(Received for publication, September 8, 1994; and in revised form, November 14, 1994)
From the
In RBL-2H3 rat tumor mast cells, cross-linking the high affinity
IgE receptor, FcR1, activates the protein-tyrosine kinases Lyn and
Syk and initiates a series of responses including protein-tyrosine
phosphorylation, inositol 1,4,5-trisphosphate synthesis, Ca
mobilization, secretion, membrane ruffling, and actin plaque
assembly. The development of chimeric receptors containing cytoplasmic
domains of individual subunits of the heterotrimeric
(
) Fc
R1 has simplified analyses of
early signaling events in RBL-2H3 cells. Here, RBL-2H3 cells were
transfected with cDNAs encoding the extracellular and transmembrane
domains of the interleukin-2 receptor
subunit (the Tac antigen)
joined to the C-terminal cytoplasmic domains of the Fc
R1
and
subunits (TT
and TT
). Both sequences contain tyrosine
activation motifs implicated in antigen receptor signal transduction.
TT
and TT
are expressed independently of the native
Fc
R1, as demonstrated by the ability of Tac cross-linking agents
to trigger the clustering and internalization through coated pits of
both chimeric receptors without co-clustering the Fc
R1. A full
range of signaling activities is induced by TT
cross-linking; the
TT
-induced responses are slower and, except for Lyn activation,
smaller than the Fc
R1-induced responses. In striking contrast,
TT
cross-linking elicits no tyrosine phosphorylation or signaling
responses, it impairs basal activities measured in secretion and
anti-PY (anti-phosphotyrosine antibody) immune complex kinase assays,
and it antagonizes Fc
R1-induced Lyn and Syk activation,
protein-tyrosine phosphorylation, and signaling responses. We
hypothesize that the isolated
subunit binds a specific kinase or
coupling protein(s) required for signaling activity, sequestering it
from the signal-transducing
subunit. Binding the same kinase or
coupling protein to the
subunit of the intact Fc
R1 may serve
instead to present it to the adjacent
subunit, resulting in
enhanced kinase activation and signaling responses.
The high affinity IgE receptor, FcR1, (
)of mast
cells and basophils belongs to the family of multichain immune system
receptors, that includes the T cell receptor (TCR), the mIg receptor of
B cells, and several members of the Fc
receptor family. These
receptors lack intrinsic enzyme activity. Instead, they recruit and
activate cytoplasmic protein-tyrosine kinases that phosphorylate
tyrosine residues in characteristic receptor subunit cytoplasmic
sequences called variously tyrosine activation motifs (TAMs), antigen
receptor homology 1 motifs, and antigen recognition and response
motifs(1, 2, 3) . TAMs are 20-25 amino
acid sequences containing two YXXL/I motifs separated by
approximately ten residues(4, 5) . They are found in
the
and
chains of the multisubunit TCR, in the Ig-
and
Ig-
chains of the mIg receptor, and in the
and
chains
of the heterotrimeric (
) Fc
R1. The
TAM-containing Fc
R1
subunit is additionally found in
association with at least two other Fc receptors, Fc
RI and
Fc
RIIIA(6, 7, 8, 9) , and in
the
subset of T cells, where it replaces the TCR
subunit(10) . Current studies suggest that the first response
of immune system cells to antigen receptor cross-linking is TAM
phosphorylation mediated by members of the Src protein-tyrosine kinase
family(11) . This primary phosphorylation provides sites for
the binding and activation of members of the Syk family of
protein-tyrosine kinases and for the binding of a range of SH2
domain-containing protein-tyrosine kinase substrates (reviewed in (3) ). Once phosphorylated by receptor-associated kinases,
these proteins initiate the downstream responses of signal
transduction.
Recently chimeric receptors containing irrelevant
extracellular and transmembrane domains and specific cytoplasmic
domains have been used to dissect the signaling activities of
individual subunits of the multichain immune system receptors. In
particular, it was found that chimeric receptors expressing the
TAM-containing cytoplasmic tails of the TCR and
chains can
both stimulate tyrosine phosphorylation and signaling responses when
transfected into T cell lines and cross-linked by antibodies to the
extracellular
domain(12, 13, 14, 15) . T cells
transfected with chimeric receptors consisting of a membrane-anchored
form of Syk in T cells also have tyrosine phosphorylating and signaling
activity following cross-linking(16) . These studies suggest
there is redundancy in the pathways that initiate TCR signaling and
that Syk activation is the critical event leading to downstream
responses. Similar studies in transfected RBL-2H3 cells have yielded
less clear-cut results. Thus, cross-linking transfected receptors
consisting of the extracellular and transmembrane domains of the Tac
antigen coupled to the cytoplasmic domains of the Fc
R1
subunit (TT
) was shown to induce tyrosine phosphorylation and
secretory responses varying from 50% of Fc
R1-mediated responses (17) to 10% of Fc
R1-mediated responses (12) to no
response at all (18) . In addition, no tyrosine phosphorylating
or secretory activity was found in association with cross-linked
chimeric receptors consisting of the extracellular and transmembrane
domains of Tac coupled to the TAM-containing C-terminal cytoplasmic
domain of the Fc
R1
subunit (TT
; (17) ).
Here, we report further studies on the distribution, associated
kinases, and signaling activities of TT and TT
expressed at
high density on RBL-2H3 cells. Our results with cells expressing
TT
provide evidence that cross-linking
alone is sufficient
to activate both Syk and Lyn and to initiate a full range of downstream
responses. In contrast, TT
cross-linking not only fails to
activate tyrosine kinases and signaling responses but it impairs
tyrosine kinase activation and signal transduction in response to
Fc
R1 cross-linking.
To observe cross-linker-induced membrane ruffling and spreading, monolayers of primed cells were incubated for 10 min at 37 °C with cross-linker (DNP-BSA or avidin), then fixed with 2% glutaraldehyde and processed for scanning electron microscopy(24) . To observe actin plaque assembly, monolayers of activated cells were fixed with paraformaldehyde and labeled with rhodamine-phalloidin(25) .
Untreated or IgE-primed cells were loaded with 2 µM fura-2/AM (Molecular Probes) for 30 min at room temperature. Cells
were primed with 2 µg/ml biotinyl-anti-Tac IgG for the last 10 min
of loading with fura-2 and activated by exchanging the room temperature
medium with fresh medium containing DNP-BSA or avidin at 35 °C.
Cells (3-10 per field in each experiment) were observed using 360
and 385 nm excitation filters and a 510 nm emission filter. At the end
of each experiment, average ratio values were calculated for a
user-defined area within each cell in background-subtracted, ratioed
images. The time resolution of ratioed images was 2-5 s. Average
ratio values were converted to [Ca]
as described in Grynkiewicz et al.(30) using a
calculated K
for fura-2/Ca
of
155 nM.
Figure 1:
The distribution of
chimeric and native receptors on transfected RBL-2H3 cells. Monolayers
of transfected cells on glass coverslips were incubated with
anti-DNP-IgE plus FITC-conjugated or biotinylated anti-Tac IgG. In A and B, TT receptors were labeled with
FITC-anti-Tac IgG for 10 min, respectively, at room temperature (A) or 37 °C (B). In C and D,
TT
receptors were labeled with anti-Tac IgG for 10 min at room
temperature, then cross-linked by 10-min incubation at 37 °C with
LRSC-conjugated goat anti-mouse IgG (C); the cells were fixed
and the IgE-primed Fc
R1 labeled with rabbit anti-IgE IgG and
FITC-anti-rabbit IgG (D). Cells in A-D were
observed by fluorescence microscopy. In E and F,
TT
(E) and TT
(F) cells were incubated for
10 min at room temperature with biotinyl-anti-Tac IgG, cross-linked for
10 min at 37 °C with avidin-conjugated 15-nm colloidal gold
particles, then fixed and processed for transmission electron
microscopy. Arrowheads point to gold particles in coated pits; arrows indicate gold particles in uncoated intracytoplasmic
vesicles. In A-D, magnification:
800. In E and F, bar = 10
µm.
Figure 2:
Secretory responses induced by receptor
cross-linking. [H]Serotonin-loaded transfected
cells were primed with anti-DNP-IgE and biotinyl-anti-Tac IgG and
activated for 20 min (A, C) or for the times indicated (B,
D) with cross-linking agents. Cross-linker-induced
[
H]serotonin release was measured as described
under ``Experimental Procedures.'' Results in B and D are corrected for spontaneous
[
H]serotonin release. In A and C, columns 1-3, TT
and TT
transfectants, respectively, were incubated for 20 min with no
addition, DNP-BSA, and avidin. In addition, TT
cells were
incubated with anti-mouse IgG (A, column 4), and TT
cells
were incubated with avidin plus DNP-BSA following a 5-min preincubation
with avidin alone (C, column 4). In B and D,
TT
and TT
cells were incubated with DNP-BSA (B and D, solid circles), avidin (B, solid triangles) or
with DNP-BSA plus avidin following a 5-min preincubation in avidin
alone (D, open triangles). Results are representative of three
separate experiments, each performed in
duplicate.
The addition of LRSC-conjugated goat anti-mouse
IgG to cross-link anti-Tac-receptor complexes on TT cells causes
the rapid clustering and internalization of chimeric receptors (Fig. 1C). Fig. 1D shows that the
noncross-linked Fc
R1 retains a uniform cell surface distribution
during the avidin-induced cross-linking, clustering, and
internalization of transfected receptors. Again, experiments with
TT
transfectants (not illustrated) yielded identical results.
Previously, we used colloidal gold labeling techniques to show that
clustering of cross-linked FcR1 is followed by their
internalization through coated pits (illustrated in (23) and (24) ). The results in Fig. 1, E and F, show that gold-labeled cross-linked TT
and TT
are
also internalized through coated pits and are later located in uncoated
intracytoplasmic vesicles.
Cells expressing TT showed a small
but consistent increase in the rate of basal or spontaneous secretion
in comparison with TT
-transfected cells (compare Fig. 2A, column 1, with Fig. 2C,
column 1). Although this may simply reflect variability in basal
activity between different subpopulations of RBL-2H3 cells, it is
striking that the TT
cells selected by Jouvin et al.(17) showed the same property.
TT cross-linking
agents caused a decrease in basal secretion in TT
cells (compare Fig. 2C, columns 1 and 3). TT
cross-linking also resulted in a 30-50% reduction in
Fc
R1-mediated secretion measured over 20 min in
TT
-transfected cells (compare Fig. 2C, columns 2 and 4). The reduction in Fc
R1-mediated secretion due
to TT
cross-linking is apparent throughout the time course of
Fc
R1-induced secretion (Fig. 2D). Control
experiments established that the binding of biotinyl-anti-Tac IgG to
TT
receptors, without subsequent TT
cross-linking, does not
alter either basal or Fc
R1-mediated secretion. The addition of
avidin to TT
transfectants without prior priming with
biotinyl-anti-Tac IgG also has no effect on basal or Fc
R1-mediated
secretion. These results provided the first indication that chimeric
receptors can specifically affect the signaling activity of the native
receptor.
Figure 3:
Cross-linker-induced ruffling and
spreading in transfected RBL-2H3 cells. Transfected cells cultured on
glass coverslips were primed with anti-DNP-IgE and biotinyl-anti-Tac
IgG, rinsed in Hanks'-BSA medium, and incubated for 10 min at 37
°C in medium alone (A), in DNP-BSA (B), or in
avidin (C, D). The cells were fixed with 2% glutaraldehyde and
processed for scanning electron microscopy as described under
``Experimental Procedures.'' Cells in A-C are
TT transfectants. Cells in D are TT
transfectants. Bar = 10 µm.
Figure 4:
Cross-linker-induced actin plaque assembly
in transfected cells. The experiment is the same as described in Fig. 3except that cells were fixed after activation in 2%
paraformaldehyde, 0.02% saponin, and F-actin was fluorescence labeled
using rhodamine-phalloidin. The fluorescence photomicrographs
illustrate F-actin distribution at the ventral cell surface. Cells are
TT transfectants incubated in medium alone (A), in
DNP-BSA (B), or in avidin (C) and TT
transfectants incubated in avidin (D). Magnification:
800.
Figure 5:
Cross-linker-induced protein-tyrosine
phosphorylation measured by flow cytometry. IgE and
biotinyl-anti-Tac-primed TT (A) and TT
(B)
cells were incubated in suspension with DNP-BSA and/or avidin for
various times, then fixed and labeled with rabbit anti-PY antibody
followed by FITC-anti-rabbit IgG. The mean fluorescence intensity per
cell was measured for 10,000 cells/sample by flow cytometry as
described under ``Experimental Procedures.'' Data are
corrected for mean fluorescence intensity in parallel unstimulated
samples and are representative of three similar experiments. Activation
was with DNP-BSA (A and B, open circles), avidin (A and B, open triangles), anti-mouse IgG (A, closed triangles) and DNP-BSA plus avidin following 5-min
preincubation with avidin alone (B, solid
circles).
Figure 6:
Cross-linker-induced protein-tyrosine
phosphorylation in TT transfectants. IgE plus
biotinyl-anti-Tac-primed TT
transfectants were incubated at 37
°C for 5 min with no addition (lane 1) or for the times
indicated with DNP-BSA (lanes 2-4) or avidin (lanes
4 and 5), then lysed, centrifuged, and 100 µg of
supernatant protein separated by SDS-PAGE. Proteins were transferred to
nitrocellulose membranes and tyrosine phosphorylated species were
detected by incubation with rabbit anti-PY followed by
S-protein A as described under ``Experimental
Procedures.'' Radiolabeled proteins were detected by
autoradiography. The migration positions of Syk, Lyn, and the Fc
R1
subunit are indicated; the
subunit is further indicated by
a small arrow.
Cross-linking IgE-primed FcR1 with
0.1 µg/ml DNP-BSA causes an increase in total anti-PY-reactive
protein that reaches its highest levels by 2 min after cross-linking
and declines by 10 min (Fig. 5, A and B). The
addition of avidin or anti-mouse IgG to unprimed TT
or TT
transfected cells caused no change in anti-PY reactivity (not
illustrated). However, cross-linking biotinyl-anti-Tac-primed TT
receptors with avidin or anti-mouse IgG increases total tyrosine
phosphorylation (Fig. 5A). Like TT
-induced
secretion, the TT
-induced protein-tyrosine phosphorylation
response is typically slower and smaller than the response to Fc
R1
cross-linking.
In contrast with TT transfectants, there is no
detectable increase in anti-PY-reactive proteins when avidin is added
to biotinyl-anti-Tac-primed TT
transfected cells (Fig. 5B). Furthermore, the addition of TT
cross-linking agents 5 min before DNP-BSA substantially reduces the
Fc
R1-mediated protein-tyrosine phosphorylation response (Fig. 5B).
The results of anti-PY immunoblotting
showed that a very similar array of proteins is tyrosine phosphorylated
in response to FcR1 and TT
cross-linking. In particular, a
major tyrosine-phosphorylated band at 72 kDa was absent from resting
cell lysates (lane 1) but present under both cross-linking
conditions (lanes 2-6). This protein was previously
identified as the protein-tyrosine kinase,
Syk(37, 38) . One protein, the Fc
R1
subunit, is tyrosine-phosphorylated only in response to Fc
R1 and
not TT
cross-linking. Consistent with previous
results(17) , we were unable to detect a new anti-PY-reactive
band corresponding to TT
itself in avidin-activated cells.
Anti-PY-reactive Lyn is observed by immunoblotting in resting as well as activated cells (Fig. 6, lanes 1-6). It has been established that the activation and inactivation of Src kinases are both mediated by tyrosine phosphorylation, although on distinct tyrosine residues (reviewed in (39) ). Thus the anti-PY reactivity observed here in resting cells may reflect Lyn phosphorylation on an inhibitory tyrosine that is recognized on immunoblots by our polyclonal anti-PY antibody.
As expected from the
flow cytometry results (Fig. 5), avidin-induced TT
cross-linking caused no detectable protein-tyrosine phosphorylation in
anti-PY immunoblotting experiments; DNP-BSA-induced Fc
R1
cross-linking in TT
cells yielded a pattern of protein tyrosine
phosphorylation that was indistinguishable from DNP-BSA-activated
TT
cells; and the Fc
R1-induced protein-tyrosine
phosphorylation response was reduced substantially when TT
was
cross-linked 5 min before the addition of DNP-BSA (not illustrated).
The reduction in Fc
R1-induced phosphorylation due to concomitant
TT
cross-linking resulted from a proportional decrease in the
phosphorylation of all protein species, not a targeted decrease in the
phosphorylation of specific proteins.
No
1,4,5-IP was synthesized in response to TT
cross-linking. Furthermore, TT
cross-linking reduced
Fc
R1-induced 1,4,5-IP
synthesis by more than 50% (data
not shown).
Figure 7:
Cross-linker-induced Ca
responses in transfected cells. TT
(A-C) and
TT
(D-F) transfected RBL-2H3 cells were loaded with
fura-2 and the [Ca
]
of
individual cells was monitored by ratio imaging microscopy as described
under ``Experimental Procedures.'' In each panel,
[Ca
]
versus time is plotted for three cells representing typical responses for
the indicated transfectant and experimental conditions. Table 1gives the total numbers of cells examined under each
condition. Extracellular Ca
was present throughout
the experiments illustrated in A, B, D, E, F, and as indicated
in C. Treatments were: A, IgE-primed TT
transfected cells activated with 0.1 µg/ml DNP-BSA; B and C, biotinyl-anti-Tac-primed TT
transfectants activated
with 25 µg/ml avidin; D, IgE-primed TT
transfected
cells stimulated with 0.1 µg/ml DNP-BSA; E,
biotinyl-anti-Tac-primed TT
transfectants incubated with 25
µg/ml avidin; F, IgE- and biotinyl-anti-Tac-primed TT
transfectants treated with avidin 5 min before activation with 0.1
µg/ml DNP-BSA.
Fura-2-labeled TT transfectants incubated with 0.1 µg/ml
DNP-BSA in the presence of extracellular Ca
responded
after a short lag (Table 1) with an abrupt rise in
[Ca
]
followed by a plateau that
decreased slowly over time in most cells (Fig. 7A).
Thirty-two of 33 TT
transfectants incubated with 25 µg/ml
avidin in the presence of extracellular Ca
also
showed Ca
responses. However, the average lag time
before the onset of avidin-activated Ca
responses was
almost four times longer than than the lag time before DNP-BSA-induced
responses (Table 1). In addition, the Ca
responses to avidin were quite heterogeneous (Fig. 7B). Although in some cells TT
cross-linking
led to Ca
responses that were indistinguishable from
optimal antigen, in others the response was smaller and in some cases
consisted of Ca
oscillations superimposed on a small
base line increase in [Ca
]
.
This pattern of increased lag times and smaller responses is typical of
cells stimulated with suboptimal concentrations of antigen (around 1
ng/ml DNP-BSA).
The two phases of the Ca response, Ca
stores release and Ca
influx, can be observed separately by activating cells
sequentially in the absence and presence of extracellular
Ca
. When activation was in Ca
-free
medium, the lag time from the addition of avidin to the onset of
responses was again longer and more variable than the lag time for
DNP-BSA-treated cells (Table 1). The traces in Fig. 7C show that both components of the Ca
response
were activated by TT
cross- linking. One avidin-treated cell
showed a single Ca
spike due to Ca
stores release during incubation in nominally
Ca
-free medium followed by a sustained elevation of
[Ca
]
when complete medium was
added. This resembles a typical response to optimal amounts of
DNP-BSA.
Two other cells showed smaller responses to
TT
cross-linking characterized by two or three Ca
oscillations when stimulation was in nominally
Ca
-free medium and a lower plateau when complete
medium was added (Fig. 7C). This pattern is again
typical of cells activated with suboptimal concentrations of antigen
(approximately 1 ng/ml DNP-BSA).
The lag times (Table 1) and Ca responses of TT
transfectants to DNP-BSA (Fig. 7D) were not
significantly different from those of TT
transfectants (Fig. 7B) and nontransfected cells.
In 17
of 18 cells, TT
cross-linking caused no Ca
responses at all (Fig. 7E). One cell showed a
single Ca
spike resembling the spontaneous
oscillations that occur occasionally in unstimulated cells. TT
cross-linking 5 min prior to the addition of DNP-BSA increased the
average lag time for the onset of the Fc
R1-induced Ca
response from 35 to 51 s (Table 1), a difference that is
significant at p < 0.05 using Student's t test. However, the magnitude of the Ca
response
in DNP-BSA and avidin cross-linked TT
cells was not reduced
compared with TT
cells activated with DNP-BSA alone (Fig. 7F).
Figure 8:
Immune complex kinase assays. IgE plus
anti-Tac-primed RBL-2H3 transfectants were activated, lysed, and their
supernatants immunoprecipitated with anti-Syk (A) or anti-PY (B) antibodies. Kinase activities in the immunoprecipitates
were measured as described under ``Experimental Procedures.''
Sources of immune complexes were: lane 1, unstimulated TT
cells; lane 2, DNP-BSA-treated (1 min) TT
cells; lane
3, avidin-treated (5 min) TT
cells; lane 4,
unstimulated TT
cells; lane 5, DNP-BSA-treated (1 min)
TT
cells; lane 6, avidin-treated (5 min) TT
cells; lane 7, avidin (5 min) pretreated TT
cells stimulated
with DNP-BSA plus avidin (1 min). In A, a large arrowhead identifies Syk and small arrowheads point to a set of
unknown phosphoproteins. In B, an arrowhead again
identifies Syk and dashes point to the 53- and 56-kDa isoforms
of Lyn. The identity of Lyn in anti-PY assays is verified by its
co-electrophoresis with autophosphorylated Lyn from an anti-Lyn
immunoprecipitate (B, lane 8). Results are representative of
three replicate assays.
TT cross-linking causes no increase in
Syk activity measured in in vitro kinase assays (Fig. 8A, lane 6). TT
cross-linking 5 min
prior to the addition of DNP-BSA has very little effect on
Fc
R1-induced Syk activation (Fig. 8A, lane 7).
Although anti-Syk immune complexes from resting cells or from cells
activated through the TT receptor do not support Syk
phosphorylation, they do phosphorylate an unidentified high molecular
weight species and a series of unknown lower molecular weight proteins (arrowheads in Fig. 8A, lanes 1, 4,
and 6). In contrast, anti-Syk immune complexes from cells
activated through the Fc
R1 and TT
receptors support Syk
phosphorylation but the unknown phosphoproteins are significantly less
prominent (Fig. 8A, lanes 2, 3, 5, and 7). The
simplest interpretation is that Syk associates in resting cells with an
uncharacterized active kinase and its substrates. These proteins either
dissociate from Syk or the kinase is inhibited in response to TT
and Fc
R1 cross-linking.
Anti-PY immunoprecipitates from resting TT and
TT
cells do not phosphorylate Syk but support a modest
phosphorylation of the 53-kDa isoform of Lyn (Fig. 8B, lanes
1 and 4). Fc
R1 cross-linking causes a large increase
in the phosphorylation of Syk and of the 53- and 56-kDa isoforms of Lyn
in both TT
and TT
cells (Fig. 8B, lanes 2 and 5). In addition, a series of co-precipitated kinase substrates
are phosphorylated in anti-PY immune complexes prepared from
DNP-BSA-treated cells.
The results in Fig. 8B, lane
3, confirm that TT cross-linking increases the Syk
phosphorylating activity of anti-PY immune complexes. PhosphorImager
analysis of this experiment showed that Syk phosphorylation following 5
min of TT
cross-linking was 21% of Syk phosphorylation measured
after 1 min of Fc
R1 cross-linking. TT
cross-linking also
increases the tyrosine phosphorylation of Lyn in anti-PY immune complex
kinase assays (Fig. 8C, lane 3). In this case,
PhosphorImager analysis showed that phosphorylation of the 53- and
56-kDa Lyn isoforms following 5 min of TT
cross-linking was,
respectively, 46 and 145% (average 117% calculated from the sum of
pixels in both bands) of Lyn phosphorylation induced by 1 min of
Fc
R1 cross-linking. An unidentified higher molecular weight
protein species also shows increased phosphorylation in anti-PY immune
complexes from avidin-activated TT
cells.
Remarkably, TT
cross-linking causes a marked decrease in the basal phosphorylation of
Syk, Lyn, and all other proteins in anti-PY in vitro kinase
assays (Fig. 8B, lane 7). Furthermore, TT
cross-linking reduces the Fc
R1-stimulated phosphorylating activity
of Lyn and Syk in anti-PY immune complexes by approximately 30%
(illustrated in Fig. 8B, lane 6, and quantified by
analyses of PhosphorImager data).
This work was initiated to explore the separate or
synergistic functions of the individual subunits of the multichain
FcR1. RBL-2H3 cells that express the native Fc
R1 at high
density were transfected with chimeric receptors consisting of the
extracellular and cytoplasmic domains of the Tac antigen joined to the
cytoplasmic tails of the Fc
R1
and
subunits. Because
these cytoplasmic sequences both contain TAMs, the chimeric receptors
were predicted to respond to cross-linking agents by tyrosine kinase
activation and some or all of the signaling responses that normally
follow Fc
R1 cross-linking. Indeed, Letourneur and Klausner (12) and Jouvin et al.(17) had previously
demonstrated cross-linker-induced protein-tyrosine phosphorylation,
secretion, and Ca
mobilization responses to TT
cross-linking in transfected RBL-2H3 cells. We determined that the
transfected and native receptors are expressed at similar high
densities on the cell surface and that they redistribute independently
after cross-linking into clusters that are internalized through coated
pits. These results established that there is no intermixing of
chimeric and native receptor subunits in the transfectants used for
these studies.
We found that TT cross-linking activates all the
biochemical and functional responses that are associated with Fc
R1
cross-linking, including protein-tyrosine phosphorylation,
1,4,5-IP
synthesis, secretion, ruffling, spreading and
actin plaque assembly. These responses to TT
cross-linking show a
slower onset than Fc
R1-induced responses and their final magnitude
is only around 50% of the maximum responses to Fc
R1 cross-linking.
TT
cross-linking also stimulates Ca
stores
release and Ca
influx responses resembling the
responses of nontransfected cells to suboptimal concentrations of
DNP-BSA.
Only Fc
R1 and not TT
cross-linking
causes Fc
R1
subunit tyrosine phosphorylation. With this
exception, the major tyrosine-phosphorylated proteins detected by
anti-PY immunoblotting are the same when cells are activated by TT
and Fc
R1 cross-linking. Of course, it remains possible that minor
substrates for protein-tyrosine phosphorylation differ when cells are
activated through these different receptors. Indeed, preliminary
experiments using the more sensitive method of
[
P]orthophosphate labeling and
anti-phosphotyrosine immunoprecipitation indicate a simpler pattern of
protein tyrosine phosphorylation in response to TT
as compared
with Fc
R1 cross-linking (work in progress). Surprisingly, no
cross-linker-induced phosphorylation of TT
protein itself was
detected in this or a previous study(17) . These results
confirm and substantially extend the analysis of TT
receptor
signaling activities begun by Letourneur and Klausner (12) and
Jouvin et al.(17) . They contradict the report (18) that TT
cross-linking does not activate transfected
RBL-2H3 cells.
The TT-activated signaling pathway was explored
further by in vitro immune complex kinase assays. Syk
activation by TT
cross-linking was demonstrated with both anti-Syk
and anti-PY immune complexes. The extent of TT
-induced Syk
activation was around one fifth of the Syk activation response to
Fc
R1 cross-linking in the same cells. Although cross-linking alone
can activate Syk(16) , studies in T cells indicate that
phosphotyrosine residues in the TAMs of cross-linked receptors enhance
the activation of Syk family members by providing docking sites for
their tandem SH2 domains(3) . Thus it is possible that the
smaller activation of Syk by TT
cross-linking than by Fc
R1
cross-linking is due in part to very low levels of avidin-induced
TT
receptor phosphorylation (below the limits of detection in our
assays) in comparison with the strong DNP-BSA-induced phosphorylation
of TAMs in the Fc
R1
and
subunits demonstrated here and
previously(34, 37, 43) . Complementary
pharmacological studies showed that the Syk-selective tyrosine kinase
inhibitor, piceatannol, abolishes signaling responses to both Fc
R1 (37) and TT
cross-linking (this study), indicating that
Syk activation is a critical event linking both the native and
transfected receptors to downstream responses. An uncharacterized
active kinase and several substrates co-precipitated with Syk from
resting but not stimulated cells, raising the possibility that the
inactive form of Syk exists in a complex with other proteins. Further
study of these co-precipitated proteins may reveal mechanisms involved
not only in receptor-mediated Syk activation but also in the modulation
of basal Syk activity that occurs through the cell cycle(44) .
The extent of Lyn activation measured in anti-PY immune complex
kinase assays was fairly similar between avidin-activated and
DNP-BSA-activated TT cells. These results suggest that
cross-linked TT
is a more effective activator of Lyn than of Syk.
Previously, Eiseman and Bolen (18) reported that TT
cross-linking does not activate Lyn. However, the transfected cells
used by these investigators had unusually small responses to Fc
R1
cross-linking, suggesting a general impairment of their signaling
capacity. Substantial Lyn activation by TT
cross-linking was also
unexpected based on evidence (17) that Lyn protein associates
specifically with TT
and does not co-precipitate with TT
. To
reconcile our results with Jouvin et al.(17) , we
propose that Lyn may require only a transient or indirect interaction
with cross-linked
for activation.
If full, even though
submaximal, signaling activity resides in the FcR1
subunit,
what is the function of the Fc
R1
subunit? The Fc
R1
subunit C-terminal cytoplasmic domain sequence incorporates a TAM
and Lyn has been observed previously to associate with the TT
construct(17) . In addition, it has been shown that deletion or
mutation of the Fc
R1
subunit C-terminal cytoplasmic domain
drastically reduces cross-linker-induced receptor phosphorylation and
signaling responses(17, 23, 45) . Other
recent studies have described specific mutations in the Fc
R1
subunit transmembrane domain that appear to be linked to atopy in
humans (reviewed in (9) ). All of these reports point to an
important role for the
subunit in Fc
R1-mediated signaling.
On the other hand, it has also been shown that a human Fc
R1
composed of just an
complex is capable of
signaling (45) and that a
-less form of the Fc
R1 is
commonly expressed on Langerhans cells (46) . Furthermore, our
data indicate that cross-linking chimeric receptors containing the
cytoplasmic domain of the isolated
subunit can activate both Lyn
and Syk and elicit all the signaling responses normally induced by
Fc
R1 cross-linking. None of these results is consistent with an
essential signaling function for the
subunit.
We approached
FcR1
subunit function through detailed studies of the
signaling activities of TT
transfectants. There is no Lyn
activation in response to TT
cross-linking, establishing that any
Lyn associated with the isolated
subunit (17) is
catalytically inactive. TT
transfectants also show no Syk
activation, protein-tyrosine phosphorylation, or biochemical or
functional responses to cross-linking agents. The absence of
TT
-induced Syk activation, secretion, and protein-tyrosine
phosphorylation was observed previously(17) . Based on
spectrofluorimetric analyses in fura-2-labeled cell suspensions, Jouvin et al.(17) reported a small Ca
mobilization response to TT
cross-linking. However,
spectrofluorimetric studies are prone to mixing artifacts and problems
due to dye leakage. Our single cell analyses clearly show that TT
cross-linking does not induce either Ca
stores
release or Ca
influx responses. These results
demonstrate that the presence of a TAM in the sequence of a
transmembrane protein is not per se sufficient to couple
receptor cross-linking to cell activation. Kim et al.(47) made a similar observation for the B cell mIg
receptor, where both the Ig-
and Ig-
chains of the receptor
contain TAMs but Ig-
triggers tyrosine kinase activation much more
efficiently that Ig-
.
Further study indicated that TT
cross-linking not only fails to stimulate signaling responses, but
actually reduces some of these responses to below basal levels. This
was indicated by the reduction in basal secretion when TT
cross-linking agents were added to TT
-transfected cells (Fig. 2C) and by the marked reductions in both Lyn and
Syk basal kinase activities in anti-PY immune complexes isolated after
TT
cross-linking (Fig. 8C). Cross-linking TT
also has a negative effect on Fc
R1-mediated signaling,
demonstrated by the substantial inhibition of Fc
R1-activated
anti-PY immune complex kinase activity, protein-tyrosine
phosphorylation, 1,4,5-IP
synthesis and secretion, and by
the delayed onset of Ca
responses, in cells
co-stimulated with TT
cross-linkers.
The mechanism by which
TT cross-linking impairs basal and Fc
R1-mediated signal
transduction is not known with certainty. One possibility is that
TT
cross-linking activates a protein-tyrosine phosphatase that
antagonizes basal and Fc
R1-activated protein-tyrosine
phosphorylation, thus suppressing signaling responses. The loss of
anti-PY-reactive proteins due to phosphatase activation would provide a
direct explanation for the ability of TT
cross-linking agents to
reduce the basal and Fc
R1-enhanced phosphorylation of Syk and Lyn
in anti-PY immune complex kinase assays. However, RBL-2H3 cells have a
high constitutive level of protein-tyrosine phosphatase activity as
demonstrated by the ability of protein-tyrosine phosphatase inhibitors
to mimic Fc
R1-induced cell activation (25, 28, 48) and by the rapid reversal of
Fc
R1-mediated protein-tyrosine phosphorylation as soon as
cross-linking is arrested (43) . This high background activity
reduces the likelihood that a specific
subunit-activated
phosphatase controls signaling activity.
Alternatively, it is
possible that the cross-linked TT receptor inhibits signaling by
binding and sequestering a kinase or a coupling protein that is
required by the signal-transducing
subunit. Since TT
cross-linking alone reduces signaling responses to below basal levels,
this hypothesis implies that even basal secretory and other responses
are controlled by receptor-dependent pathways that operate at a low
level in the absence of receptor cross-linking. There is presently no
evidence in RBL-2H3 cells for a non-kinase intermediate that couples
the cross-linked Fc
R1 to kinase signaling by providing a docking
platform for 5H2-domain proteins. Nevertheless, there is precedent for
this hypothesis in the insulin receptor system where an accessory
protein, IRS1, couples insulin binding to the amplification of a
tyrosine kinase signaling cascade (reviewed in (49) ). Kinase
sequestration is also possible. In particular, the association of Lyn
with the isolated Fc
R1
subunit in TT
transfectants (17) may form a complex that has no catalytic activity of its
own and reduces the amount of Lyn that can be activated by Fc
R1
cross-linking.
The hypothesis that the subunit binds a key
signaling element suggests a means by which this subunit, that lacks
independent signaling activity, may nevertheless modulate the signaling
activity of the intact Fc
R1. We propose that the putative
TT
-associated kinase or coupling protein associates with the
subunit of the intact Fc
R1 as well as with the isolated
subunit. Whereas binding of this protein to the isolated
subunit
sequesters it from the signal-transducing
subunit and prevents
cell activation, we hypothesize that binding the same protein to the
subunit of the intact Fc
R1 may serve instead to present it
to the adjacent
subunit, enhancing kinase activation and signal
transduction. A simple extension of this hypothesis can additionally
explain the full, but submaximal, signaling activity of the TT
receptor. We propose that the same kinase or coupling protein is
activated by association with cross-linked TT
, but is not
topographically restricted in this case by binding to
. The result
is a less efficient signaling complex and slower and smaller signaling
responses.