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Blood, 1 September 2001, Vol. 98, No. 5, pp. 1489-1497
IMMUNOBIOLOGY
Role for lipid rafts in regulating interleukin-2
receptor signaling
Mina D. Marmor and
Michael Julius
From Sunnybrook and Women's College Health Sciences
Centre and the Department of Immunology, University of Toronto,
Ontario, Canada.
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Abstract |
Lipid rafts are plasma membrane microdomains characterized by a
unique lipid environment enriched in gangliosides and cholesterol, leading to their insolubility in nonionic detergents. Many receptors are constitutively or inducibly localized in lipid rafts, which have
been shown to function as platforms coordinating the induction of
signaling pathways. In this report, the first evidence is provided for
a role of these lipid microdomains in regulating interleukin-2 receptor
(IL-2R) signaling. It is demonstrated that antibody- or ligand-mediated
immobilization of components of lipid rafts, glycosyl-phosphatidyl-inositol-anchored proteins, and the GM1 ganglioside, respectively, inhibit IL-2-induced proliferation in T
cells. IL-2R is shown to be constitutively enriched in rafts and
further enriched in the presence of immobilized anti-Thy-1. In
contrast, IL-2R and IL-2R , as well as JAK1 and JAK3, are found in
soluble membrane fractions, and their localization is not altered by
anti-Thy-1. IL-2-mediated heterotrimerization of IL-2R chains is
shown to occur within soluble membrane fractions, exclusively, as is
the activation of JAK1 and JAK3. As predicted by these results, the
disruption of lipid raft integrity did not impair IL-2-induced
signaling. Thus, the sequestration of IL-2R within lipid
microdomains restricts its intermolecular interactions and regulates
IL-2R signaling through impeding its association with IL-2R and
IL-2R.
(Blood. 2001;98:1489-1497)
© 2001 by The American Society of Hematology.
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Introduction |
Lipid rafts are plasma membrane microdomains
postulated to function in signaling and membrane
trafficking.1-3 Lipid rafts are enriched in gangliosides
(glycosphingolipids) and cholesterol, which form liquid-ordered domains
of decreased membrane fluidity. The long, saturated acyl chains of
gangliosides impart a high degree of order further stabilized by
intercalating cholesterol molecules, leading to the insolubility of
lipid rafts in nonionic detergents. Lipid rafts can be isolated based
on their detergent insolubility and low-buoyancy density using
discontinuous sucrose gradient ultracentrifugation of nonionic
detergent lysates. Lipid rafts are not artifacts of detergent
extraction. They have been detected in living cells using chemical
cross-linking and fluorescence resonance energy
transfer.4,5
The modification of proteins with saturated acyl groups can result in
their localization within lipid rafts. Thus, these microdomains are
enriched in glycosyl-phosphatidyl-inositol anchored proteins (GPI-AP)
and in many signaling molecules such as Src family protein tyrosine
kinases (PTKs), the adaptor protein LAT, heterotrimeric and small
G-proteins, and phosphoinositides.3 In addition, several
transmembrane receptors are inducibly recruited to or stabilized within
lipid rafts, including T-cell receptor (TCR), B-cell receptor
(BCR), and Fc RI.2 Subsequent activation of signaling molecules in lipid rafts may facilitate signaling through these immunoreceptors. Lipid rafts may act to segregate molecules in
the plasma membrane and to regulate signaling through the spatial coordination of intermolecular associations.
Stimulation of T cells through TCR results in the activation of
multiple signaling pathways, leading to interleukin-2 (IL-2) responsiveness and the secretion of IL-2 and resulting in autocrine cell growth.6 The high-affinity receptor for IL-2 is
composed of the IL-2R chain, which functions solely in IL-2 binding,
and IL-2R and IL-2Rc, which contribute to IL-2
binding and mediate signal transduction.7 IL-2-induced
proliferation requires activation of the Janus family kinases JAK1 and
JAK3, which are constitutively associated with IL-2R and IL-2R,
respectively.8,9 Ligand-induced IL-2R aggregation leads to
the juxtaposition of JAK1 and JAK3, resulting in their phosphorylation
and activation. Subsequent phosphorylation of tyrosine residues in
receptor chains leads to the SH2-domain-mediated recruitment of signal
transducer and activator of transcription (STAT) proteins STAT5a and
STAT5b.7 JAK-mediated phosphorylation of STAT proteins
leads to their dimerization by SH2 domain-phosphotyrosine interactions
and their translocation to the nucleus, where STAT proteins regulate
gene transcription. Signaling through the IL-2R also induces the
recruitment and activation of phosphatidylinositol 3 kinase (PI3K),
which is implicated in IL-2-mediated proliferation and survival
through its downstream effector protein kinase B/Akt.10,11
In addition, the tyrosine phosphorylation of IL-2R results in the
recruitment of Shc and Grb2 and activation of the Ras/MAPK pathway.
Lck, Syk, and Pyk-2 also associate with IL-2R; however, the
functional outcome of these interactions remains unclear.7
Similar to many other receptors, IL-2R is not randomly distributed in
the lipid bilayer. IL-2R, IL-2R, and IL-2R chains appear to
form pre-existing complexes on the surfaces of T cells, brought closer
by ligand binding,12 resulting in the aggregation of
IL-2R and IL-2R required for signaling.13,14 In
addition, IL-2R has been found in cell surface clusters that appear
to correspond to lipid rafts.15 In this study, we
demonstrate that components of lipid rafts modify signaling through
IL-2R and characterize the involvement of lipid rafts in IL-2R signaling.
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Materials and methods |
Cells, antibodies, and flow cytometry
It has been reported that 2.10 is an IL-2-dependent,
CD4 T-cell clone.16 Stable 2.10 clonal
variants expressing or lacking GPI-AP were isolated through the sorting
of Thy-1+ and Thy-1 cells on a FACStar Plus
(Becton Dickinson, Mountain View, CA). The CTLL-2 T-cell line
was obtained from American Type Culture Collection (Rockville,
MD). Primary CD8+ T cells were purified from the
lymph nodes of C57Bl/6 mice as described previously.17 The
CD8+ T-cell preparations were consistently found to be
greater than 95% CD8+TCR+ when assessed
by flow cytometry.
Antibodies used in this study and described previously18
include monoclonal antibodies (mAbs) specific for Thy-1 (30H12, M5/49,
and 5-3.2.1), CD4 (GK1.5, rat IgG2b mAb isotype control and H129, rat
IgG2a mAb isotype control), Ly6A/E (D7), TcR-C (H57-597), and CD48
(5-8A10). In addition, anti-CD45 [M18919] and anti-CD5
[53-7.320] were used. Normal hamster immunoglobulin G
(IgG) (Jackson Immunoresearch, Westgrove, PA) was used as an isotype
control for anti-CD48. Unless otherwise indicated, anti-Thy-1 refers
to the 30H12 mAb.
Flow cytometric analysis was performed by labeling cells with the
indicated antibodies for 10 minutes, followed by 3 washes. Expression
of GPI-AP was determined using anti-Thy-1 or anti-Ly6A/E followed by
fluorescein isothiocyanate (FITC)-mouse anti-rat  or
FITC-conjugated anti-CD48. Before analysis on a FACScalibur (Becton
Dickinson), cells were resuspended in 1 µg/mL 7-amino-actinomycin-D (7AAD; Sigma, St Louis, MO). Plots shown exclude dead cells based on
forward- versus side-scattering profiles and on positive staining with 7AAD.
Cellular DNA content was determined by the Vindelov
method.21 Cells cultured as indicated were harvested,
pelleted, and resuspended in Vindelov solution (3.4 mM Tris HCl, pH
7.6, 10 mM NaCl, 0.1% vol/vol NP-40, 50 µg/mL propidium iodide
[Sigma], and 20 µg/mL RNase A [Boehringer Mannheim Laval, QC,
Canada]). The proportion of cells with subdiploid DNA content was
assessed by flow cytometry on a FACScalibur, using doublet
discrimination in the FL2 channel.
Proliferation assays
Purified cholera toxin subunit (CT; Sigma) and all mAbs used
in this study were diluted to 10 µg/mL in Hanks balanced salt solution without CaCl2 or MgCl2, except
anti-TCR, which was used at 1 µg/mL. CT or antibody solutions were
incubated in wells of 96-well plates for 1 hour at 37°C. After 2 washes with Hanks balanced salt solution, 2 × 104 2.10 or CTLL-2 cells or 5 × 104 primary T cells were added in
serum-free medium.16 After 20 hours, each culture was
pulsed for 6 hours with 1 µCi of 3H-thymidine and
thymidine uptake assessed using a Topcount Microplate scintillation
counter (Canberra Packard, Meriden, CT). Where indicated, primary
CD8+ T cells were stimulated for 20 hours with anti-TCR.
Viable cells were isolated on a Lympholyte M gradient (Cedarlane, ON,
Canada) and cultured as described above. Supernatant from the X630
hybridoma transfected with complementary DNA (cDNA) encoding IL-2 was
used as a source of cytokine.22 The activity of
IL-2-containing supernatant was quantitated by bioassay, and 1 U was
defined as resulting in half-maximal proliferation of
5 × 103 CTLL-2 cells cultured for 48 hours.
Isolation of lipid rafts
Lipid rafts were isolated by discontinuous sucrose density
gradient ultracentrifugation. Cells were lysed at
2 × 107 cells/mL in TKM buffer (50 mM Tris, pH 7.4, 25 mM KCl, 5 mM MgCl2, and 1 mM EDTA) containing 0.5% wt/vol
Brij58 and protease inhibitors leupeptin (2.5 µg/mL), aprotinin (2.5 µg/mL), and Pefabloc (1 mM), all from Boehringer Mannheim. Lysates
were incubated on ice for 30 minutes, mixed with an equal volume of
80% wt/vol sucrose in TKM, and overlaid with 5.5 mL 36% sucrose
followed by 2.5 mL 5% sucrose. The gradients were subjected to
ultracentrifugation at 250 000g for 16 to 18 hours in an
SW41 rotor (Becton Dickinson), and 1-mL fractions were collected from
the top of the gradient.
Immunoprecipitations and immunoblotting
Protein or glycolipid content of fractions isolated from
sucrose density gradients was determined by immunoblotting. GM1
was detected using CT conjugated to horseradish-peroxidase (HRP). Fyn,
Thy-1, and CD45 were detected using anti-Thy-1 and anti-CD45 followed by rabbit anti-rat IgG-HRP (Sigma) and anti-Fyn serum (provided by Dr A. Veillette, IRCM, Montreal, QC, Canada)
followed by protein-A-HRP (ICN, Costa Mesa, CA).
The localization of IL-2R chains was assessed in CTLL-2 cells, which
were starved for 16 hours in 1.25 U/mL IL-2 (unstimulated) or after
stimulation with 400 U/mL IL-2. Cells were pelleted and lysed in
TKM/Brij58, and lipid rafts were isolated. To assess the effect of
immobilized anti-Thy-1 on the localization of IL-2R chains, rafts were
isolated from CTLL-2 cells cultured for 16 hours with 15 U/mL IL-2 in
flasks coated with anti-Thy-1 or control mAbs. As a positive control
for immunoblotting, immunoprecipitation of IL-2R, IL-2R, and
IL-2R was performed as described previously.18 Immunoprecipitates or 150 µL-fractions from sucrose gradients were
resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis was performed using polyclonal rabbit
anti-IL-2R, IL-2R, and IL-2R (Santa Cruz Biotechnology, Santa
Cruz, CA) followed by Protein-A-HRP.
To determine the localization of JAK1 and JAK3, equal amounts
of pooled lipid raft and soluble fractions as assessed by
GM1 immunoblottingwere diluted 4-fold in TX-100 buffer containing 50 mM HEPES, pH 7.5, 1% Triton X-100, 150 mM NaCl, 10% glycerol, 1.5 mM
MgCl2, 1 mM Na2VO4, and protease
inhibitors. Immunoprecipitations were performed using anti-JAK1
(Transduction, Lexington, KY) and JAK3-specific antisera (UBI, Lake
Placid, NY) and were collected using Protein-A- or
Protein-G-Sepharose beads, respectively. Proteins were resolved on
SDS-PAGE gels run in parallel, and immunoblot analysis was performed
using antiphosphotyrosine [4G1023] and anti-JAK1 followed
by HRP-goat, anti-mouse IgG (GaMIg-HRP; Sigma), or JAK3-specific
antisera followed by Protein-A-HRP.
To determine the effect of disrupting lipid rafts on IL-2R-induced
signaling, 107 cells/mL were incubated in 10 mM
methyl--cyclodextrin (MCD; Aldrich, Milwaukee, WI) for 20 minutes at
37°C.24 Where indicated, 400 U/mL IL-2 was added, and
cells were incubated for an additional 10 minutes. Cells were pelleted
and lysed in TX-100 buffer, and immunoprecipitation and immunoblot
analyses of JAK1 and JAK3 from postnuclear lysates were performed as
described above. To assess the effect of MCD on TCR-induced signaling,
MCD-treated 2.10 were pelleted and incubated at 107
cells/mL with 2.5 µg/mL biotin-labeled anti-TCR-C and 10 mM MCD
for 45 minutes on ice. Cells were pelleted, resuspended to 4 × 106 cells/mL, and warmed to 37°C before
stimulation with 20 µg/mL streptavidin for 30 seconds. Phospholipase
C (PLC)1 was immunoprecipitated from post-nuclear lysates
using a mixture of mouse mAbs (UBI). Immunoblot analysis was performed
on gels run in parallel using antiphosphotyrosine or anti-PLC1
revealed by GaMIg-HRP.
Sodium iodide 125-IL-2 binding assays
CTLL-2 cells maintained in IL-2 were harvested, and IL-2 bound
to its receptor was dissociated by a 1-minute incubation in 10 mM
sodium citrate, 150 mM NaCl, pH 4.0. Cells were washed twice in
phosphate-buffered saline (PBS) with 3% fetal calf serum and 0.1%
azide before the addition of 5 × 1010 M sodium iodide
125 (125I)-labeled IL-2 (NEN, Boston, MA). After 30 minutes on ice, cells were washed twice in PBS with 0.1% azide and
incubated for 10 minutes in 2 mM disuccinimidyl suberate (DSS)
(Pierce, Rockford, IL). Cells were then incubated in 5 mM ammonium
acetate for 1 minute to quench unreacted DSS, washed twice in PBS with
0.1% azide, and lysed in TKM/Brij58. Sucrose density gradient
centrifugation was performed, and proteins from fractions corresponding
to lipid rafts and soluble membranes, as assessed by GM1
immunoblotting, were resolved by SDS-PAGE. Gels were fixed in 40%
methanol and 10% acetic acid, dried, and autoradiographed at
 70°C.
| |
Results |
Immobilized mAbs specific for GPI-AP inhibit IL-2-induced
proliferation
We recently demonstrated that in the presence of immobilized mAbs
specific for GPI-AP, anti-TCR-induced proliferation was inhibited,
despite the production of IL-2.18 In addition,
IL-2-induced signaling was inhibited in these circumstances. These
results were consistent with a signaling defect in the responsiveness of T cells to endogenously produced IL-2. To confirm that GPI-AP inhibited signaling through IL-2R, we determined the effect of mAb
specific for GPI-AP on T-cell proliferation in response to exogenous IL-2.
The IL-2-dependent 2.10 T-cell clone (Figure
1A) and the CTLL-2 T-cell line (Figure
1B) were cultured in wells coated with anti-Thy-1 or an
isotype-matched control mAb. Immobilized anti-Thy-1 inhibited the
proliferation of both 2.10 and CTLL-2 cells in response to IL-2 over a
wide range of concentrations. Analysis of the growth-inhibitory effects
of anti-Thy-1 was extended to primary T cells, which require stimulation through TCR to induce expression of the high-affinity IL-2R
and acquire responsiveness to IL-2. Thus, unstimulated CD8+
lymph node T cells did not proliferate in response to exogenous IL-2
(Figure 1C). Primary CD8+ T cells were stimulated with
anti-TCR, harvested, and restimulated with exogenous IL-2. As seen in
Figure 1C, exogenous IL-2 induced the proliferation of activated
primary T cells, which were inhibited by anti-Thy-1.
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| Figure 1.
Anti-Thy-1 inhibits IL-2-induced T-cell proliferation.
The 2.10 T-cell clone (A) or the CTLL-2 T-cell line (B) were cultured
in wells precoated with mAbs specific for Thy-1 ( ) or an mAb isotype
control ( ) and the indicated concentration of IL-2. (C) Resting or
TcR-stimulated CD8+ lymph node T cells were cultured in
wells precoated with anti-Thy-1 or an isotype control in the presence
of 15 U/mL IL-2. Uptake of 3H-thymidine was assessed after
20 hours of culture.
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Anti-TCR-induced proliferation, but not IL-2 production, was inhibited
by mAb specific for multiple GPI-AP.18 Therefore, we
determined the effect of GPI-AP in addition to Thy-1 on T-cell proliferation induced by exogenous IL-2. Stable GPI+ and
GPI clonal variants of 2.10 were established by FACS
sorting of cells based on the expression of Thy-1. GPI
2.10 were shown to lack expression of all GPI-AP because of a deficiency in PIG-P, a protein required for GPI anchor
biosynthesis.25 The GPI-AP Thy-1, Ly6A/E, and CD48 are
expressed on GPI+ but not on GPI 2.10 T
cells, as determined by flow cytometry (Figure
2A-B). IL-2-induced proliferation of
GPI+ 2.10 (Figure 2C) and CTLL-2 cells (data not shown) is
inhibited by several mAbs specific for Thy-1 and by mAb specific for
Ly6A/E and CD48. Proliferation of the GPI clonal variant
is unaffected by these mAbs, as predicted for cells that lack
expression of these proteins, confirming that inhibition of
proliferation is not caused by nonspecific toxic effects of the mAb
preparations. In addition, immobilized mAbs specific for GPI-AP do not
inhibit TCR-induced signaling, as assessed by the production of
IL-2,18 demonstrating that not all signaling pathways are
inhibited in these circumstances. IL-2-induced proliferation is not
affected by isotype controls or by mAbs specific for the transmembrane
proteins CD45 and CD5 (Figure 2C). In addition, IL-2-induced
proliferation of GPI+, but not GPI, 2.10 was
inhibited by mAbs specific for Qa-2, CD55, and CD73, albeit to a lower
extent, which in turn correlated with a decreased level of
surface expression of these GPI-APs as detected by flow cytometry (data
not shown). Thus, immobilized mAbs specific for all GPI-APs tested
inhibit T-cell proliferation in response to exogenous IL-2.
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| Figure 2.
IL-2-induced proliferation is inhibited by mAbs
specific for the GPI-anchored proteins Thy-1, Ly-6A/E, and CD48.
The expression of Thy-1, Ly6A/E, and CD48 on GPI+ (A) and
GPI (B) variants of the 2.10 T-cell clone was analyzed by
flow cytometry. The first histogram represents staining with secondary
antibodies alone. (C) GPI+ and GPI 2.10 T
cells were cultured in wells precoated with the indicated mAbs and 15 U/mL IL-2. Uptake of 3H-thymidine is represented as the
percentage of the proliferative response to IL-2 in the absence of
added mAbs.
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Immobilized mAbs specific for GPI-AP result in the dissociation of
IL-2-mediated survival and proliferation
The 2.10 T-cell clone is IL-2-dependent and undergoes apoptotic
cell death on cytokine withdrawal. Although IL-2-mediated proliferation was inhibited by immobilized mAbs specific for Thy-1, IL-2 still supports cell survival in these circumstances. Figure 3A demonstrates that most cells undergo
apoptosis by 24 hours after the withdrawal of IL-2. In the presence of
IL-2, cells cultured with anti-Thy-1 or an isotype control mAb remain
viable, despite the growth inhibition mediated by anti-Thy-1 in
cultures set up in parallel (Figure 3B). Similar results are observed
using mAbs specific for CD48 or Ly6A/E and using CTLL-2 cells (data not
shown). The increase in the number of control cells undergoing
apoptosis at 48 hours is consistent with IL-2 use and catabolism by the proliferating cells. In the presence of immobilized anti-Thy-1, IL-2
is not used to aid proliferation, and it continues to support cell
survival (Figure 3A).
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| Figure 3.
Anti-Thy-1 inhibits IL-2-induced proliferation but not
cell survival.
(A) GPI+ 2.10 T cells were cultured in the presence of
immobilized anti-Thy-1 or an mAb isotype control, in the presence or
absence of 15 U/mL IL-2. After 20 hours, the viability of cells in each
condition was assessed by flow cytometry. (B) 3H-thymidine
uptake in response to 15 U/mL IL-2 was assessed in cultures set up in
parallel to those in panel A.
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The ability of IL-2 to support survival but not proliferation in the
presence of immobilized mAbs specific for GPI-AP may be a result of
residual signaling through IL-2R. Alternatively, survival may result
from distinct signals induced through IL-2R that are differentially
affected by GPI-AP. The activation of PI3K and its downstream effector
protein kinase B have been implicated in IL-2-mediated
survival.10,11 However, in the presence of immobilized
anti-Thy-1, IL-2-induced PI3K activation, as determined by protein
kinase B phosphorylation levels, was decreased relative to controls
(data not shown). This result, in addition to our previous finding that
immobilized anti-Thy-1 inhibits IL-2-induced IL-2R
heterotrimerization,18 is consistent with the inhibition of all signaling pathways induced through IL-2R, and it suggests that
viability may be mediated by residual signaling.
Another component of lipid rafts, the GM1 ganglioside, inhibits
IL-2-induced proliferation
The ability of all GPI-APs tested to inhibit IL-2R-induced
proliferation, despite their unrelated protein moieties, suggests that
a characteristic imparted by the GPI anchor is critical for this
inhibition. GPI anchoring results in the localization of proteins to
lipid rafts, and we hypothesized that this localization of GPI-AP
underlay their inhibitory capacity. Therefore, we determined the effect
of immobilizing another component of lipid rafts, the GM1 ganglioside,
on IL-2-induced proliferation.
To establish that plasma membrane compartmentalization occurs in cells
expressing or lacking GPI-AP, lipid rafts were isolated from
GPI+ and GPI 2.10 lysed in buffer containing
Brij58, a weak nonionic detergent that preserves lipid rafts. Fractions
were collected after discontinuous sucrose density gradient
ultracentrifugation, and the localization of proteins and glycolipids
was assessed by immunoblotting. In both GPI+ and
GPI clonal variants, the transmembrane tyrosine
phosphatase CD45 is found almost exclusively in soluble membranes
whereas the GM1 ganglioside, detected using the subunit of cholera
toxin (CT), is found almost exclusively in lipid rafts (Figure
4A-B). In addition, most Fyn and Thy-1
are found within lipid rafts. As predicted, Thy-1 cannot be detected in
immunoblots of fractions isolated from GPI cells (Figure
4B).
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| Figure 4.
Proliferative responses of T cells to TCR and IL-2 are
inhibited by the lipid raft component GM1.
(A-B) Plasma membrane compartmentalization into lipid rafts in the
presence or absence of GPI-anchored proteins. Lysates of
GPI+ (A) or GPI (B) 2.10 T cells were
subjected to discontinuous sucrose density gradient
ultracentrifugation. Fractions (Fr) were collected from the top
of the gradient, and the distribution of CD45, Fyn, Thy-1, and GM1 was
analyzed by immunoblotting. Fractions corresponding to lipid rafts and
soluble membranes are indicated. (C-D) Immobilized CT inhibits anti-TCR
and IL-2-induced proliferation. GPI+ and GPI
2.10 T cells were cultured in wells precoated with CT ( ),
anti-Thy-1 ( ), or an isotype control ( ) mAb and coimmobilized
anti-TcR-C (C) or in the presence of 15 U/mL IL-2 (D).
3H-thymidine uptake was assessed after 20 hours of
culture.
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Immobilized CT inhibited the proliferation of both GPI+ and
GPI cells in response to IL-2 produced endogenously on
stimulation with anti-TCR (Figure 4C) or provided exogenously (Figure
4D). Immobilized mAbs specific for Thy-1 inhibited anti-TCR and
IL-2-induced proliferation of GPI+ but not
GPI cells. The effects of immobilizing GM1 using CT
appeared identical to those of immobilizing GPI-AP using mAbs because
IL-2-induced proliferation but not cell survival or TCR-induced
production of IL-2 was inhibited (data not shown). These results are
consistent with the ability of components of lipid rafts to modify
IL-2R signaling in T cells.
IL-2R is enriched in lipid rafts, but IL-2R signaling occurs
in soluble membranes
The ability of components of lipid rafts to inhibit IL-2-induced
proliferation suggests that lipid rafts may play a role in the
regulation of IL-2R signaling. As a first step in assessing the role of
lipid rafts in IL-2R signaling, we determined the localization of IL-2R
chains. CTLL-2 cells are maintained in IL-2; therefore, to minimize
potential effects of IL-2 on the distribution of its receptor chains,
cells were cultured for 16 hours in low concentrations of IL-2. In
these circumstances, IL-2R signaling, assessed by the tyrosine
phosphorylation of JAK1 and JAK3, was not detectable.
CTLL-2
cells were treated no further (unstimulated) or were stimulated for 10 minutes with 400 U/mL IL-2 before lysis, and membranes were
fractionated by sucrose density gradient ultracentrifugation. Immunoblot analysis of fractions from these gradients revealed that a
large proportion (58%) of IL-2R was localized in lipid rafts
(Figure 5). In contrast, most IL-2R
and IL-2R were detected in soluble membranes. No significant
differences in the localization of IL-2R, IL-2R, or IL-2R were
detected on stimulation of cells with IL-2.
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| Figure 5.
IL-2R, but not IL-2R or IL-2R, is enriched in
lipid rafts.
CTLL-2 cells were left untreated or were stimulated for 10 minutes with
IL-2 before lysis. Lysates were subjected to discontinuous sucrose
density gradient ultracentrifugation, and fractions (Fr) were
collected and separated by SDS-PAGE. Localization of IL-2, IL-2R,
and IL-2R chains was analyzed by immunoblotting. The final lane in
each blot consists of immunoprecipitations of IL-2R, IL-2R, or
IL-2R chains as positive controls for immunoblotting.
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JAK1 and JAK3 kinases are constitutively associated with the IL-2R
and IL-2R chains, respectively,8,9 and their activation after stimulation by IL-2 is critical for signaling through the IL-2R.
Therefore, we assessed the localization of JAK1 and JAK3. The kinases
were immunoprecipitated from pooled fractions of sucrose density
gradients derived from CTLL-2 cells corresponding to lipid rafts and
soluble membranes, as determined by blotting for GM1. Figure 6
demonstrates that JAK1 and JAK3 are found in soluble membranes. In
addition, JAK1 and JAK3 molecules involved in IL-2R signaling, assessed
by IL-2-induced tyrosine phosphorylation, were found in soluble
membranes. JAK1 and JAK3, and IL-2R and IL-2R, were not detected
in lipid rafts at multiple additional time points after stimulation
with IL-2 (1, 3, 9, or 27 minutes; data not shown). Similar
results were observed using 2.10 (data not shown).
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| Figure 6.
JAK1 and JAK3 are localized in detergent-soluble
membranes.
CTLL-2 cells were left untreated or were stimulated for 10 minutes with
IL-2 before lysis and sucrose density gradient ultracentrifugation.
JAK1 and JAK3 were immunoprecipitated from pooled fractions of sucrose
gradients corresponding to lipid rafts (R) and soluble membranes (S).
Immunoprecipitations were split in 2 and resolved by SDS-PAGE, and
immunoblotting was performed using phosphotyrosine (PY)-, JAK1-, or
JAK3-specific antibodies.
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IL-2 binding to receptor chains can be assessed using labeled cytokine.
CTLL-2 cells were incubated with 125I-labeled IL-2, which
was subsequently chemically cross-linked to bound receptor chains.
Fractions from sucrose density gradients corresponding to lipid rafts
and soluble membranes were determined by blotting for GM1 (Figure 7A).
Proteins in these fractions were separated by SDS-PAGE, and proteins
cross-linked to 125I-labeled IL-2 were visualized by
autoradiography. 125I-labeled IL-2 was cross-linked to
IL-2R, IL-2R, and IL-2R chains in soluble membranes but not in
lipid rafts (Figure 7B). This result is consistent with the presence of
the signaling complex, composed of IL-2 and IL-2R, IL-2R, and
IL-2R in soluble membranes exclusively.
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| Figure 7.
The heterotrimeric receptor complex composed of IL-2
bound to IL-2R, IL-2R, and IL-2R is detected in
detergent-soluble membranes.
(A) CTLL-2 cells were incubated with 125I-labeled
IL-2, and bound IL-2 was cross-linked to cell-surface proteins. After
lysis, sucrose density gradient ultracentrifugation was performed.
Fractionation (Fr) into lipid rafts and soluble membranes was
determined by immunoblotting for GM1. (B) Proteins in fractions
corresponding to lipid rafts and soluble membranes were resolved by
SDS-PAGE, and proteins cross-linked to 125I-labeled-IL-2
were visualized by autoradiography. Bands corresponding to
125I-IL-2 cross-linked to IL-2R, IL-2R, and IL-2R
are indicated.
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Additional support for IL-2R signaling occurring within soluble
membranes was derived by assessing the effect of disrupting the
integrity of lipid rafts. This was achieved using
methyl--cyclodextrin (MCD), which results in the extraction of
cholesterol by forming inclusion complexes within a hydrophobic
cyclodextrin cavity.24 MCD did not inhibit IL-2-induced
tyrosine phosphorylation of JAK1 and JAK3 in CTLL-2 cells (Figure
8A-B). As a positive control for the
disruption of lipid rafts by MCD, we assessed the effects on a
TCR-induced signaling pathway known to be dependent on lipid rafts.26 Although IL-2-induced tyrosine phosphorylation
of JAK1 in 2.10 was unaffected by MCD (Figure 8C), TCR-induced tyrosine phosphorylation of PLC1 was virtually ablated (Figure 8D).
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| Figure 8.
Disruption of lipid raft integrity does not affect IL-2-induced
tyrosine phosphorylation of JAK1 and JAK3.
(A-B) CTLL-2 cells were left untreated or were incubated with 10 mM MCD
before stimulation with IL-2. JAK1 and JAK3 were immunoprecipitated
from postnuclear lysates. Immunoprecipitations were split in 2 and
resolved by SDS-PAGE, and immunoblotting was performed using
phosphotyrosine (PY)-, JAK1-, or JAK3-specific antibodies. (C-D) 2.10 cells were left untreated or were incubated with 10 mM MCD. (C) Cells
were stimulated with IL-2, and JAK1 immunoprecipitated from postnuclear
lysates was immunoblotted using anti-PY or anti-JAK1. (D) Cells were
stimulated with anti-TCR, and PLC1 immunoprecipitated from
postnuclear lysates was immunoblotted using anti-PY or anti-PLC1.
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Thus, although IL-2R was enriched in lipid rafts, the signaling
components of the IL-2R, IL-2R, and IL-2R chains, and of JAK1 and
JAK3 phosphorylated in response to IL-2, were not found in lipid rafts.
The active heterotrimeric receptor complex composed of IL-2 bound to
IL-2R, IL-2R, and IL-2R was not detected in lipid rafts. In
addition, IL-2R-induced signaling was not affected by the disruption
of lipid rafts. Taken together, these results support the conclusion
that IL-2R signaling occurs in soluble membranes.
Immobilized anti-Thy-1 results in an increased proportion of
IL-2R in lipid rafts
The ability of lipid raft components to block IL-2R signaling
suggests that rafts can regulate signaling through IL-2R, despite evidence that signaling occurs in soluble membranes. Lipid rafts may
regulate IL-2R signaling by segregating elements of the receptor complex in the plasma membrane. IL-2 may result in the dissociation of
IL-2R from lipid rafts and its interaction with IL-2R and IL-2R in soluble membranes to initiate signaling. The inhibition of
IL-2R signaling, observed on immobilization of GPI-AP or GM1 in lipid
rafts, may be owing to impairment of the mobility of IL-2R, thus
preventing its dissociation from rafts. IL-2R may be in a dynamic
equilibrium between lipid rafts and soluble membranes, and
immobilization of lipid rafts components may shift this equilibrium by
trapping IL-2R chains in lipid rafts. In either case, the prediction
would follow that a greater proportion of IL-2R would be present in
rafts in these circumstances. We therefore assessed whether
immobilization of components of lipid rafts affected the distribution
of IL-2R.
Figure 9 demonstrates that the proportion
of IL-2R localized in lipid rafts was higher in cells that had
been cultured in the presence of immobilized anti-Thy-1, relative to
an isotype-matched mAb (61.4% ± 15.8% vs 24.6% ± 6.0%). In
contrast, IL-2R was not enriched in lipid rafts in the presence or
absence of anti-Thy-1, and the proportion of IL-2R in rafts was
less than 5% in all experiments. Thus, in the presence of immobilized
anti-Thy-1, IL-2R was further enriched in lipid rafts and is
segregated from IL-2R and IL-2R localized in soluble membranes.
This demonstration is consistent with our earlier report that
IL-2-induced receptor heterotrimerization was inhibited by
anti-Thy-1.18
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| Figure 9.
The proportion of IL-2R in lipid rafts is increased
in cells cultured in the presence of immobilized anti-Thy-1.
(A) CTLL-2 cells were cultured in the presence of immobilized
anti-Thy-1 or an isotype-matched control (ctrl) mAb before lysis and
sucrose density gradient ultracentrifugation. The localization of
IL-2R and IL-2R chains was assessed by immunoblotting the
fractions of the sucrose gradients corresponding to lipid rafts and
soluble membranes. (B) The proportion of IL-2R and IL-2R in lipid
rafts and soluble membranes was quantified by scanning densitometry.
Data shown are represented as the percentages of the total IL-2R or
IL-2R localized in lipid rafts and are the averages from 3 independent experiments.
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Discussion |
These results demonstrate that immobilized mAbs specific for
GPI-AP inhibit IL-2-induced proliferation of primary T cells and in 2 T-cell lines, GPI+ 2.10 and CTLL-2 cells. Multiple GPI-APs,
including Thy-1, Ly6A/E, and CD48, can mediate this effect, suggesting
that the ability to affect IL-2R signaling reflects a common feature of
these proteins. An important characteristic imparted by the GPI anchor
is localization to lipid rafts. Lipid rafts are detergent-insoluble
microdomains in the plasma membrane that are enriched in cholesterol
and glycosphingolipids, including the ganglioside GM1.3
Consistent with the notion that their localization to lipid rafts
underlies the capacity of GPI-AP to modify IL-2R signaling,
immobilization of GM1 using the subunit of CT also inhibits
IL-2-induced proliferation.
The ability of multiple components of lipid rafts to modify cellular
responsiveness to IL-2 suggests that lipid rafts can regulate IL-2R
signaling, notwithstanding the fact that IL-2R signaling does not
appear to occur in these microdomains. Although IL-2R was enriched
in lipid rafts isolated by sucrose density gradient
ultracentrifugation, the IL-2R and IL-2R chains responsible for
signal transduction were found in detergent-soluble membranes before
and after stimulation of cells with IL-2. In addition, the Janus family
kinases phosphorylated in response to IL-2, JAK1, and JAK3 were not
detected in lipid rafts. Disruption of lipid rafts using MCD did
not perturb IL-2R-induced signaling. Finally, in cells proliferating
in response to IL-2, the active heterotrimeric receptor complex
composed of IL-2R, IL-2R, and IL-2R bound to
125I-labeled IL-2 was detected only in soluble membranes.
The function of lipid rafts in the coordination of signaling may be
2-fold. Several membrane receptors are inducibly recruited to or
stabilized within these domains, including TCR, BCR, and FcRI, and
the subsequent activation of critical signaling molecules enriched in
rafts may facilitate signaling.2 However, lipid rafts may
also function to segregate signaling molecules in resting cells,
maintaining low levels of signaling in the absence of stimulation. Thus, though the disruption of lipid rafts resulted in decreased TCR/CD3-induced signaling,26 increased basal levels of
tyrosine phosphorylation were observed on treatment with
MCD.27 In addition, MCD resulted in activation of the
Ras-ERK pathway.27,28 Furthermore, the localization of Src
PTKS in rafts may segregate these kinases from their substrates.
Palmitoylation of Fyn, which results in its localization to lipid
rafts, prevented its ability to phosphorylate a nonlipid raft resident
chimeric Ig molecule.29 Src, which is not
palmitoylated, and a nonpalmitoylated mutant of Fyn were able to
phosphorylate Ig, suggesting that membrane compartmentalization regulates protein-protein interactions.
Thus, lipid rafts may be involved in the spatial regulation of
intermolecular associations in the plasma membrane. In the context of
TCR or BCR signaling, rafts result in a segregation of enzymes and
substrates, and signaling is initiated on the regulated association of
receptors with lipid rafts.2 In the context of IL-2R
signaling, lipid rafts may mediate the segregation of receptor chains.
Lipid raft components can affect the initiation of signaling in both
receptor systems by interfering with the regulated assembly of
molecules. The first indications that lipid rafts played a role in
TCR/CD3-induced signaling were the demonstrations that mAbs specific
for GPI-AP could inhibit or potentiate signaling through the antigen
receptor [reviewed in 30], possibly by modifying the
association of TCR/CD3 with lipid rafts. The demonstration herein that
components of rafts modify IL-2 responsiveness similarly implicates
lipid rafts in the regulation of IL-2R signaling. Lipid rafts may also
be involved in regulating signaling through multiple cytokine
receptors, as suggested by our preliminary evidence that the
immobilization of GPI-AP inhibits the responsiveness of T cells to
IL-4 and IL-15, of B cells to IL-7, and of mast cells to IL-3 (M.D.M.
and M.J., unpublished observations, 2000).
Fluorescence resonance energy transfer analysis has revealed that
IL-2R, IL-2R, and IL-2R chains are loosely associated in
resting T-cell lymphoma lines.12 On IL-2 binding, the
chains are brought closer, consistent with strengthened interactions or
movement of the chains in the plasma membrane. IL-2R may be localized at the periphery of lipid rafts, where it can maintain a
loose association with IL-2R and IL-2R. Binding of IL-2 and the
subsequent strengthening of the interactions among IL-2R, IL-2R,
and IL-2R may result in the dissociation of IL-2R from lipid
rafts and the initiation of signaling in soluble membranes. Alternatively, signaling may be mediated only by IL-2R in soluble membranes. We propose that the inhibition of IL-2-induced
proliferation observed on immobilization of GPI-AP or GM1 reflects
effects on the mobility of IL-2R. These inhibitory effects may
involve steric hindrance of the association of IL-2R with IL-2R
and IL-2R, blocking a ligand-induced dissociation of IL-2R from
lipid rafts or through a shift in the equilibrium such that IL-2R
becomes trapped in lipid rafts. In support of its altered mobility, the proportion of IL-2R in lipid rafts is 2.5-fold higher in cells cultured in the presence of immobilized anti-Thy-1 than in controls.
No significant differences in the membrane distribution of IL-2R
were detected on the stimulation of cells with IL-2 for 1 to 27 minutes
(Figure 7 and data not shown). Because the levels of IL-2R chain
expression on cell surfaces exceed those of IL-2R and IL-2R
chains,31 only a fraction of available IL-2R will dissociate from lipid rafts and participate in signaling in
detergent-soluble membranes. In these acute experiments, the change in
the distribution of IL-2R is not detectable using Western blot
analysis. In contrast, the difference in the proportion of IL-2R in
lipid rafts in Figure 7 and the isotype control in Figure 9 may reflect
ligand-induced changes in the localization of IL-2R with prolonged
exposure to IL-2. In Figure 7, lipid rafts were isolated from
"starved" CTLL-2 cells, cultured for 16 hours in minimal
concentrations of IL-2 compatible with sustaining survival. In these
circumstances, the proportion of IL-2R in lipid rafts varied between
37% and 70% in 8 experiments. Results presented in Figure 9 are
derived from rafts isolated from CTLL-2 cells in log phase response to IL-2, circumstances in which 24.6% ± 6.0% of IL-2R was
localized in rafts. This difference might have resulted from a shift in the equilibrium of IL-2R between rafts and soluble membranes as a
consequence of prolonged stimulation with IL-2 and the resultant dissociation of IL-2R from rafts. Further, the IL-2R complex is
internalized on IL-2 binding. IL-2R is subsequently recycled to the
plasma membrane,32 and it is unknown whether recycling IL-2R molecules localize to lipid rafts or soluble membranes. Thus,
though the dynamic equilibrium of IL-2R localization is altered in
the presence of ligand, the time needed to observe a re-equilibration
of IL-2R likely reflects constraints imposed by receptor physiology
(re-use) and the relative abundance of IL-2R chains.
This report confirms the findings of 2 recent reports demonstrating the
localization of IL-2R in lipid rafts,15,33 and it
extends the analysis to IL-2R, IL-2R, and ligand binding. Moreover, these results provide the first demonstration of a functional role for lipid rafts in the regulation of IL-2R signaling. Field et
al33 demonstrated that in transfected Chinese hamster
ovary cells, IL-2R became associated with TX-100 insoluble domains on cross-linking. In contrast, we observed an IL-2-independent enrichment of IL-2R in lipid rafts. This apparent contradiction likely reflects the differing detergents used (0.5% Brij58 vs 0.05%
TX-100) and the differing detergent-to-cell ratios, and it highlights a
limitation imposed by the methodology used to examine lipid raft
constituents. Specifically, weak associations of proteins with lipid
rafts may be disrupted using detergents. Field et al33,34
also observed that FcRI aggregation was required for its association
with TX-100-insoluble domains. However, recent data suggest that
FcRI localization in rafts is constitutive but weak and that it is
strengthened and rendered detergent resistant on cross-linking.
High-resolution immunogold labeling and electron microscopy revealed
that in unstimulated cells, monomeric FcRI is distributed in small
clusters that also contain the Src family PTK Lyn and that likely
represent lipid rafts.35 Similarly, the association of
IL-2R with lipid rafts is likely constitutive but weak, and we have
observed that though it is maintained in the presence of Brij58, 0.5%
TX-100 disrupts the association of IL-2R, but not of Fyn and Thy-1,
with lipid rafts (data not shown). Consistent with this interpretation,
relative to other nonionic detergents, Brij58 has been shown to better
preserve associations of proteins with lipid rafts.36
Using immunogold labeling and electron microscopy, Vereb et
al15 demonstrated that IL-2R exists in clusters in the
presence or absence of IL-2. Clusters of IL-2R colocalized with
clusters of CD48; moreover, the cluster size was modulated by MCD,
suggesting they represented lipid rafts. The present study demonstrates
that only IL-2R is enriched in lipid rafts isolated in the presence
of Brij58. However, it remains possible that IL-2R and IL-2R
associate with rafts even more weakly than IL-2R and that this
association is sensitive to Brij58. Electron microscopic analyses of
IL-2R, IL-2R, and IL-2R before and after stimulation with IL-2
should provide a definitive picture of the membrane
compartmentalization of IL-2R.
A recent report detected interferon receptors JAK1 and JAK3 in caveolae
isolated from mouse embryonic fibroblasts.37 Caveolae, flask-shaped membrane invaginations, are a subset of lipid rafts. Although these 2 plasma membrane domains share common features, including detergent insolubility, they can be separated experimentally and show differing protein composition and morphology.1 In addition, lipid rafts are present in cells, including lymphocytes, that
do not express caveolin, a cholesterol-binding integral membrane protein essential for caveolae formation.1 The caveolar
localization of JAKs may not be relevant to their subcellular
localization in lymphocytes because many molecules localize to caveolae
through a direct interaction with caveolin. Indeed, examination of the sequences of all JAK kinases reveals the presence of a caveolin binding
motif,  XXXXX, where represents any of the aromatic amino
acids tyrosine, phenylalanine, or tryptophan.38
Furthermore, Takaoka et al37 isolated caveolae using a
detergent-free method. Because differential results with regard to the
localization of some proteins have been observed |
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Abstract
Introduction