Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tyrosine phosphorylation of the high affinity
immunoglobulin (Ig)E receptor (FcRI) by the Src
family kinase Lyn is the first known biochemical step
that occurs during activation of mast cells and basophils
after cross-linking of Fc
RI by antigen. The hypothesis that specialized regions in the plasma membrane, enriched in sphingolipids and cholesterol, facilitate the
coupling of Lyn and Fc
RI was tested by investigating
functional and structural effects of cholesterol depletion on Lyn/Fc
RI interactions. We find that cholesterol depletion with methyl-
-cyclodextrin substantially reduces stimulated tyrosine phosphorylation of Fc
RI
and other proteins while enhancing more downstream
events that lead to stimulated exocytosis. In parallel to
its inhibition of tyrosine phosphorylation, cholesterol depletion disrupts the interactions of aggregated Fc
RI
and Lyn on intact cells and also disrupts those interactions with detergent-resistant membranes that are isolated by sucrose gradient ultracentrifugation of lysed
cells. Importantly, cholesterol repletion restores receptor phosphorylation together with the structural interactions. These results provide strong evidence that
membrane structure, maintained by cholesterol, plays a
critical role in the initiation of Fc
RI signaling.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE first known biochemical event in antigen stimulation of mast cells and basophils is tyrosine phosphorylation of the and
subunits of the high affinity IgE receptor (Fc
RI) by the Src family kinase Lyn;
however, the mechanism by which these proteins interact
is not fully understood. A common view is that this receptor-kinase coupling occurs strictly via protein-protein
interactions. For example, in the transphosphorylation
model, antigen-induced cross-linking causes Lyn, which is
bound weakly to one receptor, to phosphorylate immune tyrosine activation motifs on a juxtaposed receptor,
thereby initiating signal transduction (Jouvin et al., 1994
;
Pribluda et al., 1994
). We recently presented a different
view that the plasma membrane structure plays an integral
role in facilitating coupling between Lyn and Fc
RI. In
particular, we proposed that Fc
RI tyrosine phosphorylation occurs within specialized regions in the plasma membrane, enriched in sphingolipids and cholesterol (Field et
al., 1997
; Sheets et al., 1999
). Membrane structures of this
composition have been isolated from many cell types and primarily characterized on the basis of their resistance to
solubilization by nonionic detergents such as Triton X-100
(TX-100)1 and their consequent buoyancy in sucrose density gradients. These isolated detergent-resistant membranes (DRMs) (also referred to in the literature as DIGs
[detergent insoluble glycolipid domains], GEMs [glycolipid-enriched membranes], and membrane rafts) are enriched in cholesterol, sphingomyelin, glycosphingolipids, and saturated glycerophospholipids, as well as dually
acylated Src family kinases (e.g., Lyn, Fyn, Yes) and glycosylphosphatidylinositol (GPI)-anchored proteins (for review see Brown and London, 1998a
; Simons and Ikonen,
1997
). They are postulated to represent plasma membrane
domains that may function as centers for signal transduction and membrane trafficking, although their nature on
the cell surface is controversial (Brown and London,
1998a
; Edidin, 1997
).
In initial studies we found that cross-linking of FcRI increases the percentage of cellular Lyn recovered in DRMs
from TX-100-lysed RBL-2H3 mast cells, suggesting that
Fc
RI aggregation causes an alteration of DRMs that may
be involved in Fc
RI-mediated signaling (Field et al.,
1995
). With sufficiently low concentrations of TX-100 to
lyse the cells, aggregated (but not monomeric) Fc
RI also
associate with DRM vesicles, and only this population of receptors is phosphorylated upon cross-linking (Field et
al., 1997
). Furthermore, fluorescence microscopy on intact
cells revealed that cross-linking of Fc
RI induces co-redistribution with DRM components, including a GD1b ganglioside (Pierini et al., 1996
), as well as Lyn and the GPI-anchored protein Thy-1 (Holowka, D., E.D. Sheets, and
B. Baird, manuscript in preparation), and with saturated
phospholipid analogues (Thomas et al., 1994
). Together, these results are consistent with the view that specialized
membrane domains function in cells to facilitate coupling
between aggregated Fc
RI and Lyn.
The detergent resistance of these membrane structures
has been hypothesized to depend upon their lipid phase
(Brown and London, 1998b). Cholesterol, which profoundly affects phase behavior of lipids, was found to be a
major lipid component of isolated DRMs derived from
MDCK cells (Brown and Rose, 1992
). In subsequent studies, Brown and colleagues found that model membranes
with compositions similar to DRMs are not solubilized by
TX-100, and this detergent resistance was observed to correlate with cholesterol concentrations that induce formation of the liquid-ordered (Lo) phase (Schroeder et al.,
1994
; Ahmed et al., 1997
; Schroeder et al., 1998
). This
phase results from cholesterol having a gel phase-like ordering effect on the saturated and near-saturated acyl
chains of glycerophospholipids and sphingolipids; yet the
lipids retain a high degree of lateral and rotational mobility, similar to lipids in the fluid liquid crystalline phase
(Brown and London, 1998a
,b). The relevance of this structure for biological membranes is supported by recent electron spin resonance measurements that showed parameters characteristic of the Lo phase for DRM vesicles
isolated from RBL cells (Ge et al., 1999
). Overall, these
results support the hypothesis that ordered lipid domains
coalescing on the plasma membrane after Fc
RI aggregation serve to co-localize Fc
RI and Lyn and thereby initiate receptor phosphorylation and signaling.
As reported here, we tested this hypothesis by using
methyl--cyclodextrin (M
CD) to selectively deplete cholesterol from RBL-2H3 cells, and we investigated the functional and structural effects of the M
CD treatment on
Fc
RI/Lyn interactions. This reagent has been used recently for efficient removal of cholesterol from a variety of
cell types (Kilsdonk et al., 1995
; Yancey et al., 1996
; Christian et al., 1997
; Gimpl et al., 1997
; Scheiffele et al., 1997
;
Friedrichson and Kurzchalia, 1998
; Keller and Simons, 1998
; Varma and Mayor, 1998
). We find that cholesterol
depletion substantially reduces stimulated tyrosine phosphorylation of Fc
RI and other substrates in RBL cells.
Furthermore, we find that cholesterol depletion selectively
disrupts the structural interactions between aggregated
Fc
RI and Lyn both for DRM vesicles from lysed cells that are isolated on sucrose gradients and for intact cells as assessed by confocal fluorescence microscopy. When cholesterol levels are repleted, these functional effects and
molecular associations are restored. These results provide
strong evidence that cholesterol is required for effective
functional coupling between aggregated Fc
RI and Lyn,
and they are consistent with an important structural role
for liquid-ordered membrane domains in this coupling.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cholesterol Depletion and Repletion
RBL-2H3 cells were maintained and harvested as previously described
(Pierini et al., 1996). Mouse monoclonal IgE specific for 2,4-dinitrophenyl
(DNP) (Liu et al., 1980
) was purified as previously described (Subramanian et al., 1996
); biotinylated and iodinated (Field et al., 1995
) or FITC-labeled (Pierini et al., 1996
) IgE was used to sensitize cells in some experiments. Mouse monoclonal anti-1,5-dansyl IgE was affinity purified as previously described (Weetall et al., 1993
). Other RBL cell membrane components were labeled with AA4 mAb (a gift from Dr. Reuben Siraganian, National Institutes of Health, Bethesda, MD), specific for the
-galactosyl GD1b ganglioside derivative; OX-7 (PharMingen), specific for the GPI-anchored protein Thy-1; and anti-Lyn (Upstate Biotechnology, Inc., and Santa Cruz Biotechnology) as previously described (Field et
al., 1995
; Pierini et al., 1996
). Transferrin receptors (TfRs; CD71) were labeled with a monoclonal antibody from PharMingen, followed by Cy3-goat anti-mouse
chain (Southern Biotechnology Associates).
To remove cholesterol, suspended cells (2-4 × 106 cells/ml) were incubated for 1 h at 37°C in the presence or absence of 10 mM MCD (Sigma
Chemical Co.) in BSA-containing buffered saline solution (BSA/BSS:
20 mM Hepes, pH 7.4, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM
MgCl2, 5.6 mM glucose, and 1 mg/ml BSA), then washed with BSA/BSS
before stimulation. For some experiments, cholesterol (Avanti Polar Lipids) was added back to cholesterol-depleted cells (2 × 106 cells/ml) in
BSA/BSS by incubation for 2 h at 37°C with indicated concentrations of
M
CD/cholesterol (8:1, mol/mol) complexes. These complexes were prepared similarly to a previously described procedure (Racchi et al., 1997
).
In brief, cholesterol in a chloroform solution was dried under nitrogen in a
glass culture tube precleaned with ethanolic KOH. An appropriate volume of sterile-filtered 300 mM M
CD in BSA/BSS was added to the tube,
and the resulting suspension was vortexed and bath sonicated until the
suspension clarified. The complex was then incubated in a rocking water
bath overnight at 37°C to maximize formation of soluble complexes.
In Vivo Tyrosine Phosphorylation Assays
Suspended RBL-2H3 cells that had been sensitized with anti-DNP IgE
(Chang et al., 1995) and cholesterol depleted/repleted or not were stimulated at a density of 106 cell/ml with multivalent DNP-BSA (1 µg/ml; Xu
et al., 1998a
) at 37°C for indicated times, lysed by addition of 5× SDS
sample buffer (50% glycerol, 0.25 M Tris, pH 6.8, 5% SDS, 0.5% bromphenol blue) and boiled for 5 min and centrifuged for 5 min at 13,000 g.
Equal numbers of cell equivalents of lysates (typically, 8 × 103 cell equivalents) were electrophoresed on 12% nonreduced SDS polyacrylamide
gels, transferred to Immobilon-P membranes (Millipore Corp.), and
probed with horseradish peroxidase-conjugated antiphosphotyrosine (4G10-HRP; Upstate Biotechnology, Inc.). Enhanced chemiluminescence (Pierce) was used for detection. Phosphorylation as a function of stimulation time was quantified after scanning blots and analyzing with Un-Scan-It (Silk Scientific) and Igor Pro (WaveMetrics).
Quantitative Measurements of FcRI
We determined the effect of MCD on the amount of Fc
RI associated
with RBL-2H3 cells by two different methods. For some experiments, biotinylated 125I-IgE was bound to Fc
RI under saturating conditions, and
these labeled cells were used to monitor the loss of IgE-Fc
RI complexes after treatment with or without M
CD as described above. The state of
125I-IgE released from the cells during M
CD treatment was assessed by
collecting the supernatants after cell pelleting (200 g, 5 min) and subjecting these to a high speed centrifugation (250,000 g, 45 min, 4°C). Gamma
counting indicated that 54% of the 125I-IgE was pelleted during the second
centrifugation, in contrast to only 6% of the 125I-IgE that could be pelleted
under these conditions for untreated cells. In another set of experiments,
Fc
RI were saturated with FITC-IgE, and cells were treated with or without M
CD. Receptor-bound FITC-IgE was measured on washed cells
with steady-state fluorimetry as previously described (Xu et al., 1998b
).
Examination of these cells by fluorescence microscopy in the presence or
absence of 15 mM NH4Cl to neutralize endosomes (Xu et al., 1998b
)
showed no evidence for internalization of FITC-IgE after M
CD treatment.
To examine the relationship between the density of anti-DNP IgE on
the cells and antigen-stimulated tyrosine phosphorylation, FcRI were saturated with mixtures of anti-DNP IgE and antidansyl IgE in percentage
mixtures of 30:70, 50:50, and 100:0. Washed cells were stimulated with 1 µg/ml DNP-BSA at 37°C for various times and tyrosine phosphorylation
was analyzed. Competition binding between each of these unlabeled antibodies and FITC-labeled anti-DNP IgE was carried out under identical
conditions. With steady-state fluorimetry to quantify the amount of cell-bound FITC-IgE, we confirmed that the percentages of anti-DNP IgE and
antidansyl IgE bound to Fc
RI in the tyrosine phosphorylation experiments were identical to the percentages added with an uncertainty of ± 8%. Furthermore, fluorimetry experiments with mixtures of FITC anti-DNP IgE and unlabeled antidansyl IgE showed that DNP-BSA binding
and cross-linking induced internalization of FITC-IgE-Fc
RI proportionally to the amount of FITC-IgE bound in the range of 30-100% occupancy
by this IgE (data not shown; Xu et al., 1998a
,b).
Sucrose Gradients
Cholesterol-depleted/repleted or untreated cells were fractionated on sucrose step gradients as previously described (Field et al., 1997), except
that the concentration of TX-100 during lysis was 0.04% instead of 0.05%.
Aliquots (200 µl) were removed from the top of the gradient, and
-radiation of biotinylated 125I-IgE was counted. The gradient fractions were then
pooled as indicated, boiled with SDS sample buffer, and blotted as described above, except that the primary antibody was rabbit anti-Lyn antibody (Upstate Biotechnology, Inc.) and the secondary antibody was
HRP-conjugated donkey anti-rabbit Ig (Amersham Pharmacia Biotech).
To determine the location of GD1b in the gradients, cells labeled with 125I-AA4 and biotinylated IgE were analyzed as previously described (Field
et al., 1995
). In some experiments, the pooled gradient fractions were electrophoresed on 12% nonreduced SDS-acrylamide gels and subsequently
were silver stained (Daiichi Silver Stain II; Owl Separation Systems).
Immunodepletion of FcRI
Anti-DNP IgE-sensitized cells were stimulated for the indicated times
with DNP-BSA (1 µg/ml) and lysed on ice with TX-100 lysis buffer [10 mM
Tris, pH 8.0, 50 mM NaCl, 1 mM Na3VO4, 30 mM sodium pyrophosphate,
10 mM sodium glycerophosphate, 0.02 U/ml aprotinin, 0.01% NaN3, 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, and 0.2% TX-100] followed by
addition of 10 µM DNP-aminocaproyl-L-tyrosine, as previously described
(Harris et al., 1997). After centrifugation at 13,000 g for 5 min to remove
insoluble material, lysates were incubated with or without 20 µg/ml rabbit
anti-IgE (Menon et al., 1984
). After incubation with protein A-agarose
beads (Pierce), samples were centrifuged to pellet the beads and supernatants were removed, boiled in SDS sample buffer, electrophoresed, and blotted with 4G10-HRP as described above. Rabbit anti-IgE/IgE-Fc
RI complexes are bound to the protein A-agarose, which result in selective
depletion of the Fc
RI
and
bands.
Degranulation Assays
The degranulation response that occurs after stimulation with the antigen
DNP-BSA or the calcium ionophore A23187 (Calbiochem-Novabiochem) for 1 h at 37°C was carried out as described previously (Harris et al., 1997),
except that cells were treated with or without M
CD immediately before stimulation.
Confocal Immunofluorescence Microscopy
Cells sensitized with FITC-IgE were treated with or without MCD, then
washed and incubated with cytochalasin D (1 µg/ml) for 5 min at room
temperature to prevent antigen-stimulated IgE-Fc
RI internalization and
to sustain Lyn co-redistribution with patched IgE-Fc
RI at the cell surface. The cells were then stimulated with 1.7 µg/ml DNP-BSA at room
temperature for 20 min and subsequently fixed with cold methanol for
Lyn labeling or with formaldehyde for GD1b or TfR labeling as previously
described (Pierini et al., 1996
). Confocal fluorescence microscopy was performed as previously described (Pierini et al., 1996
). Cross correlation
analysis of the co-redistribution of Lyn or TfR with antigen-cross-linked FITC-IgE-Fc
RI were carried out on equatorial images of individual cells
using a computational procedure similar to that previously described
(Stauffer and Meyer, 1997
). Peak values can be calculated from this analysis to a correlation coefficient (
) (Eq. 1; Barlow, 1989
), and these values
were averaged for 7-13 cells from each sample for numerical comparison
of the degree of co-redistribution.
![]() |
(1) |
In Eq. 1, xi and yi are the intensities at each equatorial point of the FITC and Cy3 fluorescence, respectively, and < x > and < y > are the corresponding average values.
Lipid Extractions and Analyses
RBL-2H3 cells were depleted of cholesterol (or not) and repleted (or not)
as described above. After treatment, the cells were washed with BSA/
BSS, and then resuspended in methanol. The cell lysate was homogenized
with a Duall ground glass tissue grinder, transferred to an ethanolic KOH-cleaned glass vial, and an equal volume of chloroform was added, followed by vigorous vortexing and probe sonication. The samples were
rocked overnight at room temperature, then centrifuged at 800 g for 5 min, and the supernatants were transferred to a new glass vial. Chloroform/methanol (1:1, vol/vol) was added to the pellets and the suspension
was vortexed vigorously and centrifuged as described above. Supernatants
consisting of the total lipid extracts were combined and stored under nitrogen at 20°C.
The extent of cholesterol depletion or repletion was measured using a
colorimetric cholesterol oxidase assay (Boehringer Mannheim) that quantifies total free cholesterol. Total lipid extracts from paired samples of
MCD-treated and untreated cells were assayed simultaneously in these
determinations. Thin layer chromatography of the total lipid extracts was
carried out on silica gel 60 plates (EM Science) as previously described
(Cartwright, 1993
), with iodine detection. The developing solvent for polar lipids was chloroform/methanol/glacial acetic acid/water (60:50:1:4,
vol/vol), and for neutral lipids, hexane/diethyl ether/glacial acetic acid (90:
10:1, vol/vol) (Cartwright, 1993
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cholesterol Depletion Inhibits Antigen-stimulated
Tyrosine Phosphorylation of FcRI
Because cholesterol is a critical component of liquid-ordered DRMs, we investigated whether reduction of the
cholesterol content of the RBL-2H3 mast cells affects the
tyrosine phosphorylation of FcRI
and
subunits. For
this purpose, we incubated the cells with 10 mM M
CD
for 1 h at 37°C to deplete cellular cholesterol before stimulation with multivalent antigen. Fig. 1 a shows a representative antiphosphotyrosine Western blot of lysates from control and M
CD-treated cells that have been stimulated
with an optimal dose of antigen, DNP-BSA, for 0-30 min
at 37°C. For the control cells, stimulated tyrosine phosphorylation is maximal after 2 min of stimulation with 1 µg/ml
DNP-BSA, and then declines over time as previously reported (Pribluda and Metzger, 1992
; Xu et al., 1998a
). For
the M
CD-treated cells, there is a substantial reduction in
stimulated tyrosine phosphorylation in all bands detected,
including the
and
subunits of Fc
RI that are readily observed under these conditions when the 4G10 mAb is
used to detect phosphotyrosine. Fig. 1 b quantifies the
time course of tyrosine phosphorylation of Fc
RI
in this
experiment. By this method of analysis, we found that
maximal Fc
RI
tyrosine phosphorylation is inhibited 95 ± 4% by pretreatment with M
CD in six separate experiments under these optimal conditions. The identity of
Fc
RI
and
in the antiphosphotyrosine blots of whole
cell lysates was confirmed by specific immunodepletion of
these bands with anti-IgE (Fig. 1 c).
|
To further characterize the cells under conditions of
MCD treatment, we determined that untreated cells contain 6.7 ± 0.7 nmol free cholesterol/106 cells (n = 5), and,
after incubation with 10 mM M
CD at 37°C for 1 h, the
amount of free cholesterol was determined to be 2.7 ± 1.0 nmol/106 cells (n = 5). Thus, the fraction of free cholesterol remaining after treatment is 0.40 ± 0.15 as compared
with untreated cells, consistent with previously described
levels of cholesterol depletion for a variety of cell types
under similar conditions (Kilsdonk et al., 1995
; Yancey et al.,
1996
; Christian et al., 1997
; Gimpl et al., 1997
; Scheiffele et al.,
1997
; Friedrichson and Kurzchalia, 1998
; Keller and Simons, 1998
; Varma and Mayor, 1998
). TLC analysis of cellular lipid extracts showed that there was no detectable difference in phospholipid composition before and after
M
CD treatment, and this is also consistent with previous
reports from other laboratories for other cell types (Kilsdonk et al., 1995
; Yancey et al., 1996
; Christian et al., 1997
;
Gimpl et al., 1997
). In particular, we found that the
amounts of phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin present in the total lipid extracts
from M
CD-treated cells were not obviously different
from untreated cell lipids (data not shown). The amounts
of two other unidentified lipid species (one polar lipid species and one neutral lipid species) observed with TLC also
were unaffected by M
CD treatment.
To further investigate the basis for the dramatic reduction in tyrosine phosphorylation of FcRI
, we measured
the amount of Fc
RI before and after M
CD treatment
using 125I-IgE. Under our optimal conditions for inhibition
of tyrosine phosphorylation (10 mM M
CD for 1 h at
37°C), we observed a 70 ± 6% (n = 6) loss of receptor-bound IgE from the cells that could be recovered in the supernatants of the cell washes after M
CD treatment. Similarly, 64 ± 7% (n = 2) of the ganglioside GD1b was lost
from the cells due to M
CD treatment as detected using 125I-AA4 mAb. The loss of Fc
RI is probably due to vesicle shedding caused by the M
CD treatment because a
substantial fraction of 125I-IgE in the post-wash supernatant after M
CD treatment can be pelleted by high speed
centrifugation as membrane vesicles that are detectable by
phase contrast microscopy (data not shown).
In steady-state fluorescence measurements of FITC-IgE
bound to MCD-treated cells, 65 ± 10% (n = 3) IgE-
Fc
RI loss was observed. Qualitatively consistent results
were visualized with FITC-IgE and Cy3-AA4 in fluorescence microscopy of labeled cells, and a similar reduction
in the cell surface expression of the GPI-anchored protein
Thy-1 was also observed (data not shown). Despite these substantial reductions in Fc
RI and the outer leaflet markers for DRMs in these cells after M
CD treatment, the
plasma membrane expression of Lyn was only modestly
reduced as assessed from fluorescence microscopy and
Western blot analysis of sucrose gradient fractions (see below). Silver-stained polyacrylamide gels of RBL cell lysates showed no significant alterations in the amounts or
composition of proteins detected in this manner (data not shown).
These results suggest that the inhibition of antigen-stimulated tyrosine phosphorylation is due in part to a reduction in the amount of FcRI available for phosphorylation
in the M
CD-treated cells. To assess the magnitude of this
effect, we determined the relationship between Fc
RI tyrosine phosphorylation and the effective concentration of
this receptor in the plasma membrane by comparing the
amount of Fc
RI
tyrosine phosphorylation for cells in
which 30% of the receptors were occupied by anti-DNP
IgE, and 70% were occupied by anti-1,5-dansyl IgE, which
does not bind DNP ligands (Weetall et al., 1993
; see Material and Methods). Using the same conditions for stimulation and analysis as was used for the M
CD-treated cells,
we determined that at the time point for maximal stimulation DNP-BSA caused 66% (n = 2) less tyrosine phosphorylation of Fc
RI
for the cells occupied with 30%
anti-DNP IgE compared with those occupied with 100%
anti-DNP IgE. Likewise, when Fc
RI on cells were occupied by 50% anti-DNP IgE, DNP-BSA caused 41% (n = 2) less tyrosine phosphorylation of Fc
RI
than those occupied with 100% anti-DNP IgE. Assuming that this approximately proportional relationship between receptor
number and
phosphorylation is valid for the M
CD-treated cells over this range of anti-DNP IgE densities,
then the expected reduction in tyrosine phosphorylation of Fc
RI
due to 70% loss of Fc
RI should be ~70%.
Thus, the actual reduction in this value (95 ± 4%) represents an 83% inhibition of the stimulated Fc
RI
tyrosine
phosphorylation expected for the amount of cross-linked
receptors present.
Because of the nearly complete inhibition of stimulated
FcRI tyrosine phosphorylation caused by M
CD-mediated cholesterol depletion, we examined the effects of this
treatment on cellular degranulation as measured by release of
-hexosaminidase. Fig. 2 summarizes the results
from three separate experiments and shows that cholesterol depletion does not significantly inhibit degranulation stimulated by an optimal dose of antigen. Furthermore,
cholesterol depletion actually enhances the amount of degranulation observed in response to stimulation by the
Ca2+ ionophore, A23187, without altering the amount of
-hexosaminidase released in unstimulated cells. These results indicate that reduction in cellular cholesterol enhances one or more of the downstream events that follow
Ca2+ elevation and lead to degranulation. They also demonstrate that cholesterol depletion under these conditions
is not cytotoxic. As seen in Fig. 1, tyrosine phosphorylation of Fc
RI is usually inhibited more strongly than stimulated phosphorylation of some other substrates (e.g., the
stimulated bands in the range of 70-100 kD) that are
known to be dependent on activation of the tyrosine kinase Syk (Zhang et al., 1996
). These results indicate that
relatively small amounts of stimulated tyrosine phosphorylation can result in substantial degranulation responses in the M
CD-treated cells. Consistent with this,
stimulation of maximal degranulation requires effective
cross-linking of only ~10% of the IgE receptors on untreated RBL-2H3 cells (Fewtrell, 1985
).
|
Cholesterol Depletion Disrupts the Interactions of
Cross-linked IgE-FcRI and Lyn with DRM Vesicles
To determine the importance of cholesterol in DRM interactions, we examined the distributions of IgE-FcRI and
Lyn across sucrose gradients of cholesterol-depleted or
untreated RBL cells. As previously demonstrated for untreated cells (Field et al., 1997
), monomeric IgE-Fc
RI
(Fig. 3 a,
) is found predominantly in the 40% sucrose region of the gradient where cytoplasmic and detergent-solubilized membrane proteins are characteristically observed, and a large percentage of cross-linked IgE-Fc
RI
(Fig. 3 a,
) is located in the low density region of the gradient where DRM vesicles are found (Field et al., 1997
;
Scheiffele et al., 1997
; Wolf et al., 1998
). After M
CD
treatment, monomeric IgE-Fc
RI (Fig. 3 a,
) is located
in the 40% sucrose region, similar to those in the untreated cells. However, cross-linked IgE-Fc
RI (Fig. 3 a,
) no longer float to the DRM region, but rather appear in the 50-60% sucrose region where aggregates of IgE-
Fc
RI characteristically locate in the absence of interactions with DRMs (Field et al., 1997
). These results are representative of four experiments, and they show that ~60%
reduction in cholesterol almost completely prevents the
association of cross-linked IgE-Fc
RI with DRMs.
|
As shown in Fig. 3 b (top), the distribution of Lyn in the
gradient fractions of stimulated (+ sAv) and unstimulated
( sAv) cells that were not treated with M
CD is qualitatively similar to that observed by Field et al. (1995)
, who
used higher concentrations of TX-100. In the present experiments, it is notable that the p56 isoform of Lyn is selectively enriched in the low density, DRM region of the
gradient (fractions 4-9), whereas the p53 isoform is located predominately in the 40% sucrose region (fractions 10-18). After cholesterol depletion, Lyn no longer localizes in the low density region of the gradient for both stimulated and unstimulated cells, whereas the distribution of
p53 Lyn in the 40% sucrose region remains essentially unchanged from the untreated cells (Fig. 3 b, middle). In the
cholesterol-depleted cells, it appears that p56 Lyn is relatively enriched in the gradient pellet (bottom fraction),
both for the stimulated and the unstimulated cells. Overall, the total amount of Lyn detected in the gradients is
only moderately reduced in the M
CD-treated cells compared with untreated control cells.
The loss of both aggregated FcRI and Lyn from the low
density region of the gradient raises the question of
whether DRMs are disrupted entirely in the cholesterol-depleted cells. To address this question, we investigated
the distribution of other DRM markers in sucrose gradients after lysis of cholesterol-depleted cells. As shown in
Fig. 3 c, AA4-labeled GD1b from cholesterol-depleted cells is found almost completely in the low density region
of the gradients, indicating that DRMs still exist in some
form after ~60% cholesterol depletion. The shift in GD1b
distribution to a slightly higher density after cholesterol
depletion suggests an increase in the protein/lipid ratio,
consistent with the substantial loss of cholesterol, a major
lipid component of the DRM (Brown and Rose, 1992
).
These same trends are observed for both stimulated (Fig. 3
c, open symbols) and unstimulated (Fig. 3 c, closed symbols) cells. Another DRM marker, the GPI-anchored protein Thy-1 (Dráberová and Dráber, 1993
; Field et al., 1995
;
Surviladze et al., 1998
), localizes similarly to GD1b in the
sucrose gradients before and after cholesterol depletion
(data not shown). These results indicate that 60% cholesterol depletion can prevent the interactions of some proteins (Fc
RI and Lyn) with DRMs without eliminating
DRM structure.
Cholesterol Depletion Prevents the Redistribution of
Lyn with Cross-linked IgE-FcRI on Intact Cells
To evaluate how cholesterol depletion affects FcRI on intact cells, we used confocal fluorescence microscopy to examine the redistributions of Lyn and GD1b with cross-linked IgE-Fc
RI. Representative images in Fig. 4, a and
b, show that both monomeric IgE-Fc
RI (left panels) and
Lyn (right panels) are uniformly distributed in the plasma
membrane in the absence and presence of M
CD, respectively. When IgE-Fc
RI is aggregated by antigen at 22°C
for 20 min, small patches of these are formed, and these
patches often cluster together on one side of the cell. As
see in Fig. 4 c, the concentration of Lyn is enhanced in
these regions of patched receptors in the absence of
M
CD treatment. For M
CD-treated cells, IgE-Fc
RI
also redistributes into patches after aggregation by antigen
(Fig. 4 d, left), indicating that lateral mobility is not impeded by cholesterol depletion; however, Lyn does not redistribute with IgE-Fc
RI under these conditions (Fig. 4
d). As indicated in the first line of Table I, these differences are statistically significant when quantified by cross
correlation analysis of multiple cells. Thus, cross-link-
dependent interactions between Fc
RI and Lyn on the cell
surface are largely prevented by cholesterol depletion, consistent with the loss of interactions of these proteins
with DRMs in the sucrose gradient analyses of lysed cells
described above.
|
|
Fig. 4 e shows that, as previously observed (Pierini et al.,
1996), cross-linking of IgE-Fc
RI at the cell surface results in co-redistribution of the GD1b ganglioside that is labeled
by Cy3-AA4 mAb. For M
CD-treated cells, we find that
co-redistribution of this outer leaflet DRM marker with
cross-linked IgE-Fc
RI is reduced compared with control
cells but not completely disrupted as was the case for Lyn.
Fig. 4 f shows an example of this variability, in which one
cell exhibits partial co-redistribution of the labeled ganglioside, and the other shows a complete lack of co-redistribution with patched IgE-Fc
RI. Under these conditions,
20% of the cells exhibited detectable co-redistribution
of labeled ganglioside, whereas no detectable co-redistribution of Lyn with IgE-Fc
RI patches was observed.
From these results, it appears that the interaction between
Lyn and Fc
RI is more sensitive to cholesterol depletion
than is the interaction between the ganglioside and Fc
RI
in the intact cells, although both are substantially prevented. The difference observed may be related to the
greater sensitivity of the Lyn-DRM interactions than
GD1b-DRM interactions, as shown in Fig. 3 (see Discussion).
As a further control, we compared the distribution of
the transferrin receptor (CD71) to FITC-IgE-FcRI cross-linked under the same conditions as above. Previous studies showed that this transmembrane protein does not associate with isolated DRMs (Melkonian et al., 1999
), nor
does it co-redistribute with other DRM-associated proteins when simultaneously but separately cross-linked on
BHK and Jurkat T cells (Harder et al., 1998
). As seen in
Fig. 4, g and h, TfRs do not co-redistribute with cross-linked IgE-Fc
RI, and they remain evenly distributed
around the periphery of the cell, often in tiny clusters that
may reflect interactions with coated pits. Quantitative
analysis of the cross correlation of TfR with cross-linked
IgE-Fc
RI show no appreciable colocalization between
these molecules whether or not the cells have been depleted of cholesterol (see Table I). These results support
the significance of the co-redistribution of Lyn with cross-linked IgE-Fc
RI described above, as well as its inhibition
by cholesterol depletion with M
CD.
Cholesterol Repletion Restores FcRI Tyrosine
Phosphorylation Together with the Association of
Cross-linked Fc
RI and Lyn with DRMs
To investigate the reversibility of cholesterol effects on the
functional and structural interactions of FcRI with Lyn,
we restored cholesterol levels in M
CD-treated cells by
incubating them with cholesterol-M
CD complexes. The
efficiency of repletion is dependent upon the incubation
period of the cells with the complex, the molar ratio of
cholesterol to M
CD, and the final concentration of
M
CD (Christian et al., 1997
; Sheets, E.D., unpublished results). To optimize repletion, we used several dilutions
of cholesterol-M
CD complexes prepared as described in
Materials and Methods. In our sequential depletion/repletion experiment, cells were initially left untreated (control
samples) or incubated with M
CD to lower cholesterol
levels as described above. During the second step, the cells
were incubated for 2 h at 37°C at the indicated dilution of
cholesterol/M
CD, 3 mM M
CD only, or buffer only. We
found that (a) the cholesterol levels of cells depleted by
exposure to M
CD in the first step did not change during
the subsequent incubation in the absence of M
CD; (b)
the presence of 3 mM of M
CD during the second step
also did not cause additional cholesterol depletion, nor did
it alter the distribution of IgE-Fc
RI in the sucrose gradients; and (c) under optimal conditions of cholesterol repletion used (3-6 mM M
CD; 8:1, mol/mol M
CD/cholesterol), the cholesterol content of the repleted cells was
3.0-3.5-fold higher than that in the untreated control cells.
Furthermore, TLC analyses of total lipid extracts indicated that cholesterol was the only lipid that changed detectably during the depletion/repletion treatments (data
not shown).
As shown in Fig. 5, repletion of cholesterol in MCD-treated cells results in partial restoration of antigen-stimulated tyrosine phosphorylation. In the experiment shown,
maximal recovery of stimulated tyrosine phosphorylation
of Fc
RI
and other bands was achieved when 1:50 dilution of the preformed 8:1 M
CD/cholesterol complexes
was used to give a final concentration of 6 mM M
CD during the repletion step (Fig. 5, lane 10). As seen in Fig.
5, lane 6, cells that had been treated with M
CD alone
during both the depletion and repletion steps have no detectable
phosphorylation, and only a very small amount
of stimulated tyrosine phosphorylation is seen in the
higher molecular weight bands. Similar results to these
were obtained in three separate experiments. Under the conditions of cholesterol depletion/repletion, loss of IgE-
Fc
RI was determined to be 77 ± 4% (n = 5), and, based
upon the proportional relationship between receptor number and
phosphorylation (above), this leads us to expect
that optimal restoration of Fc
RI
phosphorylation should be ~23% of the stimulated control in Fig. 5, lane 2. The somewhat smaller restoration that is apparent (Fig. 5,
lanes 10 and 12) suggests that other factors, such as the
loss of other outer-leaflet DRM components during cholesterol depletion noted above, may reduce the maximum
restoration achievable (see Discussion).
|
Repletion of cholesterol also results in restoration of
cross-link-dependent association of IgE-FcRI with isolated DRMs. When lysates of cholesterol-repleted cells
are analyzed on sucrose gradients, cross-linked IgE-Fc
RI
(Fig. 3 a,
) migrates to the low density sucrose region,
whereas uncross-linked IgE-Fc
RI (Fig. 3 a,
) is found
in the 40% sucrose region, similar to the gradient distributions from control cells (Fig. 3 a,
and
). Cross-
linked IgE-Fc
RI from cholesterol-repleted cells migrate
at slightly lower densities in the sucrose gradients than this
complex in the control cells, suggesting that the average
density of DRMs in repleted cells is lower than in control
cells, possibly due to a decrease in the protein/lipid ratio
resulting from an increased cholesterol content. Furthermore, as shown in Fig. 3 b (bottom), p56 Lyn also migrates
to the low density region of the gradient (fractions 1-6) after cholesterol repletion in cells with both cross-linked and
uncross-linked Fc
RI. These results, in parallel with the
restoration of stimulated Fc
RI tyrosine phosphorylation (Fig. 5), provide strong evidence that cholesterol is important for functional coupling of Fc
RI with Lyn and for
their mutual association with DRMs.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results demonstrate that cholesterol plays a critical
role in the initial step of FcRI signaling: antigen-stimulated tyrosine phosphorylation of this receptor by the Src
family tyrosine kinase Lyn. In parallel with loss of this
stimulated phosphorylation (Fig. 1), reduction of cellular
cholesterol by M
CD causes the loss of association of
both Lyn and cross-linked Fc
RI with DRMs isolated after cell lysis by TX-100 (Fig. 3). Restoration of the cholesterol content of the depleted cells using preformed cholesterol-M
CD complexes restores the association of Lyn
and cross-linked IgE-Fc
RI with DRMs (Fig. 3) and also
causes partial restoration of antigen-stimulated tyrosine
phosphorylation in the cells (Fig. 5). These results support
the hypothesis that interactions of cross-linked IgE-Fc
RI with DRMs are important for the initial coupling of Fc
RI
and Lyn that results in receptor phosphorylation. On the
cell surface, the association of Lyn with aggregated IgE-
Fc
RI is lost as the result of cholesterol depletion (Fig. 4),
indicating that the interactions detected in isolated DRMs
are relevant to those occurring in intact cells. Furthermore, these microscopy results argue against a direct interaction of cross-linked Fc
RI with Lyn as the basis for association of receptors with DRMs, and they support the view that the Lo structure of the plasma membrane is important
for Fc
RI-Lyn interactions.
An initially surprising finding in our studies is the apparently selective loss of FcRI and outer leaflet plasma
membrane components of DRMs due to cholesterol depletion by M
CD. As indicated by Western blot analysis
and fluorescence microscopy, there is a smaller loss of Lyn
due to cholesterol depletion, and there is no detectable
loss of other cellular proteins by silver stain analysis of
whole cell lysates (data not shown). The mechanism by
which cholesterol depletion causes this selective loss is not yet known, but it is interesting to speculate that vesicles
containing these components may pinch off from the cells
in a mechanism that depends on their local structural environment in the plasma membrane. In a recent study by
Ilangumaran and Hoessli (1998)
, a similar preferential release of DRM components by M
CD treatment was characterized in lymphocytes and endothelial cells, and evidence for release of these components in membrane
vesicles was described. Our results suggest that Fc
RI may
preferentially associate with DRM components on intact
cells even in the absence of receptor cross-linking. Consistent with this, Basciano et al. showed that pre-binding of
AA4 mAb or its Fab fragment to the
-galactosyl GD1b
antigen on RBL-2H3 cells can effectively inhibit the subsequent binding of IgE to Fc
RI (Basciano et al., 1986
). By
varying the cell surface density of antigen-specific IgE in
the range of 30-100%, we show that the loss of Fc
RI due
to cholesterol depletion cannot account for the nearly
complete inhibition of Fc
RI tyrosine phosphorylation that is observed in these cells. Furthermore, the partial
restoration of antigen-stimulated tyrosine phosphorylation without an increase in Fc
RI expression after cholesterol repletion strengthens the evidence that cellular cholesterol critically regulates the coupling of the remaining
Fc
RI and Lyn.
An important observation by fluorescence microscopy
is that cholesterol depletion does not prevent antigen-dependent aggregation of IgE-FcRI on the cell surface,
even though it prevents the co-redistribution of Lyn and
inhibits stimulated tyrosine phosphorylation. We previously showed that the cholesterol-binding polyene antibiotic, filipin, prevents anti-IgE-mediated patching of IgE-
Fc
RI (Feder et al., 1994
), indicating that it may prevent
aggregation of the receptor necessary to initiate signaling.
Unlike M
CD, which extracts cholesterol into a water-soluble complex that can be washed away, filipin forms complexes with cholesterol in the membrane that appear to restrict lateral diffusion of at least some membrane proteins,
making this reagent less useful for studying the role of
cholesterol in signaling by receptors that must aggregate in response to their ligands to be effective. The rapidity with
which M
CD can reduce cell cholesterol by substantial
amounts without significantly compromising cell integrity,
as evidenced by our degranulation results, and the capacity to restore stimulated tyrosine phosphorylation by reintroduction of cholesterol via M
CD complexes, make this
an extremely valuable tool for investigating the role of
cholesterol in a wide variety of receptor systems.
Recent studies used MCD to investigate the role of
cholesterol in signaling by other receptors. Pike and Miller
(1998)
showed that cholesterol depletion by M
CD inhibits EGF- and bradykinin-stimulated phosphatidylinositol
turnover, which can be restored by cholesterol repletion
with M
CD-cholesterol complexes. These receptors belong to the families of intrinsic tyrosine kinase receptors
and G protein-coupled receptors, respectively, suggesting
the potentially general importance of cholesterol and the
Lo structure it confers on the plasma membrane in mediating receptor signaling. Interestingly, cholesterol depletion by M
CD does not inhibit EGF-stimulated tyrosine
phosphorylation of its receptor (Pike, L.J., Y. Liu, K.N.
Chung, and J.A. Heuser. 1998. FASEB J. 12:A1278 [abstr.]), which probably occurs via a transphosphorylation
mechanism (Weiss and Schlessinger, 1998
). Rather, cholesterol depletion appears to affect the compartmentalization of phosphatidylinositol 4,5-bisphosphate, the primary
phospholipase C substrate in the plasma membrane, as revealed by its reduced localization with DRMs in sucrose
gradients (Pike and Miller, 1998
). In contrast, our results
with IgE receptors indicate a role for cholesterol in the initial signaling step in which these receptors are phosphorylated by Lyn, and this finding may have general relevance
for other receptors that function by interacting with Src
family kinases. Indeed, Xavier et al. (1998)
and Moran and
Miceli (1998)
showed that pretreatment of T cells with
M
CD inhibits T cell receptor for antigen-mediated Ca2+
mobilization and tyrosine phosphorylation, respectively,
providing evidence for an important role for cholesterol in
the function of this related multichain immune recognition
receptor family member.
Our degranulation results (Fig. 2) suggest that normal
levels of cholesterol may negatively regulate downstream
signaling or the exocytotic process in the RBL-2H3 cells.
The enhancement of degranulation stimulated by Ca2+
ionophore, as well as the lack of inhibition of antigen-stimulated degranulation, despite the dramatic inhibition of tyrosine phosphorylation by cholesterol depletion, are consistent with this explanation. In addition, preliminary
experiments on the effects of cholesterol depletion on
Ca2+ mobilization by antigen indicate that this activity is
inhibited less than is stimulated tyrosine phosphorylation
of FcRI, consistent with differential effects of cholesterol
depletion on different signaling steps (Holowka, D., and
E.D. Sheets, unpublished results). Membrane structural
changes involved in exocytosis may also be affected. As
described above, the proportionality of Fc
RI tyrosine phosphorylation with the number of receptors cross-linked in the range of 30-100% does not hold for more
downstream signaling events, as only a small fraction of
Fc
RI needs to be cross-linked to achieve maximal degranulation. In future experiments, it will be interesting
to explore the effects of cholesterol depletion on specific downstream signaling pathways and exocytic membrane events through bypassing receptor-mediated signaling with alternate means of activation.
The results described here are consistent with the hypothesis that cross-linked IgE-FcRI interact with Lyn-containing DRM domains on the cell surface and that
these structural interactions are integral to the initiation of
signal transduction. Monomeric Fc
RI probably interact
dynamically with DRM components, and these transient
complexes may exist in the plasma membrane of unstimulated cells as small clusters similar to those recently described for certain GPI-anchored proteins (Friedrichson
and Kurzchalia, 1998
; Varma and Mayor, 1998
). In this hypothesis, cross-linking of IgE-Fc
RI on the cell surface
causes them to cluster with DRM components, thereby
creating larger Lo regions containing Fc
RI and Lyn that are segregated from more fluid regions of the plasma
membrane. It is likely that most transmembrane proteins
are more readily accommodated by phospholipids in the
more fluid liquid crystalline phase, and these would then
segregate from the Lo regions containing Fc
RI and DRM
components. For example, the tyrosine phosphatase CD45
is a transmembrane protein that was shown to be largely
excluded from DRMs on T cells (Rodgers and Rose, 1996
)
and has been recently shown to negatively regulate the Src
family kinase Lck (D'Oro and Ashwell, 1999
). Our microscopy results (Fig. 4 and Holowka, D., E.D. Sheets, and
B. Baird, manuscript in preparation) indicate that segregated DRM domains occupy a large percentage of the cell surface (20-50%) as detectable within the limits of optical
resolution, and earlier studies indicated that DRM phospholipids represent a similarly large percentage of plasma
membrane phospholipids (Mescher and Apgar, 1985
).
Thus, segregation of certain proteins from others may be
more important for signaling promoted by DRM interactions of cross-linked Fc
RI and Lyn than an increased localized concentration of these DRM-associated proteins
within domains.
In our model, cholesterol is an essential component for
the Lo phase, and its 60% reduction, as in the studies presented here, appears to most greatly affect the association
of the transmembrane protein FcRI and the inner leaflet
component Lyn with DRMs (Figs. 3 and 4). Outer leaflet
DRM components are retained in the low-density, TX-100-insoluble membrane vesicles (Fig. 3), and they still
maintain a small but detectable association with cross-linked IgE-Fc
RI on intact cells (Fig. 4 f and data not
shown), which may reflect the continued presence of an Lo
environment in the outer leaflet under conditions of diminished cholesterol. It is possible that sphingomyelin and
other sphingolipids enriched in the outer leaflet of the
plasma membrane (Devaux, 1991
) cause a preferential retention of cholesterol in this leaflet of the bilayer under
conditions of limiting cholesterol, since this particular class
of phospholipids may interact preferentially with cholesterol (Brown, 1998
). Thus, cholesterol-dependent associations at the inner leaflet of the plasma membrane may be
more sensitive to cholesterol depletion than are such interactions in the outer leaflet. As an alternative explanation
for our results, it is possible that cholesterol serves as a
critical boundary lipid for Fc
RI that facilitates a direct interaction with Lyn. However, this explanation would not
account for the association of these components with
DRMs and the correlation between loss of this structural
association and loss of functional coupling. The involvement of DRMs in functional coupling between Fc
RI and
Lyn as a means of promoting the proximity of these proteins while excluding transmembrane tyrosine phosphatases such as CD45 is an attractive hypothesis that warrants further examination.
![]() |
Footnotes |
---|
Received for publication 4 November 1998 and in revised form 11 March 1999.
Address correspondence to Barbara Baird or David Holowka, Department of Chemistry & Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY 14853-1301. Tel.: (607) 255-4095 or (607) 255-6140. Fax: (607) 255-4137. E-mail: bab13{at}cornell.edu or dah24{at}cornell.edu
We thank Eric Holowka for carrying out the cross correlation analysis, using software developed in our laboratory by Paul Pyenta.
Supported by National Institutes of Health grants AI09838 (E.D. Sheets) and AI22449.
![]() |
Abbreviations used in this paper |
---|
BSA/BSS, BSA-containing buffered
saline solution;
DRM, detergent-resistant membrane;
GPI, glycosylphosphatidylinositol;
HRP, horseradish peroxidase;
Lo, liquid-ordered;
MCD, methyl-
-cyclodextrin;
TfR, transferrin receptor;
TX-100, Triton
X-100.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Ahmed, S.N., D.A. Brown, and E. London. 1997. On the origin of sphingolipid/ cholesterol-rich detergent-insoluble cell membranes: physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes. Biochemistry 36: 10944-10953 |
2. | Barlow, R.J. 1989. Statistics: A Guide to the Use of Statistical Methods in the Physical Sciences. John Wiley and Sons, Chichester, UK. 204 pp. |
3. |
Basciano, L.K.,
E.H. Berenstein,
L. Kmak, and
R.P. Siraganian.
1986.
Monoclonal antibodies that inhibit IgE binding.
J. Biol. Chem.
261:
11823-11831
|
4. | Brown, D.A., and E. London. 1998a. Functions of lipid rafts in biological membranes. Annu. Rev. Cell Biol. 14: 111-136 |
5. | Brown, D.A., and E. London. 1998b. Structure and origin of ordered lipid domains in biological membranes. J. Membr. Biol. 164: 103-114 |
6. | Brown, D.A., and J.K. Rose. 1992. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68: 533-544 |
7. |
Brown, R.E..
1998.
Sphingolipid organization in biomembranes: what physical
studies of model membranes reveal.
J. Cell Sci.
111:
1-9
|
8. | Cartwright, I.J. 1993. Separation and analysis of phospholipids by thin layer chromatography. In Methods in Molecular Biology. Vol. 19. J.M. Graham and J.A. Higgins, editors. Humana Press, Totowa, NJ. 153-167. |
9. |
Chang, E.Y.,
Y. Zheng,
D. Holowka, and
B. Baird.
1995.
Alteration of lipid
composition modulates Fc![]() |
10. | Christian, A.E., M.P. Haynes, M.C. Phillips, and G.H. Rothblat. 1997. Use of cyclodextrins for manipulating cellular cholesterol content. J. Lipid Res. 38: 2264-2272 [Abstract]. |
11. | Devaux, P.F.. 1991. Static and dynamic lipid asymmetry in cell membranes. Biochemistry 30: 1163-1173 |
12. |
D'Oro, U., and
J.D. Ashwell.
1999.
Cutting edge: the CD45 tyrosine phosphatase is an inhibitor of Lck activity in thymocytes.
J. Immunol.
162:
1879-1883
|
13. | Dráberová, L., and P. Dráber. 1993. Thy-1 glycoprotein and src-like protein-tyrosine kinase p53/56lyn are associated in large detergent-resistant complexes in rat basophilic leukemia cells. Proc. Natl. Acad. Sci. USA. 90: 3611-3615 [Abstract]. |
14. | Edidin, M.. 1997. Lipid microdomains in cell surface membranes. Curr. Opin. Struct. Biol. 7: 528-532 |
15. | Feder, T.J., E.-Y. Chang, D. Holowka, and W.W. Webb. 1994. Disparate modulation of plasma membrane protein lateral mobility by various cell permeabilizing agents. J. Cell. Physiol. 158: 7-16 |
16. | Fewtrell, C. 1985. Activation and desensitization of receptors for IgE on tumor basophils. In Calcium in Biological Systems. R.P. Ruben, G.B. Weiss, and J.W. Putney, editors. Plenum, New York. 129-136. |
17. |
Field, K.A.,
D. Holowka, and
B. Baird.
1995.
Fc![]() |
18. |
Field, K.A.,
D. Holowka, and
B. Baird.
1997.
Compartmentalized activation of
the high affinity immunoglobulin E receptor within membrane domains.
J.
Biol. Chem.
272:
4276-4280
|
19. | Friedrichson, T., and T.V. Kurzchalia. 1998. Microdomains of GPI-anchored proteins in living cells revealed by crosslinking. Nature 394: 802-805 |
20. | Ge, M., K.A. Field, R. Aneja, D. Holowka, B. Baird, and J. Freed. 1999. ESR characterization of liquid ordered phase of detergent resistant membranes from RBL-2H3 cells. Biophys. J. In press. |
21. | Gimpl, G., K. Burger, and F. Fahrenholz. 1997. Cholesterol as modulator of receptor function. Biochemistry 36: 10959-10974 |
22. |
Harder, T.,
P. Scheiffele,
P. Verkade, and
K. Simons.
1998.
Lipid domain structure of the plasma membrane revealed by patching of membrane components.
J. Cell Biol.
141:
929-942
|
23. |
Harris, N.T.,
B. Goldstein,
D. Holowka, and
B. Baird.
1997.
Altered patterns of
tyrosine phosphorylation and Syk activation for sterically restricted cyclic
dimers of IgE-Fc![]() |
24. | Ilangumaran, S., and D.C. Hoessli. 1998. Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane. Biochem. J. 335: 433-440 |
25. |
Jouvin, M.-H.E.,
M. Adamczewski,
R. Numerof,
O. Letourneur,
A. Vallé, and
J.-P. Kinet.
1994.
Differential control of the tyrosine kinases Lyn and Syk by
the two signaling chains of the high affinity immunoglobulin E receptor.
J.
Biol. Chem.
269:
5918-5925
|
26. |
Keller, P., and
K. Simons.
1998.
Cholesterol is required for surface transport on
influenza virus hemagglutinin.
J. Cell Biol.
140:
1357-1367
|
27. |
Kilsdonk, E.P.C.,
P.G. Yancey,
G.W. Stoudt,
F.W. Bangerter,
W.J. Johnson,
M.C. Phillips, and
G.H. Rothblat.
1995.
Cellular cholesterol efflux mediated
by cyclodextrins.
J. Biol. Chem.
270:
17250-17256
|
28. |
Liu, F.-T.,
J.W. Bohn,
E.L. Ferry,
H. Yamamoto,
C.A. Molinaro,
L.A. Sherman,
N.R. Klinman, and
D.H. Katz.
1980.
Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization.
J.
Immunol.
124:
2728-2737
|
29. |
Melkonian, K.A.,
A.G. Ostermeyer,
J.Z. Chen,
M.G. Roth, and
D.A. Brown.
1999.
Role of lipid modifications in targeting proteins to detergent-resistant
membrane rafts. Many raft proteins are acylated, while few are prenylated.
J. Biol. Chem.
274:
3910-3917
|
30. | Menon, A.K., D. Holowka, and B. Baird. 1984. Small oligomers of immunoglobulin E (IgE) cause large-scale clustering of IgE receptors on the surface of rat basophilic leukemia cells. J. Cell Biol. 98: 577-583 [Abstract]. |
31. | Mescher, M.F., and J.R. Apgar. 1985. The plasma membrane `skeleton' of tumor and lymphoid cells: a role in cell lysis? Adv. Exp. Med. Biol. 184: 387-400 |
32. | Moran, M., and M.C. Miceli. 1998. Engagement of GPI-linked CD48 contributes to TCR signals and cytoskeletal reorganization: a role for lipid rafts in T cell activation. Immunity 9: 787-796 |
33. |
Pierini, L.,
D. Holowka, and
B. Baird.
1996.
Fc![]() |
34. |
Pike, L.J., and
J.M. Miller.
1998.
Cholesterol depletion delocalizes phosphatidylinositol bisphosphate and inhibits hormone-stimulated phosphatidylinositol turnover.
J. Biol. Chem.
273:
22298-22304
|
35. | Pribluda, V.S., and H. Metzger. 1992. Transmembrane signaling by the high-affinity IgE receptor on membrane preparations. Proc. Natl. Acad. Sci. USA. 89: 11446-11450 [Abstract]. |
36. |
Pribluda, V.S.,
C. Pribluda, and
H. Metzger.
1994.
Transphosphorylation as the
mechanism by which the high-affinity receptor for IgE is phosphorylated
upon aggregation.
Proc. Natl. Acad. Sci. USA.
91:
11246-11250
|
37. | Racchi, M., R. Baetta, N. Salvietti, P. Ianna, G. Franceschini, R. Paoletti, R. Fumagalli, S. Govoni, M. Trabucci, and M. Soma. 1997. Secretory processing of amyloid precursor protein is inhibited by increase in cellular cholesterol content. Biochem. J. 322: 893-898 |
38. | Rodgers, W., and J.K. Rose. 1996. Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domains. J. Cell Biol. 135: 1515-1523 [Abstract]. |
39. |
Scheiffele, P.,
M.G. Roth, and
K. Simons.
1997.
Interaction of influenza virus
haemagglutinin with sphingolipid-cholesterol membrane domains via its
transmembrane domain.
EMBO (Eur. Mol. Biol. Organ.) J.
16:
5501-5508
|
40. |
Schroeder, R.,
E. London, and
D. Brown.
1994.
Interactions between saturated
acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and
cells show similar behavior.
Proc. Natl. Acad. Sci. USA.
91:
12130-12134
|
41. |
Schroeder, R.J.,
S.N. Ahmed,
Y. Zhu,
E. London, and
D.A. Brown.
1998.
Cholesterol and sphingolipid enhance the Triton X-100 insolubility of glycosylphosphatidylinositol-anchored proteins by promoting the formation of
detergent-insoluble ordered membrane domains.
J. Biol. Chem.
273:
1150-1157
|
42. | Sheets, E.D., D. Holowka, and B. Baird. 1999. Membrane organization in immunoglobulin E receptor signaling. Curr. Opin. Chem. Biol. 3: 95-99 . |
43. | Simons, K., and E. Ikonen. 1997. Functional rafts in cell membranes. Nature 387: 569-572 |
44. |
Stauffer, T.P., and
T. Meyer.
1997.
Compartmentalized IgE receptor-mediated
signal transduction in living cells.
J. Cell Biol.
139:
1447-1454
|
45. | Subramanian, K., D. Holowka, B. Baird, and B. Goldstein. 1996. The Fc segment of IgE influences the kinetics of dissociation of a symmetrical bivalent ligand from cyclic dimeric complexes. Biochemistry 35: 5518-5527 |
46. | Surviladze, Z., L. Dráberová, L. Kubínová, and P. Dráber. 1998. Functional heterogeneity of Thy-1 membrane microdomains in rat basophilic leukemia cells. Eur. J. Immunol. 28: 1847-1858 |
47. | Thomas, J.L., D. Holowka, B. Baird, and W.W. Webb. 1994. Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins. J. Cell Biol. 125: 795-802 [Abstract]. |
48. | Varma, R., and S. Mayor. 1998. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature 394: 798-801 |
49. |
Weetall, M.,
D. Holowka, and
B. Baird.
1993.
Heterologous desensitization of
the high affinity receptor for IgE (Fc![]() |
50. | Weiss, A., and J. Schlessinger. 1998. Switching signals on or off by receptor dimerization. Cell 94: 277-280 |
51. |
Wolf, A.A.,
M.G. Jobling,
S. Wimer-Macklin,
M. Ferguson-Maltzman,
J.L. Madera,
R.K. Holmes, and
W.I. Lencer.
1998.
Ganglioside structure dictates
signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia.
J. Cell Biol.
141:
917-927
|
52. | Xavier, R., T. Brennan, Q. Li, C. McCormack, and B. Seed. 1998. Membrane compartmentation is required for efficient T cell activation. Immunity 8: 723-732 |
53. |
Xu, K.,
B. Goldstein,
D. Holowka, and
B. Baird.
1998a.
Kinetics of multivalent
antigen DNP-BSA binding to IgE-Fc![]() ![]() |
54. |
Xu, K.,
R.M. Williams,
D. Holowka, and
B. Baird.
1998b.
Stimulated release of
fluorescently labeled IgE fragments that efficiently accumulate in secretory
granules after endocytosis in RBL-2H3 mast cells.
J. Cell Sci.
111:
2385-2396
|
55. |
Yancey, P.G.,
W.V. Rodrigueza,
E.P.C. Kilsdonk,
G.W. Stoudt,
W.J. Johnson,
M.C. Phillips, and
G.H. Rothblat.
1996.
Cellular cholesterol efflux mediated
by cyclodextrins. Demonstration of kinetic pools and mechanism of efflux.
J.
Biol. Chem.
271:
16026-16034
|
56. | Zhang, J., E.H. Berenstein, R.L. Evans, and R.P. Siraganian. 1996. Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells. J. Exp. Med. 184: 71-79 [Abstract]. |