(Received for publication, September 24, 1996, and in revised form, December 3, 1996)
From the Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York 14853-1301
The earliest known step in the activation of the
high affinity IgE receptor, FcRI, is the tyrosine phosphorylation of
its
and
subunits by the Src family tyrosine kinase, Lyn. We
report here that aggregation-dependent association of
Fc
RI with specialized regions of the plasma membrane precedes its
tyrosine phosphorylation and appears necessary for this event. Tyrosine
phosphorylation of
and
occurs in intact cells only for Fc
RI
that associate with these detergent-resistant membrane domains, which
are enriched in active Lyn. Furthermore, efficient in vitro
tyrosine phosphorylation of Fc
RI subunits occurs only for
those associated with isolated domains. This association and in
vitro phosphorylation are highly sensitive to low concentrations
of detergent, suggesting that lipid-mediated interactions with Lyn are
important in Fc
RI activation. Participation of membrane domains
accounts for previously unexplained aspects of Fc
RI-mediated
signaling and may be relevant to signaling by other multichain immune
receptors.
The plasma membrane contains specialized regions that have distinct compositions and can serve unique functions in the regulation of cell surface receptor activation. For example, caveolae have been shown to associate with certain signaling proteins (1, 2) and have been implicated in receptor activation (3-6), vesicular transport (7, 8), and the uptake of small molecules (9). Compositionally related membrane domains, which lack the invaginated morphology of caveolae as well as the membrane protein caveolin, have also been identified and biochemically separated from caveolae (10). These membrane domains, like caveolae, are resistant to solubilization in nonionic detergents such as Triton X-100, are enriched in sphingolipids and glycosylphosphatidylinositol-linked proteins, and are associated with palmitoyl-anchored signaling molecules including Src family tyrosine kinases (10-14). Detergent-resistant membrane domains isolated from rat basophilic leukemia (RBL)1 cells, a mast cell line, contain at least 30% of the cellular Lyn, a Src family tyrosine kinase, and no detectable caveolin (15).
Aggregation of FcRI on mast cells and basophils by multivalent
antigens leads to phosphorylation of immunoreceptor tyrosine-based activation motifs within the
and
receptor subunits by Lyn (16-19). This initiates a signaling cascade culminating in secretion of inflammatory mediators and cytokines that play an important role in
the allergic response (20, 21). The molecular mechanism by which
aggregation of Fc
RI initiates its phosphorylation by Lyn is
incompletely understood. Selective binding of Lyn directly to
unphosphorylated Fc
RI
(22) has been proposed to mediate an
initial trans-phosphorylation of aggregated Fc
RI (23),
but this does not account for the capacity of Fc
RI lacking the
subunit (24, 25) or chimeric receptors containing only the
cytoplasmic tail (26-28) to become tyrosine-phosphorylated upon aggregation. The involvement of detergent-resistant membrane domains in
Fc
RI signaling was recently suggested by the observation that aggregation of Fc
RI on RBL cells significantly increased the amount
of active Lyn associated with these structures (15). Furthermore,
fluorescence microscopy studies showed that aggregation of Fc
RI at
the surface of intact cells co-redistributes ganglioside-enriched membrane patches that are related to the isolated membrane domains (29,
30). The aggregation-dependent association of Fc
RI with these less fluid regions of the membrane (30, 31) is also consistent
with decreased lateral and rotational mobility of aggregated Fc
RI
(reviewed in Ref. 32). In the present study, we establish conditions
for preserving the interaction of aggregated Fc
RI with these
membrane domains following cell lysis, and we demonstrate the
importance of this interaction to the initial step in signaling, the
tyrosine phosphorylation of Fc
RI.
RBL-2H3 cells were lysed in 10 mM Tris, pH 8.0, 50 mM NaCl, 10 mM EDTA, 1 mM Na3VO4, 30 mM pyrophosphate, 10 mM glycerophosphate, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (Calbiochem, San Diego, CA), 0.02 units/ml aprotinin, 0.01% (w/v) NaN3, and 0.05% (v/v) Triton X-100. The lysates were then diluted 1:1 in 80% sucrose and analyzed by ultracentrifugation as described (15). In some experiments, the lysis buffer contained 0.025% Triton X-100, and 0.025% Triton X-100 was also present in the 80% sucrose solution used to dilute the lysate. The two lysis procedures yielded identical results.2 Sucrose solutions contained 25 mM Tris, pH 7.5, 125 mM NaCl, and 2 mM EDTA.
ImmunoblottingElectrophoresis of samples was carried out
on 12.5% acrylamide SDS gels under nonreducing conditions, and semidry
transfer to Immobilon P (Millipore, Bedford, MA) was performed as
described (15). Anti-phosphotyrosine immunoblots were performed using 0.1 µg/ml monoclonal antibody 4G10 conjugated to horseradish
peroxidase (UBI, Lake Placid, NY) and Supersignal ECL substrate
(Pierce, Rockford, IL). For the results in Fig. 2, tyrosine
phosphorylation of the FcRI
subunit was quantified from
anti-phosphotyrosine immunoblots of post-nuclear supernatants of
106 RBL cells lysed in 0.2% Triton X-100. The prominent
34-kDa band detected in these blots after Fc
RI stimulation was
identified as
based on selective immunodepletion by IgE-specific
agarose beads.2 This band was quantified with a 256 gray-scale scanner (Umax Vista-S6E) and NIH Image software.
Immunoprecipitations
After adjusting the sucrose fractions
to 0.2% Triton X-100 to extract FcRI from the membrane domains,
Fc
RI was immunoprecipitated for 90 min with
trinitrophenyl-conjugated Sepharose 4B (which efficiently binds
anti-DNP IgE). The immunoprecipitates were washed twice with 0.2%
Triton X-100 and once in lysis buffer lacking detergent prior to
elution by boiling in nonreducing SDS sample buffer.
Kinase assays were performed by
adding kinase buffer (20 mM Tris, pH 7.6, 10 mM
MgCl2, 1 mM ATP, and 1 mM
Na3VO4) to the sucrose fractions and incubating
at 37 °C for 15 min. The reaction was quenched either with 5 × nonreducing SDS sample buffer or by adding 50 mM EDTA
followed by immunoprecipitating FcRI.
In order to determine if the interaction of FcRI with membrane
domains is involved in the activation of this immunoreceptor, we
developed conditions that preserve this association during the
isolation of these complexes by equilibrium sucrose density ultracentrifugation. As shown in Fig. 1A,
limiting amounts of Triton X-100 used for cell lysis preserve the
association of aggregated Fc
RI (
,
) with the
detergent-resistant membrane domains which migrate as low density
vesicles (fractions 3-7). In 29 separate experiments, 54 ± 7%
of biotin-IgE Fc
RI complexes aggregated with streptavidin associate
with the membrane domains (
). Significant but lesser amounts of
antigen-aggregated receptors associate (
; 11 ± 1%,
n = 6), most likely reduced by the partial reversal of IgE-antigen binding during the overnight ultracentrifugation. In
contrast, monomeric Fc
RI (
) is nearly absent from the membrane domains (3 ± 1%, n = 26) and found almost
entirely in the 40% sucrose fractions containing solubilized proteins
(fractions 10-16). The association of Fc
RI with isolated membrane
domains depends on its aggregation at the cell surface, as less than
5% association is seen for Fc
RI aggregated after cell lysis or for
Fc
RI aggregated with antigen on cells and then dissociated with
monovalent hapten after lysis.2 The interaction between
aggregated Fc
RI and the membrane domains is very sensitive to the
detergent:cell lipid ratio during solubilization and
ultracentrifugation, as indicated by its disruption when concentrations of Triton X-100 greater than 0.05% are used (15). This sensitivity is
similar to that observed by Pribluda et al. (23) for Fc
RI coupling to Lyn in cell lysates, and it contrasts with
cytoskeleton-mediated detergent insolubility of aggregated Fc
RI
(33-35), which is not disrupted by high Triton X-100
concentrations.
Although this reduction in Triton X-100 used for cell lysis
dramatically increases the amount of aggregated FcRI that remains associated with detergent-resistant membrane domains, these domains are
otherwise very similar to those isolated after lysis in high Triton
X-100 (
0.2%). When directly compared, domains from low and high
detergent lysis conditions contain the same fraction of cellular Lyn,
and neither has detectable amounts of Src.2 In addition,
both preparations contain a similar spectrum of tyrosine kinase
substrates as revealed in in vitro tyrosine kinase assays
(Ref. 15 and as described below), and both contain similar amounts of
cellular protein (<2% of the total).2 By these criteria,
the domains obtained using 0.05% Triton X-100 for cell lysis appear to
be identical to other membrane domains previously described that do not
contain caveolin (10, 13-15). Furthermore, the aggregation-dependent
association of Fc
RI with membrane domains shows selectivity among
transmembrane cell surface receptors, as Fc
RI but not Type I
interleukin-1 receptors, both expressed on Chinese hamster ovary cells,
associate with membrane domains following
aggregation.3
Association of FcRI with membrane domains does not require tyrosine
phosphorylation of the receptor subunits. As shown in Fig.
1B, RBL cells permeabilized with Streptolysin O in the
presence of excess EDTA to inhibit kinase activity show a similar
amount of aggregation-dependent association of Fc
RI with
domains as intact cells (Fig. 1A). As previously shown with
broken cells (36), stimulated tyrosine phosphorylation of Fc
RI
and other substrates is prevented by EDTA in these permeabilized
cells.2
The presence of Lyn and aggregated FcRI within the same subregions
of the plasma membrane suggests that domain-associated Lyn could be
responsible for the initial phosphorylation of the immunoreceptor
tyrosine-based activation motifs. Fc
RI associates with membrane
domains very rapidly at 37 °C (
, Fig.
2A) and is more than 50% complete within
30 s, whereas substantially less than 50% of the maximal tyrosine
phosphorylation of Fc
RI
occurs during this time (
, Fig.
2A). The amount of
tyrosine phosphorylation declines
after 2 min at 37 °C, and domain-associated receptor also decreases
between 5 and 30 min in parallel with its internalization.2
At 4 °C, the association of Fc
RI with domains occurs more slowly (
, Fig. 2B), but is clearly more rapid than the
tyrosine phosphorylation during the first 5 min (
, Fig.
2B). Fc
RI internalization and downstream signaling such
as Ca2+ mobilization and phosphatidylinositol hydrolysis do
not occur at 4 °C (37), indicating that they are not required for
domain association. These results demonstrate that association of
Fc
RI with membrane domains on cells is an early,
aggregation-dependent event that is sufficiently rapid to
mediate receptor tyrosine phosphorylation.
Evidence for FcRI tyrosine phosphorylation occurring within membrane
domains of intact cells is shown in Fig. 3. Stimulation of biotin-IgE-sensitized RBL cells with streptavidin dramatically increases the tyrosine phosphorylation of many proteins. When lysates
of these cells are analyzed by sucrose gradient ultracentrifugation, most of the proteins with enhanced tyrosine phosphorylation are found
with the solubilized proteins at 40% sucrose (fractions 11-16), as
expected (15). Associated with the membrane domains (fractions 3-8)
after stimulation are tyrosine-phosphorylated proteins of approximately
90, 53/56, 45, 34, and 25-30 kDa. The 53/56-kDa doublet was identified
as Lyn by reprobing the blot with rabbit anti-Lyn (UBI).2
Significantly, the 45-, 34-, and 25-30-kDa bands appear only with
stimulation and are markedly enriched in membrane domains relative to
the other fractions. The domain-associated proteins of 34 and 25-30
kDa correspond to phosphorylated
and
2 Fc
RI subunits, respectively, as identified by immunoprecipitating Fc
RI from the sucrose gradient fractions (Fig. 3B). Fig. 3
clearly shows that the tyrosine-phosphorylated
and
subunits are almost entirely associated with membrane domains. The
majority of other tyrosine kinase substrates phosphorylated as the
result of Fc
RI aggregation are located in the solubilized protein
fractions, presumably because they are cytosolic or associated with
membranes that are solubilized in 0.05% Triton X-100. Syk, the
ZAP-70-related tyrosine kinase responsible for phosphorylating the
majority of substrates downstream of Fc
RI (19, 38, 39), is also
found exclusively in these soluble fractions,2 as expected
because activated Syk is localized primarily in the cytosol after
receptor stimulation (40, 41).2 Thus, following Fc
RI
aggregation on cells, Lyn phosphorylates the
and
subunits of
domain-associated receptors. This apparently leads to a transient
association and the activation of Syk, followed by Syk-mediated
phosphorylation of downstream substrates, most of which are not stably
associated with membrane domains.
Additional support for the involvement of these domains in initiating
FcRI activation comes from in vitro tyrosine kinase assays performed on sucrose fractions, followed by immunoprecipitation of Fc
RI in the presence of 0.2% Triton X-100 (which releases Fc
RI from membrane domains). Fig. 4A shows
that aggregated Fc
RI associated with membrane domains isolated after
cell lysis in 0.05% Triton X-100 (MD+) are efficiently
tyrosine-phosphorylated in vitro, whereas receptors in the
sucrose fractions containing solubilized proteins (40+ and 40
) are
not phosphorylated, and membrane domains from unstimulated cells
(MD
) also show no phosphorylated Fc
RI.
Streptavidin-aggregated Fc
RI from cells lysed in 0.2% Triton
X-100 migrate at a high density (50-70% sucrose) in these gradients
(15). When in vitro tyrosine kinase assays are performed on
these high density sucrose fractions (HD+), a relatively
small amount of
subunit phosphorylation is seen. This fraction does contain a small amount of Lyn that may be responsible for the phosphorylation detected,2 but it is not known whether this
represents Lyn directly associated with Fc
RI, Lyn contaminating this
fraction from the 40% sucrose fraction, or fragments of membrane
domains which remain receptor-associated in 0.2% Triton X-100.
Consistent with the last possibility, the in vitro
phosphorylation in the HD+ fraction shows a Triton X-100 sensitivity
similar to that of the MD+ fraction (see below).
When in vitro tyrosine kinase assays are performed on
membrane domains, stimulated FcRI phosphorylation is highly
sensitive to the concentration of Triton X-100 present. As shown in
Fig. 4B, addition of submicellar (0.01%) Triton X-100 to
the membrane domains causes a slight enhancement of Fc
RI
and
tyrosine phosphorylation, but higher Triton X-100 concentrations
dramatically reduce this phosphorylation. The association of Lyn with
membrane domains, as well as its activity toward the exogenous
substrate, enolase, is not significantly affected by Triton
X-100,2 and neither is phosphorylation of Lyn itself or the
45-kDa substrate (Fig. 4B). The exquisite sensitivity of
Fc
RI in vitro phosphorylation to Triton X-100 indicates
that lipid-mediated association of these receptors with the membrane
domains is required for this activation step. Fig. 4C
supports this conclusion, demonstrating that treatment of isolated
membrane domains with 0.05% Triton X-100 (
) dissociates the
aggregated receptors from these membrane domains as indicated by
reanalysis on a second sucrose gradient. Treatment of the same domains
with 0.01% Triton X-100 (
) does not dissociate the receptor, although it does cause a slight change in the distribution of the
receptor within the gradient relative to untreated membrane domains
(
). Thus, Fc
RI associated with membrane domains is functionally coupled to Lyn, and this association is easily disrupted by
detergent.
The involvement of membrane domains in this early step of FcRI
activation provides a new model in which the initial phosphorylation of
the
and
subunits by Lyn is mediated by lipid-protein
interactions. Although previous results have explained how the
phosphorylation of Fc
RI
and
and subsequent events proceed
after the association of active Lyn with a receptor cluster (23, 25,
38, 39), the structural basis for the initial interaction between Lyn
and Fc
RI in the activation process has remained poorly defined.
Several studies that detected association of Lyn with unstimulated
Fc
RI utilized methods that could stabilize the association of these receptors with membrane domains, including chemical cross-linking (18)
or low detergent:cell lipid ratios (23). We find that unstimulated
receptors do not co-isolate with the Lyn-containing domains to a large
extent (Fig. 1), although weak and/or transient interactions could
occur on intact cells. The size and stability of the domains on the
surface of intact, unstimulated cells are unknown. These domains are
likely to be small and dynamic in composition, but appear to coalesce
together with aggregated Fc
RI (29, 30). Thus, localization of Lyn
within membrane domains could serve to sequester this kinase away from
Fc
RI prior to receptor aggregation and, in turn, provide a pool of
active or readily stimulated Lyn for aggregated Fc
RI that stably
associate with the domains. Support for this aspect of the model comes
from our observation that isolated membrane domains contain abundant
tyrosine kinase activity even in the absence of Fc
RI activation
(15), as well as from other studies on Src family members that
associate with detergent-resistant membrane domains. These other
investigations have found that Fyn, Lck, and Fgr associated with
isolated membrane domains show higher specific activity in
vitro than soluble forms of these kinases (42, 43), possibly
because of the capability for kinases concentrated within domains to
trans-autophosphorylate
readily.4
Our results demonstrate that aggregation of FcRI causes its rapid
and efficient association with specialized domains in the plasma
membrane that are enriched in the tyrosine kinase, Lyn. Fc
RI
associated with membrane domains are rapidly tyrosine-phosphorylated in
intact cells, and this phosphorylation is also observed in vitro preferentially for receptors associated with membrane
domains. The interaction of Fc
RI with these specialized membrane
domains does not depend on the
subunit,3 and thus can
account for the initiation of receptor signaling independent of
specific protein-protein interactions between the
subunit and Lyn
(24-28). In addition, this receptor-membrane domain association may
facilitate coupling to processes such as Ca2+ mobilization,
lipid metabolism, and exocytic vesicle fusion. Recent evidence
indicates that specialized membrane domains, including caveolae, are
involved in the signaling of other cell surface receptors such as
certain growth factor receptors (3, 5) and
glycosylphosphatidylinositol-linked mitogenic receptors (12, 44, 45).
Receptor-domain interactions also may be important for other multichain
immune recognition receptors that utilize Src family kinases during
their initial signaling steps (46, 47).