1 Dipartimento di Fisiologia Generale ed Ambientale, University of Bari, Via
Amendola 165/A, 70126 Bari, Italy
2 Centro CNR Biomembrane and Dipartimento di Scienze Biomediche, Via G. Colombo
3, 35100 Padova, Italy
3 Sezione di Medicina Interna ed Oncologia, Dipartimento di Medicina Clinica e
Sperimentale, Università degli Studi, Policlinico Monteluce, 06100
Perugia, Italy
4 Forschungsinstitut für Molekulare Pharmakologie, 13125 Berlin,
Germany
Author for correspondence (e-mail:
g.valenti{at}biologia.uniba.it)
Accepted 10 July 2002
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Summary |
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These results represent the first evidence for the functional involvement of VAMP-2 in cAMP-induced AQP2 exocytosis in renal cells.
Key words: Aquaporin, SNARE, Tetanus toxin, Trafficking, Exocytosis
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Introduction |
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To investigate whether SNAREs participate in exocytosis of AQP2, we used
AQP2-transfected CD8 cells (Valenti et
al., 1996), which are an ideal model for studying the molecular
basis of exocytosis in non-excitable tissue
(Valenti et al., 1998
;
Valenti et al., 2000
;
Tamma et al., 2001
), and we
have determined the expression of SNAREs. In addition, we have investigated
the effect of tetanus neurotoxin (TeNT) on cAMP-induced AQP2 translocation in
intact CD8 cells. The present study provides clear evidence that the SNARE
protein VAMP/synaptobrevin-2 (VAMP-2) is functionally involved in
cAMP-stimulated AQP2 translocation in renal collecting duct cells.
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Materials and Methods |
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Rabbit polyclonal antibodies specifically directed against the external C-loop of AQP2 were raised using a peptide (IRGDLAVNALSNSTT) reproducing the C-loop of the human AQP2.
Cell culture
The study was performed on AQP2-transfected renal CD8 cells
(Valenti et al., 1996). This
cell line was established by stably transfecting the RC.SV3 rabbit cortical
collecting duct with cDNA encoding rat AQP2. This cell model system possesses
the key properties of principal cells and has been useful to clarify some key
events triggered by vasopressin action
(Valenti et al., 1998
;
Valenti et al., 2000
;
Tamma et al., 2001
). CD8 cells
were grown at 37°C, as described previously, in a hormonally defined
medium (Valenti et al., 1996
).
Confluent monolayers were used at days 3-5 after plating.
RNA isolation, RT-PCR and cDNA sequencing
Total RNA was extracted from confluent CD8 cells or rat brain, lung or
kidney by the TRIzol extraction method (TRIzol reagent, Life Technologies,
Gaithersburg, MD). The RNA was then used to amplify fragments of the cDNA of
the SNARE isoforms syntaxin-1, -3, -4, VAMP-2 and SNAP-23 by degenerate RT-PCR
employing the GeneAmp RNA PCR Core kit (Perkin-Elmer, Branchburg, NJ). The
degenerate primers (Table 1)
were designed on the basis of the SNARE nucleotide sequences available in the
GenBank database (web site:
www.ncbi.nlm.nih.gov/Entrez/nucleotide.html).
A positive control was performed by using primers specific for
ß-actin cDNA, BAF (5'-CAGATCATGTTTGAGACCTT-3') and
BAR (5'-CGGATGTCMACGTCACACACTT-3'). PCRs were performed with the
following program: (95°C, 3 minutes)x1 cycle, [94°C, 1 minute;
49 or 59°C (depending on the non-degenerate or degenerate primers used,
respectively) for 1 minute; (72°C, 30-50 seconds)]x32 cycles,
(72°C, 10 minutes)x1 cycle. The SNARE cDNAs amplified from the CD8
cells were cloned into the EcoRI/EcoRI site of the pCR2.1
vector (TA cloning kit, Invitrogen, San Diego, CA) following a TA strategy.
The sequence of the cloned DNA fragments was assessed by sequencing. Sequence
alignments were performed by using the Lasergene program (DNASTAR, UK).
|
Construction of the GFP/AQP2 expression plasmid and transfection
Total RNA extracted from rat kidney was employed to amplify the AQP2 cDNA
by using the specific primers AQP2 RAT-FW
5'-ATGTGGGAACTCAGATCCATA-3' and AQP2 RAT-REV
5'-TTCTTGAGGCTCACTGCACT-3'. The AQP2 cDNA was then cloned into the
pcDNA3.1/NT-GFP-TOPO vector (GFP Fusion TOPO TA Expression kit, Invitrogen) in
frame and at the 3' end of the GFP coding region. The resulting plasmid,
pcDNA3.1-AQP2, was then used to transfect the RC.SV3 rabbit cortical
collecting duct cell line by employing the Lipofectin Reagent (Gibco BRL) as
previously described (Valenti et al.,
1996).
Subcellular fractionation
Cells grown to confluency on 25 cm2 flask were washed in PBS,
scraped with a rubber policeman and homogenized with a glass/Teflon
homogenizer in ice-cold buffer containing 250 mM sucrose, 10 mM Tris pH 7.5, 1
mM PMSF, 1 µg/ml leupetin and pepstatin. For the preparation of the
low-speed pellet, enriched in plasma membranes (LS), and the high-speed
pellet, enriched in intracellular vesicles (HS), the cell suspension was
centrifuged at 700 g for 10 minutes at 4°C. The
supernatant was centrifuged at 17,000 g for 45 minutes at
4°C. The LS was recovered in PBS and the supernatant was spun at 200,000
g in a Beckman Rotor 60 2Ti for 60 minutes at 4°C. The
final pellet (HS) was recovered in PBS. Cell membranes were stored at
-20°C until used for immunoblotting studies. For LS and HS membrane
preparations from rabbit brain or rabbit kidney, the organs were removed, cut
into small slices and homogenized following the same procedure used for CD8
cells.
Western blotting
Membranes were solubilized in Laemmli buffer at 60°C for 10 minutes and
subjected to SDS-polyacrylamide gel electrophoresis (13% or 15%
polyacrylamide). Gels were transferred to Immobilon-P membrane (Millipore),
blocked in blotting buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.4 and 1% Triton
X-100) containing 5% non-fat dry milk for 1 hour. The membranes were incubated
with the first antibody at the dilutions reported in the legends for 2 hours
at room temperature in blotting buffer, washed in several changes of the same
blotting buffer. For the detection of syntaxin-1, syntaxin-4, SNAP-25, SNAP-23
and AQP2, the membranes were incubated with goat anti-rabbit IgG conjugated to
peroxidase (Sigma, 1:5000 dilution). For the detection of VAMP-2, membranes
were incubated with goat anti-mouse IgG conjugated to peroxidase (Sigma,
1:5000 dilution). Immunoreactive proteins were revealed with ECL-plus
chemiluminescence reaction (Amersham, Life Science).
Light and confocal microscope immunocytochemistry
Cells grown on glass coverslips were left in basal condition or stimulated
with 10-4 M forskolin (FK), a cAMP- elevating agent, for 15 minutes
at 37°C. Cells were then fixed in a fixative containing 2%
paraformaldehyde, 10 mM sodium periodate and 75 mM lysine (PLP) for 20 minutes
at room temperature. In one set of experiments, cells were permeabilized with
0.1% Triton X-100 for 10 minutes, whereas, in parallel experiments, the
permeabilzation step was omitted (see Results section). After blocking in 0.1%
gelatin in PBS for 5 minutes, cells were incubated at room temperature for 2
hours either with the anti-AQP2 antibody raised against the peptide
reproducing the C-terminus (AQP2 C-term, 1:100 dilution) or with the anti-AQP2
antibody raised against the peptide reproducing the loop C (AQP2 C-loop, 1:50
dilution).
After washing for 3x5 minutes with 0.1% gelatin, cells were incubated for 1 hour with FITC-conjugated secondary antibodies and then sequentially washed twice for 1 minute in 2.7% NaCl (high-salt PBS) and twice in regular PBS. Coverslips were mounted in 50% glycerol in 0.2 M Tris-HCl, pH 8.0 containing 2.5% n-propyl gallate as an anti-quenching agent. The slides were examined by confocal microscopy (MRC-1024 Bio-Rad equipped with a Krypton/Argon mixed gas laser). A specific software (Imaris 2.7, Bitplane, CH) was used for acquisition and processing of confocal images.
Double labeling of AQP2 and VAMP-2 was performed in cells transiently transfected with GFP-tagged AQP2. Briefly, GFP-tagged AQP2-transfected cells grown on coverslips, were fixed in ice-cold methanol for 5 minutes and incubated with the polyclonal anti VAMP-2 antibody (1:100 dilution). Cells were then stained with a goat anti-rabbit Cy3-conjugated secondary antibody (1:200 dilution).
To monitor the internalization of TeNT-FITC (see below), CD8 cells were incubated with the fluorescent toxin for 1 hour. Cells were then fixed with a solution containing 4% paraformaldehyde in PBS for 20 minutes at room temperature and examined with a Leica photomicroscope equipped for epifluorescence.
Digital images were obtained by a cooled CCD camera interfaced to the microscope (Princeton Instruments, NJ).
Tetanus toxin purification
The single-chain toxin was purified from cultures of Clostridium
tetani (Harvard strain) by the extraction procedure of Ozutsumi et al.
(Ozutsumi et al., 1985). The
crude toxin solution was chromatographed on DEAE-cellulose (Whatman) and
Ultrogel ACA-34 (LKB), followed by high-performance size exclusion liquid
chromatography analysis on a TSK G4000SW column (LKB) equilibrated with 0.1 M
sodium phosphate (pH 6.8). TeNT concentration was determined
spectrophotometrically at 280 nm with an
1mg/ml of 1.55. The di-chain
form of TeNT was obtained by nicking single-chain toxin with tosylsulfonyl
phenylalanyl chloromethyl ketone-treated trypsin (Serva) at 25°C for 60
minutes with a toxin-to-protease ratio of 1,000:1 (wt/wt). Proteolysis was
terminated by addition of soybean trypsin inhibitor at a final
protease-to-inhibitor ratio of 1:4 (wt/wt). TeNT was kept at 4°C, or after
being frozen in liquid nitrogen, was stored at -80°C at a protein
concentration of 2 to 10 mg/ml in 10 mM sodium
4-(2-hydroxyethyl)-piperazine-1-ethansulfonate (pH 7.4). TeNT was conjugated
to FITC (Pierce) by following the supplier's recommendations, and the
conjugate (TeNT-FITC) was purified by chromatography on a Sephadex G-25
column.
Treatments with tetanus neurotoxin
To investigate the effect of TeNT on VAMP-2, the toxin was employed either
in vitro on crude membrane samples or in vivo in intact CD8 cells. For the in
vitro experiments, TeNT was incubated with 10 mM DTT for 2 hours at room
temperature before use. Membrane fractions from rabbit brain (15 µg) and
CD8 cells (15 µg) were treated with TeNT (500 nM) for 1 hour at 37°C,
and the reaction was stopped by the addition of 1% SDS. Samples were then
resolved by SDS-PAGE and blotted with appropriate antibodies.
Treatment of intact CD8 cells with TeNT
CD8 cells were grown to confluency in 10 mm Petri dishes. Cells were
incubated in the presence or in the absence of whole TeNT (100 nM, for 3 hours
at 37°C in the medium). Cells were then washed in PBS Ca2+ and
Mg2+, scraped and centrifuged at 11,000 g for 10 minutes.
The pellet was resuspended in 30 µl distilled water and passed through a 29
G needle syringe to break the cells. The cell suspension was solubilized in
Laemmli buffer and loaded into 15% polyacrylamide gel. Proteins were
transferred and subjected to western blotting using monoclonal antibodies
(1:100 dilution) against human VAMP-2.
Quantitation of AQP2 cell-surface immunoreactivity
Cells were grown to confluence in 24 multiwells (Falcon, NJ, USA). For each
experimental condition, six wells, corresponding to approximately
4x106 cells in total, were tested. In experiments employing
TeNT, cells were preincubated with TeNT (100 nM, whole molecule) for 3 hours
at 37°C in the culture medium. Cells were then washed with PBS and either
left under control conditions or stimulated with 10-4M forskolin
for 15 minutes at 37°C in PBS Ca2+, Mg2+. In
parallel, cells were treated under the same experimental conditions without
TeNT pretreatment. Cells were brought to 4°C and immediately fixed in
freshly made fixative containing 2% paraformaldehyde, 10 mM sodium periodate
and 75 mM lysine (PLP) at 4°C for 20 minutes. Cells were washed in
ice-cold PBS and saturated for 15 minutes with 0.1% gelatin in PBS
(PBS-gelatin). Subsequently cells were incubated with AQP2 C-loop antibodies
(1:300 dilution in PBS-gelatin) for 1.5 hours at 4°C, washed three times
in PBS-gelatin and incubated with goat anti-rabbit IgG peroxidase-conjugated
for 1 hour at 4°C. After three washes in icecold PBS, cells were incubated
with a solution containing 3.69 mM o-phenylenediamine, in phosphate-citrate
buffer (pH 5.0) in the presence of 0.012% H2O2.
The solution was incubated for 15 minutes at room temperature, cells were
immediately transferred to 4°C and the colored solution removed. The
samples were read in a spectrophotometer at 450 nm. Cells in each well were
solubilized in 200 µl formic acid, and the protein content was determined
with the Bradford protein assay (Bradford,
1976). The peroxidase activity was expressed as OD/mg
proteins.
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Results |
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|
Expression and distribution of SNAREs in AQP2-transfected CD8
cells
To analyze the expression of the detected SNAREs proteins, western blot
experiments were carried out using specific antibodies. Membrane samples
isolated from CD8 cells or from rabbit brain and rabbit kidney used as
positive controls were immunoblotted with anti-VAMP-2, syntaxin-1A,
syntaxin-4, SNAP-23 and SNAP-25 antibodies. CD8 cells were fractionated to
separate a plasma-membrane-enriched fraction (LS) and a membrane fraction
enriched in intracellular vesicles (HS). VAMP-2 stained a 18 kDa band in both
brain and CD8 cells membrane fractions, with a stronger signal in
intracellular vesicle membranes (HS), a fraction in which AQP2 is enriched in
control cells (Fig. 2). Similar
results were obtained in membrane fractions obtained from the rabbit
brain.
|
By contrast, syntaxin-1 and syntaxin-4 antibodies labeled a distinct 36 kDa band corresponding to the expected molecular mass of syntaxins, displaying a stronger signal in a plasma membrane fraction (LS) both in CD8 and rabbit brain. This suggests that syntaxin-1A and syntaxin-4 are mainly present in the plasma membrane of CD8 cells (Fig. 2).
Our results are in agreement with those of others
(Nielsen et al., 1995;
Mandon et al., 1996
), who
found VAMP-2 in the kidney inner medulla and syntaxin-4 enriched in a membrane
fraction from inner medullary collecting duct cells. SNAP-25 was not detected
in CD8 cells, whereas it was highly expressed in rabbit brain
(Fig. 2). SNAP-25 is in fact
present principally in the brain, although Shukla et al. reported that
SNAP-25-associated Hrs-2 protein colocalizes with AQP2 in rat kidney
collecting duct principal cells (Shukla et
al., 2001
). On the other hand, the SNAP-23, a homologous SNAP-25
protein that binds to multiple syntaxins and synaptobrevins, is ubiquitously
expressed. SNAP-23 was detected in CD8 cells as well as in rabbit kidney and
found enriched in the low-speed pellet from both membrane samples
(Fig. 2).
Cleavage of VAMP-2 by tetanus neurotoxin
To directly address the issue of the functional involvement of SNAREs in
AQP2 targeting, we took advantage of the use of clostridial neurotoxins that
specifically cleave target SNAREs. This susceptibility afforded a specific
strategy to probe the function of VAMP-2 in AQP2 traffic.
TeNTs specifically hydrolyze rat VAMP-2 in synaptic vesicles but not VAMP-1
(Link et al., 1992). To test
whether TeNT was efficient in cleaving in vitro the VAMP-2-like protein
expressed in CD8 cells, a membrane fraction enriched for intracellular
vesicles was incubated with the toxin (500 nM) for 1 hour. As a control,
membranes from the rabbit brain were run at the same time. Membrane samples
were subjected to western blotting using anti-VAMP-2 antibodies. As shown in
Fig. 3 (in vitro cleavage, left
panel), toxin treatment of both membrane preparations from CD8 cells and
rabbit brain revealed complete cleavage of VAMP-2. This result further
supports the conclusion that the VAMP/synaptobrevin-like protein expressed in
CD8 cells is a VAMP-2-like protein. The cleavage fragment of about 12 kDa was
not detectable under our experimental conditions, probably because of the
reduced ability of the fragment to bind the nitrocellulose membrane during
blotting as described previously (Link et
al., 1992
).
|
As a next step, we investigated whether TeNT was able to cleave VAMP-2 in
intact CD8 cells. We first checked whether TeNT can be internalized in intact
CD8 cells. To this end, fluorescein isothiocyanate (FITC) was conjugated to
TeNT and purified by chromatography on a Sephadex G-25 column as described
previously (Matteoli et al.,
1996). Intact cells were exposed to TeNT-FITC, fixed and examined
by fluorescence microscopy. As shown in
Fig. 3 (cleavage in intact
cells, right panel), TeNT-FITC was internalized in intracellular structures
(TeNT-FITC) as assessed by fluorescence detection. Western blots of
solubilized CD8 cells exposed to TeNT in the medium for 3 hours revealed that
the internalized toxin was able to cleave the endogenously expressed
VAMP-2.
Analysis by western blotting with AQP2 antibodies revealed that cells pretreated with TeNT for 3 hours had the same amount of AQP2 as untreated control cells, indicating that the toxin did not affect the expression of AQP2 (data not shown).
Characterization of the anti-AQP2 C-loop antibody
We next examined the effect of TeNT treatment on AQP2 distribution in
resting CD8 cells and in cells that had been treated with the cAMP elevating
agent forskolin (FK). To determine the amount of AQP2 inserted in the apical
plasma membrane, we generated an antibody against a peptide reproducing the
external C-loop of human AQP2 (IRGDLAVNALSNSTT), and the cell-surface
expression of AQP2 was monitored by determining the protein immunoreactivity.
The antibody recognized both the glycosylated and non-glycosylated AQP2 from
both rat kidney and CD8 cells, with a stronger expression in a membrane
fraction enriched in intracellular vesicles
(Fig. 4A). The specificity of
the immune serum was assessed by preincubation of antiserum with a 100-fold
molar excess of immunizing peptide (Fig.
4A). To further confirm the recognition of an extracellular
epitope by the AQP2 C-loop antibody, non-permeabilized FK-stimulated CD8 cells
were stained with the AQP2 C-loop antibody or with the conventional AQP2
C-terminus antibody recognizing an epitope located in the cytosolic C-terminus
and examined by confocal microscopy. Under these experimental conditions
(Fig. 4B, -Triton X-100),
immunofluorescence staining demonstrated that only the AQP2 C-loop antibody
stained the cell surface of FK-stimulated CD8 cells, whereas no staining was
observed with the AQP2 C-terminal antibody
(Fig. 4B and xz reconstruction
in the inset). These data are fully consistent with the efficacy of the AQP2
C-loop antibody in recognizing an external epitope of the AQP2 protein. By
contrast, in permeabilized control cells, both antibodies stained
intracellular vesicles, giving a similar staining of the AQP2-bearing vesicles
(Fig. 4B, +Triton X-100).
|
Determination of cell-surface AQP2 immunoreactivity
The anti AQP2 C-loop antibody was therefore employed to monitor the AQP2
density on the plasma membrane in cells grown to confluency in ELISA
multiwells. In non-permeabilized cells, the antibody is expected to
cross-react only with AQP2 inserted into the plasma membrane
(Fig. 5A). After stimulation of
untreated cells with the cAMP-elevating agent forskolin, the immunodetectable
AQP2 on the cell surface increased by approximately two-fold compared with
that present in the plasma membrane in control cells
(Fig. 5B, -TeNT). By contrast,
TeNT pretreatment completely abolished FK-stimulated AQP2, targeting the
apical plasma membrane, as assessed by quantification of cell surface
immunoreactivity (Fig. 5B,
+TeNT). In control cells, TeNT treatment was found to have no effect on the
AQP2 density found at the cell surface.
|
Moreover double labeling of AQP2 and VAMP-2 in cells AQP2 transiently transfected with GFP-tagged AQP2 showed a partial colocalization of VAMP-2 in AQP2-bearing vesicles (Fig. 5C).
Overall, these results indicate that VAMP-2 is functionally involved in cAMP-induced AQP2 targeting to the plasma membrane.
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Discussion |
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In the present study, evidence is presented that directly supports the
hypothesis that the v-SNARE protein VAMP-2 is involved in the targeting of
AQP2 vesicles to the apical plasma membrane. Forskolin-induced translocation
of AQP2 to the apical membrane was abolished by TeNT in intact CD8 cells
(Fig. 5). This toxin
proteolytically cleaves VAMP-2 at a single site
(Link et al., 1992). It has
been suggested that VAMP-2 plays a major role in the mechanism of vesicle
trafficking (Sollner et al.,
1993
). This evidence is mainly formed on the basis of the ability
of TeNT and of botulinum neurotoxins to cleave VAMP-2 at different sites
(Schiavo et al., 1995
), which
results in the inhibition of neurotransmitter release in neuronal and
neuroendocrine cells (Hunt et al.,
1994
). The regulatory role of synaptobrevin isoforms has also been
proved in the regulated exocytosis of GLUT4 in adipocytes, in exocytosis of
histamine in enterochromaffin-like cells, in sperm acrosome reaction and in
H+-ATPase trafficking in the inner medullary collecting duct
(Banerjee et al., 2001
;
Banerjee et al., 1999
;
Foster and Klip, 2000
;
Hohne-Zell et al., 1997
;
Martin et al., 1998
;
Schulz et al., 1997
). Thus,
SNARE proteins might fulfil a universal role in vesicle-membrane fusion in
neuronal and non-neuronal cells.
In renal cells, by quantitative double immunolabeling, VAMP-2 has been
shown to colocalize with AQP2-containing vesicles
(Nielsen et al., 1995),
supporting a role for VAMP-2 in vasopressin-regulated vesicular trafficking.
In the same study, the authors demonstrated that TeNT caused a complete
cleavage of VAMP-2 in the crude membrane fraction enriched for intracellular
vesicles from kidney inner medulla
(Nielsen et al., 1995
). Jo and
colleagues (Jo et al., 1995
)
reported that purified papillary AQP2-containing endosomes from rat kidney
possess VAMP-2. The authors demonstrated that endosomes fuse, in vitro, by
means of an ATP-dependent process that is significantly inhibited when
endosomes are preincubated with either anti-VAMP-2 antibody or TeNT.
Despite the fact that these observations strongly support a functional involvement of VAMP-2 in AQP2-regulated redistribution, direct evidence for this role in intact renal cells has so far been lacking. We attempted to demonstrate the role of VAMP-2 in polarized sorting of AQP2 by determining the effect of clostridial TeNT on cAMP-induced translocation of AQP2 in intact renal CD8 cells. Central to this study was the demonstration that TeNT can enter the cells and is able to cleave the endogenously expressed VAMP-2. TeNT treatment in intact CD8 cells completely abolished cAMP-stimulated AQP2 targeting of the plasma membrane, as assessed by quantification of the cell-surface immunoreactivity of the anti-AQP2 antibody raised against a peptide reproducing the extracellular AQP2 C-loop. These results represent the first evidence for the functional involvement of VAMP-2 in cAMP-induced AQP2 redistribution in renal cells. In contrast to the effect of the toxin on cAMP-induced translocation of AQP2 to the apical membrane, the amount of cell-surface AQP2 in resting cells is not reduced by exposure to toxin (Fig. 5). This observation would suggest that the constitutive delivery of AQP2 may be VAMP-2 independent.
In neurons, VAMP-2 forms a complex with two plasma-membrane-associated
SNAREs: syntaxin and SNAP-25. These proteins bind together in a parallel
manner (Hanson et al., 1997) to
form a four-helix bundle, with two helices contributed by SNAP-25 and one each
by VAMP and syntaxin (Sutton et al.,
1998
). The formation of the SNARE complex is by itself sufficient
to bring the two interacting membranes close enough to fuse
(Weber et al., 1998
).
Among the known syntaxin isoforms, only syntaxin-1 and syntaxin-4 bind to
VAMP-2 with high affinity (Calakos et al.,
1994; Pevsner et al.,
1994
). Syntaxin-4 has been localized in the apical plasma membrane
of collecting duct principal cells (Mandon
et al., 1996
), suggesting that it may represent the counterpart
protein interacting with VAMP-2 found localized in AQP2-containing vesicles
(Jo et al., 1995
;
Nielsen et al., 1995
). In this
study, the presence of VAMP-2, syntaxin-1A and syntaxin-4 have been
demonstrated by RT-PCR and western blotting in CD8 cells. VAMP-2 was found
enriched in intracellular vesicles, whereas both syntaxin-1A and syntaxin-4
were found enriched in a plasma membrane fraction. Further studies will
address whether the v-SNARE VAMP-2 interacts with syntaxin-1A or syntaxin-4 to
mediate AQP2 targeting.
SNAP-25 has been implicated at a late step in fusion
(Banerjee et al., 1996;
Mehta et al., 1996
;
Rossi et al., 1997
). In inner
medullary collecting duct cells, treatment with botulinum toxin E, which
cleaves rat SNAP-23, reduced the amount of H+-ATPase translocated
to the apical membrane by about 52%, demonstrating that SNAP-23 has a critical
role in the regulation of H+-ATPase exocytosis
(Banerjee et al., 2001
). We
speculate that SNAP-23 detected in a plasma-membrane-enriched fraction of CD8
cells acts similarly in renal cells. Further investigation is needed to
evaluate the role of the specific SNAREs in the dynamic of complex
formation.
In summary, the data reported in this study provide clear evidence that the v-SNARE VAMP-2 is directly involved in the docking and fusion of AQP2-containing vesicles with the plasma membrane in intact AQP2-transfected CD8 cells and provide the basis for understanding the mechanism and regulation of AQP2 trafficking in response to a cAMP-elevating agent.
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Acknowledgments |
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Footnotes |
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