The low-affinity receptor for IgG, FcγRIIB, 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 FcγRIIB 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 FcγRIIB association with SHIP in vivo and for FcγRIIB-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, FcγRIIB-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 FcγRIIB1 phosphorylation following coaggregation with B cell Ag receptor. Thus, we conclude that multiple sites in FcγRIIB contribute uniquely to transduction of FcγRIIB-mediated inhibitory signals.

It has long been known that IgG-containing immune complexes can suppress humoral immune responses (1). This inhibition is dependent on the Fc portion of IgG (1, 2) and on the low-affinity Fc receptors expressed by B cells (3), FcγRIIB1, and FcγRIIB1′ (4). Coaggregation of FcγRIIB 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). FcγRIIB-deficient mice exhibit increased humoral and IgG1-mediated passive cutaneous anaphylactic responses (7) and are hypersensitive to collagen-induced arthritis (8).

FcγRIIB mediates its effects in part by modulating intermediary events in BCR signaling. BCR signaling events that are inhibited by FcγRIIB 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 FcγRIIB1-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 FcγRIIB2-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 FcγRIIB 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 FcγRIIB ITIM-derived phosphopeptides in vitro and phosphorylated FcγRIIB 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 FcγRIIB 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 FcγRIIB (23, 24, 26), and studies in SHIP-deficient DT40 cells have shown SHIP to be required for FcγRIIB-mediated inhibition of calcium mobilization (29). However, the role of SHP-1 in FcγRIIB-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 FcγRIIB 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 FcγRIIB-Dok chimeras and Dok knockout mice, Tamir et al. (35) and Yamanashi et al. (36) have shown that FcγRIIB-mediated inhibition of the Ras pathway and B cell proliferation occurs via Dok.

CD19 dephosphorylation, another downstream effect of coligation of BCR and FcγRIIB (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 PLCγ (9, 32, 33, 34, 37).

Thus, taken together, previous studies indicate that at least three functional pathways emanate from FcγRIIB, 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 FcγRIIB-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), FcγRIIB-dependent, anti-BCR-induced apoptosis was markedly enhanced in DT40 cells expressing ITIM tyrosine to phenylalanine FcγRIIB mutants, and apoptosis mediated by FcγRIIB cross-linking alone required only the transmembrane domain of this receptor (38). Finally, in the study that established the sufficiency of the ITIM in FcγRIIB function, the chimeric receptor that contained the ITIM was less inhibitory than the wild-type (WT) FcγRIIB (17). These findings suggest that some FcγRIIB inhibitory functions require regions in addition to the ITIM and may even be ITIM independent.

To address the structural basis of FcγRIIB-mediated inhibitory signaling, we generated B lymphoma cells (IIA1.6) (39) expressing various mutants of FcγRIIB1 and its splice variants, FcγRIIB1′ and FcγRIIB2 (Fig. 1). We report here that maximal FcγRIIB-mediated inhibitory signaling requires elements of FcγRIIB in addition to the ITIM and define four sites in FcγRIIB that function in inhibitory signaling.

FIGURE 1.

Diagrammatic representation of FcγRIIB isoforms and mutants used in this study.

FIGURE 1.

Diagrammatic representation of FcγRIIB isoforms and mutants used in this study.

Close modal

The murine B cell lymphoma line A20, the FcγRIIB variant IIA1.6, and FcγRIIB 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 FcγRIIB1 was obtained from M. Hogarth, that for FcγRIIB2 from J. Ravetch, and that for FcγRIIB1′ 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 FcγRIIB expression using flow cytometry in conjunction with 2.4G2 (anti-FcγRII and anti-FcγRIII extracellular domain staining).

Immunological reagents used and their sources include: monoclonal (2.4G2; American Type Culture Collection, Manassas, VA) and polyclonal (40) anti-FcγRIIB, 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).

Cells were harvested, resuspended in IMDM at 4 × 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 2× 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 α1-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-FcγRIIB and were developed using enhanced chemiluminescence (ECL).

Cells were harvested, resuspended at 2 × 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.

Cells were harvested and resuspended at 1 × 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 β-glycerophosphate, 10 mM sodium pyrophosphate, 1 mM EDTA, 1 mM PMSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml α1-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.

Cells were harvested, resuspended in IMDM at 2 × 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 2× 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.

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 × 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.

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.

FcγRIIB1 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 FcγRIIB phosphorylation suggest that ITIM is the principal, but probably not the only, tyrosyl-phosphorylated site (17). To determine which tyrosines in the FcγRIIB tail become phosphorylated subsequent to FcγRIIB coaggregation with the BCR, we evaluated the phosphorylation of various FcγRIIB 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 FcγRIIB by extracellular staining; however, the antiserum generated against the entire FcγRIIB1 cytoplasmic tail has different sensitivities for the different FcγRIIB variants. Surprisingly, a truncated FcγRIIB1 (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 FcγRIIB1 phosphorylation. However, Y235 is an unlikely phosphorylation site, since it lies within the predicted transmembrane domain of FcγRIIB1.

FIGURE 2.

Multiple FcγRIIB1 tyrosines are phosphorylated following coaggregation of FcγRIIB and BCR. Expression-matched IIA1.6 transfectants (4 × 107 cells/0.5 ml/sample) bearing the indicated FcγRIIB variants were stimulated for 30 s with 40 μg of RAMIg (i), equimolar F(ab′)2 RAMIg (f), or no ligand (u) before detergent lysis and immunoprecipitation with anti-FcγRIIB (2.4G2). Immunoprecipitates were analyzed by SDS-PAGE, electrophoretic transfer, and serial immunoblotting with anti-phosphotyrosine (AB2) and anti-FcγRIIB (rabbit anti-mouse FcγRIIB tail). Data shown are representative of three experiments.

FIGURE 2.

Multiple FcγRIIB1 tyrosines are phosphorylated following coaggregation of FcγRIIB and BCR. Expression-matched IIA1.6 transfectants (4 × 107 cells/0.5 ml/sample) bearing the indicated FcγRIIB variants were stimulated for 30 s with 40 μg of RAMIg (i), equimolar F(ab′)2 RAMIg (f), or no ligand (u) before detergent lysis and immunoprecipitation with anti-FcγRIIB (2.4G2). Immunoprecipitates were analyzed by SDS-PAGE, electrophoretic transfer, and serial immunoblotting with anti-phosphotyrosine (AB2) and anti-FcγRIIB (rabbit anti-mouse FcγRIIB tail). Data shown are representative of three experiments.

Close modal

Mutational analysis suggests that Y326 is also a major site of tyrosine phosphorylation. When the ITIM tyrosine is mutated to glycine in the B1′ isoform (B1′ Y281G), leaving only Y298, the equivalent of the B1 isoform Y326, the receptor is phosphorylated to at least half the level of WT B1′ (B1′ WT) (Fig. 2).

These data suggest that multiple FcγRIIB tyrosines are phosphorylated following coligation of FcγRIIB1 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.

Previous studies have shown that SHIP binds phosphorylated FcγRIIB1 in vivo and phosphopeptides corresponding to the ITIM in vitro (23, 24, 26). To determine whether the FcγRIIB1 phosphorylated ITIM is the only site required for binding SHIP, we examined SHIP coimmunoprecipitation with the various FcγRIIB mutants following FcγRIIB coaggregation with BCR.

As shown in Fig. 3 A, SHIP coimmunoprecipitated with WT FcγRIIB1 (B1WT) and with FcγRIIB1′ (B1′ WT) following coligation of FcγRIIB and BCR in cells transfected with FcγRIIB variants as well as in A20 cells, which express endogenous FcγRIIB1. 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 FcγRIIB that is sufficiently stable for detection by coimmunoprecipitation.

FIGURE 3.

The FcγRIIB ITIM core and C-terminal 16 residues participate in regulation of SHIP association with FcγRIIB (A), SHIP phosphorylation (B), and SHIP hydrolysis of PI(3,4,5)P3 (C and D). A, IIA1.6 transfectants (4 × 107 cells/0.5 ml/sample) bearing the indicated FcγRIIB variants were stimulated 30 s with 40 μg of RAMIg (i), equimolar F(ab′)2 RAMIg (f), or no ligand (u) before detergent lysis and immunoprecipitation with anti-FcγRIIB (2.4G2). Immunoprecipitates were analyzed by SDS-PAGE, electrophoretic transfer, and serial immunoblotting with anti-SHIP (rabbit anti-mouse SHIP), anti-phosphotyrosine (AB2), and anti-FcγRIIB (rabbit anti-mouse FcγRIIB tail). Data shown are representative of three experiments. B, IIA1.6 transfectants (2 × 107 cells/0.5 ml/sample) bearing the indicated FcγRIIB variants were stimulated for 60 s with 20 μg of RAMIg (i), equimolar F(ab′)2 RAMIg (f), or no ligand (u) before detergent lysis and immunoprecipitation with anti-SHIP (rabbit anti-mouse SHIP). Immunoprecipitates were analyzed by SDS-PAGE, electrophoretic transfer, and serial immunoblotting with anti-phosphotyrosine (AB2) and anti-SHIP. Data shown are representative of three experiments. C, 32P-labeled IIA1.6 and B1 WT transfectants (1 × 107 cells/0.2 ml/sample) were stimulated with 10 μg of RAMIg at the indicated time points and immediately lysed with methanol/chloroform (2/1, v/v). Lipids were extracted from the cell lysates, deacylated, and fractionated by HPLC. The PI(3,4,5)P3-containing fractions were collected and quantitated by liquid scintillation. D, 32P-labeled IIA1.6 transfected with the indicated FcγRIIB variants was stimulated for 1 min with 10 μg of RAMIg (I), 6.25 μg F(ab′)2, or no ligand (U). Cells were lysed, and lipids were extracted and quantitated as described in C. Data from two representative experiments are shown.

FIGURE 3.

The FcγRIIB ITIM core and C-terminal 16 residues participate in regulation of SHIP association with FcγRIIB (A), SHIP phosphorylation (B), and SHIP hydrolysis of PI(3,4,5)P3 (C and D). A, IIA1.6 transfectants (4 × 107 cells/0.5 ml/sample) bearing the indicated FcγRIIB variants were stimulated 30 s with 40 μg of RAMIg (i), equimolar F(ab′)2 RAMIg (f), or no ligand (u) before detergent lysis and immunoprecipitation with anti-FcγRIIB (2.4G2). Immunoprecipitates were analyzed by SDS-PAGE, electrophoretic transfer, and serial immunoblotting with anti-SHIP (rabbit anti-mouse SHIP), anti-phosphotyrosine (AB2), and anti-FcγRIIB (rabbit anti-mouse FcγRIIB tail). Data shown are representative of three experiments. B, IIA1.6 transfectants (2 × 107 cells/0.5 ml/sample) bearing the indicated FcγRIIB variants were stimulated for 60 s with 20 μg of RAMIg (i), equimolar F(ab′)2 RAMIg (f), or no ligand (u) before detergent lysis and immunoprecipitation with anti-SHIP (rabbit anti-mouse SHIP). Immunoprecipitates were analyzed by SDS-PAGE, electrophoretic transfer, and serial immunoblotting with anti-phosphotyrosine (AB2) and anti-SHIP. Data shown are representative of three experiments. C, 32P-labeled IIA1.6 and B1 WT transfectants (1 × 107 cells/0.2 ml/sample) were stimulated with 10 μg of RAMIg at the indicated time points and immediately lysed with methanol/chloroform (2/1, v/v). Lipids were extracted from the cell lysates, deacylated, and fractionated by HPLC. The PI(3,4,5)P3-containing fractions were collected and quantitated by liquid scintillation. D, 32P-labeled IIA1.6 transfected with the indicated FcγRIIB variants was stimulated for 1 min with 10 μg of RAMIg (I), 6.25 μg F(ab′)2, or no ligand (U). Cells were lysed, and lipids were extracted and quantitated as described in C. Data from two representative experiments are shown.

Close modal

The ITIM tyrosine and the 16 C-terminal residues are required for stable FcγRIIB 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 FcγRIIB association is required for phosphorylation. To determine whether the FcγRIIB1-mediated increase in SHIP phosphorylation depends solely on the ITIM, we examined SHIP phosphorylation following coligation with BCR in the various FcγRIIB 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 FcγRIIB1 significantly enhanced this phosphorylation by >2-fold, as seen in cells expressing WT FcγRIIB1 (B1 WT; Fig. 3 B). Unexpectedly, the truncated FcγRIIB1 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 FcγRIIB cooperate in binding of SHIP following coaggregation of FcγRIIB1 and BCR.

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 Cγ (9). SHIP recruited to BCR-coaggregated FcγRIIB may inhibit signaling in part via hydrolysis of PI(3, 4, 5)P3. To determine the effect of FcγRIIB coaggregation on BCR-mediated generation of PI(3, 4, 5)P3 and the requirement for FcγRIIB1 structural elements in these effects, we analyzed lipid levels in stimulated 32P-labeled IIA1.6 cells and FcγRIIB 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. 3 C). Coaggregation of FcγRIIB1 with BCR in WT FcγRIIB1 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, FcγRIIB1 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 FcγRIIB1, 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. 3 D). Thus, this effect requires the FcγRIIB1 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.

Another consequence of FcγRIIB 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 FcγRIIB1 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 FcγRIIB cytoplasmic tail, including the ITIM, is not required for CD19 dephosphorylation.

FIGURE 4.

The region of the FcγRIIB cytoplasmic tail distal from residue 237 is not required for CD19 dephosphorylation. Expression-matched IIA1.6 transfectants (1 × 107 cells/0.5 ml/sample) bearing the indicated FcγRIIB variants were stimulated for 30 s with 20 μg of RAMIg (i), equimolar F(ab′)2 RAMIg (f), or no ligand (u) before detergent lysis and immunoprecipitation with anti-CD19 (rabbit anti-mouse CD19). Immunoprecipitates were analyzed by SDS-PAGE, electrophoretic transfer, and serial immunoblotting with anti-phosphotyrosine (AB2) and anti-CD19. Data shown are representative of three experiments.

FIGURE 4.

The region of the FcγRIIB cytoplasmic tail distal from residue 237 is not required for CD19 dephosphorylation. Expression-matched IIA1.6 transfectants (1 × 107 cells/0.5 ml/sample) bearing the indicated FcγRIIB variants were stimulated for 30 s with 20 μg of RAMIg (i), equimolar F(ab′)2 RAMIg (f), or no ligand (u) before detergent lysis and immunoprecipitation with anti-CD19 (rabbit anti-mouse CD19). Immunoprecipitates were analyzed by SDS-PAGE, electrophoretic transfer, and serial immunoblotting with anti-phosphotyrosine (AB2) and anti-CD19. Data shown are representative of three experiments.

Close modal

Localization of SHIP activation and CD19 dephosphorylation functions to different regions of FcγRIIB 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 FcγRIIB1 (17).

Not surprisingly, maximum inhibition of BCR-mediated calcium release from intracellular stores and influx from extracellular space were seen with WT FcγRIIB1 (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.

FIGURE 5.

Inhibition of BCR-mediated calcium mobilization is partially dependent on the FcγRIIB regions that regulate SHIP. Intracellular free calcium levels ([Ca2+]i) were monitored following stimulation of indo-1-loaded IIA1.6 (1 × 106 cells/1 ml/sample) expressing the various FcγRIIB variants. Immediately before stimulation, EGTA was added to chelate extracellular Ca2+ ([Ca2+]o) to 60 nM to measure the release of intracellular calcium stores only. Analysis was begun, and resting [Ca2+]i was established before adding 15 μg of RAMIg at the indicated time point (arrow). [Ca2+]o was increased to 1.3 mM at the indicated time point (arrow) to allow measurement of calcium influx.

FIGURE 5.

Inhibition of BCR-mediated calcium mobilization is partially dependent on the FcγRIIB regions that regulate SHIP. Intracellular free calcium levels ([Ca2+]i) were monitored following stimulation of indo-1-loaded IIA1.6 (1 × 106 cells/1 ml/sample) expressing the various FcγRIIB variants. Immediately before stimulation, EGTA was added to chelate extracellular Ca2+ ([Ca2+]o) to 60 nM to measure the release of intracellular calcium stores only. Analysis was begun, and resting [Ca2+]i was established before adding 15 μg of RAMIg at the indicated time point (arrow). [Ca2+]o was increased to 1.3 mM at the indicated time point (arrow) to allow measurement of calcium influx.

Close modal
Table I.

Peak and plateau [Ca2+]i values for the influx phasea

Cell linePeak [Ca2+]i (nM)Plateau [Ca2+]i (nM)
Null (IIA1.6) 260 150 
B1CT314 Y309A 155 130 
B1CT314 LL311AA 180 105 
B1CT289 170 105 
B1CT314 145 95 
B1 WT 90 80 
Cell linePeak [Ca2+]i (nM)Plateau [Ca2+]i (nM)
Null (IIA1.6) 260 150 
B1CT314 Y309A 155 130 
B1CT314 LL311AA 180 105 
B1CT289 170 105 
B1CT314 145 95 
B1 WT 90 80 
a

Values were obtained from Fig. 5.

The ITIM tyrosine mutant (B1CT314 Y309) and the ITIM double-leucine mutant (B1CT314 LL311AA) in the truncation B1CT314 were both ∼30–60% less inhibitory than B1 WT during the plateau of the influx stage. The FcγRIIB1 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 FcγRIIB1 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 FcγRIIB1-mediated inhibition of BCR-mediated calcium mobilization. This suggests that maximal FcγRIIB-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.

Coaggregation of FcγRIIB1 with BCR results in inhibition of Erk activation (12). To investigate whether the FcγRIIB1 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 FcγRIIB mutants with BCR (Fig. 6).

FIGURE 6.

The ITIM YSLL is required for FcγRIIB inhibition of BCR-mediated Erk phosphorylation. IIA1.6 (2 × 106 cells/40-μl/sample) expressing the indicated FcγRIIB variants were stimulated for 10 min with 3.2 μg of RAMIg (i), equimolar F(ab′)2 RAMIg (f), or no ligand (u) before detergent lysis. Cells lysates were analyzed by SDS-PAGE, electrophoretic transfer, and serial immunoblotting with anti-phospho-Erk and anti-Erk1 and 2. Data shown are representative of three experiments.

FIGURE 6.

The ITIM YSLL is required for FcγRIIB inhibition of BCR-mediated Erk phosphorylation. IIA1.6 (2 × 106 cells/40-μl/sample) expressing the indicated FcγRIIB variants were stimulated for 10 min with 3.2 μg of RAMIg (i), equimolar F(ab′)2 RAMIg (f), or no ligand (u) before detergent lysis. Cells lysates were analyzed by SDS-PAGE, electrophoretic transfer, and serial immunoblotting with anti-phospho-Erk and anti-Erk1 and 2. Data shown are representative of three experiments.

Close modal

BCR-mediated Erk phosphorylation was nearly abolished upon BCR coaggregation with WT FcγRIIB1 (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 FcγRIIB1-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 FcγRIIB, 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 FcγRIIB-mediated inhibition of Erk phosphorylation. Since both mutations also completely abolished FcγRIIB-mediated increased SHIP phosphorylation, this suggests that SHIP phosphorylation, but not its stable association with FcγRIIB, is required for inhibition of Erk phosphorylation. Additionally, since FcγRIIB-mediated inhibition of Erk phosphorylation is ITIM dependent, this indicates that FcγRIIB-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.

The data presented here reveal a previously unrecognized level of complexity in the signal transducing function of FcγRIIB. 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).

FIGURE 7.

A working model describing the role of FcγRIIB1 cytoplasmic tail sequences in inhibitory signaling. Multiple regions of FcγRIIB participate in complete FcγRIIB-mediated inhibitory signaling. The cytoplasmic tail of FcγRIIB1 and its major tyrosine phosphorylation sites are shown. Possible binding proteins for the non-ITIM tyrosines are indicated. For more details, see text.

FIGURE 7.

A working model describing the role of FcγRIIB1 cytoplasmic tail sequences in inhibitory signaling. Multiple regions of FcγRIIB participate in complete FcγRIIB-mediated inhibitory signaling. The cytoplasmic tail of FcγRIIB1 and its major tyrosine phosphorylation sites are shown. Possible binding proteins for the non-ITIM tyrosines are indicated. For more details, see text.

Close modal

Full involvement of SHIP in FcγRIIB-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 FcγRIIB 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 FcγRIIB1 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 FcγRIIB 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 FcγRIIB is as follows: SHIP binds to the phosphorylated FcγRIIB1 ITIM via its SH2 domain and to a Grb2 SH3 domain via its C-terminal proline-rich region. Phosphorylation of the FcγRIIB1 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 FcγRIIB-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 FcγRIIB, the C-terminal region required for the stable interaction is not required for FcγRIIB-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 FcγRIIB cytoplasmic tail. This correlates well with studies by Pearse et al. (38) showing that FcγRIIB-mediated apoptosis is mediated by the transmembrane domain of FcγRIIB. 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 FcγRIIB-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 FcγRIIB; however, the stoichiometry of this phosphorylation is so low that this seems unlikely. Another possibility is that coligation of FcγRIIB 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 FcγRIIB-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 FcγRIIB 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 FcγRIIB. However, none of the mutations to FcγRIIB 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 Cγ activation. Thus, PI(3, 4, 5)P3 levels and consequently calcium mobilization are reduced by FcγRIIB 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 FcγRIIB. 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 FcγRIIB-mediated inhibitory signaling much later than SHIP. This also conforms with the requirement for SHP-1 in FcγRIIB-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 FcγRIIB and BCR. In studies by Muta et al. (17), mutation of the ITIM tyrosine to phenylalanine did not completely ablate tyrosyl phosphorylation of FcγRIIB, 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 FcγRIIB 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 FcγRIIB 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 FcγRIIB. 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 FcγRIIB 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 FcγRIIB-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 FcγRIIB1 when the receptor binds immune complexes (16), a function that is unique to the B1 isoform.

We thus show that although the FcγRIIB ITIM is necessary for inhibitory signaling, other domains of the receptor are also required for full inhibitory signaling. In fact, regions of FcγRIIB 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 FcγRIIB-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.

We thank Barbara Vilen for her insightful advice and William Townsend and Shirley Sobus for their technical assistance.

1

This work was supported by grants from the U.S. Public Health Service.

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 fcγRIIB for the SH2 domain bearing phosphatase SHIP. Submitted for publication.

1
Chan, P. L., N. R. Sinclair.
1971
. Regulation of the immune response. V. An analysis of the function of the Fc portion of antibody in suppression of an immune response with respect to interaction with components of the lymphoid system.
Immunology
21
:
967
2
Kohler, H., B. C. Richardson, D. A. Rowley, S. Smyk.
1977
. Immune response to phosphorylcholine. III. Requirement of the Fc portion and equal effectiveness of IgG subclasses in anti-receptor antibody-induced suppression.
J. Immunol.
119
:
1979
3
Phillips, N. E., D. C. Parker.
1983
. Fc-dependent inhibition of mouse B cell activation by whole anti-μ antibodies.
J. Immunol.
130
:
602
4
Latour, S., W. H. Fridman, M. Daeron.
1996
. Identification, molecular cloning, biologic properties, and tissue distribution of a novel isoform of murine low-affinity IgG receptor homologous to human FcγRIIB1.
J. Immunol.
157
:
189
5
Ashman, R. F., D. Peckham, L. L. Stunz.
1996
. Fc receptor off-signal in the B cell involves apoptosis.
J. Immunol.
157
:
5
6
Phillips, N. E., D. C. Parker.
1984
. Cross-linking of B lymphocyte Fcγ receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis.
J. Immunol.
132
:
627
7
Takai, T., M. Ono, M. Hikida, H. Ohmori, J. V. Ravetch.
1996
. Augmented humoral and anaphylactic responses in FcγRII-deficient mice.
Nature
379
:
346
8
Yuasa, T., S. Kubo, T. Yoshino, A. Ujike, K. Matsumura, M. Ono, J. V. Ravetch, T. Takai.
1999
. Deletion of Fcγ receptor IIB renders H-2(b) mice susceptible to collagen-induced arthritis.
J. Exp. Med.
189
:
187
9
Buhl, A. M., C. M. Pleiman, R. C. Rickert, J. C. Cambier.
1997
. Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization.
J. Exp. Med.
186
:
1897
10
Hippen, K. L., A. M. Buhl, D. D’Ambrosio, K. Nakamura, C. Persin, J. C. Cambier.
1997
. FcγRIIB1 inhibition of BCR-mediated phosphoinositide hydrolysis and Ca2+ mobilization is integrated by CD19 dephosphorylation.
Immunity
7
:
49
11
Kiener, P. A., M. N. Lioubin, L. R. Rohrschneider, J. A. Ledbetter, S. G. Nadler, M. L. Diegel.
1997
. Co-ligation of the antigen and Fc receptors gives rise to the selective modulation of intracellular signaling in B cells: regulation of the association of phosphatidylinositol 3-kinase and inositol 5′-phosphatase with the antigen receptor complex.
J. Biol. Chem.
272
:
3838
12
Tridandapani, S., G. W. Chacko, J. R. Van Brocklyn, K. M. Coggeshall.
1997
. Negative signaling in B cells causes reduced Ras activity by reducing Shc-Grb2 interactions.
J. Immunol.
158
:
1125
13
Sarmay, G., G. Koncz, J. Gergely.
1996
. Human type II Fcγ receptors inhibit B cell activation by interacting with the p21ras-dependent pathway.
J. Biol. Chem.
271
:
30499
14
Bijsterbosch, M. K., G. G. Klaus.
1985
. Crosslinking of surface immunoglobulin and Fc receptors on B lymphocytes inhibits stimulation of inositol phospholipid breakdown via the antigen receptors.
J. Exp. Med.
162
:
1825
15
Wilson, H. A., D. Greenblatt, C. W. Taylor, J. W. Putney, R. Y. Tsien, F. D. Finkelman, T. M. Chused.
1987
. The B lymphocyte calcium response to anti-Ig is diminished by membrane immunoglobulin cross-linkage to the Fcγ receptor.
J. Immunol.
138
:
1712
16
Amigorena, S., C. Bonnerot, J. R. Drake, D. Choquet, W. Hunziker, J. G. Guillet, P. Webster, C. Sautes, I. Mellman, W. H. Fridman.
1992
. Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes.
Science
256
:
1808
17
Muta, T., T. Kurosaki, Z. Misulovin, M. Sanchez, M. C. Nussenzweig, J. V. Ravetch.
1994
. A 13-amino-acid motif in the cytoplasmic domain of FcγRIIB modulates B-cell receptor signalling. [Published erratum appears in 1994 Nature 369:340.].
Nature
368
:
70
18
Cambier, J. C..
1997
. Inhibitory receptors abound?.
Proc. Natl. Acad. Sci. USA
94
:
5993
19
Daeron, M., S. Latour, O. Malbec, E. Espinosa, P. Pina, S. Pasmans, W. H. Fridman.
1995
. The same tyrosine-based inhibition motif, in the intracytoplasmic domain of FcγRIIB, regulates negatively BCR-, TCR-, and FcR-dependent cell activation.
Immunity
3
:
635
20
Malbec, O., D. C. Fong, M. Turner, V. L. Tybulewicz, J. C. Cambier, W. H. Fridman, M. Daeron.
1998
. Fc epsilon receptor I-associated Lyn-dependent phosphorylation of Fcγ receptor IIB during negative regulation of mast cell activation.
J. Immunol.
160
:
1647
21
Chan, V. W., C. A. Lowell, A. L. DeFranco.
1998
. Defective negative regulation of antigen receptor signaling in Lyn-deficient B lymphocytes.
Curr. Biol.
8
:
545
22
D’Ambrosio, D., K. L. Hippen, S. A. Minskoff, I. Mellman, G. Pani, K. A. Siminovitch, J. C. Cambier.
1995
. Recruitment and activation of PTP1C in negative regulation of antigen receptor signaling by FcγRIIB1.
Science
268
:
293
23
D’Ambrosio, D., D. C. Fong, J. C. Cambier.
1996
. The SHIP phosphatase becomes associated with FcγRIIB1 and is tyrosine phosphorylated during ‘negative’ signaling.
Immunol. Lett.
54
:
77
24
Ono, M., S. Bolland, P. Tempst, J. V. Ravetch.
1996
. Role of the inositol phosphatase SHIP in negative regulation of the immune system by the receptor Fc(γ)RIIB.
Nature
383
:
263
25
Sato, K., A. Ochi.
1998
. Superclustering of B cell receptor and FcγRIIB1 activates Src homology 2-containing protein tyrosine phosphatase-1.
J. Immunol.
161
:
2716
26
Fong, D. C., O. Malbec, M. Arock, J. C. Cambier, W. H. Fridman, M. Daeron.
1996
. Selective in vivo recruitment of the phosphatidylinositol phosphatase SHIP by phosphorylated FcγRIIB during negative regulation of IgE-dependent mouse mast cell activation.
Immunol. Lett.
54
:
83
27
Vely, F., S. Olivero, L. Olcese, A. Moretta, J. E. Damen, L. Liu, G. Krystal, J. C. Cambier, M. Daeron, E. Vivier.
1997
. Differential association of phosphatases with hematopoietic co-receptors bearing immunoreceptor tyrosine-based inhibition motifs.
Eur. J. Immunol.
27
:
1994
28
Famiglietti, S. J., K. Nakamura, J. C. Cambier.
2000
. Unique features of SHIP, SHP-1 and SHP-2 binding to FcγRIIb revealed by surface plasmon resonance.
Immunol. Lett.
68
:
35
29
Ono, M., H. Okada, S. Bolland, S. Yanagi, T. Kurosaki, J. V. Ravetch.
1997
. Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling.
Cell
90
:
293
30
Chacko, G. W., S. Tridandapani, J. E. Damen, L. Liu, G. Krystal, K. M. Coggeshall.
1996
. Negative signaling in B lymphocytes induces tyrosine phosphorylation of the 145-kDa inositol polyphosphate 5-phosphatase, SHIP.
J. Immunol.
157
:
2234
31
Fluckiger, A. C., Z. Li, R. M. Kato, M. I. Wahl, H. D. Ochs, R. Longnecker, J. P. Kinet, O. N. Witte, A. M. Scharenberg, D. J. Rawlings.
1998
. Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation.
EMBO J.
17
:
1973
32
Takata, M., T. Kurosaki.
1996
. A role for Bruton’s tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-γ2.
J. Exp. Med.
184
:
31
33
Falasca, M., S. K. Logan, V. P. Lehto, G. Baccante, M. A. Lemmon, J. Schlessinger.
1998
. Activation of phospholipase Cγ by PI 3-kinase-induced PH domain-mediated membrane targeting.
EMBO J.
17
:
414
34
Li, T., D. J. Rawlings, H. Park, R. M. Kato, O. N. Witte, A. B. Satterthwaite.
1997
. Constitutive membrane association potentiates activation of Bruton tyrosine kinase.
Oncogene
15
:
1375
35
Tamir, I., J. C. Stolpa, C. D. Helgason, K. Nakamura, P. Bruhns, M. Daeron, J. C. Cambier.
2000
. The RasGAP-binding protein p62dok is a mediator of inhibitory FcγRIIB signals in B cells.
Immunity
12
:
347
36
Yamanashi, Y., T. Tamura, T. Kanamori, H. Yamane, H. Nariuchi, T. Yamamoto, D. Baltimore.
2000
. Role of the rasGAP-associated docking protein p62(dok) in negative regulation of B cell receptor-mediated signaling.
Genes Dev.
14
:
11
37
Buhl, A. M., J. C. Cambier.
1999
. Phosphorylation of CD19 Y484 and Y515, and linked activation of phosphatidylinositol 3-kinase, are required for B cell antigen receptor-mediated activation of Bruton’s tyrosine kinase.
J. Immunol.
162
:
4438
38
Pearse, R. N., T. Kawabe, S. Bolland, R. Guinamard, T. Kurosaki, J. V. Ravetch.
1999
. SHIP recruitment attenuates FcγRIIB-induced B cell apoptosis.
Immunity
10
:
753
39
Jones, B., J. P. Tite, C. A. Janeway, Jr.
1986
. Different phenotypic variants of the mouse B cell tumor A20/2J are selected by antigen- and mitogen-triggered cytotoxicity of L3T4-positive, I-A-restricted T cell clones.
J. Immunol.
136
:
348
40
Nakamura, K., J. C. Cambier.
1998
. B cell antigen receptor (BCR)-mediated formation of a SHP-2-pp120 complex and its inhibition by FcγRIIB1-BCR coligation.
J. Immunol.
161
:
684
41
Gold, M. R., R. Aebersold.
1994
. Both phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase products are increased by antigen receptor signaling in B cells.
J. Immunol.
152
:
42
42
Damen, J. E., L. Liu, M. D. Ware, M. Ermolaeva, P. W. Majerus, G. Krystal.
1998
. Multiple forms of the SH2-containing inositol phosphatase, SHIP, are generated by C-terminal truncation.
Blood
92
:
1199
43
Burshtyn, D. N., W. Yang, T. Yi, E. O. Long.
1997
. A novel phosphotyrosine motif with a critical amino acid at position −2 for the SH2 domain-mediated activation of the tyrosine phosphatase SHP-1.
J. Biol. Chem.
272
:
13066
44
Gupta, N., A. M. Scharenberg, D. A. Fruman, L. C. Cantley, J. P. Kinet, E. O. Long.
1999
. The SH2 domain-containing inositol 5′-phosphatase (SHIP) recruits the p85 subunit of phosphoinositide 3-kinase during FcγRIIb1-mediated inhibition of B cell receptor signaling.
J. Biol. Chem.
274
:
7489
45
Songyang, Z., S. E. Shoelson, J. McGlade, P. Olivier, T. Pawson, X. R. Bustelo, M. Barbacid, H. Sabe, H. Hanafusa, T. Yi, et al
1994
. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav.
Mol. Cell. Biol.
14
:
2777
46
Osborne, M. A., G. Zenner, M. Lubinus, X. Zhang, Z. Songyang, L. C. Cantley, P. Majerus, P. Burn, J. P. Kochan.
1996
. The inositol 5′-phosphatase SHIP binds to immunoreceptor signaling motifs and responds to high affinity IgE receptor aggregation.
J. Biol. Chem.
271
:
29271
47
Aman, M. J., S. F. Walk, M. E. March, H. P. Su, D. J. Carver, K. S. Ravichandran.
2000
. Essential role for the C-terminal noncatalytic region of SHIP in FcγRIIB1-mediated inhibitory signaling.
Mol. Cell. Biol.
20
:
3576
48
Nadler, M. J. S., B. Chen, J. S. Anderson, H. H. Wortis, B. G. Neel.
1997
. Protein-tyrosine phosphatase SHP-1 is dispensable for FcγRIIB-mediated inhibition of B cell antigen receptor activation.
J. Biol. Chem.
272
:
20038