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(Received for publication, April 22, 1997, and in revised form, May 28, 1997)
From the ABSTRACT Glycosylphosphatidylinositol (GPI)-anchored
proteins can deliver costimulatory signals to lymphocytes, but the
exact pathway of signal transduction involved is not yet characterized.
GPI-anchored proteins are fixed to the cell surface solely by a
phospholipid moiety and are clustered in distinct membrane domains that
are formed by an unique lipid composition requiring cholesterol. To elucidate the role of membrane lipids for signal transduction via
GPI-anchored proteins, we studied the influence of reduced cellular
cholesterol content on calcium signaling via GPI-anchored CD59
and CD48 in Jurkat T cells. Lowering cholesterol by different inhibitors of cellular cholesterol synthesis suppressed calcium response via GPI-anchored proteins by about 50%, whereas stimulation via CD3 was only minimally affected (<10%). The decrease in overall calcium response via GPI-anchored proteins was reflected by inhibition of calcium release from intracellular stores. Cell surface expression of GPI-anchored proteins was not changed quantitatively by lowering cellular cholesterol, and neither was the pattern of immunofluorescence in microscopic examination. In addition, the distribution of
GPI-anchored proteins in detergent-insoluble complexes remained
unaltered. These results suggest that cellular cholesterol is an
important prerequisite for signal transduction via GPI-anchored
proteins beyond formation of membrane domains.
INTRODUCTION Activation of lymphocytes is the first step in any immune
reaction. Apart from the specific interaction of major
histocompatibility complex-coupled antigens with the T cell receptor
complex, costimulatory factors are effective modulators of the immune
response. Several glycosylphosphatidylinositol
(GPI)1-anchored proteins can
provide costimulatory signals to T cells (1-5). On human lymphocytes,
CD59 (5, 6), CD55 (decay accelerating factor (7)), CD52 (8), and CD73
(9, 10) were shown to be involved in activation. On murine T cells,
evidence for signal-transducing GPI-anchored proteins include Ly-6
(11-14), the major histocompatibility complex Ib molecule Qa-2 (15,
16), Thy-1 (17, 18), and sgp60, the murine analogue to CD48 (19-22). A
variety of responses could be elicited by cross-linking of different GPI-anchored proteins on T lymphocytes, which included early events such as rise in intracellular calcium (6, 10, 11, 16), and protein
tyrosine phosphorylation (1, 4, 23, 24). In addition, several late
events have been observed as interleukin-2 production and proliferation
(6, 7, 9, 11-13, 15, 16, 19, 21). Thus, GPI-anchored proteins may play
a role in modulating the immune response.
GPI-anchored proteins are linked to the plasma membrane by a
phospholipid moiety residing in the outer leaflet of the lipid bilayer
(25). Since GPI-anchored proteins have no direct connection to the
cytoplasm, the mechanism by which signal transduction can occur is not
yet elucidated. However, GPI-linked proteins are clustered in membrane
domains (26-28) that may be involved in T lymphocyte activation by
mediating the association with Src family tyrosine kinases
p56lck and p59fyn (1, 5, 26, 28-31).
GPI-anchored protein complexes exhibit a particular lipid composition
enriched in cholesterol and sphingolipids (32) that appears to be
important for their clustering in living cells and the insolubility of
GPI-anchored protein complexes in mild non-ionic detergents in
vitro (32-35). Cellular transport and function of GPI-anchored
folate receptor and alkaline phosphatase was abolished by sequestrating
plasma membrane cholesterol by specific detergents (35-38).
Accordingly, exogenously added CD59 was prevented from entering GPI
complexes by filipin (5), and internalization of endogenous CD59 was
partially inhibited by nystatin in lymphocytes (39), which suggests
that membrane cholesterol plays an important role for the distribution
and function of GPI-anchored proteins.
Here we studied the influence of membrane cholesterol on signal
transduction via GPI-anchored CD59 and CD48 in T cells. To this end,
cellular cholesterol content was reduced by culture in the presence of
cholesterol synthesis inhibitors. Subsequently the rise in cytoplasmic
calcium concentration was analyzed following cross-linking of
GPI-anchored proteins or stimulation via the T cell receptor-CD3
complex, which served as a control. The calcium response was chosen for
quantitative analysis of signal transduction since this parameter can
be measured with high reliability. We show that cholesterol lowering
particularly inhibits signal transduction via GPI-anchored proteins,
whereas no changes were found in the distribution of these proteins in
detergent-insoluble membrane domains.
EXPERIMENTAL PROCEDURES All reagents were obtained from
Sigma unless stated otherwise. Antibodies were used as follows: mouse
monoclonal antibodies MEM-43 (IgG2a), MEM-43/5 (IgG2b), MEM-125 (IgM),
MEM-129 (IgM, all four CD59), MEM-102 (IgG1, CD48), MEM-57 (IgG2a),
MEM-92 (IgM, both CD3; all generated in the lab of Václav
Ho The human T cell line
Jurkat E6-1 (American Type Culture Collection, Rockville, MD) was
grown under standard conditions in RPMI 1640 medium supplemented with
10% heat-inactivated bovine calf serum (HyClone, Logan, UT),
penicillin/streptomycin (50 units/ml and 50 µg/ml, respectively, Life
Technologies, Inc.), and 2 mM glutamine (Life Technologies,
Inc.) at 37 °C in humidified atmosphere in the presence of 5%
CO2. For modifications of cellular lipids, cells were
incubated for 3 days in serum-free Iscove's modified Dulbecco's
medium (Life Technologies, Inc.) supplemented with 0.4% (w/v) bovine
serum albumin (fraction V), 1 mg/liter transferrin, 8.1 mg/liter
monothioglycerol, 2 mM glutamine, and antibiotics as above
(40). Cellular cholesterol synthesis was blocked by addition of 2 µM lovastatin (Merck), an inhibitor of the
3-hydroxy-3-methylglutaryl-coenzyme A reductase, or squalestatin 1 (50 µg/ml) (Glaxo Wellcome), an inhibitor of squalene synthase, both
generous gifts from the respective companies. The drugs were added to
the culture medium from stock solutions in double distilled water (for
squalestatin), or dimethyl sulfoxide (maximal solvent concentration
0.2%), respectively. Dimethyl sulfoxide was used as solvent control
instead of lovastatin and had no effect on signal transduction (not
shown). Sodium mevalonate was generated from mevalonolactone (Fluka
Chemie AG, Buchs, Switzerland) by hydrolysis in NaOH. Cell viability
was >90% as determined by trypan blue exclusion, and since INDO-1
also served as a vital dye only living cells were assessed with respect
to their calcium response.
Jurkat cells were labeled
with the fluorescent Ca2+ indicator indo-1-AM (2 µM) (Molecular Probes, Inc., Eugene, OR) by incubation at
37 °C for 30 min in serum-free culture medium. Cells were primed with about 10 µg of antibodies to GPI-anchored proteins or
Hanks'-buffered salt solution including 1% bovine serum albumin for
20 min followed by a 7 min preequilibration period at 37 °C.
Subsequently, measurement of [Ca2+]i by flow
cytometry was started at 37 °C constant temperature, and after
60 s 20 µg cross-linking F(ab Cells were
prepared for stimulation of calcium response as described above. After
preequilibration, SKF 96365 hydrochloride (Calbiochem) was added at
optimal concentration (100 µM) to completely inhibit
calcium entry from the medium (42). Two min afterward, cells were
stimulated by antibodies as described, and changes in cytoplasmic
calcium concentration were determined by flow cytometry over a 3-min
period.
106 cells were treated with
0.5 units of phosphatidylinositol-specific phospholipase C (PI-PLC)
(Boehringer Mannheim) for 60 min at 37 °C in Hanks'-buffered salt
solution including 0.5% bovine serum albumin. Cells were
simultaneously loaded with INDO-1 for subsequent analysis of calcium
response.
Cells were washed
three times in Hanks'-buffered salt solution and lysed for 30 min on
ice with lysis buffer containing 20 mM Tris-HCl, pH 8.2, 140 mM NaCl, 1% Nonidet P-40 (Pierce), 10 µg/ml
aprotinin (Bayer, Leverkusen, Germany), 5 mM
iodoacetamide, 10 µg/ml leupeptin, and 0.4 mM Pefabloc
(both Boehringer Mannheim), 1 µM pepstatin, and 0.1 mM
N Indirect immunofluorescence for cell surface expression
of GPI-anchored proteins on unfixed cells was performed by standard procedures with tetramethylrhodamine isothiocyanate-labeled second antibody for flow cytometric analysis. For microscopic examination, cells were first fixed for 20 min in 4% formaldehyde followed by 0.1 M glycine before immunofluorescence staining. Photographs were taken from immunofluorescence with tetramethylrhodamine
isothiocyanate-labeled GAM Ig, since fluorescein isothiocyanate
fluorescence faded before adequate exposure of the film (Kodak EPH1600)
could be achieved. For determination of cholesterol content, 3 × 107 cells were twice extracted with chloroform/methanol
(2:1). Dried lipid extracts were dissolved in ethanol, and cholesterol
was quantified by a commercial enzymatic colorimetric test according to
the manufacturers instructions (Boehringer Mannheim).
RESULTS Cross-linking of
GPI-anchored proteins CD59 or CD48 resulted in a marked rise in
intracellular calcium as measured by flow cytometry (Fig.
1). The signal via CD59 quantified by
INDO-1 fluorescence ratio was consistently at least half of that
elicited by CD3, which was used as a positive control. No calcium
signal was obtained by cross-linking of antibodies directed to CD147 or
CD99 molecules, which were well expressed on the Jurkat cell line used,
or with isotype controls (not shown). Addition of CD59 or CD48
antibodies without cross-linking had no direct effect on the
cytoplasmic calcium concentration (not shown).
To test for the specificity of the calcium response for GPI-anchored
proteins, cells were treated with PI-PLC to cleave the glycoproteins
from their phospholipid anchor. Following treatment, CD59 or CD48 could
no longer be detected on the cell surface and the calcium signal was
essentially abolished, whereas surface expression and calcium response
elicited via CD3 remained unchanged (Fig.
2, A and B).
Cellular cholesterol content was lowered by blocking
endogenous cholesterol biosynthesis by lovastatin, an inhibitor of
3-hydroxy-3-methylglutaryl-coenzyme A reductase catalyzing the reaction
to mevalonate (44). Treatment of cells with 2 µM
lovastatin for a 3-day period decreased cellular cholesterol content by
28% compared with the solvent control (1.71 ± 0.09 nmol
versus 1.24 ± 0.15 nmol/106 cells). Under
the same conditions, lovastatin inhibited CD59-driven calcium response
by 52 ± 9%, whereas the response via CD3 was only minimally
affected (7 ± 8% inhibition; Fig.
3, A and B). The
inhibition by lovastatin could be reversed by added mevalonate (7.5 mM) showing that no unspecific toxicity was responsible for the drug effect. Accordingly, lovastatin did not alter calcium response
of Jurkat cells when added to serum-supplemented medium (91 and 97% of
solvent control for CD59 and CD3, respectively), presumably because of
the presence of serum lipoproteins providing exogenous cholesterol
(data not shown).
Lovastatin inhibits production of mevalonate that can be further
processed not only to cholesterol but also to other lipophilic products
(44) including farnesyl and geranylgeranyl moieties needed for
prenylation of proteins, e.g. of the Ras family or G-protein
The inhibitory effect of cholesterol depletion on
calcium response is not restricted to CD59 but shared by CD48, another
GPI-anchored protein (Fig. 5). Thus,
inhibition by cholesterol depletion seems to be characteristic for
signal transduction via GPI-anchored proteins.
Immunofluorescence analysis of lovastatin-treated cells revealed no
alterations in cell surface expression of GPI-anchored proteins CD59
and CD48, and CD3 (Fig. 6). The
expression of at least two different epitopes on CD59 (epitope 1:
MEM-43; epitope 2: MEM-43/5, MEM-125 (46)) was unaltered upon blockade
of cellular cholesterol synthesis, suggesting absence of gross
conformational changes of the protein part of CD59 (Fig. 6). The
markedly diminished surface fluorescence detected by monoclonal
antibodies MEM-125, MEM-129 (CD59), and MEM-92 (CD3) compared with
other antibodies of the same antigen is due to their different isotype
(IgM).
Transient release of calcium from
intracellular stores into the cytoplasm precedes capacitative calcium
entry from the environment leading to further activation of T
lymphocytes (47, 48). To test whether inhibition of the release of
intracellularly sequestered calcium underlies the total decrease in
calcium response, cells were treated with SKF 96365 prior to
stimulation. The maximal concentration of cytoplasmic calcium achieved
following stimulation via GPI-anchored CD59 was decreased by about 40%
after blockade of cholesterol synthesis (Fig.
7A). In contrast, stimulation
via CD3 yielded no difference between lovastatin-treated and untreated cells (Fig. 7B).
Cholesterol lowering may interfere with clustering of
GPI-anchored proteins and distribution in detergent-insoluble
complexes. Cells fixed in formaldehyde prior to indirect
immunofluorescence to avoid artificial clustering by antibodies, showed
a punctate pattern of CD59 and CD48 expression (Fig.
8, A and C) that
was maintained after pretreatment of cells with lovastatin (Fig. 8, B and D). Investigating the impact of cholesterol
depletion on the distribution of CD59 in large detergent-insoluble
complexes, cells cultured with or without lovastatin were lysed in
buffer containing non-ionic detergents Brij-58 or Nonidet P-40 and
analyzed by size fractionation and Western blotting. Thereby most of
GPI-anchored proteins CD59 and CD48 were found in large membrane
complexes although after lysis with Nonidet P-40 a significant amount
of CD59 was recovered in lower molecular weight fractions (Fig.
9). However, the distribution of
GPI-anchored proteins was identical irrespective of whether cells have
been cholesterol depleted or not. Thus, the influence of cholesterol
depletion on signal transduction via GPI-anchored proteins seems not to
be due to destruction of GPI-anchored protein complexes.
DISCUSSION The obtained data show that cholesterol lowering in living cells
by metabolic intervention effectively inhibits signal transduction via
GPI-anchored proteins without changing their clustering or distribution
in large detergent-insoluble complexes. In contrast, signaling via the
T cell receptor (TCR)-CD3 complex was hardly affected by cholesterol
deprivation.
Since GPI-anchored proteins lack a transmembrane domain, the clustering
of these proteins to specific membrane regions seems to be prerequisite
for signal transduction. Cholesterol is an essential component for the
formation of these detergent-insoluble complexes (5, 35-37, 39) that
mediate the association with possible signal-transducing molecules,
e.g. Src family kinases p56lck and
p59fyn (1, 29-31) or G-proteins (49). It has been shown
that exogenously added fluorescent CD59 needs to aggregate into
clusters before signal transduction is enabled (5). Lowering
cholesterol by 60% in living epithelial cells by blocking its
endogenous synthesis resulted in strong inhibition of folate uptake via
the GPI-linked folate receptor along with variable differences in
clustering of these receptors (50). In our study, reduction in
lymphocyte cholesterol content by about 30% did not disclose any
alterations in the distribution of GPI-anchored proteins when examined
by immunofluorescence or gel filtration of membrane complexes. In addition, lowering cell cholesterol had no influence on overall cell
surface expression of GPI-anchored proteins as quantitated by flow
cytometry in contrast to previous publications (51-53). Though
delipidation may cause some conformational changes in GPI-anchored proteins (54) this seems to be unlikely in our experiments, since
antibodies against at least two different epitopes on CD59 (46) bound
to cholesterol-depleted and control cells to a similar extent (Fig. 5).
Thus, in contrast to rather harsh treatments with sterol-sequestrating
detergents, which are able to destroy detergent-insoluble complexes
in situ (5, 36, 37, 39), the signal-transducing function of
these proteins seems to be particularly sensitive to a disturbed
cholesterol availability.
Although GPI-anchored proteins partly share signaling events with the
TCR, there may be unique steps in the signaling pathways for either
type of activation. Signal transduction via the TCR-CD3 complex but
also distinct signaling events induced via GPI-anchored proteins
including interleukin-2 secretion, CD69 expression, and activation of
nuclear factor- Our findings, that activation of calcium response in T cells via
GPI-anchored proteins and the TCR is differentially affected by
cholesterol depletion, supports the concept of separate signaling pathways to be involved. Moreover, since activation via CD59 and CD48
were suppressed to a similar extent, cholesterol lowering seems to
impair signaling pathways common to GPI-anchored proteins. Absence of
structural homologies between both GPI-linked molecules beyond the
phospholipid anchor suggests that the GPI-anchor itself could play an
important role in signal transduction via these proteins, as proposed
previously by others (30, 61, 63, 64).
Rescue by appropriate downstream products of cholesterol synthesis
proves that the drugs applied are actually effective via inhibition of
cholesterol biosynthesis. Such inhibition of cholesterol synthesis in
the endoplasmic reticulum may directly interfere with more subtle
associations of lipids and proteins during aggregation to
detergent-insoluble complexes. How a disturbed lipid composition can
alter the signal transduction process remains to be elucidated. Conceivably, spatial relationships between GPI-anchored proteins and
candidate signal-transducing molecules like the Src family protein
tyrosine kinases could play a role in this process as well as changes
in lateral mobility of components within the complexes.
In conclusion, our data suggest that cholesterol lowering affects
signal transduction via GPI-anchored proteins in a very early stage up
to the release of calcium without apparent structural alterations.
Thus, lipids may play an intriguing role in signal transduction via
these surface molecules whose exact molecular mechanisms remain to be
elucidated.
FOOTNOTES ACKNOWLEDGEMENTS We are grateful to Samuel Godár and Dr.
Winfried Pickl for helpful discussions, and to Dr. Otto
Majdic for the provision of antibodies.
REFERENCES
Volume 272, Number 31,
Issue of August 1, 1997
pp. 19242-19247
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
§,,,
ej
í
and Department of Internal Medicine III,
University of Vienna, Währinger Gürtel 18-20, A-1090
Vienna, Austria, ¶ Institute of Immunology, Vienna International
Research Cooperation Center, University of Vienna, Brunnerstrasse 59, A-1235 Vienna, Austria and Institute of Molecular Genetics,
Academy of Sciences of the Czech Republic, Víde
ská
1083, 14220 Prague 4, Czech Republic
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
ej
í and partly obtained from Monosan, Uden, The
Netherlands), BRIC 216 (IgG1, CD55) (Haran Sera-Lab, Crawley Down,
Sussex, UK), OKT3 (IgG2a, CD3) (Ortho Pharmaceuticals, Raritan, NJ),
monoclonal antibody AAA6 (IgG1, CD147), and monoclonal antibody 0662 (IgG3, CD99; kindly provided by O. Majdic and A. Bernard,
respectively); F(ab
)2-fragments of goat-anti-mouse (GAM)
IgG (Sigma or Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA), fluorescein isothiocyanate-labeled F(ab)2-fragments
of GAM Ig (Dako, Glostrup, Denmark), tetramethylrhodamine isothiocyanate-labeled F(ab)2-fragments of GAM IgG
(Accurate Chemical & Science Corp., Westbury, NY), GAM IgG labeled with horseradish peroxidase (Bio-Rad).
)2-fragments of GAM
IgG were added and measurement continued for another 5 min unless stated otherwise. For CD3 stimulation 2 µg of OKT3 was added directly without cross-linking. Cross-linking did not result in a strong calcium
response in any OKT3 concentration with absence of calcium response by
adding the primary antibody solely. Flow cytometric analysis was
performed on a FACStarplus (Becton Dickinson, San Jose,
CA) using excitation by argon laser with 50-milliwatt multiline UV,
emission 530 nm (Fl1, calcium-free indo-1) and 395 nm (Fl2, calcium
bound form). The fluorescence ratio Fl2/Fl1 (FR), which is a direct
estimate of the cytoplasmic calcium concentration (41), was computed in
real time by a pulse processing unit and expressed as arbitrary units.
The FR of the unstimulated control (FRus) was set to about 200 arbitrary units. For quantitation of stimulation, maximal FR was
measured at 1-3 min after adding the stimulating antibody. Since CD3
stimulation of cells often induced a calcium response somewhat higher
than stimulation via GPI-anchored proteins, results from modified cells were usually expressed as percentage of that achieved with the solvent
control to allow comparability: relative stimulation in percent of
control was calculated as (FRs - FRus)/(FRco - FRus) × 100%, with
FRco representing FR of the solvent control, and FRs representing FR of
the sample. Data are presented as means ± S.D. unless stated
otherwise.
-p-tosyl-L-lysine
chloromethyl ketone,
N-p-tosyl-L-phenylalanine chloromethyl ketone, N-CBZ-L-phenylalanine chloromethyl
ketone, and quercetin. Lysates were then applied to Sepharose 4B
minigel filtration columns as described earlier (26) with fractions 4 and 5 referring to the void volume. Aliquots of the fractions 3 to 11 were separated by 12% non-reducing SDS-polyacrylamide gel
electrophoresis (43) and blotted on nitrocellulose membrane (Hybond
ECL, Amersham International, UK) using a semidry blotting system
(C.B.S. Scientific Co., Inc., Del Mar, CA). Membranes were developed
according to standard Western blotting procedures, and detection was
performed by the chemiluminescence system from Boehringer Mannheim.
Fig. 1.
Calcium response after stimulation via
GPI-anchored proteins. Jurkat T cells were stimulated at time 0 via CD59 (MEM-43; 
) and CD48 (MEM-102; 
), or via CD3 (OKT3; 
)
as positive control. CD147 (AAA6; 
) and CD99 (0662; 
) served as
negative controls. Original tracings of INDO-1 fluorescence ratio
measured by flow cytometry are given. Expression of surface molecules
as estimated by immunofluorescence (in arbitrary units): 176 (CD59,
MEM-43), 67 (CD48, MEM-102), 119 (CD3, OKT3), 245 (CD147, AAA6), 204 (CD99, 0662), and 4 (phosphate-buffered saline control).
[View Larger Version of this Image (22K GIF file)]
Fig. 2.
PI-PLC treatment abolishes expression and
calcium responses via GPI-anchored proteins. A, calcium
response elicited in Jurkat cells pretreated with or without PI-PLC and
subsequently stimulated via CD59 (MEM-43), CD48 (MEM-102), or CD3
(OKT3). Calcium responses without enzyme pretreatment were set to
100%. B, analysis of surface expression of GPI-anchored
proteins (CD59, CD48) and CD3 after pretreatment with or without
PI-PLC. Results from one of two independent experiments are
depicted.
[View Larger Version of this Image (26K GIF file)]
Fig. 3.
Inhibition by lovastatin of CD59 but not of
CD3 induced calcium response. Jurkat cells were incubated for 3 days in serum-free medium with lovastatin (2 µM), an
inhibitor of cellular cholesterol synthesis, and mevalonate (7.5 mM) to rescue from this inhibition, as indicated.
Subsequently, cells were stimulated via CD59 (MEM-43) or CD3 (OKT3).
A, calcium responses are expressed as percentage of the
response elicited in untreated cells (solvent control = 100%).
Means ± S.D. from eight independent experiments are given.
B, calcium responses expressed as INDO-1 fluorescence ratio
(in arbitrary units (a.u.)) from a single experiment. Note that the absolute rise in cytosolic calcium in the control
(i.e. without addition of lovastatin or mevalonate) was
almost identical with CD59 and CD3 in this experiment.
[View Larger Version of this Image (48K GIF file)]
subunits. To further prove lovastatin's inhibitory effect to be
due to cholesterol lowering, squalestatin 1, an inhibitor of the
squalene synthase, was also tested for its effect on signal transduction via GPI-anchored proteins. Squalestatin 1 blocks cholesterol biosynthesis at a step distal to mevalonate synthesis and,
therefore, does not affect side products of the mevalonate pathway
(45). Squalestatin 1 inhibited signal transduction via CD59 similarly
to lovastatin (47 ± 14% inhibition; Fig.
4) with minimal effect on CD3 signaling
(2 ± 3% inhibition). The associated block in cholesterol
synthesis downstream from mevalonate could not be reverted by
mevalonate but only by cholesterol.
Fig. 4.
Inhibition by squalestatin of CD59 but not
CD3 induced calcium response. Jurkat T cells were incubated for 3 days in serum-free medium supplemented with squalestatin (50 µM), an inhibitor of cellular cholesterol synthesis, and
mevalonate (7.5 mM), or cholesterol (25 µg/ml) to test
for possible rescue from this inhibition, as indicated. Cells were
stimulated for calcium response via CD59 (MEM-43) or CD3 (OKT3), and
the response of the untreated control was set to 100%. Means ± S.D. from three independent experiments.
[View Larger Version of this Image (50K GIF file)]
Fig. 5.
Inhibition by cholesterol depletion of
calcium response via different GPI-anchored proteins. Jurkat T
cells were incubated for 3 days in serum-free medium containing 2 µM lovastatin and 7.5 mM mevalonate as
indicated. Cells were stimulated for calcium response via CD59
(MEM-43), CD48 (MEM-102), or CD3 (OKT3), which served as a control. The
response of the untreated control was set to 100%. Means ± S.D.
from three independent experiments are depicted.
[View Larger Version of this Image (52K GIF file)]
Fig. 6.
Cholesterol depletion does not alter surface
expression of GPI-anchored proteins. Jurkat cells were incubated
with solvent (black bars), lovastatin (2 µM)
(speckled bars), or lovastatin and mevalonate (7.5 mM) (hatched bars) for 3 days in serum-free medium. Cell surface immunofluorescence was determined using various antibodies to CD59 (MEM-43, MEM-43/5, MEM-125, MEM-129), CD48 (MEM-102), and CD3 (OKT3, MEM-92, MEM-57). Data from one of three independent determinations are shown.
[View Larger Version of this Image (39K GIF file)]
Fig. 7.
Cholesterol depletion inhibits release of
calcium from intracellular stores when induced by GPI-anchored proteins
but not after stimulation by CD3. Cells were grown in serum-free
medium for 3 days in the absence () or presence () of 2 µM lovastatin; a third sample of cells cultured in
presence of lovastatin and 7.5 mM mevalonate was stimulated
via CD59 to rescue from the lovastatin-induced inhibition (). SKF
96365 (100 µM) was added to inhibit calcium entry and
followed by GAM cross-linking antibody in the case of cells primed with
anti-CD59 (MEM-43; panel A) or by antibody against CD3
(OKT3; panel B), respectively. Calcium response reflecting release of intracellular calcium stores was monitored for almost 3 min
following stimulation. Data from one of three experiments are
shown.
[View Larger Version of this Image (21K GIF file)]
Fig. 8.
Cholesterol lowering fails to affect
distribution of GPI-anchored protein on the cell surface. Cells
cultured for three days in serum-free medium with (panels B
and D) or without (panels A and
C) lovastatin were fixed with formaldehyde and subsequently stained for surface expression of CD59 (MEM-43; panels A and
B) and CD48 (MEM-102; panels C and D)
by indirect immunofluorescence using tetramethylrhodamine
isothiocyanate-labeled goat-anti-mouse IgG. No fluorescence was
detected on phosphate-buffered saline controls (not shown).
Bar, 5 µm.
[View Larger Version of this Image (75K GIF file)]
Fig. 9.
Cholesterol lowering fails to influence
distribution of GPI-anchored proteins in detergent-insoluble
complexes. Jurkat T cells were incubated for 3 days in serum-free
medium with (
) or without (+) 2 µM lovastatin as
indicated. Cells were lysed in lysis buffer containing 1% of the
non-ionic detergent Brij-58 or Nonidet P-40 (NP-40),
respectively. Subsequently, detergent-insoluble complexes were
separated by minigel chromatography with fractions 4 and 5 corresponding to the void volume and with IgM and IgG eluting in
fractions 7 and 9, respectively (26). The proteins of each fraction
were separated by non-reducing polyacrylamide gel electrophoresis and
identified by Western blotting with antibodies against CD59 (MEM-43/5)
or CD48 (MEM-102) as indicated. Results from one of three independent
analyses are depicted.
[View Larger Version of this Image (37K GIF file)]
B, require expression of the TCR-
chain (14,
55-58). On the other hand, induction of calcium response (55),
activation of p56lck, protein kinase C, and ERK-2,
expression of CD25 (58), and inhibition of CD3-driven interleukin-2
production (57, 59) were shown to be independent of the expression of
TCR-. Furthermore, activation of murine T cells via GPI-anchored
Thy-1 but not via CD3 requires expression of p59fyn (60).
Thus, signaling via GPI-anchored proteins seems to differ from TCR/CD3
signaling with regard to the involvement of the TCR- chain and
particular Src protein kinases. Despite these differences, both
signaling pathways were shown to interact with each other since
adequate expression of GPI-anchored proteins is needed for signaling
via the TCR-CD3 complex (61, 62). It appears from this that T cell
activation via GPI-anchored proteins differs from TCR/CD3-driven
activation, and each pathway may require components of the other for
optimal stimulation (2, 61).
§
To whom correspondence should be addressed. Tel.: 43-1-40400-4338;
Fax: 43-1-40400-7790; E-mail: Thomas.Stulnig{at}akh-wien.ac.at.
1
The abbreviations used are: GPI,
glycosylphosphatidylinositol; GAM, goat anti-mouse; PI-PLC,
phosphatidylinositol-specific phospholipase C; TCR, T cell receptor;
FR, fluorescence ratio.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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 D. J. Dietzen, K. L. Page, T. A. Tetzloff, A. Bohrer, and J. Turk Inhibition of 3-Hydroxy-3-Methylglutaryl Coenzyme A (HMG CoA) Reductase Blunts Factor VIIa/Tissue Factor and Prothrombinase Activities via Effects on Membrane Phosphatidylserine Arterioscler. Thromb. Vasc. Biol., March 1, 2007; 27(3): 690 - 696. [Abstract] [Full Text] [PDF]
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T. B. Nicholson and C. P. Stanners Specific inhibition of GPI-anchored protein function by homing and self-association of specific GPI anchors J. Cell Biol., November 20, 2006; 175(4): 647 - 659. [Abstract] [Full Text] [PDF]
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 S. M. L. Smith, Y. Lei, J. Liu, M. E. Cahill, G. M. Hagen, B. G. Barisas, and D. A. Roess Luteinizing Hormone Receptors Translocate to Plasma Membrane Microdomains after Binding of Human Chorionic Gonadotropin Endocrinology, April 1, 2006; 147(4): 1789 - 1795. [Abstract] [Full Text] [PDF]
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 I. Kansau, C. Berger, M. Hospital, R. Amsellem, V. Nicolas, A. L. Servin, and M.-F. Bernet-Camard Zipper-Like Internalization of Dr-Positive Escherichia coli by Epithelial Cells Is Preceded by an Adhesin-Induced Mobilization of Raft-Associated Molecules in the Initial Step of Adhesion Infect. Immun., July 1, 2004; 72(7): 3733 - 3742. [Abstract] [Full Text] [PDF]
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 P. Danthi and M. Chow Cholesterol Removal by Methyl-{beta}-Cyclodextrin Inhibits Poliovirus Entry J. Virol., January 1, 2004; 78(1): 33 - 41. [Abstract] [Full Text] [PDF]
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 M. Nagafuku, K. Kabayama, D. Oka, A. Kato, S. Tani-ichi, Y. Shimada, Y. Ohno-Iwashita, S. Yamasaki, T. Saito, K. Iwabuchi, et al. Reduction of Glycosphingolipid Levels in Lipid Rafts Affects the Expression State and Function of Glycosylphosphatidylinositol-anchored Proteins but Does Not Impair Signal Transduction via the T Cell Receptor J. Biol. Chem., December 19, 2003; 278(51): 51920 - 51927. [Abstract] [Full Text] [PDF]
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 G. Staffler, A. Szekeres, G. J. Schutz, M. D. Saemann, E. Prager, M. Zeyda, K. Drbal, G. J. Zlabinger, T. M. Stulnig, and H. Stockinger Selective Inhibition of T Cell Activation Via CD147 Through Novel Modulation of Lipid Rafts J. Immunol., August 15, 2003; 171(4): 1707 - 1714. [Abstract] [Full Text] [PDF]
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S. Sanzone, M. Zeyda, M. D. Saemann, M. Soncini, W. Holter, G. Fritsch, W. Knapp, F. Candotti, T. M. Stulnig, and O. Parolini SLAM-associated Protein Deficiency Causes Imbalanced Early Signal Transduction and Blocks Downstream Activation in T Cells from X-linked Lymphoproliferative Disease Patients J. Biol. Chem., August 8, 2003; 278(32): 29593 - 29599. [Abstract] [Full Text] [PDF]
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N. Kahya, D. Scherfeld, K. Bacia, B. Poolman, and P. Schwille Probing Lipid Mobility of Raft-exhibiting Model Membranes by Fluorescence Correlation Spectroscopy J. Biol. Chem., July 18, 2003; 278(30): 28109 - 28115. [Abstract] [Full Text] [PDF]
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 C. B. Marta, C. M. Taylor, T. Coetzee, T. Kim, S. Winkler, R. Bansal, and S. E. Pfeiffer Antibody Cross-Linking of Myelin Oligodendrocyte Glycoprotein Leads to Its Rapid Repartitioning into Detergent-Insoluble Fractions, and Altered Protein Phosphorylation and Cell Morphology J. Neurosci., July 2, 2003; 23(13): 5461 - 5471. [Abstract] [Full Text] [PDF]
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M. Zeyda, G. Staffler, V. Horejsi, W. Waldhausl, and T. M. Stulnig LAT Displacement from Lipid Rafts as a Molecular Mechanism for the Inhibition of T Cell Signaling by Polyunsaturated Fatty Acids J. Biol. Chem., August 2, 2002; 277(32): 28418 - 28423. [Abstract] [Full Text] [PDF]
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 K. Iwabuchi and I. Nagaoka Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils Blood, July 30, 2002; 100(4): 1454 - 1464. [Abstract] [Full Text] [PDF]
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A. A. Wolf, Y. Fujinaga, and W. I. Lencer Uncoupling of the Cholera Toxin-GM1 Ganglioside Receptor Complex from Endocytosis, Retrograde Golgi Trafficking, and Downstream Signal Transduction by Depletion of Membrane Cholesterol J. Biol. Chem., May 3, 2002; 277(18): 16249 - 16256. [Abstract] [Full Text] [PDF]
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 H. Nagasawa, A. Cremesti, R. Kolesnick, Z. Fuks, and J. B. Little Involvement of Membrane Signaling in the Bystander Effect in Irradiated Cells Cancer Res., May 1, 2002; 62(9): 2531 - 2534. [Abstract] [Full Text] [PDF]
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E. Gubina, T. Chen, L. Zhang, E. F. Lizzio, and S. Kozlowski CD43 polarization in unprimed T cells can be dissociated from raft coalescence by inhibition of HMG CoA reductase Blood, April 1, 2002; 99(7): 2518 - 2525. [Abstract] [Full Text] [PDF]
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 F. Van Laethem, E. Baus, L. A. Smyth, F. Andris, F. Bex, J. Urbain, D. Kioussis, and O. Leo Glucocorticoids Attenuate T Cell Receptor Signaling J. Exp. Med., March 26, 2001; 193(7): 803 - 814. [Abstract] [Full Text] [PDF]
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K. Hossain, A. A. Akhand, M. Kato, J. Du, K. Takeda, J. Wu, K. Takeuchi, W. Liu, H. Suzuki, and I. Nakashima Arsenite Induces Apoptosis of Murine T Lymphocytes Through Membrane Raft-Linked Signaling for Activation of c-Jun Amino-Terminal Kinase J. Immunol., October 15, 2000; 165(8): 4290 - 4297. [Abstract] [Full Text] [PDF]
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 T. M. STULNIG, M. BERGER, M. RODEN, H. STINGL, D. RAEDERSTORFF, and W. WALDHÄUSL Elevated serum free fatty acid concentrations inhibit T lymphocyte signaling FASEB J, May 1, 2000; 14(7): 939 - 947. [Abstract] [Full Text]
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 S Miotti, M Bagnoli, A Tomassetti, M. Colnaghi, and S Canevari Interaction of folate receptor with signaling molecules lyn and G(alpha)(i-3) in detergent-resistant complexes from the ovary carcinoma cell line IGROV1 J. Cell Sci., January 1, 2000; 113(2): 349 - 357. [Abstract] [PDF]
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F. Y. S. Chuang, M. Sassaroli, and J. C. Unkeless Convergence of Fc{gamma} Receptor IIA and Fc{gamma} Receptor IIIB Signaling Pathways in Human Neutrophils J. Immunol., January 1, 2000; 164(1): 350 - 360. [Abstract] [Full Text] [PDF]
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K. Krauss and P. Altevogt Integrin Leukocyte Function-associated Antigen-1-mediated Cell Binding Can Be Activated by Clustering of Membrane Rafts J. Biol. Chem., December 24, 1999; 274(52): 36921 - 36927. [Abstract] [Full Text] [PDF]
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 J. E. Osheroff, P. E. Visconti, J. P. Valenzuela, A. J. Travis, J. Alvarez, and G. S. Kopf Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation Mol. Hum. Reprod., November 1, 1999; 5(11): 1017 - 1026. [Abstract] [Full Text] [PDF]
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R. D. J. Huby, R. J. Dearman, and I. Kimber Intracellular Phosphotyrosine Induction by Major Histocompatibility Complex Class II Requires Co-aggregation with Membrane Rafts J. Biol. Chem., August 6, 1999; 274(32): 22591 - 22596. [Abstract] [Full Text] [PDF]
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 S. Ilangumaran, S. Arni, G. van Echten-Deckert, B. Borisch, and D. C. Hoessli Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes Mol. Biol. Cell, April 1, 1999; 10(4): 891 - 905. [Abstract] [Full Text]
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K. A. Melkonian, A. G. Ostermeyer, J. Z. Chen, M. G. Roth, and D. A. Brown Role of Lipid Modifications in Targeting Proteins to Detergent-resistant Membrane Rafts. MANY RAFT PROTEINS ARE ACYLATED, WHILE FEW ARE PRENYLATED J. Biol. Chem., February 5, 1999; 274(6): 3910 - 3917. [Abstract] [Full Text] [PDF]
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T. M. Stulnig, M. Berger, T. Sigmund, D. Raederstorff, H. Stockinger, and W. Waldhausl Polyunsaturated Fatty Acids Inhibit T Cell Signal Transduction by Modification of Detergent-insoluble Membrane Domains J. Cell Biol., November 2, 1998; 143(3): 637 - 644. [Abstract] [Full Text] [PDF]
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S. Arni, S. A. Keilbaugh, A. G. Ostermeyer, and D. A. Brown Association of GAP-43 with Detergent-resistant Membranes Requires Two Palmitoylated Cysteine Residues J. Biol. Chem., October 23, 1998; 273(43): 28478 - 28485. [Abstract] [Full Text] [PDF]
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V. O. Rybin, X. Xu, M. P. Lisanti, and S. F. Steinberg Differential Targeting of beta -Adrenergic Receptor Subtypes and Adenylyl Cyclase to Cardiomyocyte Caveolae. A MECHANISM TO FUNCTIONALLY REGULATE THE cAMP SIGNALING PATHWAY J. Biol. Chem., December 22, 2000; 275(52): 41447 - 41457. [Abstract] [Full Text] [PDF]
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A. Cremesti, F. Paris, H. Grassme, N. Holler, J. Tschopp, Z. Fuks, E. Gulbins, and R. Kolesnick Ceramide Enables Fas to Cap and Kill J. Biol. Chem., June 22, 2001; 276(26): 23954 - 23961. [Abstract] [Full Text] [PDF]
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T. M. Stulnig, J. Huber, N. Leitinger, E.-M. Imre, P. Angelisova, P. Nowotny, and W. Waldhausl Polyunsaturated Eicosapentaenoic Acid Displaces Proteins from Membrane Rafts by Altering Raft Lipid Composition J. Biol. Chem., September 28, 2001; 276(40): 37335 - 37340. [Abstract] [Full Text] [PDF]
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