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Receptor IIB That Function in Inhibitory Signaling1
*
Department of Immunology, University of Colorado Health Sciences Center and National Jewish Medical and Research Center, Denver, CO 80262;
Department of Cell Biology, Yale University, New Haven, CT 06510; and
Laboratoire d’Immunologie Cellulaire et Clinique, Institut National de la Santé et de la Recherche Médicale, Unité255, Institut Curie, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB, functions broadly in
the immune system, blocking mast cell degranulation, dampening the
humoral immune response, and reducing the risk of autoimmunity.
Previous studies concluded that inhibitory signal transduction by
FcRIIB is mediated solely by its immunoreceptor tyrosine-based
inhibition motif (ITIM) that, when phosphorylated, recruits the
SH2-containing inositol 5'- phosphatase SHIP and the SH2-containing
tyrosine phosphatases SHP-1 and SHP-2. The mutational analysis reported
here reveals that the receptor’s C-terminal 16 residues are also
required for detectable FcRIIB association with SHIP in vivo and for
FcRIIB-mediated phosphatidylinositol 3-kinase hydrolysis by SHIP.
Although the ITIM appears to contain all the structural information
required for receptor-mediated tyrosine phosphorylation of SHIP,
phosphorylation is enhanced when the C-terminal sequence is present.
Additionally, FcRIIB-mediated dephosphorylation of CD19 is
independent of the cytoplasmic tail distal from residue 237, including
the ITIM. Finally, the findings indicate that tyrosines 290, 309, and
326 are all sites of significant FcRIIB1 phosphorylation following
coaggregation with B cell Ag receptor. Thus, we conclude that multiple
sites in FcRIIB contribute uniquely to transduction of
FcRIIB-mediated inhibitory signals. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB1, and
FcRIIB1' (4). Coaggregation of FcRIIB with B cell Ag
receptors (BCR)4 using
immune complexes or intact anti-BCR Abs causes apoptosis
(5) and inhibits blastogenesis (6) and
proliferation (3). FcRIIB-deficient mice exhibit
increased humoral and IgG1-mediated passive cutaneous anaphylactic
responses (7) and are hypersensitive to collagen-induced
arthritis (8).
FcRIIB mediates its effects in part by modulating intermediary
events in BCR signaling. BCR signaling events that are inhibited by
FcRIIB coaggregation include CD19 phosphorylation and subsequent
phosphatidylinositol 3-kinase (PI3-K) recruitment (9, 10, 11),
p21ras activation (12, 13),
phosphatidylinositol 4,5-bisphosphate (PI(4, 5)P2)
hydrolysis (14), calcium mobilization (15),
and extracellular regulated kinase (Erk) activation
(12).
The structural basis of FcRIIB1-mediated inhibitory signaling was
first approached by Amigorena et al. (16), who showed that
a 13-aa sequence in the cytoplasmic tail is necessary for splice
variant FcRIIB2-mediated inhibition of BCR-mediated calcium
mobilization as well as immune complex internalization and subsequent
Ag presentation. Muta et al. (17) later found that, when
placed in an inert receptor context, this sequence is sufficient for
inhibition of BCR-mediated calcium mobilization and that the tyrosine
contained in this sequence is phosphorylated upon coaggregation with
BCR. This 13-mer contains the consensus sequence I/VxYxxL/V, which is
found in many inhibitory receptors and is now known as an
immunoreceptor tyrosine-based inhibition motif (ITIM) (18, 19). Coaggregation of FcRIIB and BCR causes phosphorylation
of the ITIM tyrosine, presumably by BCR-associated Lyn (20, 21), resulting in association with SH2 domain-containing
effector molecules. The SH2-containing protein tyrosine phosphatases
SHP-1 and SHP-2 as well as the phosphatidylinositol 5'-phosphatase SHIP
bind FcRIIB ITIM-derived phosphopeptides in vitro and phosphorylated
FcRIIB in vivo (22, 23, 24, 25, 26). As the nonphosphorylated ITIM
peptide bound none of these SH2 domain-containing proteins,
phosphorylation of the ITIM is apparently required for association with
all three phosphatases (22). In addition, the isoleucine
in the -2 position relative to the ITIM tyrosine is required for SHP-1
and SHP-2 association, since FcRIIB ITIM phosphopeptides in which
this residue is mutated to alanine are unable to bind these two protein
phosphatases in vitro (27). This residue is not required
for SHIP association with the ITIM
(27).5
Recent studies of binding using plasmon resonance indicate that
additional, more N-terminal residues (AENTITYSLL; underline
indicates N-terminal residues) from the ITIM modulate binding to SHP-1
and SHP-2 (28).
Coimmunoprecipitation studies show that in vivo SHIP is the major
protein associated with tyrosyl-phosphorylated FcRIIB (23, 24, 26), and studies in SHIP-deficient DT40 cells have shown SHIP to
be required for FcRIIB-mediated inhibition of calcium mobilization
(29). However, the role of SHP-1 in FcRIIB-mediated
signaling cannot be excluded, as SHP-1 is reportedly required for
inhibition of proliferation in B cells from
SHP-1- (motheaten) mice
(22) and superclustering of FcRIIB and BCR. Thus, SHIP
cleavage of phosphatidylinositol 3,4,5,-triphosphate
(PI(3, 4, 5)P3) to phosphatidylinositol 3,4-bisphosphate
[PI(3, 4)P2] could abrogate the calcium
response. SHIP could also affect calcium mobilization through its
linker function, as phosphorylated SHIP binds Shc (30) and
p62dok (35). Using FcRIIB-Dok
chimeras and Dok knockout mice, Tamir et al. (35) and
Yamanashi et al. (36) have shown that FcRIIB-mediated
inhibition of the Ras pathway and B cell proliferation occurs via
Dok.
CD19 dephosphorylation, another downstream effect of coligation of BCR
and FcRIIB (10, 11), could play a role in the
down-regulation of calcium mobilization by terminating
PI(3, 4, 5)P3 synthesis (10, 31).
Following BCR stimulation, phosphorylated CD19 binds and activates
PI3-K (9), which then phosphorylates PI (4, 5)P2, yielding
PI(3, 4, 5)P3 that is required for activation of
Btk and PLC (9, 32, 33, 34, 37).
Thus, taken together, previous studies indicate that at least three
functional pathways emanate from FcRIIB, involving SHP-1/SHP-2,
SHIP/Dok, and dephosphorylation of CD19. Two of these pathways, those
involving CD19 and SHIP, target PI(3, 4, 5)P3 and
thereby affect calcium mobilization. SHIP, through its interaction with
p62dok, could also affect
p21ras and Erk activation.
On close examination, previous studies suggest that the ITIM may not
mediate all effects of FcRIIB-mediated negative signaling. In a
study by D’Ambrosio et al. (22), inhibition of the
calcium response was not completely abrogated by mutating the ITIM
tyrosine to alanine. Additionally, in a study by Ono et al.
(29), FcRIIB-dependent, anti-BCR-induced apoptosis
was markedly enhanced in DT40 cells expressing ITIM tyrosine to
phenylalanine FcRIIB mutants, and apoptosis mediated by FcRIIB
cross-linking alone required only the transmembrane domain of this
receptor (38). Finally, in the study that established the
sufficiency of the ITIM in FcRIIB function, the chimeric receptor
that contained the ITIM was less inhibitory than the wild-type (WT)
FcRIIB (17). These findings suggest that some FcRIIB
inhibitory functions require regions in addition to the ITIM and may
even be ITIM independent.
To address the structural basis of FcRIIB-mediated inhibitory
signaling, we generated B lymphoma cells (IIA1.6) (39)
expressing various mutants of FcRIIB1 and its splice variants,
FcRIIB1' and FcRIIB2 (Fig. 1
). We
report here that maximal FcRIIB-mediated inhibitory signaling
requires elements of FcRIIB in addition to the ITIM and define four
sites in FcRIIB that function in inhibitory signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine B cell lymphoma line A20, the FcRIIB variant
IIA1.6, and FcRIIB transfectants were grown in IMDM supplemented
with 5% heat-inactivated FBS (HyClone, Logan, UT), 50 U/ml penicillin,
and 50 U/ml streptomycin at 37°C with 7.5%
CO2. Transfectants were made as described
previously (4, 16, 22, 35).
Briefly, the cDNA encoding FcRIIB1 was obtained from M. Hogarth,
that for FcRIIB2 from J. Ravetch, and that for FcRIIB1' as
described previously (4), and mutants were generated by
PCR (sequences available on request). The sequences of the cloned
fragments were confirmed by dideoxy sequencing, and the cDNAs were
cloned into the expression vector pCB6 for B1 WT, B1CT314 and all its
mutations, B1CT289, and B2 WT and into the expression vector NT for B1'
WT and B1CT237. For the expression vector constructs, IIA1.6 cells were
transfected by electroporation, selected for growth in 0.5–1 mg/ml of
the appropriate section factor (G418 for pCB6 and zeocin for NT), and
sorted to equivalent high levels of FcRIIB expression using flow
cytometry in conjunction with 2.4G2 (anti-FcRII and
anti-FcRIII extracellular domain staining).
Immunological reagents used and their sources include: monoclonal
(2.4G2; American Type Culture Collection, Manassas, VA) and polyclonal
(40) anti-FcRIIB, rabbit anti-mouse (RAM) Ig
and F(ab')2 of RAM Ig (Zymed, South San
Francisco, CA), polyclonal anti-CD19 (10), polyclonal
anti-SHIP (40), protein A-Sepharose beads (Pharmacia
Biotech, Piscataway, NJ), cyanogen bromide-activated Sepharose beads
(Pharmacia Biotech), anti-phosphotyrosine (Ab2; Calbiochem, La
Jolla, CA), anti-phospho-Erk (New England Biolabs, Beverley, CA),
anti-Erk1 (New England Biolabs), and anti-Erk2 (New England
Biolabs).
Immunoprecipitation of FcRIIB
Cells were harvested, resuspended in IMDM at 4 x
107/0.5 ml/sample, and held on ice for 10 min.
They were then stimulated with 40 µl/ml of RAM Ig or equimolar
F(ab')2 RAM Ig for 30 s at 37°C and
immediately lysed with 0.5 ml of a 2x Nonidet P-40 lysis buffer (final
concentration containing 1% Nonidet P-40, 10 mM Tris (pH 7.2), 150 mM
NaCl, 10 mM sodium pyrophosphate, 1 mM EDTA, 1 mM PMSF, 2 µg/ml
aprotinin, 2 µg/ml leupeptin, 2 µg/ml
1-antitrypsin, 10 mM NaF, and 1 mM
Na3VO4). Lysates were
cleared by centrifugation at 14,000 rpm. Cleared lysates were then
incubated with 2.4G2 coupled to cyanogen bromide-Sepharose beads at 24
µg of Ab/sample for 1–12 h at 4°C. Beads were washed three times
in lysis buffer. Immunoprecipitates were eluted by boiling in reducing
Laemmli sample buffer, fractionated by 10% SDS-PAGE, and
transferred to polyvinylidene difluoride (PVDF) membranes. Membranes
were serially immunoblotted with anti-phosphotyrosine, polyclonal
anti-SHIP, and polyclonal anti-FcRIIB and were developed
using enhanced chemiluminescence (ECL).
Immunoprecipitation of SHIP
Cells were harvested, resuspended at 2 x 107/0.5 ml/sample, stimulated for 1 min, and lysed as described above. Cleared lysates were preabsorbed with 15 µl of protein A-Sepharose beads/sample for 30 min at 4°C. The preabsorbed lysates were then incubated with 5 µl of polyclonal anti-SHIP and 7 µl of protein A-Sepharose beads for 1–12 h at 4°C. Beads were washed three times in lysis buffer. Immunoprecipitates were eluted by boiling in reducing Laemmli sample buffer, fractionated by 10% SDS-PAGE, and transferred to PVDF membranes. Membranes were serially immunoblotted with anti-phosphotyrosine and polyclonal anti-SHIP and were developed using ECL.
Immunoprecipitation of CD19
Cells were harvested and resuspended at 1 x
107/0.5 ml/sample, stimulated for 30 s as
described above, pelleted, and lysed in RIPA buffer (50 mM Tris (pH
7.2), 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium
deoxycholate, 25 mM ß-glycerophosphate, 10 mM sodium pyrophosphate, 1
mM EDTA, 1 mM PMSF, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml
1-antitrypsin, 10 mM NaF, and 1 mM
Na3VO4). Lysates were
cleared by centrifugation at 14,000 rpm. Cleared lysates were
preabsorbed with 15 µl of protein A-Sepharose beads for 30 min.
Preabsorbed lysates were then incubated with 5 µl of polyclonal
anti-CD19 and 7 µl of protein A-Sepharose beads for 1–12 h.
Beads were washed three times in lysis buffer. Immunoprecipitates were
eluted by boiling in reducing Laemmli sample buffer, fractionated by
10% SDS-PAGE, and transferred to PVDF membranes. Membranes were
serially immunoblotted with anti-phosphotyrosine and polyclonal
anti-CD19 and were developed using ECL.
Detection of phosphorylated Erk
Cells were harvested, resuspended in IMDM at 2 x 106/40 µl/sample, and stimulated with 3.2 µg/sample of RAM Ig or equimolar F(ab')2 for 10 min at 37oC. Cells were immediately lysed in 2x Nonidet P-40 lysis buffer, and the lysate was cleared by centrifugation at 14,000 rpm. The cleared whole cell lysate was boiled with reducing Laemmli sample buffer, fractionated by 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were serially immunoblotted using polyclonal anti-phospho-Erk and combined anti-Erk1 and 2 Abs and developed using ECL.
Measurement of intracellular Ca2+ concentration
Cells were loaded with indo-1/AM by incubation in IMDM containing 5% FBS and 5 µM indo-1/AM (Molecular Probes, Eugene, OR) for 45 min at 37°C and washing twice. Cells were resuspended at 1 x 106/ml/sample in IMDM containing 5% FBS and monitored using a flow cytometer (model 50H; Ortho Diagnostic Systems, Raritan, NJ). Cells were stimulated with 15 µg/ml of RAM Ig at the indicated time point. To detect only release of intracellular Ca2+ stores, Ca2+ in the medium was buffered to 60 nM by adding EGTA immediately before stimulation. To detect calcium influx, CaCl2 was added back to 1.3 mM at the indicated time point.
Measurement of PI(3, 4, 5)P3 production
As described previously (41), cells were harvested, incubated at 107 cells/ml in low phosphate medium with 0.5 mCi/ml [32P]orthophosphate for 1.5 h, and washed. 32P-labeled cells were stimulated with RAM Ig and equimolar F(ab')2 at the indicated time points and lysed with methanol/chloroform (2/1, v/v). Lipids were extracted and deacylated with methanol/25% methylamine/n-butanol (45.7/42.8/11.4, v/v/v), and HPLC was used to fractionate deacylated phosphoinositides. The fractions containing the PI(3, 4, 5)P3 peak were collected and counted on a scintillation counter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB1 cytoplasmic tail occurs
at both ITIM and non-ITIM tyrosines
FcRIIB1 tyrosine residues that become phosphorylated following
coaggregation of this receptor with BCR may mediate protein–protein
interactions that propagate inhibitory signaling events. Previous
studies of FcRIIB phosphorylation suggest that ITIM is the
principal, but probably not the only, tyrosyl-phosphorylated site
(17). To determine which tyrosines in the FcRIIB tail
become phosphorylated subsequent to FcRIIB coaggregation with the
BCR, we evaluated the phosphorylation of various FcRIIB splice
isoforms and mutants in which only certain tyrosines were preserved
(Figs. 1
and 2). Note that all
transfectants were sorted to equivalent levels of FcRIIB by
extracellular staining; however, the antiserum generated against the
entire FcRIIB1 cytoplasmic tail has different sensitivities for the
different FcRIIB variants. Surprisingly, a truncated FcRIIB1
(B1CT314 Y309A) containing Y235, Y264, and Y290, but no ITIM tyrosine,
was phosphorylated comparably to the truncated WT receptor (B1CT314),
suggesting that one or more of the tyrosines outside the ITIM are also
phosphorylated. Indeed, even B1CT289, which lacks 41 C-terminal
residues, including ITIM and Y290, was detectably phosphorylated,
although the extremely low level of phosphorylation was most likely not
physiologically significant. Taken together, these data indicate
that Y235 and/or Y264 are very minor sites, whereas Y309 and Y290 are
major sites of coaggregation-induced FcRIIB1 phosphorylation.
However, Y235 is an unlikely phosphorylation site, since it lies within
the predicted transmembrane domain of FcRIIB1.
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).
These data suggest that multiple FcRIIB tyrosines are phosphorylated
following coligation of FcRIIB1 and BCR; Y290, Y309, and Y326 are
heavily phosphorylated, whereas Y235 and/or Y264 are phosphorylated at
much reduced levels. These inducibly phosphorylated residues are sites
at which additional effectors may dock. Besides the ITIM tyrosine
(Y309), only Y235 and Y326 have equivalents in the other splice
isoforms, suggesting that these isoforms, while all inhibitory, may
have partially unique functions.
Stable association of SHIP with FcRIIB1 requires both the ITIM
core and the 16 C-terminal residues in the FcRIIB1 tail
Previous studies have shown that SHIP binds phosphorylated
FcRIIB1 in vivo and phosphopeptides corresponding to the ITIM in
vitro (23, 24, 26). To determine whether the FcRIIB1
phosphorylated ITIM is the only site required for binding SHIP, we
examined SHIP coimmunoprecipitation with the various FcRIIB mutants
following FcRIIB coaggregation with BCR.
As shown in Fig. 3A, SHIP
coimmunoprecipitated with WT FcRIIB1 (B1WT) and with FcRIIB1'
(B1' WT) following coligation of FcRIIB and BCR in cells transfected
with FcRIIB variants as well as in A20 cells, which express
endogenous FcRIIB1. However, SHIP did not coimmunoprecipitate with
the truncated mutant B1CT314 or with the ITIM tyrosine mutant B1'
Y281G. This indicates that both the ITIM core YSLL and the C-terminal
16 aa residues are required for an association of SHIP with FcRIIB
that is sufficiently stable for detection by coimmunoprecipitation.
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RIIB1 tail cooperate to mediate FcRIIB1-dependent SHIP
phosphorylation
The ITIM tyrosine and the 16 C-terminal residues are required for
stable FcRIIB association with SHIP in vivo; however, in vitro
analysis has demonstrated binding of phosphorylated ITIM peptides to
SHIP (28). It is not known what avidity of FcRIIB
association is required for phosphorylation. To determine whether the
FcRIIB1-mediated increase in SHIP phosphorylation depends solely on
the ITIM, we examined SHIP phosphorylation following coligation with
BCR in the various FcRIIB transfectants. Note that the anti-SHIP
Abs used in these experiments precipitated several bands in the
110–150 kDa range; these multiple bands have been reported previously
and represent splice isoforms and/or cleavage products
(42).
Although aggregation of BCR alone caused some increase in SHIP
phosphorylation, as previously reported (12, 30),
coaggregation of FcRIIB1 significantly enhanced this phosphorylation
by >2-fold, as seen in cells expressing WT FcRIIB1 (B1 WT; Fig. 3B). Unexpectedly, the truncated FcRIIB1 lacking the
C-terminal 16 residues (B1CT314) was less able to support increased
SHIP phosphorylation than the full-length B1 (B1 WT), indicating that
the C-terminal residues play a role in this response.
Consistent with in vitro peptide binding studies (27, 28, 43), mutation of either the ITIM tyrosine (B1CT314 Y309A) or leucines to alanine (B1CT314 LL311AA) in the CT314 truncation further decreased the receptor’s ability to enhance SHIP phosphorylation. This is consistent with a critical role of SHIP SH2 domain binding to the phosphorylated ITIM.
Mutating the ITIM isoleucine (-2 position relative to the tyrosine,
B1CT314 I307A), which is required to bind SHP-1 and SHP-2, but not
SHIP, in vitro (27), to alanine in the C-terminal
truncation had a similar effect on SHIP phosphorylation as the
C-terminal truncation alone (CT314), indicating that the increased SHIP
phosphorylation depends neither on the binding of these protein
phosphatases nor on the isoleucine residue that participates in their
binding. Thus, the ITIM core YSLL and the C-terminal 16 residues of
FcRIIB cooperate in binding of SHIP following coaggregation of
FcRIIB1 and BCR.
FcRIIB1-mediated degradation of PI(3, 4, 5)P3 is
dependent upon regions of the receptor required for stable
association with SHIP
PI3-kinase-mediated production of
PI(3, 4, 5)P3 is critical step in BCR signaling,
being required for receptor-mediated activation of Bruton’s tyrosine
kinase (37) and phospholipase C (9). SHIP
recruited to BCR-coaggregated FcRIIB may inhibit signaling in part
via hydrolysis of PI(3, 4, 5)P3. To determine the
effect of FcRIIB coaggregation on BCR-mediated generation of
PI(3, 4, 5)P3 and the requirement for FcRIIB1
structural elements in these effects, we analyzed lipid
levels in stimulated 32P-labeled
IIA1.6 cells and FcRIIB transfectants. Consistent with previous
findings by Gupta et al. (44), BCR aggregation resulted in
the rapid appearance of PI(3, 4, 5)P3, which was
sustained for the duration of the experiment (Fig. 3C).
Coaggregation of FcRIIB1 with BCR in WT FcRIIB1 transfectants
resulted in apparently normal generation of
PI(3, 4, 5)P3 during the first seconds following
stimulation, but this was followed by complete loss of this lipid. In
fact, within 1 min following costimulation,
PI(3, 4, 5)P3 dropped below basal levels, becoming
virtually undetectable.
As noted earlier, FcRIIB1 could modulate BCR-mediated
PI(3, 4, 5)P3 levels by two mechanisms:
PI(3, 4, 5)P3 production may be terminated by
inactivation of PI3-K due to CD19 dephosphorylation, and SHIP may
degrade PI(3, 4, 5)P3. To assess the role in
inhibition of SHIP-interactive sites within FcRIIB1, we assessed the
ability of B1CT314 and B1CT289 to mediate the effect. Interestingly,
both these mutants exhibited a 50% reduced capacity to mediate
reduction of PI(3, 4, 5)P3 levels (Fig. 3D). Thus, this effect requires the FcRIIB1 C-terminal 16
aa required for stable association with SHIP, suggesting that high
avidity binding, probably resulting in extended dwell time at the
plasma membrane, is necessary for SHIP-mediated
PI(3, 4, 5)P3 hydrolysis.
The finding that the B1CT289 mutant, which lacks both the ITIM and the C-terminal SHIP association signals, remained competent to mediate a 50% reduction in BCR-induced PI(3, 4, 5)P3 generation was unexpected and indicated that structural information sufficient for this inhibitor lies N-terminal from Y290. As will be described below, further analysis indicates that this effect may be mediated by CD19 dephosphorylation and consequent termination of PI3-K activation.
Dephosphorylation of CD19 does not require the 93 C-terminal tail
residues of the FcRIIB1 tail and is not dependent on the ITIM
Another consequence of FcRIIB coligation with BCR is the
decreased phosphorylation of CD19, which affects
PI(3, 4, 5)P3 levels by reducing PI3-K activation
(10, 11). We therefore investigated which FcRIIB1
regions are involved in mediating this effect (Fig. 4). Surprisingly, the B1CT237 truncation,
which lacks all the cytoplasmic tail except the juxtamembrane lysine,
mediated CD19 dephosphorylation similarly to B1 WT. In addition, the
C-terminal truncation CT314 mediated CD19 dephosphorylation. These
results indicate that most of the FcRIIB cytoplasmic tail, including
the ITIM, is not required for CD19 dephosphorylation.
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RIIB1 regions required for CD19 dephosphorylation and SHIP
binding cooperate to cause maximum inhibition of the BCR-mediated
calcium response
Localization of SHIP activation and CD19 dephosphorylation
functions to different regions of FcRIIB allowed dissection of the
relative roles of these two functions in modulating BCR-mediated
calcium mobilization. The calcium response occurs in two stages,
release from intracellular stores and influx from the extracellular
space. To differentiate the two, we first buffered extracellular
calcium to resting cytosolic levels (60 nM) to allow measurement of
release from intracellular stores following stimulation. We then raised
extracellular calcium to 1.3 mM to allow measurement of influx. It was
shown previously that the influx stage is most affected by FcRIIB1
(17).
Not surprisingly, maximum inhibition of BCR-mediated calcium release
from intracellular stores and influx from extracellular space were seen
with WT FcRIIB1 (Fig. 5 and Table I). Regarding the calcium influx phase,
the truncation mutant B1CT314 mediated 20% less inhibition than that
mediated by the WT receptor during the plateau. This indicates that the
C-terminal 16 residues implicated in SHIP binding,
PI(3, 4, 5)P3 degradation, and SHIP phosphorylation
also participate in inhibition of calcium influx.
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30–60% less inhibitory than B1 WT during the plateau of the influx
stage. The FcRIIB1 truncation lacking the ITIM (B1CT289) was
similarly inhibitory (Fig. 5 and Table I). Thus, the ITIM YSLL, which
is also involved in SHIP binding and phosphorylation, participates in
inhibiting calcium mobilization.
Interestingly, the B1CT289 truncation mediated inhibition of calcium
mobilization as well as both B1CT314 Y309A and B1CT314 LL311AA (Fig. 5
and Table I), indicating that some inhibition of calcium mobilization
is independent of the FcRIIB1 tail portion distal from residue 289,
including the ITIM. Thus, the region required for CD19
dephosphorylation and the regions required for SHIP phosphorylation and
binding are all implicated in FcRIIB1-mediated inhibition of
BCR-mediated calcium mobilization. This suggests that maximal
FcRIIB-mediated inhibition of BCR-mediated calcium mobilization
requires both SHIP hydrolysis of PI(3, 4, 5)P3 and
CD19 dephosphorylation-mediated inhibition of
PI(3, 4, 5)P3 synthesis.
The ITIM core YxLL sequence, but not the C-terminal 16 residues, is required to inhibit phosphorylation of Erk1 and Erk2
Coaggregation of FcRIIB1 with BCR results in inhibition of Erk
activation (12). To investigate whether the FcRIIB1
structural elements that affect SHIP phosphorylation and binding and
CD19 dephosphorylation also affect Erk activity, we examined Erk1 and
Erk2 phosphorylation following coaggregation of various FcRIIB
mutants with BCR (Fig. 6).
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RIIB1 (B1 WT). Erk phosphorylation was
similarly inhibited by the C-terminal truncation mutant B1CT314,
suggesting that the 16 C-terminal residues that are required to stably
bind SHIP are not required for FcRIIB1-mediated inhibition of Erk
phosphorylation. In a separate experiment the B1CT314 I307A mutation
inhibited Erk phosphorylation to a level similar to that mediated by
B1CT314, indicating that I307, required for SHP-1 and SHP-2 binding to
FcRIIB, is not required for inhibition of Erk phosphorylation. The
ITIM tyrosine (B1CT314 Y309A) and double-leucine (B1CT314 LL311AA)
mutants were both incapable of mediating inhibition of Erk
phosphorylation; the ITIM core YxLL is thus required for
FcRIIB-mediated inhibition of Erk phosphorylation. Since both
mutations also completely abolished FcRIIB-mediated increased SHIP
phosphorylation, this suggests that SHIP phosphorylation, but not its
stable association with FcRIIB, is required for inhibition of Erk
phosphorylation. Additionally, since FcRIIB-mediated inhibition of
Erk phosphorylation is ITIM dependent, this indicates that
FcRIIB-mediated CD19 dephosphorylation, capable of reducing
PI(3, 4, 5)P3 by 50%, does not affect Erk
activation. These results support the hypothesis that inhibition of the
p21ras/Erk pathway is mediated by SHIP
phosphorylation-dependent association with Shc and/or Dok. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB. At least
four distinguishable sites participate in signal transduction by this
receptor: the full ITIM consensus sequence IxYxxL, which, based on
previous studies (27), associates with SHP-1 and SHP-2;
the ITIM core sequence YSLL, which participates in recruiting SHIP,
leading to an increase in its phosphorylation, hydrolysis of
PI(3, 4, 5)P3, and inhibition of Erk activation and
calcium mobilization; the C-terminal 16 residues, which function
cooperatively with the ITIM core to promote stable SHIP binding, SHIP
phosphorylation, hydrolysis of PI(3, 4, 5)P3, and
inhibition of calcium mobilization; and a region contained within the
transmembrane and/or extracellular domains, which mediates
dephosphorylation of CD19 and inhibition of BCR-mediated
PI(3, 4, 5)P3 generation and calcium mobilization
(Fig. 7).
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RIIB-mediated signaling thus involves
two regions, the ITIM YSLL core and the C-terminal 16-aa residues. The
phosphorylated ITIM alone may mediate a transient association of SHIP,
via the SHIP SH2 domain (27, 28, 43), with FcRIIB that,
while not detectable by immunoprecipitation, is enough to facilitate an
increase in SHIP phosphorylation, probably due to transient
colocalization of SHIP in proximity to BCR-associated kinases. The 16
C-terminal residues stabilize FcRIIB1 interaction with SHIP
sufficiently to allow detection by coimmunoprecipitation and allow for
a further increase in SHIP phosphorylation. This stable interaction
appears necessary for the receptor’s ability to mediate a maximal
decrease in PI(3, 4, 5)P3 levels; stable
localization of SHIP at the plasma membrane would retain SHIP in
proximity to its substrate, PI(3, 4, 5)P3.
Interaction of SHIP with the ITIM in the absence of the C terminus,
while sufficient to mediate a partial increase in SHIP phosphorylation,
is probably insufficient to support SHIP-mediated hydrolysis of
PI(3, 4, 5)P3 due to the transience of the
interaction. Interestingly, this C-terminal region contains a tyrosine,
Y326, that is phosphorylated subsequent to coligation of BCR and
FcRIIB and may play a role in the interaction with SHIP; the
sequence in which Y326 occurs, YQNH, is similar to the Grb2 SH2 domain
binding motif, YQNY, and contains the asparagine that is critical for
Grb2 binding (45). Since SHIP contains a proline-rich
sequence in its C terminus that has been reported to bind Grb2 via one
of its SH3 domains (46), and this C terminus has recently
been reported to be necessary for SHIP’s inhibitory activity
(47), a possible mechanism for stable interaction of SHIP
with FcRIIB is as follows: SHIP binds to the phosphorylated
FcRIIB1 ITIM via its SH2 domain and to a Grb2 SH3 domain via its
C-terminal proline-rich region. Phosphorylation of the FcRIIB1 Y326
leads to formation of a trimeric complex as the Grb2 SH2 domain binds
the phosphotyrosine. The binding energy provided by the bidentate
binding of each component stabilizes the interaction. The ITIM core
YSLL is also required for the FcRIIB-mediated increase in SHIP
phosphorylation and for inhibition of Erk phosphorylation. Although the
amount of SHIP phosphorylation appears to depend on the stability of
its interaction with FcRIIB, the C-terminal region required for the
stable interaction is not required for FcRIIB-mediated inhibition of
Erk phosphorylation. Thus inhibition of Erk phosphorylation may depend
on SHIP phosphorylation, and the increase in SHIP phosphorylation
mediated by the ITIM alone, although not maximal, is sufficient for
this. This is consistent with studies suggesting that interaction of
phosphorylated SHIP and Shc may lead to inhibition of BCR-mediated Erk
activation (12) as well as with data from our laboratory
indicating that phosphorylated SHIP interaction with
p62dok leads to inhibition of BCR-mediated Erk
phosphorylation (35) .
Perhaps the most surprising observation from this study is that reduced
CD19 phosphorylation is independent of the ITIM and, in fact, requires
little if any of the FcRIIB cytoplasmic tail. This correlates well
with studies by Pearse et al. (38) showing that
FcRIIB-mediated apoptosis is mediated by the transmembrane domain of
FcRIIB. Since increased PI(3, 4, 5)P3 levels,
through the ability of PI(3, 4, 5)P3 to recruit
Akt, are a survival signal, reduced phosphorylation of CD19, leading to
decreased PI3-K activity, could be a mechanism for FcRIIB-mediated
apoptosis.
Note that at least the last six residues encoded in the transmembrane
exon (KKKQVP) are actually cytoplasmic and could interact with effector
molecules that could mediate CD19 dephosphorylation; however, the CT237
truncation has eliminated all but the juxtamembrane lysine. One
possibility is that Y235, just two residues into the actual
transmembrane region, may be exposed to the cytosol and thus become
phosphorylated and associate with the CD19 dephosphorylation machinery
subsequent to coligation of BCR and FcRIIB; however, the
stoichiometry of this phosphorylation is so low that this seems
unlikely. Another possibility is that coligation of FcRIIB with BCR
displaces CD19 from the BCR aggregate so that BCR-associated kinases
can no longer maintain the phosphorylation state of CD19. Yet another
possibility is that the extracellular or transmembrane domain could
interact with another transmembrane protein that mediates CD19
dephosphorylation. These possibilities are currently under study.
Mechanisms underlying FcRIIB-mediated reduction of
PI(3, 4, 5)P3 levels and, consequently, the calcium
response are more complicated, involving the C-terminal region, the
ITIM YSLL, and the region involved in CD19 dephosphorylation. Stable
association of SHIP with FcRIIB accounts for 50% of the initial
reduction in PI(3, 4, 5)P3 levels. However, the
other 50% is ITIM independent. Similarly, the ITIM plus the C-terminal
region accounts for 60% of the inhibition of the late phase calcium
response, with the remainder being ITIM independent. Thus, most of the
inhibition of PI(3, 4, 5)P3 levels and calcium
mobilization corresponds to SHIP phosphorylation and interaction with
FcRIIB. However, none of the mutations to FcRIIB studied here was
able to completely abrogate this inhibition. The remaining
ITIM-independent inhibitory activity could be attributed to the
dephosphorylation of CD19, causing a decrease in
PI(3, 4, 5)P3 generation and, hence, phospholipase
C activation. Thus, PI(3, 4, 5)P3 levels and
consequently calcium mobilization are reduced by FcRIIB signaling in
two ways: CD19 dephosphorylation prevents
PI(3, 4, 5)P3 generation, and SHIP degrades
it.
The isoleucine in the -2 position relative to the ITIM tyrosine
apparently plays no role in inhibition of calcium mobilization, which
is not surprising in light of previous work showing SHP-1 to be
dispensable for inhibition of the calcium response (48).
However, SHP-1 could be involved in very late signaling events leading
to apoptosis or inhibition of proliferation. This would be consistent
with the kinetics of interaction of SHP-1 and SHIP with phosphorylated
ITIM peptides from FcRIIB. SHIP has a fast on and off rate, while
SHP-1 has a slow on and off rate (28). SHP-1 may thus
accumulate at the receptor very slowly and be involved in
FcRIIB-mediated inhibitory signaling much later than SHIP. This also
conforms with the requirement for SHP-1 in FcRIIB-mediated
inhibition of B cell proliferation (22).
The other surprising outcome of this mutational analysis is that
tyrosines in addition to the ITIM tyrosine are phosphorylated following
coaggregation of FcRIIB and BCR. In studies by Muta et al.
(17), mutation of the ITIM tyrosine to phenylalanine did
not completely ablate tyrosyl phosphorylation of FcRIIB, although it
was very significantly decreased. Technical variations may account for
the difference in the amount of non-ITIM tyrosine phosphorylation
observed between our study and Muta’s. For example, phosphorylation of
non-ITIM tyrosines may be more apparent at our 30-s point than at the
3-min point in the Muta study. Another possibility is that the
different anti-phosphotyrosine Abs used may have different
sensitivities and substrate reactivities. Nevertheless, our results are
consistent with one important point from the Muta study; they showed
that the ITIM alone, in the context of a chimeric receptor, inhibits
IIA1.6 cell BCR-mediated IL-2 secretion only half as well as the entire
WT receptor, implying that FcRIIB regions besides the ITIM
contribute to its function. This correlates with the half-maximal
inhibition of PI(3, 4, 5)P3 levels and calcium
mobilization we see in cells expressing truncated receptors lacking
only the C-terminal 16 residues (B1CT314) or 41 residues, including the
ITIM (B1CT289). Although the results reported by Muta et al. did not
demonstrate any inhibition of calcium mobilization in cells expressing
an FcRIIB deletion mutant lacking 13 residues including the ITIM, it
is possible that deletion of a sequence in the middle of the receptor
may have a greater effect on the conformation than a truncation,
somehow abrogating interactions that mediate CD19 phosphorylation.
Two of the phosphorylated tyrosines that occur outside the ITIM are
also conserved in all three splice isoforms of murine FcRIIB. This
may reflect the common inhibitory function of these isoforms. We have
shown that Y326, which lies within the C-terminal region required for
stable association with SHIP, is phosphorylated following coaggregation
of FcRIIB and BCR and that Y235, which lies within the region
required for CD19 dephosphorylation, is apparently phosphorylated at
low stoichiometry. These tyrosines may be involved in protein-protein
interactions that contribute to FcRIIB-mediated inhibitory
signaling; for example, Y326 may bind Grb2. Interestingly, Y290, which
only occurs in the B1 isoform, is also heavily phosphorylated; the
region in which this residue occurs is required to prevent endocytosis
of FcRIIB1 when the receptor binds immune complexes
(16), a function that is unique to the B1 isoform.
We thus show that although the FcRIIB ITIM is necessary for
inhibitory signaling, other domains of the receptor are also required
for full inhibitory signaling. In fact, regions of FcRIIB that
regulate SHIP and CD19 lie completely outside of the ITIM and may act
via tyrosine phosphorylation. In addition, we show that both SHIP
activity and CD19 dephosphorylation contribute to complete inhibition
of BCR-mediated calcium mobilization by modulating
PI(3, 4, 5)P3 levels. The use of multiple
mechanisms in FcRIIB-mediated inhibitory signaling reflects the
redundancy found in many critical signaling pathways; this redundancy
provides additional modes for precise control of cellular responses and
backup mechanisms to ensure the fidelity of the responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John C. Cambier, National Jewish Medical and Research Center, 1400 Jackson Street, Room K1004, Denver, CO 80206.
3 Ida and Cecil Green Professor of Immunology.
4 Abbreviations used in this paper: BCR, B cell Ag receptor; PI3-K, phosphatidylinositol 3-kinase; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; Erk, extracellular regulated kinase; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; WT, wild type; RAM, rabbit anti-mouse; PVDF, polyvinylidene difluoride; ECL, enhanced chemiluminescence; ITIM, immunoreceptor tyrosine-based inhibition motif; SHP-1, SH2-containing phosphatase 1; SHP-2, SH2-containing phosphatase 2; SHIP, SH2-containing inositol 5'-phosphatase; PI(3,4,5)P3, phosphatidylinositol 3,4,5,-triphosphate.
5 P. Bruhns, F. Vely, O. Malbec, W. H. Fridman, E. Vivier, and M. Daeron. Molecular basis of the binding specificity of fcRIIB for the SH2 domain bearing phosphatase SHIP. Submitted for publication.
Received for publication November 1, 1999. Accepted for publication July 25, 2000.
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