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-Chain Dependent Recruitment of Tyrosine Kinases to Membrane Rafts by the Human IgA Receptor Fc
R1
Department of Microbiology, Dartmouth Medical School, Lebanon, NH 03756
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R, redistributes to
plasma membrane rafts after cross-linking and that tyrosine kinases are
relocated to these sites following FcR capping. We demonstrate by
confocal microscopy that FcR caps in membrane rafts by a
-chain-independent mechanism but that -chain expression is
necessary for Lyn redistribution. Immunoblotting of rafts isolated by
sucrose density gradient centrifugation demonstrated recruitment of
-chain and phosphorylated tyrosine kinases Lyn and Bruton’s
tyrosine kinase to membrane rafts after FcR cross-linking.
Time-dependent differences in Lyn phosphorylation and Bruton’s
tyrosine kinase distribution were observed between cells expressing
FcR plus -chain and cells expressing FcR only. This study
defines early FcR-triggered membrane dynamics that take place before
FcR internalization. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R)3 is a 50- to
70-kDa transmembrane glycoprotein expressed primarily by myeloid cells
including neutrophils, monocytes, and macrophages (1, 2).
Cross-linking FcR with aggregates of IgA or IgA-opsonized particles
triggers a variety of cellular responses including phagocytosis,
oxidative burst, and degranulation in neutrophils and monocytes
(3, 4). More recently, attention has turned to the role of
FcR-targeted Ag uptake and signaling following Ag
uptake.4
Despite the range of functions triggered by FcR, relatively little
is known about the initial plasma membrane events that mediate
association of FcR with downstream signaling effectors. FcR
associates with the src family member tyrosine kinase
p53/56-Lyn (5). Cross-linking FcR triggers calcium
release from intracellular stores in neutrophils
(6), and respiratory bursts are inhibited by
phosphatidylinositol (PI) 3-kinase inhibitors (7). In the
monocytic cell line U937, FcR cross-linking results in
phosphorylation of the associated FcR -chain and phospholipase C
(PLC) (8). FcR on mesangial cells mediates
phosphorylation of PLC1 that is linked to calcium mobilization
through PI phosphate hydrolysis (9).
Signaling is accomplished by FcR by its association with the FcR
-chain to form the trimer FcR/ (10). The FcR
-chain dimer is also found in the high-affinity IgG (FcRI) and
IgE (Fc
RI) receptor complexes expressed on mast cells and monocytes,
respectively (11). Arguably, many of the early signaling
events triggered by FcRI and FcR could be similar in terms of
tyrosine kinase recruitment, and indeed the high-affinity IgE receptor
FcRI has been shown to activate the tyrosine kinases Lyn and Syk
through FcR -chain (12, 13). More recently, the site of
FcRI activation in the plasma membrane has been investigated.
Cross-linked FcRI has been shown to redistribute to membrane domains
rich in glycosphingolipids and cholesterol (14). These
domains, termed membrane "rafts," have estimated average sizes
ranging from 70 nm (15) to 500 nm in diameter
(16). Rafts are characterized by detergent insolubility
and a high content of ganglioside GM-1, which is not a significant
component of other plasma membrane domains (17). The
redistribution of cross-linked FcRI to membrane rafts is significant
in that rafts are rich in signaling molecules such as tyrosine kinases
(18, 19). Recruitment of tyrosine kinases to rafts by
FcRI has now been demonstrated in confocal microscopy studies
(14).
The aim of this study was to determine whether FcR redistributed to
rafts and whether this was the site of tyrosine kinase recruitment and
phosphorylation. Further, we wished to determine whether these events
were dependent on FcR -chain expression. To circumvent the problems
caused by studying FcR function in cells expressing endogenous
-chain, we cotransfected the cDNA for FcR and -chain or FcR
alone into the B cell line A20 IIA1.6, which does not express other Fc
receptors or -chain. We show that while cross-linked FcR
colocalizes with membrane rafts irrespective of -chain, recruitment
of Lyn and Btk to the rafts and phosphorylation of these kinases was
dependent on the presence of -chain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R/-chain constructs
The pCAV vector containing the human FcR cDNA was the gift of
Dr. C. Maliszewski (Immunex, Seattle, WA). Cells of the A20 IIA1.6 B
cell line, which are Fc receptor negative (20), were
either cotransfected with pCAV/FcR cDNA and pNUT/-chain cDNA
constructs or transfected with pCAV/FcR cDNA by electroporation
using a Bio-Rad electroporator (Bio-Rad, Richmond, CA) at 250 V, 960
µF. The pNUT vector allows selection using methotrexate.
B cell transfectants and culture
Transfectants expressing FcR and -chain were cultured in
RPMI 1640 medium supplemented with 10% FBS, 40 µg/ml gentamicin, 2
mM L-glutamine, 1 mM sodium pyruvate, and 0.9 mg/ml
methotrexate. Transfectants expressing FcR and no -chain were
cultured similarly except that 0.8 mg/ml G418 was used as the selection
agent instead of methotrexate. Levels of FcR cell-surface
expression were routinely monitered by flow cytometry using a Becton
Dickinson FACScan (San Diego, CA).
Abs and fluorochromes
Anti-FcR (My43) is a mouse IgM mAb produced in our laboratory
(21). Polyclonal rabbit anti-Lyn or anti-Btk and
agarose-conjugated PY20 anti-phosphotyrosine (PY) Ab were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse transferrin
receptor (TfR) Ab (biotin-conjugated) was purchased from PharMingen
(San Diego, CA). Indocarbocyanine 3 (Cy3)-conjugated goat
anti-mouse (GAM) IgM (µ) (Caltag Laboratories, Burlingam, CA) was
labeled with Cy3 according to the manufacturers instructions (Molecular
Probes, Eugene, OR). FITC-conjugated GAM-IgM (µ) was purchased from
Caltag. Cy3-conjugated goat anti-rabbit (GAR) IgG (H+L),
HRP-conjugated GAR-IgG, and FITC-conjugated streptavidin were purchased
from Jackson ImmunoResearch Laboratories (West Grove, PA).
FITC-conjugated cholera toxin (ChTx) (subunit B) was purchased from
Sigma (St. Louis, MO).
FcR capping and endocytosis
Cells were assessed for viability by ethidium bromide exclusion then adjusted to a density of 105 cells/ml in RPMI 1640 plus 10% FBS and adhered for 90 min at 37°C to chamber slides previously coated with 0.1 mg/ml poly-L-lysine (Sigma). Slides were chilled to 4°C and gently washed once with 4°C media to remove nonadherent cells. Cells were incubated with 20 µg/ml My43 in RPMI 1640 for 1 h at 4°C, followed by washing three times with medium. Surface-bound My43 was cross-linked with Cy3- or FITC-conjugated F(ab')2-GAM µ-chain as appropriate at a concentration of 1 µg/ml. Samples were incubated for 45 min at 4°C before washing three times in 4°C media. Medium was removed and replaced with 37°C medium and incubated as indicated before fixation for 30 min at room temperature with 0.5% paraformaldehyde in PBS.
Cell-surface and intracellular staining of fixed cells
GM-1 in rafts was labeled for 45 min at room temperature with 0.1 µg/ml FITC-conjugated ChTx subunit B (22). Samples were then washed three times in PBS. For intracellular staining, cells were permeabilized with 0.5% saponin, 0.1% BSA, and 0.1% NaN3 in PBS (permeabilization buffer) for 15 min at room temperature. Anti-Lyn Ab was added at a concentration of 20 µg/ml in permeabilization buffer for 45 min before washing three times. Cy3-conjugated GAR-IgG (100 µl) was then added at 1.0 µg/ml and incubated for 45 min before washing three times in permeabilization buffer. Background staining was assessed by incubation of cells with fluorochrome-conjugated Ab alone. Specificity of Lyn staining was demonstrated with a Lyn-blocking peptide (Santa Cruz Biotechnology).
Laser scanning confocal fluorescence microscopy
Cover slips were mounted using Prolong Antifade (Molecular
Probes), and cells were analyzed with a Bio-Rad MRC1000 laser
scanning system equipped with a Kry/Arg laser and beam splitter to
allow simultaneous two- and three-color imaging. Codistribution of
FcR and GM-1 or FcR and Lyn was assessed by selecting cells with
capped cell-surface FcR. Images were then assessed for
codistribution of GM-1 or Lyn At least 100 cells from random fields
were imaged and counted for each time point. Images were analyzed using
Adobe PhotoShop 4.0 software (Mountain View, CA).
Isolation of detergent-insoluble membrane domains
Isolation of rafts was performed by the method of Fra et al.
with modifications (23). One hundred million cells were
resuspended to 5 ml in culture medium and incubated at 4°C with
HRP-conjugated ChTx (5 µg/ml) followed by PBS washing. Cells were
lysed for 30 min in detergent extraction buffer (25 mM Tris, pH 7.6,
150 mM NaCl, 5 mM EDTA, 20 µg/ml each of chymostatin, leupeptin,
antipain, pepstatin, 40 mM
Na3VO4/200 mM NaF, 0.05%
Triton X-100 (all from Sigma)) and adjusted to 1.5 M sucrose in 20 mM
Tris, pH 7.5. Samples (3 ml) were added to 13-ml ultracentrifuge tubes
and then overlaid with 7 ml of 1.2 M sucrose followed by a layer of
0.15 M sucrose. Samples were centrifuged at 38,000 rpm in a Beckman
SW41 rotor for 18 h at 4°C. One-milliliter fractions were
carefully withdrawn using a pipettor and assayed for peroxidase
activity by luminol chemiluminescence (Amersham-Pharmacia Biotech,
Piscataway, NJ) using an EG&G Berthold Microlumat 96V chemiluminometer.
For FcR cross-linking, cells were chilled to 4°C and incubated
with My43 followed by F(ab')2-GAM µ-chain
before lysis. After washing, cells were warmed to 37°C and reactions
were stopped by the addition of ice-cold PBS and placing tubes in an
ice water bath. Cells were then pelleted and lysed by addition of 1 ml
ice-cold detergent lysis buffer followed by 30 min further incubation
before ultracentrifugation.
SDS-PAGE and immunoblotting
Raft fractions were adjusted to equal protein concentrations and equal amounts of protein resolved by SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose membranes and incubated overnight at 4°C with 5% nonfat dry milk and 0.5% Tween 20 in PBS. Membranes were incubated with Abs to Lyn and Btk (0.2 µg/ml) for 2 h at room temperature and washed six times for 5 min in PBS and transferred to tubes containing 3% nonfat dry milk/0.05% Tween 20 in PBS. HRP-conjugated anti-rabbit IgG Ab was added at a 1/10,000 dilution (0.1 µg/ml) and incubated for a further 2 h at room temperature. Membranes were then washed six times for 5 min in PBS. Proteins were detected by enhanced chemiluminescence (ECL) (Amersham-Pharmacia Biotech, Piscataway, NJ).
Immunoprecipitation
Agarose-coupled anti-PY Ab (PY20; Santa Cruz Biotechnology) was incubated overnight at 4°C with raft or nonraft fractions (100 µg protein/sample) in 1% Nonidet P-40 then washed three times in 10 mM Tris, 2 mM Na3VO4, and 1% Nonidet P-40, pH 7.0, before washing once in 10 mM Tris, pH 7.6. Immunoprecipitated proteins were immunoblotted as described for Lyn and Btk. Where appropriate, immunoprecipitated proteins were treated with the tyrosine phosphatase LAR-D1. Briefly, beads were washed in 50 mM NaCl, 25 mM imidazole, 5 mM DTT, and 2.5 mM EDTA, pH 7.0, and resuspended in 20 µl buffer containing 5 U LAR-D1 (Calbiochem, San Diego, CA). Samples were incubated for 2 h at 37°C, and reactions were stopped with SDS-PAGE sample buffer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R caps in rafts independently of -chain expression
We generated
FcR+/-chain+ and
FcR+/-chain- IIA1.6
transfectants. Flow cytometric analysis indicated the levels of FcR
cell-surface expression were comparable between
-chain+ cells and
-chain- cells (data not shown). To examine
the plasma membrane distribution of FcR, we performed confocal
microscopy analysis of -chain+ and
-chain- cells. Midsection confocal images of
-chain+ cells (Fig. 1
a1) demonstrated a punctate
staining of FcR (red) distributed evenly in the plasma membrane.
Staining of the plasma membrane ganglioside GM-1 with FITC-ChTx subunit
B (green) also showed a punctate distribution of GM-1 (Fig. 1a2). Although the punctate staining of GM-1 and FcR
could potentially be caused by aggregation through clustering of FcR
at 4°C, this is unlikely because cells fixed at 4°C before binding
of Ab to FcR showed the same distribution of FcR and GM-1 as
cells in which FcR was cross-linked at 4°C before fixation (data
not shown). Additionally, 0 min cells (Fig. 1, a1,
b1, and c1) were manipulated and fixed at 4°C
without warming, which would minimize FcR or GM-1 redistribution
caused by cross-linking of FcR. The plasma membrane distribution of
FcR and GM-1 in -chain- cells was similar
to -chain+ cells (Fig. 1, a3 and
a4). -Chain+ (Fig. 1b1)
and -chain- (Fig. 1c1) cells
costained for FcR and GM-1 at 4°C and then fixed showed some
overlap of red and green fluorescence (indicated by yellow). However,
areas of plasma membrane were observed to stain for FcR (red)
but not GM-1 and conversely GM-1 (green) but not FcR.
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R on -chain+ cells was cross-linked and
incubated at 37°C for 2 and 5 min before fixation and costaining for
GM-1. We consistently observed that cross-linked FcR capped after
warming cells to 37°C (Fig. 1
, b2 and b3). In
-chain+ cells, GM-1 was observed to cap and
its distribution coincided with that of FcR (indicated by yellow
color). In -chain+ cells, cocapping of FcR
and GM-1 persisted for at least 5 min before complete receptor
internalization by 10–20 min (Fig. 1b4). We tested the
dependence of FcR capping to rafts on expression of -chain. In
the -chain- transfectants, we observed
capping of FcR in membrane rafts within 2 min (Fig. 1c2),
which persisted at 5 min (Fig. 1c3), before receptor
internalization within 10–20 min (Fig. 1c4).
The kinetics of FcR capping, GM-1 capping, and FcR
internalization were similar in -chain+ and
-chain- cells (Fig. 1, d–g).
FcR capping was observed within 30 s in 33% of
-chain+ cells and 36% of
-chain- cells (Fig. 1d). Elevated
levels of FcR capping persisted for 5 min in both
-chain+ and -chain-
cells. GM-1 capping was observed to occur within 30 s (Fig. 1e) in both -chain + (28%) and
-chain- cells (40%). GM-1 capping persisted
for 20 min and was still observed in 18% of
-chain+ and -chain-
cells, although FcR capping was no longer observed. FcR and GM-1
cocapping were observed to have similar kinetics in
-chain+ and -chain-
cells (Fig. 1f). The percent of cocapped FcR/GM-1
returned to baseline levels within 10 min in both cell types. Similar
kinetics of FcR internalization were observed in
-chain+ and -chain-
cells (Fig. 1g). By 20 min, -chain+
and -chain- cells demonstrated equal (80%)
internalization of cell-surface FcR.
GM-1 is not internalized with FcR
To determine whether there was internalization of GM-1 when FcR
internalization was observed, cells were permeabilized with saponin to
permit detection of internal GM-1 and then labeled with FITC-ChTx. When
cells were fixed at 4°C before permeabilization and staining for
GM-1, we did not observe internal GM-1 staining (Fig. 2a). After internalization of
FcR, there was no detectable intracellular GM-1 and no noticeable
difference in cell-surface levels of GM-1 (Fig. 2b). Thus it
appears that FcR is internalized without GM-1 internalization,
suggesting that FcR leaves rafts before endocytosis.
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R is selective for rafts
To further demonstrate selectivity of FcR for rafts over other
plasma membrane domains, we compared the distribution of FcR with
that of the TfR. The TfR is excluded from rafts and has a mutually
exclusive distribution with respect to GM-1 (17). Thus, it
is a useful marker for nonraft plasma membrane. FcR was stained with
My43 plus Cy3-GAM-IgM as described. The cells were then fixed at 4°C
and permeabilized before staining for TfR with biotin-conjugated
anti-TfR Ab followed by FITC-conjugated streptavidin (Fig. 3a). Two pools of TfR were
observed in cells, a pool of intracellular transferrin and a pool of
cell-surface TfR. Almost no colocalization of FcR and TfR was
observed in cells fixed without warming to 37°C, indicating that
FcR and TfR partition into different plasma membrane domains. After
warming cells to 37°C to cap cross-linked FcR (Fig. 3b), TfR staining did not colocalize with FcR. This
confirms our observation that FcR cocaps with the raft marker GM-1
and that capped FcR is selective for membrane rafts over other
plasma membrane domains.
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R caps in a -chain-dependent manner
Temporary localization of FcR in rafts could increase the
proximity of cross-linked FcR/ complexes with raft-associated
tyrosine kinases (18). We costained
-chain+ cells for FcR and the tyrosine
kinase p53/56-Lyn (Fig. 4a). In the steady state
(formaldehyde fixed at 0 min), Lyn had a diffuse, subplasma membrane
distribution. After 2 min of FcR cross-linking at 37°C, we
observed a transient redistribution of Lyn. The distribution of Lyn
changed such that it was largely codistributed with FcR. By 5 min,
polarization of Lyn was not evident, although FcR was still capped.
This indicates that Lyn is recruited to FcR/ complexes in
membrane rafts before FcR internalization.
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-chain, but
-chain immunoreceptor tyrosine-based activation motifs are
substrates for Lyn (24). Therefore we hypothesized that
codistribution of FcR and Lyn was dependent on expression of
-chain (Fig. 4b). Although Lyn has N-terminal myristate
and palmitate chains (25), our data suggest that Lyn was
not completely partitioned into the rafts. We observed that Lyn was
evenly distributed in regions of plasma membrane in unstimulated cells
with no GM-1 staining. In -chain- cells,
there was no change in the distribution of Lyn, although FcR was
observed in a cap at 2 min and 5 min (Fig. 4b). Cells were
assessed for codistribution of FcR and Lyn after 2 min and 5 min at
37°C (Fig. 4c). -Chain+ and
-chain- cells with capped FcR were
analyzed for polarized Lyn staining, which codistributed with capped
FcR. At 2 min, when FcR capping was maximal, 70% of
-chain+ cells with capped FcR showed clear
codistribution of Lyn. In -chain- cells, at 2
min, 10% of cells demonstrated codistribution of polarized Lyn. By 5
min, the difference in FcR:Lyn copolarization between
-chain+ and -chain-
cells was less apparent than at 2 min, with 26% of
-chain+ cells showing FcR:Lyn
copolarization compared with 7% in -chain-
cells. To confirm that redistribution of Lyn to FcR caps was
occurring at membrane rafts, we performed three-color confocal
microscopy staining for FcR (red), GM-1 (green), and Lyn (blue). At
0 min, cells had an even distribution of both FcR, GM-1, and Lyn
(Fig. 5). After 2 min at 37°C, FcR,
GM-1, and Lyn had each redistributed and cocapped to a pole of the
cell.
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-Chain-dependent recruitment and activation of Lyn in membrane
rafts
Membrane rafts are resistant to solubilization by the detergent
Triton X-100. Low levels of Triton X-100 will cause dissolution of
nonraft plasma membrane, leaving intact not only membrane rafts but
associated proteins (26). We labeled the cell-surface GM-1
with HRP-conjugated ChTx (subunit B) before lysis in 0.05% Triton
X-100. After sucrose density gradient centrifugation of lysates, a
distinct opaque band was observed at the interface between the 0.15 M
and the 1.2 M sucrose layers. HRP activity as assessed by luminol
chemiluminescence was detected mainly in the fraction corresponding to
this band (Fig. 6a). Overall,
76% of the HRP activity corresponding to the raft constituent GM-1 was
distributed across fractions 3, 4, and 5, verifying that the detergent
insoluble domains were recovered by this technique. HRP-ChTx incubated
lysates were centrifuged in parallel to lysates from cells in which
FcR had been cross-linked. The ChTx-treated samples were not used in
signaling experiments described in this study but were used to
demonstrate the location of rafts in the sucrose gradients. Raft
fractions from cells where FcR had been cross-linked were adjusted
to the same protein concentration and used for immunoblotting
experiments. Each fraction from sucrose gradients of
-chain+ and -chain-
cells was immunoblotted for Lyn (Fig. 6, b and
c). In absence of FcR cross-linking, there was no
observable difference between -chain+ and
-chain- cells in the distribution of Lyn
between raft and nonraft fractions. While Lyn was observed at higher
levels in raft fractions, significant levels were observed in nonraft
fractions also. This indicates that in unstimulated cells Lyn does
partition into membrane rafts but is also found in nonraft
fractions.
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-chain+ and
-chain- unstimulated cells (Fig. 7a). In
-chain+ cells, after 30 s of FcR
cross-linking at 37°C, increased levels of Lyn were detected compared
with the 0-min samples, and additional anti-Lyn-reactive proteins
of a higher molecular mass than the p53/56 bands observed in
unstimulated cells were detected. After 2 min of FcR cross-linking,
the same banding pattern was observed, which became less pronounced
after 5 min. The higher molecular mass protein reactive with
anti-Lyn Ab suggests that phosphorylation of Lyn was occurring in
response to FcR cross-linking in -chain+
cells. In the -chain- cells, we did not
observe the appearance of anti-Lyn-reactive higher molecular mass
protein. In fact, the amount of Lyn in
-chain- cell rafts after FcR cross-linking
for 30 s, 2 min, and 5 min decreased (Fig. 7a).
The decrease in Lyn content in rafts from
-chain- cells was not due to an increase in
total protein in the samples. After FcR cross-linking, total protein
concentration in the raft fraction remained constant.
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b). Additionally, the amount
of Lyn in the rafts did not appear to increase in the absence of
vanadate suggesting that Lyn phosphorylation is necessary for its
increased levels in the rafts.
In other experiments to confirm that the higher molecular mass proteins
were phosphorylated Lyn species, we used an agarose-conjugated
anti-PY Ab to immunoprecipitate phospho-proteins from raft
fractions. These samples were then analyzed by immunoblotting with an
anti-Lyn Ab (Fig. 7c). In the
-chain+ cells, we detected a doublet of
phosphorylated Lyn at a higher molecular mass (60 and 63 kDa) in
addition to the p53/56 doublet, which was most evident in the 30-s and
2-min samples (Fig. 7c). In the
-chain- cells, these higher molecular mass
phosphorylated proteins were not observed, although
tyrosine-phosphorylated p53/56-Lyn was precipitated by the anti-PY
Ab. Inactive p53/56 Lyn has a carboxyl-terminal tyrosine phosphate
group, which is removed by CD45 to allow binding to target proteins via
its SH2 domains (27). The p53/56 bands may represent these
inactive species. A final experiment to confirm that the higher
molecular mass proteins observed in Fig. 7, a and
c represent tyrosine-phosphorylation of Lyn was to treat
rafts from -chain+ cells where FcR had been
cross-linked for 30 s at 37°C with the tyrosine-specific
phosphatase LAR-D1 (Fig. 7d). Following LAR-D1 treatment,
anti-Lyn Ab did not detect the higher molecular mass proteins
observed in the untreated immunoprecipitate.
Detection of phosphorylated -chain in rafts
Raft fractions from -chain+ and
-chain- cells were resolved by SDS-PAGE under
nonreducing conditions and immunoblotted for -chain (Fig. 8). Only -chain+
cells showed -chain as expected. At 0 min, two protein species were
detected with molecular masses of 22 kDa and 24 kDa as previously
observed for nonreduced -chain dimer (8). After FcR
cross-linking for 30 s, 2 min, and 5 min, two additional proteins
reactive with anti--chain Ab were detected with higher molecular
masses than the 22 kDa and 24 kDa proteins. There was also a
substantial increase in the level of -chain observed in rafts after
FcR cross-linking, which was maximal at 30 s and 2 min. This
data correlates with observed increases in FcR in rafts by confocal
microscopy (Fig. 1). Interestingly, by 5 min, although the same
anti--chain-reactive bands were observed, the amount of
-chain had decreased despite the fact that equal amounts of protein
were loaded.
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-Chain-dependent recruitment of Btk to membrane rafts
Btk is a member of the Tec family of protein tyrosine kinases
expressed primarily in hemopoietic cells. Btk is activated by the B
cell Ag receptor (28) and activates src family
kinases including Lyn (29). Compared with Lyn, low levels
of Btk were detected in the raft fractions of unstimulated
-chain+ and -chain-
cells (Fig. 9a). In
-chain+ cells following FcR cross-linking,
there was an increase in the levels of Btk in the raft fractions over 5
min, suggesting that Btk is recruited to the rafts on FcR ligation.
Recruitment and maintenance of Btk to the rafts is dependent on
-chain expression. In the -chain- cells, a
low level of Btk was detected in the raft fraction of unstimulated
cells, which was comparable to that observed in
-chain+ unstimulated cells. The amount of Btk
in these fractions did not increase with time, but decreased. When we
stripped and reprobed the anti-Lyn blots from our
immunoprecipitation experiments (Fig. 7c), we observed that
Btk in membrane rafts was phosphorylated (Fig. 9b). In
-chain+ cells, an increase in the level of
tyrosine-phosphorylated Btk was observed after FcR cross-linking. In
-chain- cells, low levels of phosphorylated
Btk were detected in unstimulated cells, but this decreased after
FcR cross-linking. In -chain+ cells,
increased tyrosine-phosphorylated Btk in rafts was matched by a
concomitant decrease in phosphorlyated Btk levels in the nonraft
fraction (Fig. 9c). In -chain- cells, phosphorylated Btk
levels in the nonraft fraction did not decrease but were maintained and
increased slightly on FcR cross-linking (Fig. 9d). The
minor lower band in Fig. 9c may represent slight degradation
of Btk in the samples. This data shows that in the absence of
-chain, phosphorylated Btk is not targeted from the nonraft fraction
to the raft fraction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R caps transiently in rafts before endocytosis, and
that capping is associated with -chain-dependent recruitment of
tyrosine kinases Lyn and Btk. FcR capping represents a large scale
reorganization of the plasma membrane and also results in capping of
the raft component GM-1. Within 2 min of FcR cross-linking, 20–30%
of cells showed colocalization of FcR and GM-1, and this persisted
for 5 min before FcR internalization. The mechanism of FcR
redistribution into rafts is unclear. Redistribution of FcR into
rafts occurred independently of -chain expression, showing that
neither -chain structure nor -chain-triggered signal transduction
is required. Rafts are less fluid than surrounding plasma membrane
domains because intercalation of cholesterol between the acyl chains in
rafts allows tighter packing of sphingolipid molecules
(30). Cross-linking could change the conformation of
FcR, decreasing its membrane solubility and lateral mobility, and
could alter the equilibrium of FcR entering and leaving rafts to
trap FcR in rafts.
The similar kinetics of GM-1 and FcR capping suggest that capping of
GM-1 is driven by FcR entering the rafts. In studies of association
of the IgE receptor (FcRI) with rafts (14), the
formation of several clusters of FcRI was accompanied by colocalized
clustering of GM-1, suggesting that receptor distribution drives raft
distribution. An alternative explanation is that cross-linked FcR
and associated rafts become linked with the actin cytoskeleton. The
work of Moran and Miceli (31) demonstrated that during
CD48/TCR costimulation of T cells, association of 
-chain with the
actin cytoskeleton was enhanced in a raft-dependent manner. Their
results suggest rafts are a site where signal transduction and
cytoskeletal reorganization are integrated.
While we did not observe internalization of GM-1, FcR was found to
be completely internalized 20 min after cross-linking. Cells
permeabilized then stained for GM-1 after FcR internalization showed
no internal GM-1 or colocalization with FcR-containing vesicles,
indicating that either FcR leaves rafts before internalization or
that GM-1 is excluded from rafts during the formation of the endocytic
vesicles. There is evidence that the endocytic mechanism prevents raft
internalization. In common with many other surface receptors,
endocytosis of Fc receptors occurs via clathrin-coated pits
(32). Studies in lymphoid cells have shown that plasma
membrane structures called caveolae, which have the same lipid
composition as rafts, are excluded from clathrin-coated pits
(33). If FcR associates with clathrin-coated pits for
internalization, then exclusion of raft lipids (GM-1) from
clathrin-coated pits would be consistent with our observation of the
physical separation of GM-1 and FcR after 10–20 min of
cross-linking.
We observed that Lyn redistributed to FcR capped in rafts in a
-chain-dependent manner. This suggests that redistribution of Lyn
could occur through increased association of Lyn with the
phosphorylated -chain immunoreceptor tyrosine-based activation motif
(8). We have observed by confocal microscopy that Lyn was
evenly distributed around the cell periphery and that Lyn was found in
both raft and nonraft fractions by immunoblotting This shows that
although N-terminally acylated Lyn partitions into both raft and
nonraft domains. Therefore, lipid chains do not result in exclusive
partitioning of Lyn into rafts. Our examination of rafts isolated from
-chain+ and -chain-
cells after FcR cross-linking supports this hypothesis. Equal levels
of Lyn were detected in rafts of -chain+ and
-chain- cells before FcR cross-linking,
but only -chain+ cells demonstrated an
increased level of Lyn in rafts due to Lyn -chain association. We
observed that lysis in the absence of vanadate prevented the increase
in Lyn content of the rafts normally observed in
-chain+ cells. Thus, when cells were lysed and
rafts isolated under conditions in which Lyn and other components of
the proximal signaling complex would become dephosphorylated, increased
amounts of Lyn were no longer detected in the raft fraction. The
retention in rafts of increased amounts of Lyn in the presence of
vanadate suggests that interaction between phosphorylated Lyn and the
other signaling molecules intimately associated with rafts helped to
retain Lyn in the raft fraction.
Btk activation has been implicated in FcR-triggered signal
transduction (34). We observed that the levels of Btk in
the raft fraction increased in a -chain-dependent manner and that,
like Lyn, Btk was phosphorylated following FcR ligation. Btk is not
acylated and was thus recruited into the raft fraction from the cytosol
(29). Recruitment of Btk into rafts is most likely
mediated by binding of Btk to membrane PI 3,4,5-triphospate by its
pleckstrin homology domain (35), indicating that PI
3-kinase is also activated in the raft-associated signaling complex. If
PI 3-kinase is dependent on -chain for activation, then lack of
signaling in the -chain- cells fits well with
the observed lack of recruitment of Btk to the rafts. Our data suggest
signaling is required not only to increase Btk levels in the rafts, but
to maintain them. Collectively, our data suggest that the function of
rafts is to provide an environment where phosphorylation of plasma
membrane tyrosine kinases is initiated before FcR internalization.
Lyn and Btk phosphorylation occurs within 30 s and continues for 2
min before FcR internalization, which occurs after around 5 min and
is not complete till at least 10 min. This indicates that signals
triggered by FcR in rafts activate downstream events independently
of delivery of FcR to the endosomal pathway. The novel role of
-chain in regulating Lyn and Btk localization and phosphorylation
links signal transduction and membrane localization. The importance of
integrating signaling and changes in the plasma membrane orientation is
required for understanding how FcR triggers downstream changes in
cell physiology.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Li Shen, Department of Microbiology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756. E-mail address:
3 Abbreviations used in this paper: FcR, Ig Fc-domain receptor; Btk, Bruton’s tyrosine kinase; Cy3, indocarbocyanine 3; Cy5, indodicarbocyanine 5; ChTx, cholera toxin; ECL, enhanced chemiluminescence; PY, phosphotyrosine; TfR, transferrin receptor; PI, phosphatidylinositol; PLC, phospholipase C; GAM, goat anti-mouse; GAR, goat anti-rabbit.
4 L. Shen, M. van Egmond, K. Siemasko, M. Clark, J.G.J. van de Winkel, and W.F. Wade. Presentation of ovalbumin internalised via the IgA Fc receptor (CD89) is enhanced through FcR chain signaling. Submitted for publication.
Received for publication May 6, 1998. Accepted for publication September 7, 1999.
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