From the Insitute for Medical Biochemistry,
University of Münster, von-Esmarch-Str. 56, D-48149
Münster, Federal Republic of Germany and the
§ Laboratory of Physiology, Campus Gasthuisberg, Catholic
University of Leuven, B-3000 Leuven, Belgium
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ABSTRACT |
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Annexin II is a member of a multigene family of
Ca2+-regulated, membrane-binding proteins implicated
through biochemical and perforated cell experiments in
Ca2+-triggered secretion. Within most cells annexin II
resides in a tight heterotetrameric complex with a cellular protein
ligand, p11, and complex formation is mediated via the N-terminal 14 residues of annexin II including the N-terminal acetyl group. To
analyze at the single cell level whether the annexin II-p11 complex is involved in regulated secretion, we used membrane capacitance measurements to follow exocytotic fusion events in bovine aortic endothelial cells manipulated with respect to their annexin II-p11 complex formation. Upon guanosine 5'-O-(thiotriphosphate)
(GTPS) stimulation, the endothelial cells show a significant
increase in membrane capacitance which is generally preceded by a
transient rise in intracellular Ca2+ and thus indicative of
the occurrence of Ca2+-regulated secretion. The
GTP
S-induced capacitance increase is markedly reduced in cells
loaded with a synthetic peptide, Ac1-14, which corresponds in sequence
to the N-terminal 14 residues of annexin II in their correctly
acetylated form and which is capable of disrupting preformed annexin
II-p11 complexes. The effect of the peptide is highly specific as the
nonacetylated variant, N1-14, which is incapable of disrupting annexin
II-p11, does not interfere with the GTP
S-induced increase in
membrane capacitance. These data show that intact annexin II-p11
complexes are indispensable for regulated exocytosis to occur in an
efficient manner in endothelial cells.
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INTRODUCTION |
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Changes in the intracellular Ca2+ levels are known to regulate a variety of cellular processes ranging from the control of cell architecture to intracellular communication, cellular differentiation, and cell death. These cellular responses are mediated through a large number of Ca2+-binding proteins which are thought to couple the Ca2+ stimulus (transient increases in the cytosolic Ca2+ concentration) to the different biological events most likely by displaying different properties in their Ca2+-free and their Ca2+-bound conformations. Generally, the Ca2+-binding proteins are grouped according to the structure of their principal Ca2+ binding motif with the two largest families known to date being the EF hand proteins and the annexins (1). In contrast to EF hand-type and most other Ca2+-binding proteins, annexins characteristically also display a Ca2+-dependent interaction with negatively charged phospholipids enriched in the cytosolic leaflets of cellular membranes (2-4). Due to these biochemical characteristics, annexins have been implicated in mediating and/or regulating membrane related events including membrane-membrane contacts, membrane-cytoskeleton linkages, membrane organizations, and ion currents across membranes. However, experiments addressing the problem of annexin function in living cells have been very scarce, and the biological role of annexins remains enigmatic.
Annexin II is a 36-kDa protein comprising two principal domains, a
C-terminal protein core consisting of four homologous segments of 70 amino acids, the so-called annexin repeats, and an N-terminal region
which is less structured and 30 residues in length (4). While the core
domain harbors binding sites for Ca2+, phospholipid (the
common annexin ligands) and F-actin, the N-terminal sequence contains
phosphorylation sites for pp60src and protein
kinase C as well as the binding site for an intracellular protein
ligand of 11 kDa. This p11 protein is a member of the S100 family of EF
hand-type Ca2+-binding proteins which forms obligatory
dimers (1, 5). p11 binding is mediated solely through the N-terminal
14-amino acids of annexin II which form an amphiphatic -helix with
the hydrophobic side chains and the N-terminal acetyl group of the posttranslationally modified annexin II being indispensable for high
affinity p11 interaction (6, 7). Complex formation establishes a
heterotetrameric annexin II2p112 entity, which
has altered biochemical properties when compared with monomeric annexin II, e.g. the complex but not monomeric annexin II can
aggregate chromaffin granules at low micromolar Ca2+
concentrations (8).
Within cells the annexin II-p11 complex is found in the cortical cytoskeleton underlying the plasma membrane, on early endosomal membranes as well as associated with secretory vesicles like chromaffin granules (4). Monomeric annexin II, on the other hand, appears to be at least partly cytosolic. Its complex formation with p11 and thus the cell cortex association is regulated by protein kinase C phosphorylation in the N-terminal domain (9-11). In line with the intracellular distribution, annexin II-p11 has been implicated in early endocytotic and late secretory events occurring at or close to the plasma membrane (4). A role in Ca2+-regulated exocytosis has been deduced mainly from studies using digitonin-permeabilized chromaffin cells. Here the progressive loss of secretory responsiveness to elevated Ca2+ concentrations can be slowed down by the addition of annexin II with the annexin II-p11 complex being more efficient than monomeric annexin II (12, 13). The suggested role of annexin II in Ca2+-regulated exocytosis is in line with the morphological observation that in quick-frozen deep-etched chromaffin cells the protein appears to form physical connections between the plasma membrane and the membrane of secretory granules (14). However, annexin II-p11 is probably not an obligatory and general constituent of Ca2+ regulated secretory pathways since the protein complex shows variable expression levels in different cell types with exocytotically active neuronal cells containing little annexin II and since ectopic expression of a trans-dominant annexin II-p11 mutant in rat pheochromocytoma PC12 cells does not affect the Ca2+-triggered dopamine release in a permeabilized cell system (15).
To analyze within living cells expressing considerable amounts of
annexin II-p11 whether the complex participates in regulated exocytosis
we chose bovine pulmonary artery endothelial cells as a model system
and used membrane capacitance tracings to follow exocytotic fusion
events. We show that the cells, which contain as typical secretory
granules Weibel-Palade bodies positive for von Willebrand factor,
respond to a GTPS1
stimulus by a transient rise in cytosolic Ca2+ and a
subsequent increase in cell surface area by 15%. Loading of the cells
with a synthetic peptide comprising the N-terminal 14 residues of
annexin II including the N-acetyl group (Ac1-14) significantly
interferes with the GTP
S-induced membrane capacitance increase. This
peptide harbors the entire p11 binding site and is capable of
disrupting preformed annexin II-p11 complexes. In contrast to Ac1-14,
the nonacetylated 14-mer (N1-14) does not interfere with annexin
II-p11 complex formation and also has no effect on the GTP
S induced
capacitance increase. The specific effect of the complex disrupting
annexin II peptide indicates that annexin II-p11 participates in
regulated exocytosis in endothelial cells possibly by providing
physical linkages at the level of the plasma membrane.
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EXPERIMENTAL PROCEDURES |
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Antibodies and Proteins-- Mouse monoclonal antibodies, H28 and H21, were used for the detection of annexin II and p11, respectively (16). Von Willebrand factor was stained with a polyclonal rabbit antibody (Dako). Dichlorotriazinyl aminofluorescein-coupled goat anti-mouse, rhodamine-coupled goat anti-rabbit, and Texas Red-coupled goat anti-rabbit antibodies (Dianova) were employed as secondary antibodies. All secondary antibodies were tested in control experiments by omitting the primary antibody and showed neglectable fluorescence signals on CPAE cells. Immunoblotting experiments employed horseradish peroxidase-coupled pig anti-mouse antibodies (Dako) as secondary antibodies.
The heterotetrameric annexin II-p11 complex was purified from porcine intestinal epithelium (17). Monomeric annexin II was expressed recombinantly in baculovirus-infected insect cells and purified from the cell lysate as described previously (18).Peptides--
Peptide synthesis was carried out on an automated
synthesizer (model 9050; Milligen). For N-terminal acetylation,
peptides were left on the resin, and the Fmoc
(N-(9-fluorenyl)methoxycarbonyl) group was released by
treatment with dimethylformamide. Subsequently, the free N terminus was
acetylated with acetic anhydride/N-ethyldiisopropylamine for
30 min. Removal of the protection group was then achieved by treatment
with 95% trifluoroacetic acid, 2.5% ethanediol, 2.5% anisole.
Peptides were purified by reverse-phase high performance liquid
chromatography on a preparative column (Vydac 218TP 1022) using a
linear acetonitrile gradient for elution. All products were
characterized by mass spectrometry (MALDI compact III; Kratos Analytical) and by N-terminal sequence analysis of a nonacetylated aliquot on an automated gas-phase sequenator (Knaur model 810). The
purified peptides were lyophilized, washed three times with water, and
then stored at 20 °C as lyophilized aliquots.
Cell Culture-- Experiments were performed on single, nonconfluent endothelial cells of an established cell line from bovine pulmonary artery (cell line CPAE, ATCC CCL-209). Cells were grown in Dulbecco's modified Eagle's medium containing 20% fetal calf serum, 2 mM glutamine, 100 µg/ml streptomycin, 100 units/ml penicillin, detached by exposure to 0.05% trypsin in a Ca2+- and Mg2+-free solution, reseeded on gelatin-coated coverslips, and kept in culture 2-4 days before use.
Immunofluorescence--
CPAE cells grown on fibronectin-coated
glass coverslips were washed briefly in phosphate-buffered saline and
fixed and permeabilized for 10 min in methanol at 20 °C.
Subsequently, the cells were washed three times, 5 min each, in
phosphate-buffered saline and incubated in a moist chamber with 20 µl
of the primary antibody for 30 min at 37 °C. The monoclonal
antibodies H28 and H21 were employed as undiluted hybridoma culture
supernatants, whereas the polyclonal anti-von Willebrand factor
antibody was diluted 1:100. After washing in phosphate-buffered saline
(three times, 5 min each), the cells were incubated with the respective
secondary antibodies, washed again, and mounted in Moviol. Conventional microscopy was performed using a Zeiss Axiophot photomicroscope and
Kodak Tmax 3200 film. Confocal images were taken using a Leica laser
scanning microscope.
Capacitance and Intracellular Ca2+
Measurements--
Cells were voltage clamped in the whole-cell patch
clamp mode by use of an EPC-9 patch clamp amplifier (Heka, Germany)
technique. Holding potential was 0 mV. The normal bath solution
contained (in mM): 150 NaCl, 6 KCl, 1 MgCl2,
1.5 CaCl2, 10 glucose, 11.5 HEPES, pH adjusted to 7.4 with
NaOH. The osmolarity of the solution, as measured with a vapor pressure
osmometer (Wescor 5500, Schlag, Gladbach, Germany), was 315 mOsm. The
pipette solution contained (in mM): 40 KCl, 100 potassium
aspartate, 1 MgCl2, 0.5 EGTA, 4 Na2ATP, 10 HEPES, pH adjusted to 7.2 with KOH. Under these conditions, no
K+ currents can be activated. For stimulation of
exocytosis, cells were loaded after breaking the membrane seal with 100 µM GTPS.
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(Eq. 1) |
Phospholipid Binding-- Phospholipid vesicles were prepared from bovine brain extract as described previously (19). Binding studies employing monomeric annexin II or the annexin II-p11 complex in the presence or absence of the peptides Ac1-14 and N1-14 were carried out using 0.25 mg/ml liposomes in 100 µl of a buffer containing 50 mM imidazole/HCl, pH 7.5, 150 mM NaCl, and EGTA-buffered Ca2+ to give a final free Ca2+ concentration of 250 nM. In experiments analyzing the petide effects, 5.5 pmol of annexin II-p11 were incubated with a 500-fold molar excess of the respective peptide in assay buffer for 5 min at room temperature prior to adding the liposomes. Subsequently, the mixture was incubated for 25 min at room temperature and liposome-bound protein was separated from the unbound fraction by centrifugation at 150,000 × g for 15 min. The resulting phospholipid pellet was resuspended in 20 µl of SDS sample buffer (20), whereas the supernatants obtained were dried down and then dissolved in 20 µl of SDS sample buffer. 10 µl of each sample were analyzed by SDS-polyacrylamide gel electrophoresis (20) and immunoblotting (21).
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RESULTS |
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GTPS Induced Secretion in Cultivated Endothelial
Cells--
Calf pulmonary artery endothelial (CPAE) cells were chosen
to analyze the participation of annexin II-p11 in regulated exocytosis for two major reasons. 1) Within the mammalian organism cells of the
endothelium show the strongest expression of annexin II and we have
shown previously that CPAE cells contain significant amounts of annexin
II and p11 (22). 2) Cultivated endothelial cells are capable of
responding to an external stimulus, e.g. thrombin, by
Ca2+-triggered exocytosis of Weibel-Palade bodies, the
principal storage granules of von Willebrand factor (vWF) and
P-selectin (23). Fig. 1 shows that the
CPAE cells used in this study contain vWF-positive granules with the
typical morphological appearance of Weibel-Palade bodies. Moreover,
treatment with the Ca2+ ionophore A23187 induces cell
surface appearance of vWF as revealed by immunofluorescence staining of
nonpermeabilized cells (not shown). This indicates that the cells are
capable of Ca2+-regulated exocytosis of at least
vWF-positive granules.
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The Annexin II-p11 Complex Is Involved in Regulated Exocytosis in Endothelial Cells-- Annexin II has been localized to the subplasmalemmal region of different types of cells, and this localization is thought to be of crucial importance for a role of the protein in Ca2+-regulated secretion in adrenal chromaffin cells (11). The tight association of annexin II with the cell cortex depends on its complex formation with p11 (11, 32), and this is mediated through the N-terminal domain of annexin II (33, 34). The N-terminal 14 residues of annexin II (AcSTVHEILCKLSLEG) including the N-acetyl group of the N-terminal serine harbor the entire p11 binding site, and synthetic peptides comprising this sequence (herein referred to as Ac1-14) bind p11 with high specificity and affinity. In contrast, peptides of the same sequence containing a nonmodified N terminus (N1-14) show a more than 1000-fold reduced affinity for p11 (6, 7).
Based on the high affinity of the Ac1-14/p11 interaction we analyzed whether the peptide in addition to competing with annexin II for p11 binding (6) could also disrupt preformed and lipid bound annexin II-p11 complexes and therefore could be used as a function-interfering reagent. Liposome pelleting assays were performed to elucidate whether Ac1-14 is capable of liberating p11 from liposome-bound annexin II-p11 complexes. Experiments were carried out at a Ca2+ concentration of 250 nM, i.e. at a level experienced in the GTP
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DISCUSSION |
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In this report we establish for the first time membrane
capacitance tracing as an approach to the study of regulated exocytosis in endothelial cells at the single cell level. We show that a substantial increase in cell surface area indicative of secretory vesicle fusion with the plasma membrane occurs in GTPS-stimulated endothelial cells and that in the majority of the cells this increase is preceded by a transient rise in intracellular Ca2+. This
secretory response most likely involves Weibel-Palade bodies, the
principal storage granules for von Willebrand factor and P-selectin in
endothelial cells. vWF-positive structures are present in the CPAE
cells employed (Fig. 1) and immunofluorescence analysis of nonpermeabilized endothelial cells reveals that the internally stored
vWF is released to the cell surface once the intracellular Ca2+ concentration is elevated through the action of the
Ca2+ ionophore A23187 (data not shown). However, other
populations of endothelial storage granules may also contribute to the
exocytotic response observed after GTP
S infusion and subsequent
Ca2+ elevation. These could include storage particles for
endothelin-1 and tissue-type plasminogen activator, which have been
identified recently and which are morphologically and biochemically
distinct from vWF-positive Weibel-Palade bodies (38, 39).
The capacitance measurements in combination with the use of a peptide disrupting the annexin II-p11 complex also show for the first time at the single cell level that the complex is involved in regulated exocytosis. Previously, various lines of biochemical evidence as well as data from experiments using digitonin-permeabilized cells have implicated the heterotetrameric annexin II-p11 complex in Ca2+-regulated secretion in adrenal chromaffin cells (2-4). The complex, but not monomeric annexin II, is capable of aggregating chromaffin granules at Ca2+ concentrations occurring in stimulated cells. In such simple protein-granule mixtures annexin II-p11 following vesicle aggregation is even able to display fusogenic properties, however, only in the presence of elevated concentrations of cis-unsaturated fatty acids (8). Moreover, annexin II has been identified as a component which is lost from chromaffin cells upon digitonin permeabilization and upon adding back can retard the run-down in Ca2+-triggered secretion observed in the permeabilized cells (12, 13). This effect of annexin II appears to require complex formation with p11 and phosphorylation by protein kinase C, an enzyme stimulated in activated chromaffin cells (13). Interestingly, the proposed function of annexin II-p11 in chromaffin granule exocytosis correlates with a translocation of annexin II from the cytosol to the submembraneous region observed following nicotinic stimulation of chromaffin cells (11). Such a translocation might also occur in activated endothelial cells although here the majority of annexin II (and p11) is already found in the cortical region of nonstimulated cells (Fig. 5C). Based on these immunocytochemical data it appears likely that in endothelial cell exocytosis annexin II-p11 functions in the submembraneous region, possibly by anchoring secretory vesicles in the cell cortex.
Although the mechanistic basis of the annexin II-p11 function in regulated exocytosis is not known several morphological observations point to a structural role in linking vesicles to the plasma membrane and/or the cortical cytoskeleton. An ultrastructural analysis of stimulated chromaffin cells, for example, located annexin II between the plasma membrane and the facing chromaffin granule membranes, possibly forming fine strands cross-linking the opposing membranes (14). A high resolution image of intermembrane junctions formed by annexin II-p11 was obtained recently using cryo-electron microscopy of artificial liposomes and chromaffin granules aggregated by annexin II-p11 in a Ca2+-dependent manner. The junctions observed were of constant thickness (210 Å) and showed a symmetric distribution of electron-dense material, most likely representing annexin II molecules bound to the outer leaflets of each vesicle and linked via a central p11 dimer (40). A structural role of the annexin II-p11 complex is also in line with the relative abundance of the proteins in cells where they have now been implicated in regulated secretion, i.e. chromaffin and endothelial cells. It remains to be seen whether and how annexin II-p11, as an entity linking secretory vesicle to the cell cortex, cooperates with the SNARE-based docking and fusion machinery (41) in regulated exocytosis. In contrast to the SNARE complexes, annexin II-p11 is probably not an obligatory component of the exocytotic machinery since it is not expressed to similarly high levels in all cells capable of regulated secretion and since in PC 12 cells expression of a trans-dominant annexin II-p11 mutant does not interfere with dopamine secretion triggered by Ca2+ elevation in digitonin-permeabilized cells (15). Interestingly, application of the Ac1-14 peptide disrupting the annexin II-p11 complex does not result in a complete inhibition of the exocytotic response in CPAE cells. This could mean that in vivo peptide-mediated complex disruption is not complete, e.g. because a portion of annexin II-p11 is incorporated into higher order structures and thereby protected from the peptide action. Alternatively, it remains possible that the different pathways of regulated exocytosis most likely existing in endothelial cells (see above) depend differently on annexin II-p11.
In addition to Ca2+, phosphorylation by protein kinase C most likely represents another signal regulating annexin II-p11 activities during Ca2+-triggered secretion. Chromaffin cell annexin II is phosphorylated by PKC in response to nicotinic stimulation (42), and only phosphorylated annexin II-p11 is able to restore secretion in permeabilized chromaffin cells depleted of their PKC activity (13). Moreover, in adrenergic chromaffin cells a synthetic annexin II peptide corresponding to a sequence in the N-terminal domain, which includes the major PKC acceptor site, Ser-25, inhibits both nicotine-induced recruitment of annexin II to the cell periphery and nicotine-triggered exocytosis (11). While the mechanistic consequences of the regulatory PKC phosphorylation in living cells are not known, it is interesting to note that phosphorylation by PKC of chromaffin granule-bound annexin II induces a fusion of the granule membranes (10). In addition, PKC phosphorylation of annexin II-p11 inhibits the vesicle aggregation properties displayed by the complex (43). Finally, PKC phosphorylation of monomeric annexin II at a second site, Ser-11, inhibits p11 binding and thus formation of the heterotetrameric annexin II-p11 complex (9, 18). These biochemical analyses indicate that the annexin II function is subject to multiple regulations, which could differ from cell type to cell type. In chromaffin cells activation of PKC, e.g. by nicotinic stimulation, probably induces a translocation of (phosphorylated) annexin II to the submembraneous region, whereas annexin II is already concentrated in the periphery of nonstimulated endothelial cells (Fig. 5). Complex formation with p11, which itself can be fine-tuned by PKC action, establishes the heterotetrameric entity capable of linking membrane surfaces to one another and/or to an underlying cytoskeleton in a Ca2+-regulated manner. The experimental data obtained here show for the first time that the integrity of this annexin II-p11 complex with its cross-linking activity is indispensable for efficient regulated exocytosis in endothelial cells.
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ACKNOWLEDGEMENTS |
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We thank Joachim Seemann and Klaus Weber (Max Planck Institute for Biophysical Chemistry, Göttingen) for synthesizing peptides used in this study, Geert Raskin (Leuven) for his help with the capacitance measurements, and Ulrich Kubitschek (Institute for Medical Physics and Biophysics, Münster) for help with the confocal microscopy.
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FOOTNOTES |
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* This work was supported by the EU Biomed-2 program (BMH4-CT96-0602), the F.W.O. (Flanders, G0237.95.N), and the Deutsche Forschungsgemeinschaft.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.
¶ To whom correspondence should be addressed. Tel.: 49 251 835 6722; Fax: 49 251 835 6748; E-mail: gerke{at}uni-muenster.de.
1
The abbreviations used are: GTPS, guanosine
5'-O-(thiotriphosphate); BAPTA,
1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CPAE, calf pulmonary artery endothelial; NSF,
N-ethlymaleimide-sensitive fusion protein; PKC, protein
kinase C; SNARE, soluble NSF attachment protein receptor; vWF, von
Willebrand factor.
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REFERENCES |
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