Báo cáo khóa học: Stimulation-dependent recruitment of cytosolic phospholipase A2-a to EA.hy.926 endothelial cell membranes leads to calcium-independent association pdf

Stimulation-dependent recruitment of cytosolic phospholipase A
2
-a
to EA.hy.926 endothelial cell membranes leads
to calcium-independent association
Seema Grewal, Jennifer Smith, Sreenivasan Ponnambalam and John Walker
School of Biochemistry and Molecular Biology, University of Leeds, UK
Cytosolic phospholipase A
2
-a (cPLA
2
-a) is a calcium-
activated enzyme involved in agonist-induced arachidonic
acid release. In endothelial cells, free arachidonic acid is
predominantly converted into prostacyclin, a potent vaso-
dilator and inhibitor of platelet activation. As the rate-lim-
iting step in prostacyclin production is the generation of free
arachidonic acid by cPLA
2
-a, this enzyme has become an
attractive pharmacological target and the focus of many
studies. Following stimulation with calcium-mobilizing
agonists, cPLA
2
-a translocates to intracellular phospholipid
membranes via its C2 domain. In this study, the calcium-
induced association of cPLA
2
-a with EA.hy.926 endothelial
cell membranes was investigated. Subcellular fractionation
and immunofluorescence studies showed that following

stimulation with histamine, thrombin or the calcium
ionophore A23187, cPLA
2
-a relocated to intracellular
membranes. Treatment of A23187-stimulated cells with
EGTA or BAPTA-AM demonstrated that a substantial
pool of cPLA
2
-a remained associated with membrane frac-
tions in a calcium-independent manner. Furthermore,
immunofluorescence microscopy studies revealed that cells
stimulated for periods of greater than 10 min showed a high
proportion of calcium-independent membrane-associated
cPLA
2
-a. Calcium-independent membrane association of
cPLA
2
-a was not due to hydrophobic or cytoskeletal inter-
actions. Finally, the recombinant C2 domain of cPLA
2
-a
exhibited calcium-independent membrane binding to mem-
branes isolated from A23187-stimulated cells but not those
isolated from nonstimulated cells. These findings suggest
that novel mechanisms involving accessory proteins at the
target membrane play a role in the regulation of cPLA
2
-a.
Such regulatory associations could enable the cell to dis-

criminate between the varying levels of cytosolic calcium
induced by different stimuli.
Keywords: endothelium; cPLA
2
-a; arachidonic acid;
calcium; C2 domain.
Cytosolic phospholipase A
2
-a (cPLA
2
-a) belongs to a
growing family of phospholipase A
2
enzymes that catalyse
the hydrolysis of the sn-2 fatty-acyl bond of phospholipids
to liberate free fatty acids [1]. In the endothelium, cPLA
2
-a
plays a pivotal role in releasing free arachidonic acid from
membrane phospholipids. This arachidonic acid is the
precursor for prostacyclin, a member of the eicosanoid
family of inflammatory mediators, which acts as a potent
vasodilator and inhibitor of platelet aggregation [2]. As the
rate-limiting step in the production of prostacyclin is the
generation of arachidonic acid by cPLA
2
-a, it can be seen
that cPLA
2
-a plays a crucial role in several endothelial

functions such as haemostasis, angiogenesis, control of
vascular tone and prevention of thrombosis formation.
Consequently, cPLA
2
-a has become an attractive target for
the development of novel pharmacological therapeutics
against various pathological conditions [3,4]. To date,
however, the exact mechanisms involved in the control of
this important enzyme remain unclear.
cPLA
2
-a is an 85 kDa, calcium-sensitive protein and is
subject to regulation at both the transcriptional and post-
translational level [5]. Early studies on the cloning, expres-
sion and purification of cPLA
2
-a show that purified
recombinant cPLA
2
-a binds to natural membranes in the
presence of physiological concentrations of calcium [6].
More recently, studies on cultured mammalian cells have
shown that cPLA
2
-a is present in the cytosol of resting cells
and relocates to intracellular membranes following stimu-
lation with a variety of agonists that cause an increase in
cytosolic calcium levels [7–10]. In accordance with this,
studies on platelets and endothelial cells have shown that
this membrane relocation is consistent with an increased

PLA
2
activity in membrane fractions [11,12]. Several studies
have also shown that cPLA
2
-a activity in endothelial cells is
regulated by phosphorylation [13–15], however the role of
these modifications in the translocation and membrane
association of cPLA
2
-a is unclear.
The translocation of cPLA
2
-a to membrane phospho-
lipids has been shown to be mediated by its calcium-
dependent lipid binding or C2 domain, which promotes
binding to phospholipids upon elevation of intracellular
calcium concentrations [16]. C2 domains are remark-
able modules present in over 100 proteins including the
Correspondence to J. Walker, School of Biochemistry and Molecular
Biology, University of Leeds, Leeds LS2 9JT, UK.
Fax: + 44 1133433167, Tel.: + 44 1133433119,
E-mail:
Abbreviations: cPLA
2
-a, cytosolic phospholipase A2 alpha.
Note: A departmental web site is available at .
leeds.ac.uk
(Received 3 September 2003, revised 27 October 2003,
accepted 30 October 2003)

Eur. J. Biochem. 271, 69–77 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03903.x
GTPase-activating protein, phospholipase C and the syn-
aptic vesicle protein, synaptotagmin [17]. The Ca
2+
-binding
properties of these domains allow them to act as electro-
static switches that bind phospholipid membranes in the
presence of calcium without requiring large conformational
changes [18]. Based on this, the binding of cPLA
2
-a to
membranes is believed to be reversible, with translocation
occurring only in the presence of calcium. In agreement with
this, binding of cPLA
2
-a to synthetic liposomes was shown
to be fully reversible, and the addition of an excess of the
calcium chelator, EGTA, abolished binding [19]. Recently,
however, in Chinese hamster ovary cells transfected with
GFP-cPLA
2
-a, there is evidence for a translocation of
cPLA
2
-a to membranes that persists even after calcium
levels have returned to resting levels [20].
Here, using biochemical subfractionation and immuno-
fluorescence localization studies, we investigated the relo-
cation of cPLA
2

-a to membranes in EA.hy.926 endothelial
cells. Our results demonstrate an association of cPLA
2
-a
with endothelial cell membranes that is not consistent with a
simple reversible calcium-dependent interaction of cPLA
2
-a
with phospholipids. These results imply that some mechan-
ism, other than simple C2-dependent association with lipids,
must be involved in the regulation of cPLA
2
-a.
Experimental procedures
Materials
Tissue culture media, enzymes and antibiotics were pur-
chased from Gibco BRL (Paisley, Scotland). Goat poly-
clonal antibodies to cPLA
2
-a were obtained from Santa
Cruz Biotechnology Inc. (CA, USA). Secondary fluorescein
isothiocyanate-conjugated secondary antibodies were from
Sigma and anti-goat horseradish peroxidise-conjugated Igs
were from Pierce (Cheshire, UK). All other standard
reagents and chemicals were from Sigma (Poole, Dorset,
UK) or BDH (Poole, Dorset, UK).
Cell culture
The EA.hy.926 cell line, a hybrid of human umbilical vein
endothelial cells (HUVEC) and A549 human lung carci-
noma epithelial cells [21], was a generous gift from C. J.

Edgell (University of North Carolina, USA). Cells were
cultured on plasticware at 37 °C in a humid atmosphere
containing 5% (v/v) CO
2
in air. Cells were grown in
Dulbecco’s modified Eagles medium supplemented with
10% fetal bovine serum, penicillin (100 UÆmL
)1
), strepto-
mycin (100 lgÆmL
)1
)andHAT(100l
M
hypoxanthine,
0.4 l
M
aminopterin, 16 l
M
thymidine).
Subcellular fractionation
This method was carried out as described previously [22]
and cells were grown to confluence. Medium was removed
and the cells were washed twice with prewarmed NaCl/P
i
(Dulbecco A, Oxoid Ltd, Hampshire, UK). For stimula-
tions, the cells were then incubated for the appropriate time
at 37 °Cwith5l
M
A23187 in Hepes/Tyrode’s buffer
containing 1 m

M
CaCl
2
. Cells were then washed twice with
NaCl/P
i
, scraped into ice-cold Buffer A (100 m
M
KCl,
10 m
M
Pipes, 1 m
M
NaN
3
,1m
M
phenylmethanesulfonyl
fluoride, 1 m
M
sodium orthovanadate, 50 m
M
benzamidine,
0.1 mgÆmL
)1
leupeptin) and lysed either by freeze-thawing
or by homogenization with a Dounce homogeniser. Veri-
fication of lysis was performed using Trypan Blue staining
(Sigma) according to the manufacturer’s instructions. The
cell lysate was centrifuged at 200 000 g for 10 min at 4 °C

and the resultant soluble cytosolic fraction (C1) was
removed. The insoluble pellet was washed twice in Buffer
A (generating cytosol washes C2 and C3) and finally
solubilized in Buffer B [Buffer A containing 1% (v/v) Triton
X-100]. Insoluble material was removed by centrifugation
and the Triton-soluble membrane fraction (M) was collec-
ted. Preparations were carried out at various free calcium
concentrations by adding the appropriate amounts of CaCl
2
and EGTA to Buffer A as determined by the
METLIG
pro-
gram [23]. Equivalent amounts of the subcellular fractions
were analysed by SDS/PAGE and Western blotting.
Preparation of cell cytoskeletons
This method was carried out as described previously [22].
Cells were grown to confluence, washed three times with
NaCl/P
i
and then collected by scraping into ice-cold Buffer
B. Cytoskeleton fractions were isolated by centrifugation at
200 000 g for 2 h at 4 °C. The supernatant (representing
cytosol and membrane proteins) was removed and the
insoluble pellet (representing the cytoskeletal fraction) was
solubilized in Laemmli sample buffer. Various free calcium
concentrations were maintained by adding the appropriate
amounts of CaCl
2
and EGTA to Buffer B, as determined by
the

METLIG
program. Equivalent amounts of the fractions
were analysed by SDS/PAGE and Western blotting.
Temperature-induced phase separation of Triton X-114
The separation procedure was carried out as described in
a previous report [24]. EA.hy.926 cell membrane fractions
were prepared as described above. Membrane fractions
were resuspended in 1% (w/v) Triton X-114, 150 m
M
NaCl,
10 m
M
Tris, 2 l
M
CaCl
2
,pH7.4.Sampleswerethen
incubated at 30 °C for 10 min and centrifuged at 3000 g
for 3 min. The aqueous upper phase (representing hydro-
philic proteins) was separated from the detergent-rich lower
phase (representing hydrophobic proteins) and equivalent
amounts of the two were analysed by Western blotting.
Immunofluorescence microscopy
The method for immunofluorescence microscopy was
adapted from previous reported methods [25,26]. Cells were
grown on glass coverslips overnight. Media was removed
and the cells were washed three times with prewarmed (to
37 °C) NaCl/P
i
and fixed in prewarmed 10% (v/v) formalin

in neutral buffered saline [approximately 4% (v/v) formal-
dehyde, Sigma] for 5 min. All subsequent steps were
performed at room temperature. After fixation, the cells
were permeabilized with 0.1% (v/v) Triton X-100 in NaCl/
P
i
for 5 min and fixed once again for 5 min. The cells were
then washed three times with NaCl/P
i
and incubated in
freshly prepared sodium borohydride solution (1 mgÆmL
)1
in NaCl/P
i
) for 5 min to reduce autofluorescence. Following
70 S. Grewal et al.(Eur. J. Biochem. 271) Ó FEBS 2003
three further NaCl/P
i
wash steps, the cells were blocked in
5% (v/v) rabbit serum in NaCl/P
i
for 3 h. The cells were
then incubated with primary antibody [diluted 1 : 100 into
NaCl/P
i
, 5% (v/v) serum] overnight followed by fluorescein
isothiocyanate-conjugated secondary antibody for 3 h, with
eight NaCl/P
i
washes performed in between incubations.

Sodium azide (1 m
M
) was included in all incubations to
prevent bacterial growth. The cells were then washed eight
times with NaCl/P
i
and mounted onto slides in Citifluor
mounting medium (Agar Scientific, Hertfordshire, UK).
Confocal imaging
Confocal fluorescence microscopy was performed using a
Leica TCS SP spectral confocal imaging system coupled to
a Leica DM IRBE inverted microscope. Each confocal
section was the average of four scans to obtain optimal
resolution. The system was used to generate individual
sections that were 0.485 lm thick. All figures shown in this
study represent 0.485 lm sections taken through the
nucleus.
SDS/PAGE and Western blotting
Proteins (20 lg per well) were separated on SDS/polyacryl-
amide gels using a discontinuous buffer system [27]. For
Western blot analysis, proteins were transferred to nitrocel-
lulose [28]. Subsequently, the nitrocellulose blots were
blocked in 5% (w/v) nonfat milk in NaCl/P
i
,0.1%(v/v)
Triton X-100 for 1 h. Primary antibody incubations
(1 : 1000) were carried out overnight at room temperature,
followed by 1 h incubations with the appropriate horse-
radish peroxidase-conjugated secondary antibody. For
antigenic adsorption, the antibody was incubated with its

corresponding blocking peptide (1 : 5 ratio of lg antibody
to lg antigen) for 30 mins at room temperature prior to
being incubated with the nitrocellulose blot. Immunoreac-
tive bands were visualized using an enhanced chemilumines-
cence detection kit (Pierce) according to the manufacturer’s
instructions. Following this, the developed films were
photographed and captured using the FujiFilm Intelligent
dark Box II with the Image Reader Las-1000 package. The
intensity of the bands was quantified densitometrically using
the
AIDA
(advanced image data analyzer) 2.11 software
package. In general, the average
1
and SEM from three
independent experiments were calculated.
C2 domain binding assays
The binding assays were based on those described previ-
ously [29]. Membrane fractions from nonstimulated and
A23187-stimulated cells were prepared as described above.
Membrane fractions (corresponding to approximately
100 lg of total protein) were incubated with 0.1 lg purified
C2 domain for 30 min at 30 °C.Thesamplewasthen
centrifuged at 200 000 g for 10 min at 4 °C to sediment the
membranes. Any unbound C2 domain in the supernatant
was removed whilst any bound material was solubilized
in Buffer B. Insoluble material was removed by centri-
fugation and the soluble membrane fraction was collected.
Samples were analysed by SDS/PAGE and Western
blotting.

Results
CPLA
2
-a relocates to intracellular membranes following
an elevation in cytosolic calcium concentration
The subcellular location of cPLA
2
-a in EA.hy.926 endo-
thelial cells was investigated by immunofluorescence micro-
scopy using an antibody that specifically recognizes the
a-isoform of cPLA
2
(Fig. 1A, lane 1). Antigenic adsorption
of this antibody with the appropriate blocking peptide
abolished detection of cPLA
2
-a by both Western blotting
(Fig. 1A, lane 2) and immunofluorescence microscopy (data
not shown). Using this specific antibody, a comparison of
Fig. 1. Relocation of cPLA
2
-a to intracellular
membranes following A23187-stimulation. (A)
cPLA
2
-a was detected by Western blotting of
EA.hy.926 lysates (20 lgprotein)usingagoat
polyclonal antibody (i). Also shown are con-
trol lanes corresponding to antigen-adsorbed
antibody (ii), and horseradish peroxidase

conjugated anti-(goat IgG) controls (iii). (B)
Cells were grown on coverslips and incubated
with buffer alone (i) or stimulated with 5 l
M
A23187 (ii), 10 l
M
histamine (iii) or 1 UÆmL
)1
thrombin (iv) in the presence of 1 m
M
extra-
cellular calcium for 1 min. Cells were then
fixed and permeabilized, and cPLA
2
-a was
detected using immunofluorescence micros-
copy. Scale bar, 10 lm.
Ó FEBS 2003 Calcium-independent binding of cPLA2-a (Eur. J. Biochem. 271)71
the location of cPLA
2
-a in resting and stimulated
EA.hy.926 cells was carried out. In nonstimulated cells,
cPLA
2
-a was present throughout the cytosol and the
nucleus (Fig. 1B, panel i). Following elevation of the
cytosolic calcium concentration in response to the physio-
logical stimulus histamine or thrombin, or the calcium
ionophore, A23187, a specific relocation of cPLA
2

-a to
intracellular membranes resembling the endoplasmic reti-
culum and nuclear envelope was evident (Fig. 1B, panels
ii–iv). Both secondary antibody and peptide-adsorbed
antibody controls gave no staining (data not shown)
confirming that the staining observed corresponded specifi-
cally to cPLA
2
-a. Measurement of intracellular calcium
concentrations using Fura-2-AM demonstrated that expo-
sure to either 10 l
M
histamine, 1 UÆmL
)1
thrombin or 5 l
M
A23187 in the presence of 1 m
M
extracellular calcium led to
an increase in cytosolic calcium concentration from a resting
value of 100 n
M
to approximately 1–2 l
M
(data not shown).
These values were consistent with those obtained from other
studies on endothelial cells [30,31]. Based on these findings,
future experiments were performed primarily with A23187,
to avoid the complication of agonist-specific signalling
events.

To investigate the calcium-dependency of relocation
further, fractionations were performed under the resting
and elevated calcium levels observed in endothelial cells.
Firstly, cytosol and membrane fractions were obtained from
resting cells that were lysed in the presence of various free
calcium concentrations. Analysis of the resultant samples
indicated that, with increasing concentrations of free
calcium, an increasing amount of cPLA
2
-a was found
associated with membranes (Fig. 2A). In the complete
absence of calcium, no cPLA
2
-a was present in the
membrane fraction. In contrast, all of the endogenous
cPLA
2
-a was found to be membrane-bound at calcium
concentrations of 800 n
M
or above (Fig. 2).
Subfractionation experiments were also performed in the
presence of either 100 n
M
free calcium for resting cells or
2 l
M
free calcium following A23187 stimulation. These
calcium concentrations were representative of the intracel-
lular cytosolic calcium concentration in the absence and

presence of stimulation, respectively (as described above).
The results of these fractionation studies (Fig. 3A) indicated
that under resting calcium levels of 100 n
M
,cPLA
2
-a was
predominantly cytosolic. In contrast, when cells were
stimulated with 5 l
M
A23187 for 10 min and fractionated
in the presence of 2 l
M
calcium, most of the cPLA
2
-a was
membrane-bound. The cytosolic location of lactate dehy-
drogenase confirmed that cells were lysed sufficiently.
Quantification of the relative levels of cPLA
2
-a in the
fractions showed that in resting cells 79.9% ± 6.9 of the
total amount was present in the cytosol with only
19.1% ± 6.0 present in the membrane fraction. In contrast,
following stimulation only 28.5% ± 1.9 remained in the
cytosolic fraction whereas 71.5% ± 1.9 was found to be
membrane-associated (Fig. 3B).
The association of cPLA
2
-a with membranes is EGTA-

resistant and time-dependent
To further characterize the binding of cPLA
2
-a to endo-
thelial cell membranes, the effects of the calcium chelator
EGTA on membrane association were studied. Cells were
stimulated and fractionated as above, and membrane
fractions were washed with 5 m
M
EGTA. The results
(Fig. 4) show that, as demonstrated above, only approxi-
mately 30% of the total amount of cPLA
2
-a remained
cytosolic following stimulation with A23187 and homo-
genization in the presence of 2 l
M
Ca
2+
.
2
Remarkably,
however, only 14.8% ± 2.6 of the total protein could be
eluted from the membrane pellet by washing with EGTA,
with greater than half the amount of total cPLA
2
-a
(52.5% ± 4.65) remaining tightly associated with the
membrane in a manner that resisted extraction with EGTA.
Similar results were seen following treatment of cells with

10 l
M
histamine or 1 UÆmL
)1
thrombin with minimal loss
of cPLA
2
-a from stimulated membrane fractions following
washing with EGTA (Fig. 4C).
The effects of calcium chelation on the subcellular
location of cPLA
2
-a were examined using immunofluores-
cence microscopy. Cells were stimulated with 5 l
M
A23187
for various time periods. To test the effects of reducing
cytosolic calcium levels, cells were stimulated in the same
way then the extracellular and intracellular calcium chela-
tors, EGTA and BAPTA-AM respectively, were added to
the cells
3
. After a 1 min stimulation period followed by
calcium chelation, a cytosolic staining pattern resembling
Fig. 2. Calcium dependency of membrane binding. (A) Cells were
grown to confluence in flasks, and scraped into Buffer A containing the
free calcium levels indicated. The cells were homogenized and fract-
ionated into cytosol and membrane. Fractions were separated by SDS/
PAGE and Western blotted, and cPLA
2

-a was detected. (B) The rel-
ative amount of cPLA
2
-a in each fraction was quantified and expressed
as a percentage of the total amount of cPLA
2
-a. The amounts in the
respective cytosol and membrane fractions were plotted against the
corresponding calcium concentration. The data is representative of
results obtained from three independent experiments.
72 S. Grewal et al.(Eur. J. Biochem. 271) Ó FEBS 2003
that of a nonstimulated cell was evident (Fig. 5A). In
contrast, cells stimulated for 10 min showed a high
proportion of membrane-relocated cPLA
2
-a that was
resistant to the calcium chelation. Consistent with this,
subfractionation of cells directly into EGTA following
A23187-stimulation showed that increased stimulation time
led to an increase in the EGTA-resistant pool of membrane-
bound cPLA
2
-a (Fig. 5B). Under these conditions, more
than 20% of the total cPLA
2
-a pool was found to be
EGTA-resistant when directly solubilized in a Triton/
EGTA buffer following a 10 min stimulation period
(Fig. 5C).
EGTA-resistant membrane binding is not dependent

on a change in hydrophobicity or the cytoskeletal
association of cPLA
2
-a
It was possible that the binding of calcium to the C2 domain
results in a change in the overall hydrophobicity of the
protein, allowing it to partially insert itself into the lipid
bilayer. To address this question, temperature-induced
phase separation of Triton X-114 was performed. A
solution of Triton X-114 is homogenous at temperatures
below 20 °C. Above this temperature, the solution separates
into an aqueous phase and a detergent phase. Previous
studies have shown that integral membrane proteins and
proteins with exposed hydrophobic regions partition into
the detergent phase [21]. Analysis of cPLA
2
-a in membrane
fractions prepared from resting and stimulated cells showed
that no change in hydrophobicity occurred, and all the
protein was exclusively in the aqueous phase of a Triton
X-114 solution (Fig. 6A).
The possibility that the observed EGTA-resistant
binding of cPLA
2
-a to membranes was due to a
cytoskeletal interaction was investigated. Cytoskeleton
fractions were isolated by direct solubilization and sedi-
mentation from nonstimulated and A23187-stimulated
cells. The results from these studies showed that there was
no association of cPLA

2
-a with the cytoskeletal pellet in
either resting cells isolated in EGTA or 100 n
M
calcium,
or in A23187-stimulated cells isolated in 2 l
M
calcium
(Fig. 6B).
Fig. 3. Calcium-induced relocation of cPLA
2
-a. (A) Resting EA.hy.926
cells scraped into 100 n
M
free calcium buffer, or A23187-stimulated
cells (10 min at 37 °C) scraped into 2 l
M
free calcium buffer were
homogenized and subfractionated into cytosolic (C) and membrane
(M) fractions, including intermediate wash steps (C2, C3 and M2).
Samples, including total lysates (T), were separated by SDS/PAGE
and Western blotted, and cPLA
2
-a was detected. The distribution of
the cytosolic marker, lactate dehydrogenase (LDH) in resting cells was
also determined. (B) Quantification of the amount of cPLA
2
-a present
in each of the indicated fractions, expressed as a percentage of the total
amount of cPLA

2
-a (± SEM, n ¼ 3).
Fig. 4. EGTA-resistant binding of cPLA
2
-a to EA.hy.926 cell mem-
branes. (A) Cells were stimulated with 5 l
M
A23187 for 10 min in the
presence of 1 m
M
extracellular calcium. Cells were then scraped into
lysis buffer containing 2 l
M
free calcium, homogenized and subfract-
ionated. Following removal of the cytosolic fraction, the remaining
pellet containing membrane proteins was washed twice in lysis buffer
containing 5 m
M
EGTA. The remaining pellet was solubilized in
EGTA/Triton X-100 to give the membrane fraction. The samples were
then immunoblotted to detect cPLA
2
-a. (B) Quantification of the
amount of cPLA
2
-a present in each of the indicated fractions,
expressed as a percentage of the total amount of cPLA
2
-a (± SEM,
n ¼ 3). (C) Subcellular fractionation was also carried out following

10 min stimulation with 1 UÆmL
)1
thrombin and 10 l
M
histamine, as
described above. The amount of cPLA
2
-a in the samples was detected
by Western blotting.
Ó FEBS 2003 Calcium-independent binding of cPLA2-a (Eur. J. Biochem. 271)73
The C2 domain of cPLA
2
-a demonstrates
calcium-independent binding to membranes
To determine whether the C2 domain alone was able to
confer EGTA-resistant membrane binding, in vitro binding
studies of purified recombinant C2 domain to EA.hy.926 cell
membranes were performed. The results of these studies
(Fig. 7A) show that the calcium dependency of binding of
purified C2 domain to membranes prepared from nonstim-
ulated cells is identical to that of the binding of the
endogenous protein. To determine whether this binding
was reversible, membrane fractions containing the bound C2
domain were washed with EGTA. As expected, the C2
domain could be removed from nonstimulated membrane
fractions by washing with 5 m
M
EGTA, and was found
exclusively in the EGTAwashfraction (Fig. 7B). To examine
whether any changes occurred in the membrane following

stimulation, studies were also performed using membrane
fractions isolated from A23187-stimulated cells (in the pres-
ence of 2 l
M
free calcium). Using this approach, remarkable
differences in the binding properties of the C2 domain
were observed (Fig. 7B). In contrast to the data shown
above, only a small proportion of the C2 was able to bind to
the membranes, resulting in a large pool that remained in the
soluble fraction. Furthermore, these studies showed that
the protein that did bind could not be removed from the
membrane by EGTA washing, hence was found tightly
associated with the EGTA-resistant membrane fraction.
Discussion
To date, the association of cPLA
2
-a with cellular mem-
branes has been attributed to the calcium-dependent
binding of its C2 domain to membrane phospholipids. This
domain promotes the reversible binding of proteins to
phospholipids in the presence of calcium. The results shown
here, however, demonstrate a novel mode of binding of
cPLA
2
-a to EA.hy.926 cell membranes in a manner that
resists extraction with the calcium chelator EGTA.
Using subcellular fractionation experiments, it was
observed that endogenous cPLA
2
-a binds to EA.hy.926

cell membranes in a calcium-dependent manner. At con-
centrations below 200 n
M
the protein was largely cytosolic,
whereas it was completely membrane-associated at physio-
logically elevated calcium concentrations of 800 n
M
and
above. In resting endothelial cells, the basal levels of
arachidonic acid release and prostacyclin production [32]
imply that a pool of cPLA
2
-a is constitutively membrane-
associated and catalytically active. Not surprisingly there-
fore a small proportion of cPLA
2
-a was found to be
associated with a nonstimulated membrane fraction. The
exact role and nature of this constitutively membrane-
associated cPLA
2
-a requires further investigation.
Most interestingly, more than 50% of the total cellular
pool of cPLA
2
-a relocated to membranes following stimu-
lation with A23187 and remained associated with a
membrane fraction even after extraction with EGTA.
Similar results were also seen using 10 l
M

histamine or
1UÆmL
)1
thrombin (Fig. 4). Immunofluorescence micros-
copy also confirmed that membrane-relocated cPLA
2
-a
remained associated with membranes even in the presence
of intracellular and extracellular calcium chelators. Fur-
thermore, the amount of cPLA
2
-a present in the EGTA-
resistant membrane fraction was seen to increase with
stimulation time, suggesting that prolonged activation leads
to membrane association that resists extraction by the
removal of calcium. These findings are consistent with those
published by Hirabayashi and coworkers [20] which
suggested that stimulation periods of less than 2 min caused
only partial activation and reversible relocation of cPLA
2
-a
Fig. 5. Effects of EGTA and BAPTA-AM on the relocation of cPLA
2
-a. Cells were stimulated with 5 l
M
A23187 in the presence of 1 m
M
extracellular calcium for the times indicated. (A) Cells were fixed and permeabilized, and cPLA
2
-a was detected by fluorescence microscopy. For

EGTA/BAPTA-AM treatment, cells were stimulated and processed as above. However, following stimulation, cells were washed with 5 m
M
EGTA/5 m
M
BAPTA-AM for 5 mins directly prior to fixation. Scale bar, 5 lm. (B) Cells were treated as indicated, scraped into a 5 m
M
EGTA
buffer, homogenized and subfractionated into cytosolic and membrane fractions and cPLA
2
-a was detected by immunoblotting. (C) Quantification
oftheamountofcPLA
2
-a present in each of the indicated fractions, expressed as a percentage of the total amount of cPLA
2
-a (± SEM, n ¼ 3).
74 S. Grewal et al.(Eur. J. Biochem. 271) Ó FEBS 2003
in CHO cells, whereas longer stimulations caused binding
that persisted even after reduction of cytosolic levels of
calcium to resting values. This may be a mechanism for
allowing the cell to discriminate appropriate signals from
small transient fluctuations in intracellular calcium concen-
trations. Hence, once the calcium transient exceeds a critical
level, a tight-binding state of cPLA
2
-a couldleadtoa
continuous membrane localization and arachidonic acid
production for prolonged periods, even after the calcium
levels return to their resting value.
A recently published study also demonstrates a prolonged
ionophore-stimulated, perinuclear membrane association of

wild type cPLA
2
-a for several minutes after the return of
intracellular calcium to unstimulated levels [33]. This
phenomenon was seen to be dependent on the phosphory-
lation of S505, which enhanced the hydrophobic interaction
of catalytic domain residues to membrane phospholipids.
In support of this, Evans and colleagues [34] demonstrated
that full-length cPLA
2
-a dissociated more slowly from
membranes than the C2-domain alone, also indicating that
the catalytic domain may be involved in prolonged mem-
brane binding. In the present study, however, no change in
the overall hydrophobicity of cPLA
2
-a in ionophore-treated
cells was observed by the Triton X-114 phase separation
method (Fig. 6). However it may be possible that this
method is insufficiently sensitive to detect subtle changes in
hydrophobicity.
Analysis of the calcium dependency of binding of the
purified C2 domain of cPLA
2
-a to EA.hy.926 membranes
demonstrated that it exhibited calcium-dependent mem-
brane binding properties identical to that of the endogenous
full-length protein. Interestingly, it was observed that
recombinant C2 domain could bind to nonstimulated
membranes in a reversible manner, whereas binding to

stimulated membranes was EGTA-resistant. Most import-
antly, it was noticed that only partial binding of the C2
domain to stimulated membranes occurred. This raises the
possibility that under stimulated conditions, the binding site
Fig. 7. In vitro binding of pure re-folded C2 domain to to EA.hy.926 cell
membranes. (A) Cells were grown to confluence in flasks, scraped into
EGTA buffer and fractionated into cytosol and membrane fractions.
Membrane fractions were incubated with 0.1 lgpureC2domainat
30 °C for 30 min in the presence of the indicated levels of free calcium.
Following centrifugation at 200 000 g any unbound protein (soluble)
was collected and the pellet (membrane) was solubilized. (B) Cell
membranes were prepared from resting (in EGTA) and stimulated
(5 l
M
A23187 for 10 min, in 2 l
M
calcium) cells. Membranes were
incubated with 0.1 lg pure C2 domain at 30 °Cfor30minsinthe
presence of 2 l
M
free calcium. Following centrifugation at 200 000 g
any unbound protein (soluble) was collected and the membrane pellet
was washed in 5 m
M
EGTA. Following a further centrifugation step,
the EGTA-elutable fraction was collected (EGTA wash) and the
EGTA-resistant cell pellet (membrane) was solubilized in Triton
X-100. Fractions were analysed by Western blotting using a mouse
anti-(cPLA
2

-a)mAb.
Fig. 6. Temperature-induced phase separation of Triton X-114 and
extraction of EA.hy.926 cytoskeletons. (A) Cytosol and membrane
fractions from nonstimulated cells and A23187-stimulated cells were
prepared in the presence of 100 n
M
and 2 l
M
free calcium levels,
respectively. The membrane fractions were resuspended in 1% (v/v)
Triton X-114 and temperature-induced phase separation was per-
formed. The aqueous and detergent phases were separated and made
up to equal volumes. Samples were immunoblotted for cPLA
2
-a.(B)
Triton X-100 soluble (S, representing cytosol and membranes) and
insoluble (P, representing cytoskeleton) fractions were prepared from
nonstimulated cells (in EGTA or 100 n
M
free calcium levels) and cells
stimulated with A23187 for 10 mins (in 2 l
M
free calcium). Equivalent
amounts of the fractions were immunoblotted for cPLA
2
-a.
Ó FEBS 2003 Calcium-independent binding of cPLA2-a (Eur. J. Biochem. 271)75
for cPLA
2
-a may be partially blocked or saturated by

endogenous cPLA
2
-a. These findings imply that, following
stimulation, the membrane fraction undergoes a change
that allows the anomalous binding of cPLA
2
-a. It is possible
that this may be due to a change in the protein or lipid
composition, implying that some other protein or lipid
interaction is involved in the EGTA-resistant binding of
cPLA
2
-a to membranes. Previous studies have shown that
cPLA
2
-a is able to bind to ceramide, cholesterol and
phosphatidylinositol 4,5-bisphosphate [35–37] thus it is
possible that such interactions mediate the calcium-inde-
pendent membrane associations observed here. This, and
the possible involvement of binding proteins, is further
supported by the observation that cPLA
2
-a relocates to
specific cellular membranes indicating that a specific mech-
anism for targeting is present. In particular there is no
relocation of cPLA
2
-a to the plasma membrane whereas C2
domains in other proteins (e.g. protein kinase C-c [38]),
result in these proteins moving exclusively from the cytosol

to the plasma membrane. Overexpression of the C2 domain
of cPLA
2
-a alone [8] or GFP–C2–cPLA
2
-a fusion proteins
[34,39] demonstrate that this truncated protein exhibits the
same relocation patterns as the full-length protein, indica-
ting that the targeting information or mechanism lies within
this region of the protein. C2 domains are also known to
mediate protein–protein interactions hence the presence of a
C2domainincPLA
2
-a further supports the possibility that
an accessory protein is involved in the regulation of
cPLA
2
-a. Previous far Western studies identified the inter-
mediate filament protein vimentin as an adaptor protein
that interacts with the C2 domain of cPLA
2
-a in a calcium-
dependent manner [40]. Whether vimentin plays a role in the
calcium-independent association of cPLA
2
-a with phos-
pholipids remains to be investigated. Furthermore, a grow-
ing body of evidence supports the functional coupling of
cPLA
2

-a to its downstream cyclo-oxygenase enzymes,
COX-1 and COX-2 [41,42]. It is possible that these enzymes,
which show similar subcellular localization [43,44], may
specifically interact with cPLA
2
-a and act as accessory
proteins. The identification and characterization of these
and/or other binding partners or adapter proteins would
give further insight into the novel mechanism of cPLA
2
-a
regulation identified here.
In conclusion, it has been demonstrated here that cPLA
2
-
a relocates to cellular membranes following elevations in
cytosolic free calcium concentration; however, it is able to
remain tightly associated with the membrane in a calcium-
independent manner. Prolonged association with mem-
branes, despite a return of cytosolic calcium to resting levels,
could be of physiological significance in prolonging arachi-
donate production in response to cell stimulation. These
results indicate that this novel binding is not due simply to
the calcium-dependent lipid binding capacity of the C2
domain, and that some other binding partner or accessory
protein may be involved in the regulation of cPLA
2
-a.
Acknowledgements
This work was funded by the British Heart Foundation and the

BBSRC. We thank Dr C. J. Edgell for the gift of the EA.hy.926 cells,
Dr R. Williams for purified C2 domain and Dr E. E. Morrison for
assistance with confocal imaging.
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