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Originally published In Press as doi:10.1074/jbc.M410261200 on September 28, 2004

J. Biol. Chem., Vol. 279, Issue 50, 51931-51938, December 10, 2004
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Two Distinct Tyrosine-based Motifs Enable the Inhibitory Receptor Fc{gamma}RIIB to Cooperatively Recruit the Inositol Phosphatases SHIP1/2 and the Adapters Grb2/Grap*{boxs}

Isabelle Isnardi{ddagger}§, Renaud Lesourne{ddagger}§||, Pierre Bruhns§, Wolf H. Fridman{ddagger}, John C. Cambier**, and Marc Daëron{ddagger}§{ddagger}{ddagger}

From the {ddagger}Laboratoire d'Immunologie Cellulaire et Clinique, INSERM U255, Institut de Recherches Biomédicales des Cordeliers, 75006 Paris, France, the **Department of Immunology, University of Colorado Health Sciences Center and National Jewish Medical and Research Center, Denver, Colorado 80262, and the §Unité d'Allergologie Moléculaire et Cellulaire, Institut Pasteur, 75015 Paris, France

Received for publication, September 7, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fc{gamma}RIIB are low-affinity receptors for IgG that contain an immunoreceptor tyrosine-based inhibition motif (ITIM) and inhibit immunoreceptor tyrosine-based activation motif (ITAM)-dependent cell activation. When coaggregated with ITAM-bearing receptors, Fc{gamma}RIIB become tyrosyl-phosphorylated and recruit the Src homology 2 (SH2) domain-containing inositol 5'-phosphatases SHIP1 and SHIP2, which mediate inhibition. The Fc{gamma}RIIB ITIM was proposed to be necessary and sufficient for recruiting SHIP1/2. We show here that a second tyrosine-containing motif in the intracytoplasmic domain of Fc{gamma}RIIB is required for SHIP1/2 to be coprecipitated with the receptor. This motif functions as a docking site for the SH2 domain-containing adapters Grb2 and Grap. These adapters interact via their C-terminal SH3 domain with SHIP1/2 to form a stable receptor-phosphatase-adapter trimolecular complex. Both Grb2 and Grap are required for an optimal coprecipitation of SHIP with Fc{gamma}RIIB, but one adapter is sufficient for the phosphatase to coprecipitate in a detectable manner with the receptors. In addition to facilitating the recruitment of SHIPs, the second tyrosine-based motif may confer upon Fc{gamma}RIIB the properties of scaffold proteins capable of altering the composition and stability of the signaling complexes generated following receptor engagement.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fc{gamma}RIIB are low affinity receptors for the Fc portion of IgG antibodies that are widely expressed by cells of hematopoietic origin (1). Their low affinity enables them to remain free in the presence of high concentrations of circulating IgGs and to bind immune complexes with a high avidity. Fc{gamma}RIIB are unique among Fc receptors in exhibiting inhibitory properties. Indeed, Fc{gamma}RIIB were demonstrated to negatively regulate cell activation triggered by other Fc receptors in mast cells (2), B cell receptors (BCRs)1 for antigen in B cells (3, 4), and T cell receptors for antigen in T cells (5), i.e. by all receptors containing immunoreceptor tyrosine-based activation motifs. Fc{gamma}RIIB must be coaggregated with activating receptors via IgG immune complexes in order to exert their inhibitory effects (2). The in vivo relevance of the regulatory properties of Fc{gamma}RIIB was ascertained in Fc{gamma}RIIB-deficient mice. Fc{gamma}RIIB–/– mice were shown to mount enhanced antibody responses (6), exhibit enhanced IgG- and IgE-induced anaphylactic reactions (7), be hypersensitive to collagen-induced arthritis (8, 9), and develop spontaneous systemic lupus erythematosus in the C57BL/6 background (10). Fc{gamma}RIIB are therefore likely to play major roles in the prevention of autoimmune diseases, allergies, and other inflammatory diseases.

The regulatory properties of Fc{gamma}RIIB were shown to depend on the presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its intracytoplasmic domain. This motif was defined as a tyrosine residue that is followed at position Tyr + 3 and preceded at position Tyr – 2 by hydrophobic amino acids (11). The ITIM tyrosine becomes phosphorylated by a Src family protein tyrosine kinase upon the coaggregation of inhibitory receptors with activating receptors (12), providing a docking site for SH2 domain-containing cytosolic phosphatases (13). Because of the presence of a leucine residue at position Tyr + 2 (14), Fc{gamma}RIIB were shown to recruit selectively the single SH2 domain-containing inositol 5'-phosphatases SHIP1 (15, 16) and/or SHIP2 (17). These phosphatases dephosphorylate 5'-phosphate groups in 3'-phosphorylated inositols and phosphatidylinositides (18, 19) among which phosphatidylinositol 3,4,5-triphosphate (20, 21), generated by phosphatidylinositol 3-kinase during cell activation, is a major substrate. Phosphatidylinositol 3,4,5-triphosphate enables the membrane translocation of cytosolic molecules possessing pleckstrin homology domains including Bruton's tyrosine kinase, phospholipase C{gamma}, and the GTP/GDP exchange factor Vav, which are essential for signaling (21). SHIP1 also functions as an adapter to recruit Dok-1, which itself recruits RasGAP, which inhibits Ras activation (22). Taken together, these results suggest that the primary function of Fc{gamma}RIIB is to recruit SHIP1 (and SHIP2), which dampen positive signaling. Indeed, Fc{gamma}RIIB-dependent inhibition of cell activation was abrogated in mast cells (23) and markedly inhibited in B cells (24) from SHIP1–/– mice, and inhibition of cell activation could be induced by Fc{gamma}RIIB, the intracytoplasmic domain of which was replaced by the catalytic domain of SHIP1 (25, 26). Likewise SHIP2, which is inducibly expressed in lipopolysaccharide-activated B cells, may contribute to Fc{gamma}RIIB-dependent negative regulation of the BCR-induced activation of these cells (27).

There is a general consensus that the Fc{gamma}RIIB ITIM is both necessary and sufficient for inhibition. The conclusion that it is necessary was based on the pioneer work by Amigorena et al. who showed that a 13-amino acid deletion, which was later understood to encompass the ITIM, abrogated inhibition in B cells (4). A point mutation of the ITIM tyrosine also abrogated Fc{gamma}RIIB-dependent inhibition of mast cell and T cell activation (5) and abolished (28) or reduced (29) the calcium response in B cells. The conclusion that the ITIM is sufficient was based on works by Muta et al., who showed that a chimeric molecule whose intracytoplasmic domain contained the murine Fc{gamma}RIIB ITIM retained inhibitory properties in B cells (28). More recently however, we found that a C-terminal deletion of the intracytoplasmic domain of Fc{gamma}RIIB, which left the ITIM intact, prevented SHIP1 from being coprecipitated in a detectable manner and reduced the inhibitory effect of Fc{gamma}RIIB on BCR signaling (29). We show here that this C-terminal sequence contains a second tyrosine-based motif that mediates the recruitment, via their SH2 domain, of the adapter proteins Grb2 and Grap, which interact, via their C-terminal SH3 domain, with SHIP1 and SHIP2, thus stabilizing the binding of these phosphatases to the Fc{gamma}RIIB ITIM. Supporting a critical role of this trimolecular complex in vivo, we provide evidence that adapters are necessary for Fc{gamma}RIIB to recruit phosphatases.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells—IIA1.6 cells were cultured in RPMI medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 0.5 µM 2-mercaptoethanol, and 2 mM sodium pyruvate. DT40 cells (30) purchased from the RIKEN cell bank (Tsukuba Science City, Japan), were cultured in RPMI medium supplemented with 10% fetal calf serum, 1% chicken serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 0.5 µM 2-mercaptoethanol. Culture reagents were from Invitrogen.

Antibodies—The rat anti-mouse Fc{gamma}RIIB 2.4G2 monoclonal antibody (31) was purified on protein G-Sepharose. F(ab')2 fragments and the IgG of polyclonal rat anti-mouse Ig (RatAM), F(ab')2 fragments and IgG of polyclonal rabbit anti-mouse Ig (RabAM), the IgG of polyclonal rabbit anti-chicken Ig (RabAC), FITC-labeled mouse anti-rat Ig (MARat) F(ab')2 and FITC-labeled goat anti-rabbit Ig (GARab) F(ab')2 were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA); rabbit anti-phospho-Akt, anti-Akt, anti-phospho-ERK and anti-ERK antibodies were from New England Biolabs (Beverly, MA); mouse anti-Grb2 antibodies came from BD Transduction Laboratories; mouse antiphosphotyrosine monoclonal antibodies (4G10) and rabbit anti-SHIP1, anti-Nck, and anti-Nck{beta} antibodies were obtained from Upstate Biotechnology (Lake Placid, NY); and rabbit anti-Grb2 and anti-CrkL antibodies and horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse Ig antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antibodies against recombinant extracellular domains of Fc{gamma}RIIB and mouse anti-GST antibodies were kind gifts from Prof. C. Sautès-Fridman and Dr. J.-L. Teillaud (INSERM U255, Paris, France), respectively. Rabbit anti-SHIP2 antibodies were a gift from Dr. D. Wisniewski (Memorial Sloan-Kettering Cancer Center, New York, NY). Rabbit anti-Grap antibodies were a gift from Dr. G.-S. Feng (The Burnham Institute, La Jolla, CA). Rabbit anti-SHIP1 antibodies used in DT40 cells were a gift from Dr. J. V. Ravetch (The Rockefeller University, New York, NY).

cDNA Constructs—The cDNA of mouse Fc{gamma}RIIB was modified by a point mutation of the codon coding for Val-212 (GTT -> GTA), which induced no amino acid change but created a KpnI restriction site. This cDNA encoding the entire extracellular and transmembrane domains and the first six intracytoplasmic amino acids of Fc{gamma}RIIB was inserted into an expression vector under the control of the SR{alpha} promoter in pBR322 (32) and in which a neomycin resistance gene was introduced. The cDNA encoding the mutated intracytoplasmic domain of Fc{gamma}RIIB1 Y326F was amplified using the primers 5'-AAGAAAAAGCAGGTACCAGCTCTCCCA-3' and 5'-CGAGCTCAAATGTGGAACTGAAAATCATGCTCTGTTTCTTC-3'. This cDNA was then fused to the cDNA encoding the extracellular and transmembrane domains of Fc{gamma}RIIB.

Transfectants—cDNAs were stably transfected in IIA1.6 and DT40 cells by electroporation. Transfectants were selected and cloned as described (33–35). The expression of receptors on clones remained stable. Several clones of each transfectant were used and gave similar results.

Indirect Immunofluorescence—To measure the expression of Fc{gamma}RIIB, cells were incubated with or without 10 µg/ml 2.4G2, washed, and stained with 50 µg/ml FITC-labeled MARat F(ab')2. To measure the expression of the BCR, cells were incubated with or without 10 µg/ml RabAC, washed, and stained with 50 µg/ml FITC-labeled GARab F(ab')2. Fluorescence was analyzed with the FACScalibur system (BD Biosciences).

Flow Cytometric Analysis of Calcium Mobilization—Intracellular free calcium concentration was determined by preloading 1 x 106 IIA1.6 cells with 5 mM Fluo-3 AM (Molecular Probes, Eugene, OR) in the presence of 0.2% Pluronic F-127 (Sigma) for 30 min at room temperature. Cells were washed three times in RPMI medium, resuspended at 1 x 106 cells/ml in complete medium, and intracellular free calcium concentration was monitored with a flow cytometer. After 3 min at 37 °C, IIA1.6 cells were stimulated with 45 µg/ml RabAM IgG or 30 µg/ml RabAM F(ab'), and [Ca2+2]i was measured. The mean [Ca2+]i was evaluated with FCS Assistant 1.2.9 {beta} software (BD Biosciences).

Western Blot Analysis—Material was boiled in sample buffer, fractionated by SDS-PAGE, and transferred onto Immobilon-P membranes (Millipore, Bedford, MA). Membranes were saturated with either 5% bovine serum albumin or 5% skimmed milk (Régilait, Saint-Martin-Belle-Roche, France), diluted in Western buffer (150 mM NaCl, 10 mM Tris, and 0.5% Tween 20 (Merck), pH 7.4), and incubated with the indicated antibodies followed by horseradish peroxidase-goat anti-rabbit or horseradish peroxidase-goat anti-mouse. Labeled antibodies were detected using an ECL kit (Amersham Biosciences).

Whole Cell Lysate Analysis—IIA1.6 transfectants were stimulated at 37 °C for the indicated times (Fig. 2) with 30 µg/ml intact RatAM IgG or 20 µg/ml F(ab')2 fragments of RatAM IgG and lysed by three cycles of incubation for 1 min in liquid nitrogen followed by 1 min at 37 °C in pH 8 lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 5 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride). Proteins were quantitated using a Bio-Rad protein assay, and 40 µg of proteins were treated as described in Western blot analysis.



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  		FIG. 2.
The C-terminal tyrosine (Tyr-326) of the intracytoplasmic domain of Fc{gamma}RIIB1 is mandatory for the abrogation of ERK phosphorylation and the complete inhibition of Akt phosphorylation. A, ERK phosphorylation following coaggregation of BCR with wt and mutated Fc{gamma}RIIB in IIA1.6 transfectants. Cells were unstimulated (–) or stimulated (+) with either RatAM F(ab')2 or RatAM IgG for the indicated periods of time. Cells were lysed, and proteins were fractionated by SDS-PAGE and Western blotted (WB) with anti-pERK or anti-ERK antibodies. B, Akt phosphorylation following coaggregation of BCR with wt and mutated Fc{gamma}RIIB in IIA1.6 transfectants. Cells were unstimulated (–) or stimulated (+) with either RatAM F(ab')2 or RatAM IgG for the indicated periods of time. Cells were lysed, and proteins were fractionated by SDS-PAGE and Western blotted (WB) with anti-pAkt or anti-Akt antibodies.

 
Immunoprecipitation—IIA1.6 transfectants were stimulated at 37 °C for 3 min with 30 µg/ml intact RatAM, 20 µg/ml F(ab')2 fragments of RatAM, or RabAM or treated with 100 mM pervanadate. Pervanadate was generated by mixing 1 ml of 20 mM Na3VO4 with 330 ml of 30% H2O2 followed by a 5-min incubation at room temperature, yielding a solution of 6 mM pervanadate. DT40 transfectants were stimulated at 37 °C for 0.5, 1, or 3 min with 30 µg/ml RabAC IgG. Cells were lysed in pH 7.4 lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 5 mM NaF, 5 mM sodium pyrophosphate, 0.4 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin and 1 mM phenylmethylsulfonyl fluoride). Postnuclear lysates were immunoprecipitated with 2.4G2-coated Sepharose beads or protein A-Sepharose beads (Amersham Biosciences) coupled to rabbit anti-Grb2 or anti-Grap antibodies. Eluates from immunoadsorbents were treated as described above under the subheading "Western Blot Analysis."

GST Fusion Proteins—cDNA encoding the SH2 domain of SHIP1 was amplified by PCR using as a template cDNA generated from RNA extracted from RBL-2H3 cells. cDNA encoding a Grap-containing GST fusion protein was a gift from Dr. S.E. Shoelson (Joslin Diabetes Center, Boston, MA). GST-SHIP1 SH2 and GST-Grap cDNAs were inserted in pGEX-4T-2 (Amersham Biosciences) and transfected into DH5-{alpha} Escherichia coli. Bacteria producing GST-Grb2 SH2 were a gift from Dr. I. Broutin (UMR 8015 CNRS, Paris, France). Bacteria producing GST-Grb2 were a gift from Dr. S. Latour (INSERM U429, Paris, France). All fusion proteins were produced in DH5-{alpha} E. coli following isopropyl-1-thio-{beta}-D-galactopyranoside induction, purified on glutathione-agarose beads (Sigma) and analyzed by SDS-PAGE. Soluble fusion proteins were eluted from glutathione-agarose beads with a solution of 50 mM Tris and 25 mM glutathione, pH 8. The fusion proteins used in Fig. 3C were purchased from Santa Cruz Biotechnology. GST fusion protein beads were incubated for 2 h in lysates from 1 x 107 cells. Eluates from beads were treated as described above under the subheading "Western Blot Analysis."



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  		FIG. 3.
The 16 C-terminal amino acids of the intracytoplasmic domain of Fc{gamma}RIIB1 bind in vitro to Grb2 and Grap, which coprecipitate with phosphorylated Fc{gamma}RIIB1 in vivo. A, in vitro binding of adapters to Fc{gamma}RIIB C-ter peptides. Agarose beads coated with C-ter (nonphosphorylated) or pC-ter (phosphorylated) peptides corresponding to the 16 C-terminal amino acids of Fc{gamma}RIIB were incubated with IIA1.6 cell lysate. Precipitated material was fractionated by SDS-PAGE and Western blotted with anti-SHIP1, anti-Nck{beta}, anti-Nck, anti-CrkL, anti-Grap, and anti-Grb2 antibodies. Whole cell lysate (WCL) was used as a positive control. B, in vitro binding of adapter-containing GST fusion proteins to Fc{gamma}RIIB C-ter peptides. Agarose beads coated with pC-ter or C-ter were incubated with GST-Grap or GST-Grb2. Precipitated material was fractionated by SDS-PAGE and Western blotted (WB) with anti-GST antibodies. C, in vitro binding of SH2 domain-containing GST fusion proteins to Fc{gamma}RIIB C-ter and ITIM peptides. Agarose beads coated with nonphosphorylated or phosphorylated ITIM or C-ter peptides were incubated with GST-SHIP1 SH2 or GST-Grb2 SH2. Precipitated material was fractionated by SDS-PAGE and Western blotted (WB) with anti-GST antibodies. D, coprecipitation of adapters with wt Fc{gamma}RIIB1 in IIA1.6 transfectants. Cells were stimulated (+) or not stimulated (–) with RatAM IgG or treated with pervanadate for 3 min. Cells were lysed, and Fc{gamma}RIIB were precipitated with 2.4G2. Immunoprecipitates (IP) were fractionated by SDS-PAGE and Western blotted (WB) with anti-Fc{gamma}RIIB, anti-pTyr, anti-SHIP1, anti-Grb2 and anti-Grap antibodies. WCL, whole cell lysate.

 
Peptides and in Vitro Binding of Proteins—Biotinylated peptides corresponding to the Fc{gamma}RIIB ITIM, either phosphorylated (pITIM) or not phosphorylated (ITIM), and to the Fc{gamma}RIIB 16 C-terminal amino acids, either phosphorylated (pC-ter) or not phosphorylated (C-ter), were purchased from Sigma-Genosys (The Woodlands, TX). The peptides were coupled to streptavidin-agarose beads. Beads were incubated for 2 h with lysates from 1 x 107 IIA1.6 cells or 2 x 107 DT40 cells or with soluble GST fusion proteins. Eluates from beads were treated as described under the subheading "Western Blot Analysis."


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The ITIM Is Necessary but Not Sufficient for Fc{gamma}RIIB to Recruit SHIP1—Because we reported previously that a deletion of the 16 C-terminal amino acids of the Fc{gamma}RIIB1 intracytoplasmic domain (Fc{gamma}RIIB1 {Delta}314) abolished the coprecipitation of SHIP1 with the receptor (29) and because the deleted sequence contains a tyrosine residue, we examined the respective contributions of the ITIM tyrosine and the C-terminal tyrosine of Fc{gamma}RIIB in the recruitment of SHIP1. Fc{gamma}RIIB1 bearing a point mutation of either the ITIM tyrosine (Fc{gamma}RIIB1 Y309G) or the C-terminal tyrosine (Fc{gamma}RIIB1 Y326F) were stably expressed in the Fc{gamma}R-negative variant of the murine B lymphoma A20/2J, IIA1.6. IIA1.6 transfectants expressing wild-type (wt) Fc{gamma}RIIB1 were used as positive controls (Fig. 1A). Wild-type and mutant Fc{gamma}RIIB1 were coaggregated with BCR using intact RabAM IgG antibodies that can bind both to BCR via their Fab portions and to Fc{gamma}RIIB1 via their Fc portion. Fc{gamma}RIIB1 were immunoprecipitated, and immunoprecipitates were Western blotted with anti-Fc{gamma}RIIB, anti-phosphotyrosine, and anti-SHIP1 antibodies. Wild-type and mutant Fc{gamma}RIIB1 became tyrosyl-phosphorylated following coaggregation with BCR. Compared with wt Fc{gamma}RIIB1, Fc{gamma}RIIB1 mutants were less phosphorylated. The coprecipitation of SHIP1 with phosphorylated wt Fc{gamma}RIIB1 was lost not only in cells expressing Fc{gamma}RIIB1 Y309G, as expected, but also in cells expressing Fc{gamma}RIIB1 Y326F, although the ITIM remained intact in this mutant (Fig. 1B). As observed previously (29), the coprecipitation of SHIP1 and SHIP2 was also lost in cells expressing Fc{gamma}RIIB1 {Delta}314 (supplemental Fig. S1, found in the on-line version of this article).



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  		FIG. 1.
The C-terminal tyrosine (Tyr-326) of the intracytoplasmic domain of Fc{gamma}RIIB1 is mandatory for SHIP1 coprecipitation. A, schematic representation of the Fc{gamma}RIIB mutants and peptides used in this study. Amino acid sequence of the ITIM and C-ter peptides are indicated in black and white, respectively. Histograms show the expression of wt and mutated Fc{gamma}RIIB1 assessed by indirect immunofluorescence. Bold histograms, 2.4G2 and FITC-mouse anti-rat F(ab')2; thin histograms, FITC-mouse anti-rat F(ab')2 only. EC, extracellular; TM, transmembrane; IC, intracellular. B, coprecipitation of SHIP1 with wt and mutated Fc{gamma}RIIB1 in IIA1.6 transfectants. Cells were stimulated (+) or not stimulated (–) with R2AM IgG for 3 min. Cells were lysed, and Fc{gamma}RIIB1 were precipitated with 2.4G2. Immunoprecipitates (IP) were fractionated by SDS-PAGE and Western blotted (WB) with anti-Fc{gamma}RIIB, anti-pTyr, and anti-SHIP1 antibodies.

 
This loss of a detectable coprecipitation of SHIP1 with Fc{gamma}RIIB1 Y326F was correlated with a loss of inhibition of ERK activation. ERK phosphorylation, induced upon BCR aggregation, was indeed decreased upon the coaggregation of BCR with wt Fc{gamma}RIIB1 but not upon the coaggregation of BCR with either Fc{gamma}RIIB1 Y309G or Fc{gamma}RIIB1 Y326F (Fig. 2A).

Akt phosphorylation, which was induced upon BCR aggregation, was abolished upon the coaggregation of BCR with wt Fc{gamma}RIIB1 and was partially inhibited upon the coaggregation of BCR with Fc{gamma}RIIB1 Y309G, Fc{gamma}RIIB1 Y326F (Fig. 2B), or Fc{gamma}RIIB1 {Delta}314 (supplemental Fig. S2A, available in the on-line version of this article). To understand how Fc{gamma}RIIB1 mutants could still inhibit Akt activation to some extent, we analyzed the colocalization of SHIP1 with wt or mutant Fc{gamma}RIIB1 by confocal microscopy (supplemental Fig. S2, B and C). As observed previously (36), SHIP1 colocalized with BCR-wt Fc{gamma}RIIB1 coaggregates in >80% of the cells. The colocalization of SHIP1 with either BCR-Fc{gamma}RIIB1 {Delta}314 or BCR-Fc{gamma}RIIB1 Y309G coaggregates was reduced but could still be observed in 40–50% of the cells. Altogether, these data indicate that the tyrosine contained in the C-terminal sequence of Fc{gamma}RIIB1 contributes to the recruitment of SHIP1 and to Fc{gamma}RIIB-dependent inhibition of ERK and Akt activation.

Fc{gamma}RIIB Contain a Second Tyrosine-based Motif That Binds the Adapters Grb2/Grap in Vitro, and Fc{gamma}RIIB Recruit These Adapters in Vivo—The C-terminal tyrosine of Fc{gamma}RIIB is within a consensus Grb2-binding site. Indeed, phosphorylated peptides corresponding to the 16 C-terminal amino acids of Fc{gamma}RIIB that were deleted in Fc{gamma}RIIB1 {Delta}314 (pC-ter), but not the same non-phosphorylated peptides (C-ter), precipitated Grb2 and Grap from a IIA1.6 cell lysate but not the related adapters Nck, Nck{beta}, or CrkL, which were all present in the lysate (Fig. 3A). pC-ter, but not C-ter, bound to GST fusion proteins containing Grb2 or Grap (Fig. 3B). Finally, pC-ter, but not C-ter, also bound to a GST fusion protein containing the SH2 domain of Grb2. This GST-Grb2 SH2 fusion protein failed to bind to a phosphorylated peptide corresponding to the Fc{gamma}RIIB ITIM (pITIM). Conversely, a GST fusion protein containing the SH2 domain of SHIP1 bound to pITIM but not to pC-ter (Fig. 3C). These data indicate that pC-ter can bind to the adapters Grb2 and Grap but not to SHIP1. In vitro binding results from a direct interaction of pC-ter with the two adapters and, at least for Grb2, this interaction is via its SH2 domain. Conversely, pITIM can bind to SHIP1 (and SHIP2) (14) but not to adapter molecules.

Based on the above in vitro results, we investigated whether adapter molecules would coprecipitate with phosphorylated Fc{gamma}RIIB in IIA1.6 cells. Fc{gamma}RIIB1 phosphorylation was induced either by coaggregating the receptors with BCR using RatAM IgG antibodies or by treating cells with pervanadate. The coprecipitation of SHIP1 varied with the intensity of Fc{gamma}RIIB1 phosphorylation. Neither Grb2 nor Grap coprecipitated with Fc{gamma}RIIB1 in untreated cells. Grb2, but not Grap, coprecipitated with Fc{gamma}RIIB1 in a detectable manner following coaggregation with BCR. Both Grb2 and Grap coprecipitated with Fc{gamma}RIIB1 following pervanadate treatment (Fig. 3D). Phosphorylated Fc{gamma}RIIB1 therefore recruits the adapters Grb2 and Grap in vivo.

Grb2 and Grap Interact with SHIP1/2 in Vitro and in Vivo— Although pC-ter and pITIM bound specifically to the SH2 domains of Grb2 and SHIP1, respectively (Fig. 3C), pITIM precipitated not only SHIP1 but also Grb2 when incubated with the IIA1.6 cell lysate (Fig. 4A). Grb2 was previously reported to bind SHIP1 (18) but not SHIP2 via its C-terminal SH3 domain (37). However, GST-Grb2 precipitated both SHIP1 and SHIP2 from the IIA1.6 cell lysate (Fig. 4B). We therefore analyzed the binding of SHIP1 and SHIP2 to the three domains of Grb2 (SH3-N, SH2, and SH3-C) separately. Neither GST-SH2 nor GST-SH3-N precipitated SHIP1 or SHIP2 in a detectable manner (although GST-SH3-N precipitated Sos), whereas GST-SH3-C precipitated SHIP1 and SHIP2, as did GST-Grb2 (Fig. 4B). The in vitro interactions of Grb2 with both SHIP1 and SHIP2 are therefore mediated by the C-terminal SH3 domain of Grb2.



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  		FIG. 4.
Grb2 and Grap bind in vitro and in vivo to SHIP1/2. A, in vitro binding of SHIP1 and Grb2 to Fc{gamma}RIIB C-ter and ITIM peptides. Agarose beads coated with nonphosphorylated or phosphorylated peptides corresponding to the Fc{gamma}RIIB ITIM or C-ter were incubated with IIA1.6 cell lysate. Precipitated material was fractionated by SDS-PAGE and Western blotted (WB) with anti-SHIP1 and anti-Grb2 antibodies. B, in vitro binding of SHIP1/2 to fusion proteins containing Grb2 or its domains. Agarose beads coated with GST, GST-Grb2, GST-Grb2 SH3N, GST-Grb2 SH2, or GST-Grb2 SH3C were incubated with IIA1.6 cell lysate. Precipitated material was fractionated by SDS-PAGE and Western blotted (WB) with anti-SHIP2, anti-SHIP1, and anti-Sos antibodies. C, coprecipitation of SHIP1/2 with Grb2 and Grap in IIA1.6 Fc{gamma}RIIB1 transfectants. Cells were unstimulated (–) or stimulated (+) with either RatAM F(ab and ')2 or RatAM IgG for 3 min. Cells were lysed, and Grb2 Grap were precipitated with anti-Grb2 or anti-Grap antibodies, respectively. Immunoprecipitates (IP) were fractionated by SDS-PAGE and Western blotted (WB) with anti-Grb2, anti-Grap, anti-SHIP2, and anti-SHIP1 antibodies. Whole cell lysate (WCL) was used as a positive control.

 
To confirm these in vitro data, we examined the coprecipitation of SHIP1/2 with adapter proteins in IIA1.6 cells expressing wt Fc{gamma}RIIB1. As detected by Western blotting with corresponding antibodies, Grb2 but not Grap was precipitated by anti-Grb2 antibodies, whereas Grap but not Grb2 was precipitated by anti-Grap antibodies, and comparable amounts of each adapter were precipitated in all conditions. Small amounts of SHIP1 and SHIP2 coprecipitated with Grb2 in unstimulated cells. Higher amounts of both phosphatases coprecipitated with Grb2 following BCR aggregation, and even higher amounts coprecipitated following the coaggregation of BCR with Fc{gamma}RIIB1. Neither SHIP1 nor SHIP2 coprecipitated in a detectable manner with Grap in unstimulated cells. Minute amounts of SHIP2 coprecipitated with Grap following BCR aggregation, and both SHIP1 and SHIP2 coprecipitated with Grap following the coaggregation of BCR with Fc{gamma}RIIB1 (Fig. 4C). Taken together, these results indicate that the two known SH2 domain-containing inositol 5'-phosphatases SHIP1 and SHIP2 can bind in vitro to Grb2 and can associate in vivo with Grb2 and Grap in B cells.

Two Tyrosine-based Motifs Are Required for Fc{gamma}RIIB to Recruit Either SHIP1 or Grb2—To determine the respective contributions of the two Fc{gamma}RIIB1 motifs in the binding of adapterphosphatase complexes, we constructed an in vitro model of the intracytoplasmic domain of Fc{gamma}RIIB. The C-ter peptide, phosphorylated or not, and the ITIM peptide, phosphorylated or not, were mixed in variable proportions, and a constant amount of the mixture was used to coat agarose beads. These were used to precipitate SHIP1 and Grb2 from IIA1.6 cell lysate (Fig. 5A). pITIM alone, but not ITIM, precipitated SHIP1 and a small amount of Grb2. Conversely, pC-ter alone, but not C-ter, precipitated Grb2 and a small amount of SHIP1. The amount of SHIP1 precipitated by pITIM-coated beads decreased when the beads were coated with decreasing amounts of pITIM and increasing amounts of C-ter (Fig. 5A, left), but not when beads were coated with decreasing amounts of pITIM and increasing amounts of pC-ter (Fig. 5A, right). Likewise, the amount of Grb2 precipitated by pC-ter-coated beads decreased when beads were coated with decreasing amounts of pC-ter and increasing amounts of ITIM (Fig. 5A, middle panel), but it increased when beads were coated with decreasing amounts of pC-ter and increasing amounts of pITIM (Fig. 5A, right). These results indicate that, when present on the same beads, pC-ter could enhance the in vitro binding of SHIP1 to pITIM and that, conversely, pITIM could enhance the in vitro binding of Grb2 to pC-ter.



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  		FIG. 5.
The two tyrosine-based motifs cooperate to bind SHIP1 and Grb2 in vitro and are required for Fc{gamma}RIIB to recruit either SHIP1 or Grb2 in vivo. A, in vitro binding of SHIP1 and Grb2 to beads coated with a mixture of Fc{gamma}RIIB C-ter and ITIM peptides. Four micrograms of nonphosphorylated or phosphorylated Fc{gamma}RIIB C-ter and ITIM peptides were mixed in variable proportions and bound to agarose beads. Beads were incubated in IIA1.6 cell lysate. Precipitated material was fractionated by SDS-PAGE and Western blotted (WB) with anti-SHIP1 and anti-Grb2 antibodies. B, coprecipitation of Grb2 with wt and mutated Fc{gamma}RIIB1 in IIA1.6 transfectants. Cells were stimulated (+) or not stimulated (–) with RabAM IgG for 3 min. Cells were lysed, and Fc{gamma}RIIBs were precipitated with 2.4G2. Immunoprecipitates (IP) were fractionated by SDS-PAGE and Western blotted (WB) with anti-Fc{gamma}RIIB, anti-pTyr, anti-SHIP1, and anti-Grb2 antibodies.

 
To validate these in vitro observations in vivo, we examined whether the Fc{gamma}RIIB ITIM contributes to the recruitment of Grb2 as the Fc{gamma}RIIB C-terminal motif does for the recruitment of SHIP1 following the coaggregation of Fc{gamma}RIIB1 with BCR in IIA1.6 transfectants. Both Grb2 and SHIP1 failed to coprecipitate, not only with phosphorylated Fc{gamma}RIIB1 Y326F but also with phosphorylated Fc{gamma}RIIB1 Y309G (Fig. 5B). Both the ITIM and the C-terminal motifs are therefore necessary for Fc{gamma}RIIB to cooperatively recruit SHIP1 and Grb2 as a phosphatase-adapter complex.

Grb2 or Grap Is Required for Fc{gamma}RIIB to Recruit SHIP—To investigate the respective roles of the two adapters Grb2 and Grap in this cooperative binding, we used the same in vitro model as in Fig. 5A with cell lysates from the DT40 chicken B cells. These were wt cells, Grb2-deficient cells, Grap-deficient cells, or Grb2- and Grap-deficient cells (30). Beads coated with pITIM and pC-ter required lower amounts of pITIM to precipitate SHIP from wt DT40 cell lysate (Fig. 6, right) than beads coated with pITIM and C-ter (Fig. 6, left). The same was observed in lysate from Grb2-deficient cells and lysate from Grap-deficient cells but not in lysate from Grb2- and Grap-deficient cells in which comparable amounts of SHIP were precipitated by beads coated with pITIM and pC-ter or with pITIM and C-ter (Fig. 6). Either Grap or Grb2 is therefore necessary and sufficient to support the binding of SHIP to the Fc{gamma}RIIB ITIM.



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  		FIG. 6.
Adapters are required for stabilizing the in vitro binding of SHIP to the Fc{gamma}RIIB pITIM. Twelve micrograms of nonphosphorylated or phosphorylated peptides corresponding to the Fc{gamma}RIIB C-ter and ITIM were mixed in variable proportions and bound to agarose beads. Beads were incubated in cell lysates from wt, Grb2–/–Grap+/+, Grb2+/+Grap–/–, or Grb2–/–Grap–/– DT40 cells. Precipitated material was fractionated by SDS-PAGE and Western blotted (WB) with anti-SHIP1 antibodies.

 
To confirm these in vitro data, wt and the three deficient DT40 cells were stably transfected with wt Fc{gamma}RIIB1 (Fig. 7A). Fc{gamma}RIIB1 were coaggregated with the DT40 BCR by RabAC IgG antibodies. In wt DT40 cells, SHIP coprecipitated with Fc{gamma}RIIB1 upon coaggregation with BCR. Coprecipitation was lost in Grb2/Grap doubly deficient cells (Fig. 7B). Coprecipitation was retained in Grb2 or Grap singly deficient cells, albeit in lower amount than in wt cells (Fig. 7C). One adapter is therefore necessary and sufficient for Fc{gamma}RIIB1 to recruit SHIP, but both are required for an optimal recruitment.



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  		FIG. 7.
Adapters are mandatory for Fc{gamma}RIIB1 to recruit SHIP in vivo. A, expression of BCR and Fc{gamma}RIIB1 in DT40 transfectants. Histograms show the expression of BCR or Fc{gamma}RIIB1 in DT40 cells as assessed by indirect immunofluorescence. Top, bold histograms, RabAC and FITC-GARab F(ab')2; thin histograms, FITC-GAR F(abab ')2 only. Bottom, bold histograms, 2.4G2 and FITC-MARat F(abab '); thin histograms, FITC-MARat F(ab')2 only. B, in vivo recruitment of SHIP by Fc{gamma}RIIB1 in wt or Grb2/Grap doubly deficient DT40 transfectants. Wild-type or Grb2–/–Grap–/– DT40 cells were stimulated or not stimulated with RabAC IgG for the indicated periods of time (in minutes). Cells were lysed, and Fc{gamma}RIIB were precipitated with 2.4G2. Immunoprecipitates (IP) were fractionated by SDS-PAGE and Western blotted (WB) with anti-Fc{gamma}RIIB, anti-pTyr, and anti-SHIP1 antibodies. C, in vivo recruitment of SHIP by Fc{gamma}RIIB1 in wt or deficient DT40 transfectants. Wild-type, Grb2–/–Grap+/+, Grb2+/+Grap–/–, or Grb2–/– Grap–/– DT40 cells were stimulated (+) or not stimulated (–) with RabAC IgG for 1 min. Cells were lysed, and Fc{gamma}RIIB were precipitated with 2.4G2. Immunoprecipitates (IP) were fractionated by SDS-PAGE and Western blotted (WB) with anti-Fc{gamma}RIIB, anti-pTyr, and anti-SHIP1 antibodies.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We show here the following: 1) that in addition to the ITIM, the intracytoplasmic domain of Fc{gamma}RIIB contains a second tyrosine-based motif that recruits the SH2 domain-containing adapters Grb2 and Grap; 2) that these adapters interact with the inositol-phosphatases SHIP1 and SHIP2 via their C-terminal SH3 domain; 3) that the two tyrosine-based motifs each contribute to the recruitment of both SHIP1 and Grb2 by Fc{gamma}RIIB; and 4) that adapters are necessary for Fc{gamma}RIIB to recruit SHIP1.

Fc{gamma}RIIB-dependent negative regulation is thought to depend on the phosphorylated ITIM. The Fc{gamma}RIIB ITIM was shown to be necessary on the basis of mutational analyses. The deletion of a 13-amino acid sequence containing the ITIM (4) or the point mutation of the ITIM tyrosine (5) was indeed sufficient to abrogate most of the inhibitory properties of Fc{gamma}RIIB1 in B cells, T cells, and mast cells. The Fc{gamma}RIIB ITIM was concluded to be sufficient for inhibition on the basis of a study showing that a chimeric molecule whose intracytoplasmic domain was constituted by residues 53–68 (VKFSRSAEPPAYQQGQ) of the human T cell receptor-{xi} subunit and residues 303–315 (AENTITYSLLKHP) containing the ITIM (in boldfaced type) of murine Fc{gamma}RIIB, linked by a two-serine spacer, could inhibit BCR-induced calcium mobilization and interleukin-2 secretion in IIA1.6 cells (28). The inhibition of BCR-mediated interleukin-2 secretion by this chimera was, however, half that induced by wt Fc{gamma}RIIB1, and the authors suggested that other sequences in Fc{gamma}RIIB1 might be required to maximize inhibition. One also notices that a T cell receptor-{xi} tyrosine residue was present in the construction in addition to the ITIM tyrosine, as well as another two proline and four serine residues that could potentially recruit cytosolic molecules. The conclusion that the Fc{gamma}RIIB ITIM is sufficient to account for the inhibitory properties of the receptor may therefore not be as firmly established as is usually accepted.

The inhibitory properties of Fc{gamma}RIIB could be accounted for by the ability of the receptors to recruit SHIP1 (23–25). A role of SHIP2 was also suggested in the Fc{gamma}RIIB-dependent negative regulation of lipopolysaccharide-activated B cells (27). We show here that cytosolic molecules other than the two phosphatases are recruited by phosphorylated Fc{gamma}RIIB1. These are the two adapter molecules, Grb2 and Grap, that bind to the C-terminal motif via their SH2 domains. The intracytoplasmic domain of Fc{gamma}RIIB therefore contains two tyrosine-based motifs that bind specifically the SH2 domain of SHIP1 and the SH2 domain of Grb2, respectively. The recruitment of phosphatases, however, required an intact adapter-binding motif and, conversely, the recruitment of adapters required an intact phosphatase-binding motif. These observations could be explained by a cooperative binding of phosphatases and adapters to Fc{gamma}RIIB1. Supporting this possibility, we found that Grb2 could interact with SHIP1 and SHIP2 via its C-terminal SH3 domain and that SHIP1 (38) and SHIP2 coprecipitated with Grb2 and Grap in IIA1.6 cells. Coprecipitation of phosphatases with adapters was enhanced following BCR aggregation and further enhanced following coaggregation of BCR with Fc{gamma}RIIB1. Because the interactions between SH3 domains and proline-rich sequences are not inducible per se, this finding suggests that phosphotyrosine-dependent interactions may stabilize phosphotyrosine-independent interactions when adapters and phosphatases are brought in proximity within signaling complexes. Conversely, phosphotyrosine-independent interactions may stabilize phosphotyrosine-dependent interactions. Using a model of the Fc{gamma}RIIB1 intracytoplasmic domain in which peptides containing the two SH2 domain-binding sites were bound to the same beads, we indeed found that, when phosphorylated, the C-terminal peptide enhanced the binding of SHIP1 to the phosphorylated ITIM peptide and that, conversely, when phosphorylated, the ITIM peptide enhanced the binding of Grb2 to the phosphorylated C-terminal peptide. This reciprocal enhancement of phosphatase and adapter binding suggests that the recruitment of SHIP1 and Grb2 by Fc{gamma}RIIB1 involves cooperative binding within a trimolecular complex composed of the phosphorylated receptor, the phosphatase, and the adapter.

This conclusion may not be restricted to the interactions of Fc{gamma}RIIB1, SHIP1, and Grb2. Indeed, molecules that contain two SH2 domains require the cooperative binding of these two domains to two sequences containing phosphorylated tyrosines in order to be recruited in vivo. Thus, the recruitment of the protein tyrosine kinases ZAP-70 and Syk (39, 40) or of the tyrosine phosphatase SHP-1 (41) requires the conservation of their two SH2 domains and the conservation of the two tyrosine residues of immunoreceptor tyrosine-based activation motifs in immunoreceptors (42) or of the two ITIMs in killer cell inhibitory receptors (33, 43) respectively. Moreover, molecules that contain a single SH2 domain were found to require the cooperation of other SH2 domain-containing molecules in order to be recruited (44). We wish therefore to propose that one SH2 domain alone may not be sufficient to enable stable interactions between signaling molecules.

Stable interactions between Fc{gamma}RIIB1 and SHIP1 can be operationally defined as enabling the coprecipitation of the phosphatase with the receptor. Based on our results, such an interaction would require the two SH2-binding motifs in Fc{gamma}RIIB1 and the adapter molecules. SHIP1/2-Grb2/Grap complexes would indeed bind to Fc{gamma}RIIB1 with a high avidity resulting from the combined affinities of the SHIP1/2 SH2 domain for the ITIM and the Grb2/Grap SH2 domain for the C-terminal motif. A stable interaction between Fc{gamma}RIIB1 and SHIP1 correlates with an optimal inhibition of B cell responses. However, Fc{gamma}RIIB1 Y309G and Fc{gamma}RIIB1 {Delta}314 retained some ability to inhibit Akt phosphorylation, although they failed to coprecipitate SHIP1. This inhibition could be explained by an unstable recruitment of SHIP1 directly to the ITIM of mutant Fc{gamma}RIIB1 {Delta}314 or indirectly to the C-terminal motif of Fc{gamma}RIIB1 Y309G via adapters. This is consistent with the partial colocalization of SHIP1 with mutant receptors observed in IIA1.6 cells. Whatever the mechanism of unstable interactions, adapters may stabilize the recruitment of single SH2 domain-containing phosphatases by Fc{gamma}RIIB and thus modulate Fc{gamma}RIIB signaling.

Because Grb2 and Grap are coexpressed in B cells (45), we examined their respective roles with Grb2- and/or Grap-deficient DT40 cells. Using the same in vitro model of the Fc{gamma}RIIB1 intracytoplasmic domain, we found a similar enhancement of the binding of SHIP1 to the phosphorylated ITIM peptide by the phosphorylated C-terminal peptide in cell lysates from wt DT40 cells as from IIA1.6 cells. This enhancement was abolished in Grb2/Grap doubly deficient cells. Importantly, the same result was observed in vivo as SHIP coprecipitated with Fc{gamma}RIIB1 in wt DT40 cells but not in Grb2/Grap doubly deficient DT40 cells. These experiments thus provide genetic evidence that adapters are necessary for Fc{gamma}RIIB to recruit SHIP. Noticeably, Grb2 and Grap could substitute for each other, i.e. the phosphorylated C-terminal peptide enhanced the binding of SHIP to the phosphorylated ITIM peptide and some SHIP coprecipitated with Fc{gamma}RIIB1 in both Grb2-and Grap-deficient cells. Grb2 and Grap were previously described as replacing each other in T cells where they couple the hematopoietic progenitor kinase-1 to phosphorylated proteins such as the linker of activation of T cells (30). Although SHIP1 could be recruited by Fc{gamma}RIIB1 in the presence of either Grb2 or Grap, the coprecipitation of SHIP with Fc{gamma}RIIB1 was of a lower magnitude in single deficient DT40 cells than in wt DT40 cells. Both adapters may therefore be required for an optimal in vivo recruitment of SHIP1 by Fc{gamma}RIIB.

Because Grb2 and Grap are each composed of one SH2 domain and two SH3 domains, when one or the other is recruited by Fc{gamma}RIIB via its SH2 domain, its C-terminal SH3 domain is engaged with SHIP1/2 but its N-terminal SH3 domain remains free to bind other proline-rich molecules. These molecules could either be sequestered from nearby signaling complexes and/or contribute to Fc{gamma}RIIB-derived signals (46). Grb2 associates with a variety of molecules via its N-terminal SH3 domain, and, interestingly, Grap associates with only some molecules among Grb2 partners (45). It was recently reported that Rasdependent T cell proliferation and IL-2 production were enhanced in Grap-deficient mice (47), suggesting that Grap itself could mediate negative regulation. When recruited by Fc{gamma}RIIB, adapters may thus reinforce inhibition. Finally, our work provides evidence that Fc{gamma}RIIB may have a more complex function than simply recruiting SHIP. Fc{gamma}RIIB indeed appear to function as scaffold proteins that modulate the composition of signaling complexes generated by immunoreceptors with which they are coengaged.


   FOOTNOTES
 
* This work was supported in part by INSERM, the Université Pierre et Marie Curie, and the Institut Pasteur. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at www.jbc.org) contains supplemental Figs. S1 and S2, which deal with the 16 C-terminal amino acids of the intracytoplasmic domain of Fc{gamma}RIIB1. Back

Supported by a fellowship from the Université Pierre et Marie Curie. Back

|| Supported by a fellowship from the Association pour la Recherche contre le Cancer. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Unité d'Allergologie Moléculaire et Cellulaire, Dépt. d'Immunologie, Inst. Pasteur, 25 Rue du Docteur Roux, 75015 Paris, France. Tel.: 33-1-4568-8642; Fax: 33-1-4061-3160; E-mail: daeron{at}pasteur.fr.

1 The abbreviations used are: BCR, B cell receptor; C-ter, Fc{gamma}RIIB 16 C-terminal amino acids; ERK, extracellular signal-regulated kinase; FITC, fluorescein isothiocyanate; GARab, goat anti-rabbit Ig; GST, glutathione S-transferase; ITIM, immunoreceptor tyrosine-based inhibition motif; MARat, mouse anti-rat Ig; pC-ter, phosphorylated C-ter; pITIM, phosphorylated ITIM; RabAC, rabbit anti-chicken Ig; RabAM, rabbit anti-mouse Ig; RatAM, rat anti-mouse Ig; SH, Src homology; SHIP, SH2 domain-containing inositol 5'-phosphatase; wt, wild-type. Back


   ACKNOWLEDGMENTS
 
We thank Prof. C. Sautès-Fridman and Dr. J.-L. Teillaud (INSERM U255, Paris, France) for anti-Fc{gamma}RIIB antibodies and anti-GST antibodies, respectively, Dr. D. Wisniewski (Memorial Sloan-Kettering Cancer Center, New York, NY) for anti-SHIP2 antibodies, Dr. G.-S. Feng (The Burnham Institute, La Jolla, CA) for anti-Grap antibodies, Dr. J. V. Ravetch (The Rockefeller University, New York, NY) for the anti-SHIP1 antibodies used for DT40 cells, Dr. S. E. Shoelson (Joslin Diabetes Center, Boston, MA) for cDNA encoding GST-Grap, Dr. I. Broutin (CNRS UMR 8015, Paris, France) for GST-Grb2 SH2, and Dr. S. Latour (INSERM U429, Paris, France) for GST-Grb2.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Daëron, M. (1997) Annu. Rev. Immunol. 15, 203–234[CrossRef][Medline] [Order article via Infotrieve]
  2. Daëron, M., Malbec, O., Latour, S., Arock, M., and Fridman, W. H. (1995) J. Clin. Investig. 95, 577–585[Medline] [Order article via Infotrieve]
  3. Sinclair, N. R. S. C., and Chan, P. L. (1971) Adv. Exp. Med. Biol. 12, 609–615
  4. Amigorena, S., Bonnerot, C., Drake, J., Choquet, D., Hunziker, W., Guillet, J. G., Webster, P., Sautès, C., Mellman, I., and Fridman, W. H. (1992) Science 256, 1808–1812[Abstract/Free Full Text]
  5. Daëron, M., Latour, S., Malbec, O., Espinosa, E., Pina, P., Pasmans, S., and Fridman, W. H. (1995) Immunity 3, 635–646[CrossRef][Medline] [Order article via Infotrieve]
  6. Wernersson, S., Karlsson, M. C., Dahlstrom, J., Mattsson, R., Verbeek, J. S., and Heyman, B. (1999) J. Immunol. 163, 618–622[Abstract/Free Full Text]
  7. Ujike, A., Ishikawa, Y., Ono, M., Yuasa, T., Yoshino, T., Fukumoto, M., Ravetch, J., and Takai, T. (1999) J. Exp. Med. 189, 1573–1579[Abstract/Free Full Text]
  8. Yuasa, T., Kubo, S., Yoshino, T., Ujike, A., Matsumura, K., Ono, M., Ravetch, J. V., and Takai, T. (1999) J. Exp. Med. 189, 187–194[Abstract/Free Full Text]
  9. Nakamura, A., Yuasa, T., Ujike, A., Ono, M., Nukiwa, T., Ravetch, J. V., and Takai, T. (2000) J. Exp. Med. 191, 899–905[Abstract/Free Full Text]
  10. Bolland, S., and Ravetch, J. V. (2000) Immunity 13, 277–285[CrossRef][Medline] [Order article via Infotrieve]
  11. Vivier, E., and Daëron, M. (1997) Immunol. Today 18, 286–291[Medline] [Order article via Infotrieve]
  12. Malbec, O., Fong, D., Turner, M., Tybulewicz, V. L. J., Cambier, J., C., Fridman, W. H., and Daëron, M. (1998) J. Immunol. 160, 1647–1658[Abstract/Free Full Text]
  13. D'Ambrosio, D., Hippen, K. H., Minskoff, S. A., Mellman, I., Pani, G., Siminovitch, K. A., and Cambier, J. C. (1995) Science 268, 293–296[Abstract/Free Full Text]
  14. Bruhns, P., Vély, F., Malbec, O., Fridman, W. H., Vivier, E., and Daëron, M. (2000) J. Biol. Chem. 275, 37357–37364[Abstract/Free Full Text]
  15. Ono, M., Bolland, S., Tempst, P., and Ravetch, J. V. (1996) Nature 383, 263–266[CrossRef][Medline] [Order article via Infotrieve]
  16. Fong, D. C., Malbec, O., Arock, M., Cambier, J. C., Fridman, W. H., and Daëron, M. (1996) Immunol. Lett. 54, 83–91[CrossRef][Medline] [Order article via Infotrieve]
  17. Muraille, E., Bruhns, P., Pesesse, X., Daëron, M., and Erneux, C. (2000) Immunol. Lett. 72, 7–15[CrossRef][Medline] [Order article via Infotrieve]
  18. Damen, J. E., Liu, L., Rosten, P., Humphries, R. K., Jefferson, A. B., Majerus, P. W., and Krystal, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1689–1693[Abstract/Free Full Text]
  19. Lioubin, M. N., Algate, P. A., Tsai, S., Carlberg, K., Aebersold, R., and Rohrschneider, L. R. (1996) Genes Dev. 10, 1084–1095[Abstract/Free Full Text]
  20. Scharenberg, A. M., El-Hillal, O., Fruman, D. A., Beitz, L. O., Li, Z., Lin, S., Gout, I., Cantley, L. C., Rawlings, D. J., and Kinet, J.-P. (1998) EMBO J. 17, 1961–1972[CrossRef][Medline] [Order article via Infotrieve]
  21. Bolland, S., Pearse, R. N., Kurosaki, T., and Ravetch, J. V. (1998) Immunity 8, 509–516[CrossRef][Medline] [Order article via Infotrieve]
  22. Tamir, I., Stolpa, J. C., Helgason, C. D., Nakamura, K., Bruhns, P., Daëron, M., and Cambier, J. C. (2000) Immunity 12, 347–358[CrossRef][Medline] [Order article via Infotrieve]
  23. Malbec, O., Schmitt, C., Bruhns, P., Krystal, G., Fridman, W. H., and Daëron, M. (2001) J. Biol. Chem. 276, 30381–30391[Abstract/Free Full Text]
  24. Brauweiler, A., Tamir, I., Dal Porto, J., Benschop, R. J., Helgason, C. D., Humphries, R. K., Freed, J. H., and Cambier, J. C. (2000) J. Exp. Med. 191, 1545–1554[Abstract/Free Full Text]
  25. Ono, M., Okada, H., Bolland, S., Yanagi, S., Kurosaki, T., and Ravetch, J. V. (1997) Cell 90, 293–301[CrossRef][Medline] [Order article via Infotrieve]
  26. Aman, M. J., Walk, S. F., March, M. E., Su, H. P., Carver, D. J., and Ravichandran, K. S. (2000) Mol. Cell. Biol. 20, 3576–3589[Abstract/Free Full Text]
  27. Brauweiler, A., Tamir, I., Marschner, S., Helgason, C. D., and Cambier, J. C. (2001) J. Immunol. 167, 204–211[Abstract/Free Full Text]
  28. Muta, T., Kurosaki, T., Misulovin, Z., Sanchez, M., Nussenzweig, M. C., and Ravetch, J. V. (1994) Nature 368, 70–73[CrossRef][Medline] [Order article via Infotrieve]
  29. Fong, D. C., Brauweiler, A., Minskoff, S. A., Bruhns, P., Tamir, I., Mellman, I., Daëron, M., and Cambier, J. C. (2000) J. Immunol. 165, 4453–4462[Abstract/Free Full Text]
  30. Liou, J., Kiefer, F., Dang, A., Hashimoto, A., Cobb, M. H., Kurosaki, T., and Weiss, A. (2000) Immunity 12, 399–408[CrossRef][Medline] [Order article via Infotrieve]
  31. Unkeless, J. C. (1979) J. Exp. Med. 150, 580–596[Abstract/Free Full Text]
  32. Takebe, Y., Seiki, M., Fujisawa, J. I., Hoy, P., Yokota, K., Arai, K. I., Yoshida, M., and Arai, N. (1988) Mol. Cell. Biol. 8, 466–472[Abstract/Free Full Text]
  33. Bruhns, P., Marchetti, P., Fridman, W. H., Vivier, E., and Daëron, M. (1999) J. Immunol. 162, 3168–3175[Abstract/Free Full Text]
  34. Daëron, M., Bonnerot, C., Latour, S., and Fridman, W. H. (1992) J. Immunol. 149, 1365–1373[Abstract]
  35. Takata, M., Sabe, H., Hata, A., Inazu, T., Homma, Y., Nukada, T., Yamamura, H., and Kurosaki, T. (1994) EMBO J. 13, 1341–1349[Medline] [Order article via Infotrieve]
  36. Phee, H., Rodgers, W., and Coggeshall, K. M. (2001) Mol. Cell. Biol. 21, 8615–8625[Abstract/Free Full Text]
  37. Wisniewski, D., Strife, A., Swendeman, S., Erdjument-Bromage, H., Geromanos, S., Kavanaugh, W. M., Tempst, P., and Clarkson, B. (1999) Blood 93, 2707–2720[Abstract/Free Full Text]
  38. Harmer, S. L., and DeFranco, A. L. (1999) J. Biol. Chem. 274, 12183–12191[Abstract/Free Full Text]
  39. Bu, J. Y., Shaw, A. S., and Chan, A. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5106–5110[Abstract/Free Full Text]
  40. Kurosaki, T., Johnson, S. A., Pao, L., Sada, K., Yamamura, H., and Cambier, J. C. (1995) J. Exp. Med. 182, 1815–1823[Abstract/Free Full Text]
  41. Lesourne, R., Bruhns, P., Fridman, W. H., and Daëron, M. (2001) J. Biol. Chem., 6327–6336
  42. Kimura, T., Sakamoto, H., Apellea, E., and Siraganian, R. P. (1996) Mol. Cell. Biol. 4, 1471–1478
  43. Burshtyn, D. N., Lam, A. S., Weston, M., Gupta, N., Warmerdam, P. A., and Long, E. O. (1999) J. Immunol. 162, 897–902[Abstract/Free Full Text]
  44. Yamasaki, S., Nishida, K., Sakuma, M., Berry, D., McGlade, C. J., Hirano, T., and Saito, T. (2003) Mol. Cell. Biol. 23, 2515–2529[Abstract/Free Full Text]
  45. Trüb, T., Frantz, J. D., Miyazaki, M., Band, H., and Shoelson, S. E. (1997) J. Biol. Chem. 272, 894–902[Abstract/Free Full Text]
  46. Tridandapani, S., Kelly, T., Cooney, D., Pradhan, M., and Coggeshall, K. M. (1997) Immunol. Today 18, 424–427[CrossRef][Medline] [Order article via Infotrieve]
  47. Shen, R., Ouyang, Y. B., Qu, C. K., Alonso, A., Sperzel, L., Mustelin, T., Kaplan, M. H., and Feng, G. S. (2002) Mol. Cell. Biol. 22, 3230–3236[Abstract/Free Full Text]

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