-TRPC4 or myc-
TRL. In contrast, the amount of YFP-
ERM
associated with the crude membranes was found to be much higher in cells
co-expressing myc-TRPC4 than in cells co-transfected with myc-
TRL
(Fig. 7B, right panel). These
results suggest that YFP-EBP50 and YFP-
ERM primarily associate with
membranes through their interaction with proteins of the ERM family and
myc-TRPC4, respectively.
|
Disruption of the EBP50-ERM interaction decreases the fraction of
TRPC4 associated with the plasma membrane
The effect of the disruption of the EBP50-ERM interaction on the surface
expression of TRPC4 was then investigated. To compare the plasma membrane
association of myc-TRPC4 in HEK293 cells co-expressing YFP-EBP50 or
YFP-ERM, we performed cell surface biotinylation experiments. As shown
in Fig. 8, the amount of
myc-TRPC4 present at the cell surface was significantly higher (
2.5-fold,
P<0.01, one sample t-test) in cells co-transfected with
YFP-EBP50 than in cells co-expressing the
ERM mutant. Thus, disruption
of the TRPC4-ERM interaction was found to have similar effects on the surface
expression of TRPC4 as disruption of the TRPC4-EBP50 complex by mutation of
the channel tail.
|
Disruption of the EBP50-ERM interaction prevents the translocation of
TRPC4 from the cortical actin layer to the plasma membrane
The subcellular distributions of myc-TRPC4 and myc-TRL in HEK293
cells overexpressing YFP-
ERM were studied by indirect
immunofluorescence microscopy. In about 60% of the transfected cells, the
staining pattern of myc-TRPC4 was similar to that shown in
Fig. 4J. However, in the
remaining 40%, myc-TRPC4 and YFP-
ERM were found to overlap in a
perinuclear compartment, most probably representing the Golgi apparatus as
well as in vesicles associated with filamentous structures that resembled
cytoskeletal elements (Fig.
9A,B). The fact that this reticular phenotype was not detected in
all the cells co-expressing myc-TRPC4 and YFP-
ERM may indicate that the
relative stoichiometry between the scaffold protein and its ligands is
important for this process. YFP-
ERM was expected to behave as a
dominant interfering inhibitor of the TRPC4-ERM interaction. In some cells,
the amount of mutant relative to endogenous EBP50 might, however, not have
been sufficient to completely block the interaction with the endogenous
proteins. By contrast, a large overexpression of YFP-
ERM may favor the
self-association of the scaffold protein rather than its interaction with
other target molecules. Importantly, the reticular pattern was not seen in
cells expressing YFP-
ERM alone (data not shown) or together with
myc-
TRL (Fig. 9C,D)
suggesting that YFP-
ERM and myc-TRPC4 need each other to associate with
the filamentous structures.
|
To further characterize this phenotype, cells co-expressing YFP-ERM
and myc-TRPC4 were stained with antibodies to various cytoskeletal proteins or
with Texas-Red phalloidin. The reticular distribution of YFP-
ERM
(Fig. 10A) was not found to
significantly overlap with the ß tubulin
(Fig. 10B) or vimentin (data
not shown) immunostaining patterns. Interestingly, however, a significant
proportion of the ß tubulin was observed as discrete dot-like structures
between the juxtanuclear filament mass and the cell surface
(Fig. 10B) in cells
co-expressing myc-TRPC4 and YFP-
ERM, indicating that the microtubules
were partially depolymerized. YFP-
ERM
(Fig. 10D) was found to mainly
co-localize with the cortical actin layer beneath the plasma membrane and with
actin cables within the cells (Fig.
10E), indicating that the YFP-
ERM/myc-TRPC4 complex is able
to bind to F-actin in an ERM-independent manner. The association of the
myc-TRPC4-bearing vesicles with the cortical actin suggests that they were
able to reach the submembranous compartment but not to fuse with the plasma
membrane. Thus, we propose that the interaction between EBP50 and the proteins
of the ERM family is required for the insertion of myc-TRPC4 in the plasma
membrane.
|
![]() |
Discussion |
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A previous study has demonstrated that 35S-labeled murine TRPC4
binds strongly to the first PDZ domain of NHERF (the rabbit ortholog of EBP50)
and only weakly to the PDZ2 domain (Tang
et al., 2000). Surprisingly, however, immobilized fusion proteins
containing either the first or the second PDZ domain of EBP50 were found to
precipitate myc-TRPC4 equally well from extracts of myc-TRPC4 expressing
cells. The discrepancy between our data and the previously published results
could indicate that, in our assay, the interaction between TRPC4 and the PDZ2
domain was not direct but mediated by another partner present in the cell
lysate. Alternatively, the steady state binding equilibria might not have been
established by the end of the pull-down assays in Tang's study since the
GST-fusion proteins were incubated with in vitro translated TRPC4 for only 30
minutes. Indeed, by performing surface plasmon resonance experiments, Raghuram
et al. have recently shown that both PDZ domains of EBP50 strongly interact
with the TRL motif present at the C-terminus of the cystic fibrosis
transmembrane conductance regulator (CFTR) but that the PDZ2-CFTR complex is
formed much slower than the PDZ1-CFTR interaction
(Raghuram et al., 2001
).
Deletion of the last three C-terminal amino acids (TRL) of TRPC4 was found
to completely abolish the interaction with EBP50 and to alter both the
localization and the surface expression of TRPC4 in HEK293 cells. Instead of
being evenly distributed at the cell surface, the TRL mutant
accumulated into the cell outgrowths and its expression in the plasma membrane
was 2.4 times lower than that of myc-TRPC4. Two non-exclusive hypotheses can
be proposed to explain these results.
First, despite the fact that they do not show overt cell surface polarity,
fibroblasts have apical and basolateral cognate routes for the delivery of
newly synthesized proteins from the trans-Golgi network (TGN) to the plasma
membrane (Musch et al., 1996;
Yoshimori et al., 1996
). Thus,
the PDZ-binding site may be required for the efficient sorting of TRPC4 at the
TGN level in HEK293 cells. In support of this hypothesis, deletion of the TRL
motif present at the C-terminus of CFTR causes the redistribution of the
truncation mutant from the apical to the basolateral membrane in airway
epithelial cells (Moyer et al.,
1999
; Moyer et al.,
2000
) and epithelial basolateral proteins have been shown to
accumulate into the cell outgrowths in nonpolarized cells
(Grinstein et al., 1993
;
Peranen et al., 1996
), such as
myc-
TRL. Moreover, a PDZ-domain protein called CAL (CFTR-associated
ligand), which mainly colocalizes with TGN markers in both epithelial and
nonpolarized cells, has recently been identified
(Cheng et al., 2001
). CAL has
been shown to bind to the C-terminus of CFTR and to modulate its surface
expression.
Second, interaction of the C-terminus of TRPC4 with a PDZ-domain-containing
protein may be required for the retention and the accumulation of TRPC4 at the
cell surface. Indeed, through their association with the actin-binding protein
of the ERM family, cortical scaffolds such as EBP50 or the related protein
E3KARP may anchor TRPC4 to the cytoskeleton and prevent its internalization.
In support of this hypothesis, CFTR molecules containing deletions that
inhibit their interaction with EBP50 exhibit decreased expression and
residence times in the plasma membrane of nonpolarized cells
(Moyer et al., 2000).
Alternatively, interaction of the C-terminus of TRPC4 with EBP50 may
facilitate the recycling of internalized TRPC4 molecules to the plasma
membrane, as recently shown for the ß2-adrenergic receptor
(Cao et al., 1999
).
To analyze how the actin-linker function of EBP50 influences the cell
surface expression of myc-TRPC4, an EBP50 mutant lacking the ERM-binding site
was generated and co-expressed with myc-TRPC4 in HEK293 cells. In a large
percentage of transfected cells, myc-TRPC4 and YFP-ERM were found to
overlap in a perinuclear compartment, most probably representing the Golgi
apparatus as well as in vesicles aligned along actin-filaments. YFP-
ERM
and myc-TRPC4 were found to need each other to associate with the
cytoskeleton. We speculate that YFP-
ERM first localizes to the Golgi
apparatus through its interaction with the C-terminus of myc-TRPC4 and then
mediates the anchoring of the post-Golgi vesicles containing myc-TRPC4 to the
actin filaments. Two potential mechanisms can be proposed to explain how
YFP-
ERM may regulate the transfer of myc-TRPC4 from the TGN to the
cortical actin network. First, YFP-
ERM may be responsible for the
sorting of TRPC4 in the TGN into specific vesicles, which are then transported
along the actin microfilaments towards the cell surface. It has been shown
that proteins that are sorted to the apical cognate route in fibroblasts
become incorporated into lipid rafts in the TGN
(Verkade and Simons, 1997
;
Zegers and Hoekstra, 1998
) and
that EBP50 interacts via its first PDZ domain with the broadly expressed
raft-associated protein PAG (phosphoprotein associated with
glycosphingolipid-enriched microdomains)
(Brdickova et al., 2001
). Thus,
YFP-
ERM may recruit TRPC4 into lipid rafts at the TGN through its
interaction with a raft-resident protein such as PAG. Second, YFP-
ERM
may bind via one of its PDZ domains to an actin-based motor. In support of
this hypothesis, INAD in Drosophila directly interacts with the
unconventional myosin NINAC, which has been suggested to function as an
actin-based motor (Bahler,
2000
). Recent studies demonstrate that other multi-PDZ domains
proteins including PDS-95 and LIN-10 also associate with post-Golgi vesicles
and cytoskeleton components (El-Husseini et
al., 2000
; Setou et al.,
2000
). This may indicate a common role for PDZ-domain-containing
proteins in transport of channel- or receptor-containing vesicles, ensuring
reliability of transducisome assembly.
The association of myc-TRPC4 with the cortical actin layer suggests that
TRPC4 can reach the submembranous compartment. However, the vesicles
containing the cation channel seem to be trapped there and myc-TRPC4 is not
delivered to the cell surface. We speculate that EBP50 binding to ezrin may
allow the myc-TRPC4-bearing vesicles to leave the cortical actin and fuse with
the plasma membrane. Interestingly, overexpression of CAL, which does not
possess an ERM-binding site, was also found to decrease the cell surface
expression of CFTR in COS-7 cells, in part by reducing the anterograde
trafficking of CFTR to the plasma membrane
(Cheng et al., 2001). The
effects of CAL were reversed by overexpressing EBP50
(Cheng et al., 2001
). Many
findings support the hypothesis that the EBP50-ERM complex may play a role in
the insertion of TRPC4 into the plasma membrane. First, at least two target
molecules of the ERM proteins, namely the phosphatidylinositol
(4,5)-biphosphate and the phosphatidylinositol 3-kinase (reviewed by
Bretscher et al., 2000
) have
been shown to regulate fusion events. Second, EBP50 binds to EPI64 (EBP50-PDZ
interactor of 64 kDa), a protein expressed in many cell types and containing a
rab GTPase-activating protein (GAP) domain
(Reczek and Bretscher, 2001
).
As a plasma membrane-associated rab-GAP, EPI64 would be expected to enhance
the intrinsic rate of hydrolysis of GTP on the active rab on an incoming
vesicle, perhaps providing a signal that it has reached the appropriate site.
Finally, in parietal cells, the phosphorylation of subapically located ezrin,
which allows its association with EBP50, has been correlated with the
translocation and fusion of rab11a-containing vesicles with the apical surface
(Urushidani and Forte, 1997
).
EBP50 binds only to activated ERM proteins and activation of the ERM proteins
can be promoted by stimulation of surface receptors. Thus, PDZ proteins may be
involved in determining the surface expression of TRPC4 not only in basal
conditions but also upon activation of the cells with specific stimuli.
In summary, we report that the last three C-terminal amino acids of TRPC4 comprise a PDZ-interacting domain, which controls the subcellular localization and surface expression of TRPC4 in HEK293 cells. Our data also suggest that TRPC4 insertion in the plasma membrane involves interaction with the ERM-bound scaffold EBP50. Because the EBP50 mutant that lacks the ERM-binding site was found to play a role in TRPC4 sorting at the TGN level and to be required for TRPC4 transport along actin microfilaments, we propose that another PDZ protein (such as CAL), which preferentially associates with the Golgi apparatus, may control the intracellular trafficking of TRPC4. In this view, the C-terminal domain of TRPC4 may sequentially interact with several PDZ proteins involved in targeting to the surface or insertion/retention in the plasma membrane, depending on their location within the cell.
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Acknowledgments |
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