From the Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853
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ABSTRACT |
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We recently showed that aggregation of the
high affinity IgE receptor on mast cells, Fc Multichain immune recognition receptors present on hematopoietic
cells interact with Src family protein tyrosine kinases
(PTKs)1 as an early signaling
step (1). Aggregation of the high affinity receptor for IgE, Fc The phosphorylation of Fc Lyn association with the detergent-resistant membranes (DRMs) isolated
from the RBL-2H3 mucosal mast cell line (16) occurs as in other cells
for Src family PTKs that contain a consensus site for dual fatty acid
modifications (17, 18). These low density membranes are isolated by
sucrose gradient ultracentrifugation of Triton X-100 (TX-100)-lysed
cells. In other cell types, these preparations have been shown to be
enriched in cholesterol, sphingomyelin, and gangliosides (19), as well
as a subset of membrane-associated proteins, including certain Src
family PTKs, heterotrimeric GTP-binding proteins, and
glycosylphosphatidylinositol (GPI)-linked proteins (18-21).
Preparations similar to DRMs are also referred to as
detergent-insoluble glycolipid-enriched domains (DIGs),
glycolipid-enriched membranes (GEMs), or sphingolipid-cholesterol rafts
(22, 23). Caveolae, which are flask-shaped invaginations on the plasma
membrane that contain the marker protein caveolin, can also be isolated
using similar methods and appear to contain many of the same components (24, 25). RBL cells appear to be like other hematopoietic cells,
including T- and B-cell lines (26-29), that do not contain caveolae
but do exhibit DRMs that are enriched in signaling molecules (16).
Fc In the present study, we investigated the structural basis for the
interaction of Fc Cell Lines and Antibodies--
RBL-2H3 cells, mouse monoclonal
anti-dinitrophenyl IgE, and biotinylated 125I-IgE were
previously described (6, 16). P815 mouse mastocytoma cells and Chinese
hamster ovary (CHO) cells stably transfected with wild-type or mutant
Fc
The IL-2 receptor
Primary and secondary antibodies used to form receptor complexes at the
cell surface were as follows. For IgE, affinity-purified rabbit
anti-mouse IgE (34); for Sucrose Gradient Ultracentrifugation--
Confluent cells were
harvested using EDTA and suspended at 8 × 106/ml in
buffered saline solution with 1 mg/ml bovine serum albumin. Cells
labeled with the appropriate antibody were stimulated with streptavidin
(Sigma) or secondary antibodies as indicated under "Results,"
followed by the addition of an equal volume of ice cold 2× lysis
buffer (final concentration, 25 mM Hepes, pH 7.5, 50 mM NaCl, 10 mM EDTA, 1 mM
Na3VO4, 30 mM pyrophosphate, 10 mM glycerophosphate, 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride (Calbiochem), 0.02 units/ml
aprotinin, and 0.01% (w/v) NaN3) with the indicated
concentration of Surfact-Amps TX-100 (Pierce). For aggregating Fc Electron Microscopy--
Sucrose gradient fractions containing
DRMs from either anti-IgE-stimulated or unstimulated cells lysed in
0.05% TX-100 were pooled according to the distribution of
125I-IgE bound to Fc We previously demonstrated that the PTK Lyn associates with
DRMs isolated from RBL cells, and we found that this association is
enhanced upon Fc As part of our investigation of the structural basis for this receptor
association with DRMs, we examined the detergent sensitivity. For low
detergent conditions, we use 0.05% TX-100 to lyse RBL cells at 4 × 106/ml and then dilute this lysate 1:1 with 80% sucrose
prior to sucrose gradient ultracentrifugation. Under these conditions, the micellar detergent to cell lipid ratio during the
ultracentrifugation is approximately 3 (38). To exclude some
artifactual possibilities, we added 0.025% TX-100 to all of the
sucrose gradient steps, as shown in Fig.
1 (RI, causes this
immunoreceptor to associate rapidly with specialized regions of the
plasma membrane, where it is phosphorylated by the tyrosine kinase Lyn.
In this study, we further characterize the detergent sensitivity of
this association on rat basophilic leukemia-2H3 mast cells, and we compare the capacity of structural variants of Fc
RI and other receptors to undergo this association. We show that this interaction is
not mediated by the
subunit of the receptor or the cytoplasmic tail
of the
subunit, both of which are involved in signaling. Using
chimeric receptor constructs, we found that the extracellular segment
of the Fc
RI
subunit was not sufficient to mediate this association, implicating Fc
RI
and/or
transmembrane segments. To determine the specificity of this interaction, we compared the
association of several other receptors. Interleukin-1 type I receptors
on Chinese hamster ovary cells and
4 integrins on rat basophilic leukemia cells showed little or no association with
isolated membrane domains, both before and after aggregation on the
cells. In contrast, interleukin-2 receptor
(Tac) on Chinese hamster
ovary cells exhibited aggregation-dependent membrane
domain association similar to Fc
RI. These results provide insights
into the structural basis and selectivity of lipid-mediated
interactions between certain transmembrane receptors and
detergent-resistant membranes.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
RI,
results in phosphorylation of the
and
2 subunits of
this receptor by the Src family PTK, Lyn (2, 3). Lyn phosphorylation of
the immunoreceptor tyrosine-based activation motif (ITAM) within
Fc
RI
subunits allows the ZAP-70-related PTK, Syk, to associate
with the receptor via its two Src homology 2 domains (1, 2). This
recruitment and consequent activation of Syk leads to further
downstream signaling, including phosphorylation and activation of
phospholipase C
, mobilization of intracellular calcium, and
activation of protein kinase C (4). In mast cells and basophils, which
both express Fc
RI, these cascades result in the exocytosis of
preformed granules containing histamine and other vasoactive compounds,
as well as other cellular responses.
RI by Lyn is a critical event in receptor
activation, but the mechanism by which receptor aggregation stimulates
this event is not well understood. We have proposed a novel model for
this process in which specialized membrane domains enriched in Lyn
mediate this phosphorylation that occurs after the
aggregation-dependent association of Fc
RI with these
domains (5). This model is consistent with our findings that the rapid association of Fc
RI with these domains does not depend on receptor phosphorylation (6). It is strongly supported by preferential tyrosine
phosphorylation of those receptors associated with membrane domains.
Moreover, in vitro tyrosine kinase assays reproduce this activation step within these membrane domains that can be isolated because of their resistance to detergent solubilization (6). Other
models that require a direct interaction between Lyn and the
subunit of monomeric Fc
RI (7, 8) do not explain some observations,
including the capacity of mutant and chimeric receptors lacking the
subunit to activate cells (9-14) or the difficulty in identifying the
molecular basis of this interaction (15).
RI associates with isolated DRMs when the receptor is aggregated
at the cell surface (6). Preservation of this association after cell
lysis depends on the stability of the receptor aggregate and on using a
low concentration of TX-100 in the lysate (6, 16). These interactions
can be detected at the intact cell surface, as observed with
fluorescence microscopy. For example, aggregation of Fc
RI into
patches causes co-redistribution of DiI-C16, a fluorescent lipid probe with saturated acyl chains, and this lipid analog has
reduced lateral mobility in these patches (30). Co-redistribution with
aggregated Fc
RI on intact cells is also observed for three other
membrane components isolated with DRMs (16), a GD1b
ganglioside derivative (31), the GPI-linked protein Thy-1,2
and Lyn.2
RI, a multisubunit transmembrane receptor, with
isolated DRMs. These membranes were visualized by whole-mount electron
microscopy to compare them with similar preparations from other cells.
We also compared the association of wild-type and mutant Fc
RI with
DRMs isolated from hematopoietic and nonhematopoietic cell lines.
Finally, we investigated the specificity of this interaction by
measuring the aggregation-dependent association of other
transmembrane receptors with DRMs. These results provide evidence that
the structural features of Fc
RI that mediate the detergent-sensitive
interaction with membrane domains occur selectively but not uniquely
with this receptor.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
RI were generously provided by Dr. H. Metzger (National Institutes
of Health) and were maintained as described (32). Prior to harvesting,
cells were sensitized with biotinylated 125I-IgE for 4-24
h. All stable Fc
RI transfectants expressed at least 50,000 IgE
receptors per cell at the time of the experiments except the
2 P815 cell line, which expressed approximately
18,000 receptors per cell. CHO cells stably transfected with type I
IL-1 receptors were from Dr. S. Dower (University of Sheffield, United Kingdom) and were maintained as described (33).
(Tac) and Chimera-1 receptor were transiently
expressed on CHO cells for 48 h prior to performing experiments. The IL-2 receptor
DNA was provided in a pCMV mammalian expression vector by Dr. B. Howard (National Institutes of Health). This plasmid
was transfected using LipofectAMINE (Life Technologies, Inc.) at 1 µg
of DNA per ml and 10 µl of liposomes per ml in Opti-MEM (Life
Technologies, Inc.) for 5 h in the absence of serum. The Chimera-1
DNA was constructed by ligating the DNA encoding the extracellular
portion of the Fc
RI
subunit, the DNA encoding the transmembrane
and intracellular portions of the type I IL-1 receptor, and the
pcDNA3.1 mammalian expression vector (Invitrogen, Carlsbad, CA).
The rat Fc
RI
cDNA used to make this construct was provided by
Dr. H. Metzger (National Institutes of Health). The type I IL-1
receptor DNA was cloned using reverse transcription PCR from murine
adult brain total RNA (provided by Dr. A. Trumpp, University of
California, San Francisco). Sequencing confirmed that the junction of
this chimeric receptor corresponds to base 669 of the Fc
RI
sequence (GenBankTM accession number M17153) and base 1015 of the IL-1 receptor sequence (GenBankTM accession number
M20658). The Chimera-1 plasmid was transfected using LipofectAMINE Plus
(Life Technologies) at 2 µg of DNA per ml and 3 µl of liposomes per
ml in Opti-MEM for 3 h.
4 integrin, TA-2 mouse
monoclonal antibody (35) provided by Dr. T. Issekutz (Toronto Hospital, Toronto, Ontario, Canada) and rabbit anti-mouse IgG Fc secondary antibody (Jackson ImmunoResearch, West Grove, PA); for IL-1 receptors, M5 rat monoclonal antibody (36), provided by Dr. S. Dower, and goat
anti-rat IgG secondary antibody (Fisher); for IL-2 receptor
, 3G10
mouse monoclonal antibody (Boehringer Mannheim) and rabbit anti-mouse
IgG secondary antibody (Cappel, West Chester, PA). All primary
antibodies were iodinated with chloramine T as described previously
(37). After cell harvest, the receptors were labeled with the
appropriate primary antibody for at least 30 min at 20 °C, followed
by two washes in buffered salt solution (20 mM Hepes, pH
7.4, 135 mM NaCl, 5 mM KCl, 1.8 mM
CaCl2, 1 mM MgCl2, 5.6 mM glucose). For
4 integrin experiments, RBL
cells were presaturated with IgE, and IgE was present during TA-2
binding to prevent any interaction of Fc
RI with the Fc portion of
TA-2.
RI
after cell lysis, we used rabbit anti-IgE because lysed RBL cells
appear to contain sufficient free biotin to interfere with streptavidin
aggregation.3
After incubation on ice for at least 10 min, the lysates were then
diluted with an equal volume of 80% (w/v) sucrose in 25 mM Hepes, pH 7.5, and 150 mM NaCl. Step gradients of sucrose
were formed by layering 0.25 ml of 80%, 0.5 ml of 60%, 1.5 ml of 40% (containing the cell lysate), 0.75 ml of 30%, 0.5 ml of 20%, and 0.5 ml of 10% (w/v) sucrose in Beckman Ultra-Clear centrifuge tubes
(11 × 60 mm). Centrifugation and the analysis 0.2-ml aliquots of
the gradients were performed as described (6, 16).
RI in a parallel stimulated sample.
Subsequent steps were performed with the technical assistance of
Shannon Caldwell at the Cornell Integrated Microscopy Center. Formvar carbon-coated grids (300 mesh) were suspended on the top of drops of
the sucrose fractions for 30 min to allow the adherence of DRMs. The
grids were then extensively washed, fixed with 1% gluteraldehyde for 3 min, washed, and stained for 30 s with 2% uranyl acetate. The
negatively stained DRMs were visualized with a Philips EM-201 transmission electron microscope.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
RI stimulation (16). Under these standard cell lysis
conditions with high concentrations of TX-100, Fc
RI did not
co-isolate with DRMs. However, with lower detergent lysis conditions,
similar to those identified by Pribluda et al. (7) for
enhancing the association of kinase activity with immunoprecipitated Fc
RI, we found that substantial amounts of the aggregated receptor co-purified with the low density DRMs following sucrose gradient ultracentrifugation (6).
and
). Under these conditions,
as when no TX-100 is added to the gradient (6), less than 5% of the
unaggregated Fc
RI associates with the DRM fractions (
, fractions
3-7) and greater than 50% of streptavidin-aggregated
biotin-IgE-Fc
RI complexes associate with these (
). From this
result, it appears unlikely that the plasma membrane is incompletely
solubilized or that association of aggregated receptors with DRMs
depends on the separation of DRM components from TX-100 during the
ultracentrifugation process.
View larger version (21K):
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Fig. 1.
Sucrose gradient ultracentrifugation of RBL
cell lysates. RBL cells sensitized with biotinylated
125I-IgE and suspended at 8 × 106/ml in
buffered saline solution were stimulated for 5 min at 37 °C with 10 nM streptavidin ( ) or left unstimulated (
and
)
and then lysed with an equal volume of 2× lysis buffer containing
0.1% TX-100. After 10 min on ice, the lysates were either loaded onto
sucrose gradients as described under "Experimental Procedures" (
and
) or else first treated with 2.5 µg/ml rabbit anti-IgE for an
additional 10 min prior to loading onto the gradient (
). For the
samples represented by
and
, 0.025% TX-100 was added to the
entire sucrose gradient. The 125I present in each 0.2-ml
fraction is shown as a percentage of the total 125I-IgE in
the entire gradient including the pellet (fraction 21). The right
axis indicates the percentage of sucrose used for each step in
forming the gradient (heavy line).
To investigate the requirements for DRM association of aggregated
FcRI, we lysed cells with 0.05% TX-100 prior to adding anti-IgE to
form Fc
RI aggregates. When this lysate was fractionated (Fig. 1,
), these aggregates migrated at a higher density in the sucrose
gradient, similar to aggregates formed on cells that are subsequently
lysed in 0.2% or higher TX-100 (16). In contrast, addition of anti-IgE
prior to cell lysis with 0.05% TX-100 causes greater than
50% of Fc
RI to associate with low density membranes (e.g. see Fig. 6A), similar to results using
streptavidin cross-linking (Fig. 1 (
) and Ref. 6). These results
indicate that the interactions between DRM components and aggregated
Fc
RI that occur on intact cells must be preserved during cell lysis
and sucrose gradient fractionation, as they cannot be caused by
receptor aggregation subsequent to cell lysis.
The association of FcRI with DRMs could be mediated by
protein-protein, protein-lipid, or some combination of these
interactions. By varying the amount of TX-100 used to lyse RBL cells,
we further investigated the detergent sensitivity of this interaction.
Unaggregated Fc
RI was recovered in the low density gradient
fractions when insufficient TX-100 (<0.03%) was used for cell lysis
(Fig. 2,
). Under these conditions, it
is possible that plasma membranes are not solubilized completely and
therefore migrate at this low density because of their lipid content.
With greater than 0.03% TX-100 for cell lysis, the membranes appeared
effectively solubilized, and monomeric Fc
RIs were found almost
entirely in the 40% sucrose fraction. In addition, other
membrane-bound proteins that do not associate with DRMs, including
4 integrins (e.g. see Fig. 6B) and
Src,3 were found in the 40% sucrose fractions at 0.05%
TX-100. Under these same conditions, greater than 50% of
streptavidin-aggregated biotin-IgE bound to Fc
RI remained associated
with DRMs in the sucrose gradient (Fig. 2,
). When concentrations of
TX-100 used during cell lysis were greater than 0.05%, aggregated
Fc
RIs were not retained with DRMs, and complete disruption of this
association occurred as low as 0.08% TX-100 (Fig. 2). As shown
previously, lipid-anchored markers of these DRMs (i.e.
Thy-1, GD1b gangliosides, and Lyn) remain associated even
at 0.5% TX-100 (16). This marked sensitivity for Fc
RI/DRM
association is consistent with a lipid-mediated interaction (see under
"Discussion").
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Electron microscopy of isolated, negatively stained DRMs from RBL cells lysed at 0.05% TX-100 reveals them to be vesicular structures (Fig. 3). The majority range from 50 to 200 nm in diameter; larger vesicles with diameters up to 1.4 µm were less frequently observed.3 These vesicles are qualitatively similar in appearance and size to DRM preparations from MDCK cells (25), T- and B-cell lines (28), neuroblastoma cells (26), and RBL cells, more typically lysed in 1% TX-100.4 Thus, DRMs isolated with lower TX-100 to cell lipid ratios are not ultrastructurally different from other DRMs that have been prepared with higher TX-100, and these preparations are not significantly contaminated with unsolubilized membrane sheets or organelles.
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To investigate further the structural basis of the FcRI/DRM
interaction, we used P815 mast cells and CHO cells that were stably
transfected with various Fc
RI constructs. As shown in Fig.
4, P815 cells stably expressing wild-type
2 Fc
RI subunits (wt) show
aggregation-dependent receptor association with low density
DRMs that is similar to native Fc
RI on RBL cells at 0.05% TX-100
(Fig. 2 and Ref. 6). Furthermore, cells expressing mutated versions of
the receptor lacking either the C-terminal cytoplasmic tail of the
subunit or the cytoplasmic tail of the
subunit show similar
aggregation-dependent association (Fig. 4). Interestingly, IgE receptors entirely lacking the
subunit (Fc
RI
2) also show aggregation-dependent DRM
interactions. Some differences in the magnitude of association were
observed, but the ratio of aggregated to monomeric receptors associated
with the DRMs remained fairly constant. These differences could
represent small contributions to the DRM association from the deleted
protein segments. Alternatively, this variability could be due to
differences between the transfected cell lines themselves that have
been separately subcloned. Significantly, Fc
RI in the
and
subunit cytoplasmic tail mutant cells do not activate tyrosine kinases
or other downstream signals (9), although they do become insoluble in
high concentrations of TX-100 after extensive aggregation (32).
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Nonhematopoietic CHO cells expressing wild-type FcRI showed
qualitatively similar association of this receptor with DRMs that
depends on aggregation (Fig. 5,
wild-type). However, a chimeric receptor containing the
extracellular segment of the Fc
RI
subunit and the transmembrane
and intracellular segments of the type-1 IL-1 receptor (Fig. 5,
Chimera-1) did not show significant DRM association in the
presence or absence of receptor aggregation. This chimeric receptor
demonstrates that the extracellular segment of Fc
RI
is not
sufficient to mediate this interaction. In contrast, a similar chimeric
receptor that contains the extracellular segment of the Fc
RI
subunit and the transmembrane and intracellular segments of IL-2
receptor
(p55, Tac; 39) (Fig. 5, Chimera-2) did show
aggregation-dependent association with the DRMs. The results in Figs. 4 and 5 are consistent with the transmembrane segments
of these receptors being most important for determining DRM
association. We also examined the association of Fc
RI
subunits linked to the plasma membrane by a GPI anchor. These receptors, which
lack a transmembrane segment, associated with the DRMs even in the
absence of aggregation (Fig. 5, GPI) and also remained associated to a
similar extent in 0.2% TX-100,3 conditions that would
extract aggregated, wild-type Fc
RI (Fig. 2). This is consistent with
previous studies from our laboratory (16) and others (19, 27) on
various GPI-linked proteins that show constitutive association with
DRMs isolated by this method, and it supports the view that the
membrane-anchoring region is most critical for these interactions.
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DRM association was compared for FcRI and other cell surface
receptors. Native Fc
RI on RBL cells associated with DRMs after aggregation with polyclonal anti-IgE (Fig.
6A). In contrast, the native
4 integrins on RBL cells showed only low amounts of DRM association both after binding of the monoclonal antibody TA-2 (Fig.
6B,
) and when further aggregated with a polyclonal
antibody (
). Similarly, stably transfected type-1 IL-1 receptors on
CHO cells labeled with M5 monoclonal antibody did not associate with DRMs (Fig. 6C,
), even after polyclonal antibody
aggregation (
) or streptavidin aggregation of biotinylated
M5.3 The possibility that these results are due to the
dissociation of M5 from the receptor during lysis and gradient
fractionation was excluded by labeling the IL-1 receptors with
125I-IL-1. When unlabeled M5 plus secondary antibody was
used to aggregate these receptors, the distribution of label was very similar to Fig. 6C, including the appearance of
125I-IL-1-receptor complexes in the 50-60% sucrose
fractions.3
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When we examined the association of IL-2 receptor that was
transiently expressed on CHO cells, we found that these receptors labeled with a monoclonal antibody did not associate with low density
DRMs (Fig. 6D,
). Interestingly, when these receptors were aggregated with a polyclonal antibody, significant amounts did
associate (
). In addition, some receptor aggregates also shift to
the high density sucrose fractions. As with Fc
RI, similar association with DRMs is seen if streptavidin is used to aggregate these receptors labeled with a biotinylated monoclonal
antibody.3 These results show that
aggregation-dependent association with DRMs is restricted to
certain receptors, indicating that antibody-mediated receptor
aggregation is not sufficient to cause nonspecific association or
entanglement with membrane domains at the cell surface.
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DISCUSSION |
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We previously established that the association of FcRI with
DRMs plays an important role in the initiation of signaling by this
multichain immune recognition receptor (6). Therefore, to understand
fully the activation of Fc
RI, it is necessary to characterize this
interaction. In this study, we examined the structural basis of this
aggregation-dependent association, including the
contribution of different portions of the receptor.
One of the most striking characteristics of the association of
aggregated FcRI with DRMs is the detergent sensitivity of this
interaction. Fig. 2 demonstrates that TX-100 concentrations as low as
0.08% nearly completely eliminated the association. The unusual
requirement of low concentrations of detergent presumably reflects a
weaker interaction of these membrane structures with the receptor,
which is a transmembrane protein, than with lipid-modified proteins
that can associate directly via their saturated acyl chains without
disturbing the lipid packing of the bilayer (Fig. 5 and Ref. 16). The
interaction of monomeric Fc
RI with membrane domain components may
also occur on the cell surface, but too weakly to be preserved during
lysis at TX-100 concentrations greater than 0.03%. Thus, aggregation
of Fc
RI at the plasma membrane may serve to stabilize these
preexisting interactions. We are currently investigating whether the
appearance of unaggregated Fc
RI in the low density sucrose gradient
fractions at the lowest TX-100 concentrations (Fig. 2) is due to weak
interactions with DRM. The selective detergent sensitivity that we
observed is most readily explained by a lipid-mediated association, but
we cannot rule out protein-protein interactions between Fc
RI and
other DRM components that are too weak to withstand higher detergent lysis conditions.
It is important to note that aggregation of FcRI
following detergent treatment of cells does not induce
similar associations with the DRMs (Fig. 1). Thus, interactions between
aggregated Fc
RI and DRM components must be preserved when the cells
are lysed. The ultracentrifugation of these lysates on sucrose
gradients caused the low density components to rise in the gradient,
where they can coalesce to form larger vesicles (Fig. 3), even in the presence of micellar TX-100 (Fig. 1).
The DRMs isolated from RBL cells lysed with 0.05% TX-100 appear
similar to those from 0.2 to 1% TX-100 lysates by three criteria. First, Lyn and GPI-linked proteins are selectively enriched in both of
these preparations. Greater than 50% of these lipid-linked proteins
(Fig. 5) but less than 2% of total cellular protein are found in the
low density sucrose fractions.3 Second, whole-mount
electron microscopy of the vesicles from 0.05 and 1% TX-100 lysates
reveal similar structures (Fig. 3).4 Third, the protein
compositions of the DRMs prepared with both levels of detergent are
similar as revealed by silver staining of SDS-polyacrylamide
gels.3 Thus, although it is necessary to use low detergent
concentrations to preserve interactions between aggregated FcRI and
DRMs, this membrane preparation appears to be very similar to those
isolated from other cells that lack caveolae (27, 28).
We used structural variants of FcRI to determine what portions of
the
,
, and
subunits are involved in
aggregation-dependent DRM association. Previous results
with RBL cells showed that tyrosine phosphorylation of the
and
subunit ITAMs is not necessary (6). Now we find with transfected P815
mast cells that the ITAM-containing cytoplasmic segments of these
subunits are not required for DRM association (Fig. 4). Thus, there
appears to be no involvement of proteins with Src homology 2 domains
that bind to phosphorylated ITAMs. Receptor variants lacking the entire
subunit (Fc
RI
2) also showed DRM association
(Fig. 4). This observation is consistent with the capacity of Fc
RI
2 to activate PTK signaling in these transfected P815
cells (9), as well as in U937 monocytes and transfected NIH-3T3
fibroblasts (10). Thus, the
subunit may amplify Fc
RI activation
by providing a docking site for Lyn or other signaling molecules, but
this subunit is not required for the initial step in receptor activation.
Aggregated FcRI associated with DRMs on CHO cells (Fig. 5), showing
that there is no requirement for hematopoietic cell-specific proteins
or lipids, such as the GD1b ganglioside derivatives on mast
cells that are recognized by the AA4 antibody (40, 41). This
observation is consistent with the capacity of Fc
RI to become phosphorylated by Lyn and to activate Syk when cotransfected with these
kinases in NIH-3T3 cells (2, 10). Unlike wild-type Fc
RI, GPI-linked
Fc
RI
associates with DRMs on CHO cells in the absence of any
aggregation (Fig. 5) and in both low and high levels of
TX-100,3 as seen for other GPI-linked proteins, including
Thy-1 (16). Thus, the GPI lipid tail is probably critical for mediating
aggregation-independent association of some proteins with DRMs.
DRM association under low TX-100 conditions does not occur for all
receptors after aggregation, as shown in Fig. 6 for 4 integrins and type-1 IL-1 receptors. This result enabled the use of
chimeric receptors to assess the structural portions critical for this
interaction. We found that only one (Chimera-2) of the two chimeras
that contain the extracellular segment of the Fc
RI
subunit
showed aggregation-dependent association with DRMs, indicating that this segment is not sufficient. The association of
these chimeras corresponds to that of the source receptors: IL-2
receptor
and Chimera-2 associate with DRMs, whereas IL-1 receptor
and Chimera-1 do not (Figs. 5 and 6). These results indicate that the
structural basis for IL-2 receptor
and Fc
RI association with
DRMs primarily resides with the transmembrane and/or cytoplasmic segments of these receptors. Furthermore, because Fc
RI
is not necessary for association with DRMs, and Fc
RI
is not
significantly exposed at the extracellular side of the plasma membrane
(42), it is likely that extracellular segments of Fc
RI are not
involved in this interaction.
A recent study demonstrated that the structural basis for the
association of the influenza hemagglutinin protein with
detergent-resistant membrane domains resides in its transmembrane
segment, but the amino acid residues primarily responsible for this
interaction did not suggest a predictable structural motif (43). Our
results also point to the transmembrane segments of the FcRI
and/or
subunits being primarily responsible for
aggregation-dependent association of Fc
RI with DRMs, as
extracellular Fc
RI
, cytoplasmic Fc
RI
, and Fc
RI
are
not necessary or sufficient (Figs. 4 and 5). Although it remains
formally possible that the 19-residue cytoplasmic segment of Fc
RI
is important for these interactions, this seems unlikely in light
of previous studies indicating that this short sequence is not
necessary for Fc
RI signaling (9) or detergent insolubility (32).
Covalently attached palmitic acid has been reported for the Fc
RI
and
subunits (44), and it is possible that these lipid
modifications are involved in DRM association of this receptor. More
extensive mutational analyses are required to define completely the
structural basis for Fc
RI and IL-2 receptor
interactions with DRMs.
Our results point out that caution must be used when interpreting data
from studies involving IL-2 receptor chimeras. For example,
chimeras with extracellular and transmembrane segments of IL-2 receptor
and ITAM-containing cytoplasmic segments of the Fc
RI
or
subunits (8, 12, 13, 45) or the T cell receptor
subunit (12, 45)
have been used extensively to investigate the activation of mast cells
and T cells. These studies, which showed kinase activation upon
aggregation of the chimeric receptors, suggested that ITAM sequences
were the only segments of these these receptors essential for
signaling. However, our results with DRMs in Figs. 4 and 5 would
predict that these chimeric constructs might associate with specialized
domains in the plasma membrane because of structural features normally
provided by other portions of multichain immune recognition receptors,
and they could consequently interact with domain-associated Lyn or
similar Src family PTKs. Both Fc
RI and IL-2 receptors utilize Src
family PTKs in early signaling events (46, 39), and DRM association may
represent a common mechanism for interaction of such receptors with
these kinases.
In conclusion, the present study supports a model of FcRI activation
that involves the selective interaction of this receptor with
specialized membrane domains immediately following receptor aggregation
on cells (for a review, see Ref. 5). These domains, as isolated, are
enriched in Lyn and facilitate the coupling of the receptor with this
kinase in a process that may also be utilized by other receptors. The
transmembrane segments of Fc
RI probably play the major role in this
lipid-protein interaction, which also occurs for IL-2 receptor
after aggregation. Further study will be required to test this
hypothesis more fully and to answer other important questions
concerning the structural basis for this interaction, including the
specific DRM components involved. Other questions about the role of
compartmentalization in IgE receptor signaling also remain to be
answered. These include the regulation of Lyn activity associated with
membrane domains on cells and the role of tyrosine phosphatases in the
regulation of signaling orchestrated by Fc
RI interactions with these
membrane domains.
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FOOTNOTES |
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* This work was supported by Grant AI22449 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: G. W. Hooper Foundation, Box 0552,
University of California, San Francisco, CA 94143.
§ To whom correspondence should be addressed. Tel.: 607-255-4095; Fax: 607-255-4137; E-mail: dah24{at}cornell.edu.
The abbreviations used are: PTK, protein tyrosine kinase; ITAM, immunoreceptor tyrosine-based activation motif; RBL, rat basophilic leukemia; DRM, detergent-resistant membrane; GPI, glycosylphosphatidylinositol; CHO, Chinese hamster ovary; IL, interleukin; TX-100, Triton X-100.
2 D. Holowka, unpublished observations.
3 K. A. Field, D. Holowka, and B. Baird, unpublished observations.
4 D. Reczeck and K. Field, unpublished observations.
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