Mutational analysis reveals multiple distinct sites within Fc gamma receptor IIB that function in inhibitory signaling

The low-affinity receptor for IgG, FcgammaRIIB, 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 FcgammaRIIB 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 FcgammaRIIB association with SHIP in vivo and for FcgammaRIIB-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, FcgammaRIIB-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 FcgammaRIIB1 phosphorylation following coaggregation with B cell Ag receptor. Thus, we conclude that multiple sites in FcgammaRIIB contribute uniquely to transduction of FcgammaRIIB-mediated inhibitory signals.     

Engagement of CD22 on B cells with the monoclonal antibody epratuzumab stimulates the phosphorylation of upstream inhibitory signals of the B cell receptor

The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr⁸²² on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr⁸⁰⁷, a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr²⁹² on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.     

The chicken leukocyte receptor complex: a highly diverse multigene family encoding at least six structurally distinct receptor types

The chicken Ig-like receptors (CHIR) have been described as two Ig domain molecules with long cytoplasmic tails containing inhibitory motifs. In this study, we demonstrate that CHIR form a large family, with multiple members showing great sequence variability among members as well as a great diversity in domain organization and properties of the transmembrane and cytoplasmic segments. We characterize various novel receptor types with motifs indicative of inhibitory, activating, or both functions. In addition to the inhibitory receptors with two ITIM, receptors with a single immunoreceptor tyrosine-based switch motif or receptors lacking a cytoplasmic domain were isolated. Activating receptors with a short cytoplasmic domain and a transmembrane arginine assembled with the newly identified chicken FcepsilonRIgamma chain. Three bifunctional receptor types were characterized composed of one or two C2-type Ig-like domains, a transmembrane region with a positively charged residue and combinations of cytoplasmic motifs such as ITIM, immunoreceptor tyrosine-based switch motif, and YXXM. RT-PCR revealed distinct expression patterns of individual CHIR. All receptor types shared a conserved genomic architecture, and in single Ig domain receptors a pseudoexon replaced the second Ig exon. Southern blot analyses with probes specific for the Ig1 domain were indicative of a large multigene family. Of 103 sequences from the Ig1 domain of a single animal, 41 unique sequences were obtained that displayed extensive variability within restricted Ig regions. Fluorescence in situ hybridization localized the CHIR gene cluster to microchromosome 31 and identified this region as orthologous to the human leukocyte receptor complex.     

The pseudo-immunoreceptor tyrosine-based activation motif of CD5 mediates its inhibitory action on B-cell receptor signaling

Genetic studies revealed that CD5 could be a negative regulator of the B-cell antigen receptor (BCR). We explore here the effect of human CD5 on BCR-triggered responses. B cells were obtained expressing a chimera composed of extracellular and transmembrane domains of Fcgamma type IIB receptor fused to CD5 cytoplasmic domain (CD5cyt). Coligation of the chimera with the BCR induces CD5cyt tyrosine phosphorylation. A rapid inhibition of BCR-induced calcium response is observed, as well as a partial but delayed inhibition of phospholipase Cgamma-1 phosphorylation. Activation of extracellular regulated kinase-2 is also severely impaired. Moreover, at the functional level, interleukin-2 production is abolished. Src homology 2 domain-bearing tyrosine phosphatase SHP-1 and Src homology 2 domain-bearing inositol 5'-phosphatase SHIP usually participate in negative regulation of the BCR. We show that they do not associate with the phosphorylated CD5 chimera. We finally demonstrate that the pseudo-immunoreceptor tyrosine based activation motif present in CD5cyt is involved because its deletion eliminates the inhibitory effect of the chimera, both at biochemical and functional levels. These results demonstrate the inhibitory role of CD5 pseudo-immunoreceptor tyrosine based activation motif tyrosine phosphorylation on BCR signaling. They further support the idea that CD5 uses mechanisms different from those already described to negatively regulate the BCR pathway.     

Dendritic cell immunoactivating receptor,a novel C-type lectin immunoreceptor,acts as an activating receptor through association with Fc receptor gamma chain

An increasing number of C-type lectin receptors are being discovered on dendritic cells, but their signaling abilities and underlying mechanisms require further definition. Among these, dendritic cell immunoreceptor (DCIR) induces negative signals through an inhibitory immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail. Here we identify a novel C-type lectin receptor, dendritic cell immunoactivating receptor (DCAR), whose extracellular lectin domain is highly homologous to that of DCIR. DCAR is expressed similarly in tissues to DCIR, but its short cytoplasmic portion lacks signaling motifs like ITIM. However, a positively charged arginine residue is present in the transmembrane region of the DCAR, which may explain its association with Fc receptor gamma chain and its stable expression on the cell surface. Furthermore, cross-linking of DCAR in the presence of gamma chain activates calcium mobilization and tyrosine phosphorylation of cellular proteins. These signals are mediated by the immunoreceptor tyrosine-based activating motif (ITAM) of the gamma chain. Thus, DCAR is closely related to DCIR, but it introduces activating signals into antigen-presenting cells through its physical and functional association with ITAM-bearing gamma chain. The identification of this activating immunoreceptor provides an example of signaling via a dendritic cell-expressed C-type lectin receptor.     

Src homology 2 domain-containing protein-tyrosine phosphatases, SHP-1 and SHP-2, are required for platelet endothelial cell adhesion molecule-1/CD31-mediated inhibitory signaling

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a newly assigned member of the Ig immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. To test whether PECAM-1 is capable of delivering inhibitory signals in B cells and the functional requirement of protein-tyrosine phosphatases (PTPs) for this inhibitory signaling, we generated chimeric Fc gamma RIIB1-PECAM-1 receptors containing the extracellular and transmembrane portions of murine Fc gamma RIIB1 and the cytoplasmic domain of human PECAM-1. These chimeric receptors were stably expressed in chicken DT40 B cells either as wild-type or mutant cells deficient in SHP-1(-/-), SHP-2(-/-), SHIP(-/-), or SHP-1/2(-/-) and then assessed for their ability to inhibit B cell Ag receptor (BCR) signaling. Coligation of wild-type Fc gamma RIIB1-PECAM-1 with BCR resulted in inhibition of intracellular calcium release, suggesting that the cytoplasmic domain of PECAM-1 is capable of delivering an inhibitory signal that blocks BCR-mediated activation. This PECAM-1-mediated inhibitory signaling correlated with tyrosine phosphorylation of the Fc gamma RIIB1-PECAM-1 chimera, recruitment of SHP-1 and SHP-2 PTPs by the phosphorylated chimera, and attenuation of calcium mobilization responses. Mutational analysis of the two tyrosine residues, 663 and 686, constituting the immunoreceptor tyrosine-based inhibitory motifs in PECAM-1 revealed that both tyrosine residues play a crucial role in the inhibitory signal. Functional analysis of various PTP-deficient DT40 B cell lines stably expressing wild-type chimeric Fc gamma RIIB1-PECAM-1 receptor indicated that cytoplasmic Src homology 2-domain-containing phosphatases, SHP-1 and SHP-2, were both necessary and sufficient to deliver inhibitory negative regulation upon coligation of BCR complex with inhibitory receptor.     

Ligand stimulation of CD155alpha inhibits cell adhesion and enhances cell migration in fibroblasts

CD155 (poliovirus receptor) localizes in cell-matrix adhesions and cell-cell junctions, but its role in the regulation of cell adhesion and cell motility has not been investigated. We identified a conserved immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic domain of human CD155alpha. The ITIM was tyrosine-phosphorylated upon binding of anti-CD155 monoclonal antibody D171, poliovirus, and DNAM-1 (CD226) to human CD155alpha, and recruited SH2-domain-containing tyrosine phosphatase-2 (SHP-2). After CD155alpha stimulation with its ligands, cell adhesion was inhibited and cell motility was enhanced, effects that were associated with the phosphorylation of ITIM by Src kinases and accompanied by dephosphorylation of focal adhesion kinase and paxillin. These effects were abolished by introducing a point-mutation in Y398F into the ITIM of CD155alpha and by coexpression of a dominant negative SHP-2 mutant with CD155alpha. These results suggest that CD155alpha plays a role in the regulation of cell adhesion and cell motility.     

Recruitment of Grb2 and SHIP1 by the ITT-like motif of TIGIT suppresses granule polarization and cytotoxicity of NK cells

Activating and inhibitory receptors control natural killer (NK) cell activity. T-cell immunoglobulin and ITIM (immunoreceptor tyrosine-based inhibition motif) domain (TIGIT) was recently identified as a new inhibitory receptor on T and NK cells that suppressed their effector functions. TIGIT harbors the immunoreceptor tail tyrosine (ITT)-like and ITIM motifs in its cytoplasmic tail. However, how its ITT-like motif functions in TIGIT-mediated negative signaling is still unclear. Here, we show that TIGIT/PVR (poliovirus receptor) engagement disrupts granule polarization leading to loss of killing activity of NK cells. The ITT-like motif of TIGIT has a major role in its negative signaling. After TIGIT/PVR ligation, the ITT-like motif is phosphorylated at Tyr225 and binds to cytosolic adapter Grb2, which can recruit SHIP1 to prematurely terminate phosphatidylinositol 3-kinase (PI3K) and MAPK signaling, leading to downregulation of NK cell function. In support of this, Tyr225 or Asn227 mutation leads to restoration of TIGIT/PVR-mediated cytotoxicity, and SHIP1 silencing can dramatically abolish TIGIT/PVR-mediated killing inhibition.     

CD22 regulates B cell receptor-mediated signals via two domains that independently recruit Grb2 and SHP-1

Recognition of antigen by the B cell antigen receptor (BCR) determines the subsequent fate of a B cell and is regulated in part by the involvement of other surface molecules, termed coreceptors. CD22 is a B cell-restricted coreceptor that gets rapidly tyrosyl-phosphorylated and recruits various signaling molecules to the membrane following BCR ligation. Although CD22 contains three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), only the two carboxyl-terminal ITIM tyrosines are required for efficient recruitment of the SHP-1 phosphatase after BCR ligation. Furthermore, Grb2 is inducibly recruited to CD22 in human and murine B cells. Unlike SHP-1, Grb2 recruitment to CD22 is not inhibited by specific doses of the Src family kinase-specific inhibitor PP1. The tyrosine residue in CD22 required for Grb2 recruitment (Tyr-828) is distinct and independent from the two ITIM tyrosines required for efficient SHP-1 recruitment (Tyr-843 and Tyr-863). Individually both Lyn and Syk are required for maximal phosphorylation of CD22 following ligation of the BCR, and together Lyn and Syk are required for all of the constitutive and induced tyrosine phosphorylation of CD22. We propose that the cytoplasmic tail of CD22 contains two domains that regulate signal transduction pathways initiated by the BCR and B cell fate.     

CD72 negatively regulates signaling through the antigen receptor of B cells

The immunoreceptor tyrosine-based inhibition motif (ITIM) is found in various membrane molecules such as CD22 and the low-affinity Fc receptor for IgG in B cells and the killer cell-inhibitory receptor and Ly-49 in NK cells. Upon tyrosine phosphorylation at the ITIMs, these molecules recruit SH2 domain-containing phosphatases such as SH2-containing tyrosine phosphatase-1 and negatively regulate cell activity. The B cell surface molecule CD72 carries an ITIM and an ITIM-like sequence. We have previously shown that CD72 is phosphorylated and recruits SH2-containing tyrosine phosphatase-1 upon cross-linking of the Ag receptor of B cells (BCR). However, whether CD72 modulates BCR signaling has not yet been elucidated. In this paper we demonstrate that expression of CD72 down-modulates both extracellular signal-related kinase (ERK) activation and Ca2+ mobilization induced by BCR ligation in the mouse B lymphoma line K46micromlambda, whereas BCR-mediated ERK activation was not reduced by the ITIM-mutated form of CD72. Moreover, coligation with CD72 with BCR reduces BCR-mediated ERK activation in spleen B cells of normal mice. These results indicate that CD72 negatively regulates BCR signaling. CD72 may play a regulatory role in B cell activation, probably by setting a threshold for BCR signaling.     

Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of B and T lymphocyte attenuator required for association with protein tyrosine phosphatases SHP-1 and SHP-2

B and T lymphocytes express receptors providing positive and negative co-stimulatory signals. We recently identified a novel co-stimulatory molecule, B and T lymphocyte attenuator (BTLA), which exerts inhibitory effects on B and T lymphocytes. The cytoplasmic domain of murine and human BTLA share three conserved tyrosine-based signaling motifs, a Grb-2 recognition consensus, and two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Phosphorylation of the cytoplasmic domain of BTLA induced the association with the protein tyrosine phosphatases SHP-1 and SHP-2. Association of SHP-1 and SHP-2 to other receptors can involve recruitment to either a single receptor ITIM or to two receptor ITIMs. Here, we analyzed the requirements of BTLA interaction with SHP-1 and SHP-2 in a series of murine and human BTLA mutants. For human BTLA, mutations of either Y257 or Y282, but not Y226, abrogated association with both SHP-1 and SHP-2. For murine BTLA, mutation of either Y274 or Y299, but not Y245, also abrogated association with both SHP-1 and SHP-2. These results indicate that for both murine and human BTLA, association with SHP-1 or SHP-2 requires both of conserved ITIM motifs and does not involve the conserved Grb-2 consensus. Thus, similar to the bisphosphoryl tyrosine-based activation motif (BTAM) by which the Grb-2 associated binder (Gab1), PDGF receptor, and PECAM-1 recruit SHP-2, BTLA also relies on dual ITIMs for its association with the phosphatases SHP-1 and SHP-2.     

FcR-like 2 Inhibition of B cell receptor-mediated activation of B cells

FcR-like (FCRL) 2 is a transmembrane protein with immunomodulatory potential that is preferentially expressed by memory B cells in humans. It has two consensus ITIMs in addition to a putative ITAM sequence in its cytoplasmic domain. We have confirmed the cellular distribution of FCRL2 and analyzed its functional potential to show that coligation with the BCR leads to tyrosine phosphorylation of its ITIM motifs and subsequent Src homology region 2 domain-containing phosphatase-1 recruitment to facilitate inhibition of BCR signaling. Mutational analysis indicates that the tyrosine residues in both inhibitory motifs of FCRL2 are required for complete inhibition of BCR signaling, whereas tyrosines in the putative activation motif are dispensable for signal modulation. These findings suggest a negative immunomodulatory function for FCRL2 in the regulation of memory B cells.     

Functional requirements for signaling through the stimulatory and inhibitory mouse NKR-P1 (CD161) NK cell receptors

The NK cell receptor protein 1 (NKR-P1) (CD161) molecules represent a family of type II transmembrane C-type lectin-like receptors expressed predominantly by NK cells. Despite sharing a common NK1.1 epitope, the mouse NKR-P1B and NKR-P1C receptors possess opposing functions in NK cell signaling. Engagement of NKR-P1C stimulates cytotoxicity of target cells, Ca2+ flux, phosphatidylinositol turnover, kinase activity, and cytokine production. In contrast, NKR-P1B engagement inhibits NK cell cytotoxicity. Nonetheless, it remains unclear how different signaling outcomes are mediated at the molecular level. Here, we demonstrate that both NKR-P1B and NKR-P1C associate with the tyrosine kinase, p56(lck). The interaction is mediated through the di-cysteine CxCP motif in the cytoplasmic domains of NKR-P1B/C. Disrupting this motif leads to abrogation of both stimulatory and inhibitory NKR-P1 signals. In addition, mutation of the consensus ITIM (LxYxxL) in NKR-P1B abolishes both its Src homology 2-containing protein tyrosine phosphatase-1 recruitment and inhibitory function. Strikingly, engagement of NKR-P1C on NK cells obtained from Lck-deficient mice failed to induce NK cytotoxicity. These results reveal a role for Lck in the initiation of NKR-P1 signals, and demonstrate a requirement for the ITIM in NKR-P1-mediated inhibition.     

The B cell inhibitory Fc receptor triggers apoptosis by a novel c-Abl family kinase-dependent pathway

The inhibitory Fc receptors function to regulate the antigen-driven activation and expansion of lymphocytes. In B cells, the Fc gammaRIIB1 is a potent inhibitor of B cell antigen receptor (BCR) signaling when coligated to the BCR by engagement of antigen-containing immune complexes. Inhibition is mediated by the recruitment of the inositol phosphatase, SHIP, to the Fc gammaRIIB1 phosphorylated tyrosine-based inhibitory motif (ITIM). Here we show that BCR-independent aggregation of the Fc gammaRIIB1 transduces an ITIM- and SHIP-independent proapoptotic signal that is dependent on members of the c-Abl tyrosine kinase family. These results define a novel Abl family kinase-dependent Fc gammaRIIB1 signaling pathway that functions independently of the BCR in controlling antigen-driven B cell responses.     

KIR2DL5 can inhibit human NK cell activation via recruitment of Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2)

Human NK cells use class I MHC-binding inhibitory receptors, such as the killer cell Ig-like receptor (KIR) family, to discriminate between normal and abnormal cells. Some tumors and virus-infected cells down-regulate class I MHC and thereby become targets of NK cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatases, Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) and SHP-2, to two phosphorylated cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). KIR2DL5 is a type II member of the KIR2D family with an atypical extracellular domain and an intracytoplasmic domain containing one typical ITIM and one atypical ITIM sequence. Although KIR2DL5 structure is expressed by approximately 50% of humans and is conserved among primate species, its function has not been determined. In the present study, we directly compared functional and biochemical properties of KIR2DL5, KIR3DL1 (a type I KIR with two ITIMs), and KIR2DL4 (the only other type II KIR, which has a single ITIM) in a human NK-like cell line. Our results show that KIR2DL5 is an inhibitory receptor that can recruit both SHP-1 and SHP-2, and its inhibitory capacity is more similar to that of the cytoplasmic domain of KIR2DL4 than KIR3DL1. Interestingly, inhibition of NK cell cytotoxicity by KIR2DL5 was blocked by dominant-negative SHP-2, but not dominant-negative SHP-1, whereas both dominant-negative phosphatases can block inhibition by KIR3DL1. Therefore, the cytoplasmic domains of type II KIRs (2DL4 and 2DL5) exhibit distinct inhibitory capacities when compared with type I KIRs (3DL1), due to alterations in the canonical ITIM sequences.     

NKp44 triggers NK cell activation through DAP12 association that is not influenced by a putative cytoplasmic inhibitory sequence

NKp44 (NCR2) is a member of the natural cytotoxicity receptor (NCR) family that is expressed on activated human NK cells. We dissected structural attributes of NKp44 to determine their contributions to receptor function. Our results demonstrate that surface expression and NK cell activation by NKp44 is mediated through noncovalent association with the immunoreceptor tyrosine-based activation motif-containing protein, DAP12. Physical linkage to DAP12 requires lysine-183 in the NKp44 transmembrane domain. Intriguingly, the cytoplasmic domain of NKp44 also contains a sequence that matches the immunoreceptor tyrosine-based inhibitory motif (ITIM) consensus. By expressing a chimeric receptor in an NK-like cell line, we found that this ITIM-like motif from NKp44 lacks inhibitory capacity in a redirected cytotoxicity assay. The NKp44 cytoplasmic tyrosine was efficiently phosphorylated in the chimeric receptor upon treating the cells with pervanadate, but it was unable to recruit ITIM-binding negative effector phosphatases. We also generated NK-like cell lines expressing epitope-tagged wild-type or tyrosine to phenylalanine mutant (Y238F) versions of NKp44 and compared their capacities to induce activation marker expression, promote IFN-gamma production, or stimulate target cell cytotoxicity. We did not detect any tyrosine-dependent reduction or enhancement of NK cell activation through wild-type vs. Y238F mutant NKp44. Finally, the cytoplasmic tyrosine-based sequence did not provide a docking site for the AP-2 clathrin adaptor, nor did it potentiate receptor internalization. In summary, all activating properties and surface expression of NKp44 are mediated through its association with DAP12, and the putative ITIM in the NKp44 cytoplasmic domain does not appear to attenuate activating function.     

Molecular basis of the recruitment of the SH2 domain-containing inositol 5-phosphatases SHIP1 and SHIP2 by fcgamma RIIB

FcgammaRIIB are single-chain low affinity receptors for IgG that negatively regulate immunoreceptor tyrosine-based activation motif-dependent cell activation. They bear one immunoreceptor tyrosine-based inhibition motif (ITIM) that becomes tyrosyl-phosphorylated upon coaggregation of FcgammaRIIB with immunoreceptor tyrosine-based activation motif-bearing receptors and that recruits SH2 domain-containing inositol 5-phosphatases (SHIPs) in vivo. Synthetic FcgammaRIIB ITIM phosphopeptides, however, also bind SH2 domain-containing protein-tyrosine phosphatases (SHPs) in vitro. To identify SHIP-binding sites, we exchanged residues between the FcgammaRIIB ITIM and the N-terminal ITIM of a killer cell Ig-like receptor that does not bind SHIPs. Loss of function and gain of function substitutions identified the Y+2 leucine, in the FcgammaRIIB ITIM, as determining the binding of both SHIP1 and SHIP2, but not the binding of SHP-1 or SHP-2. Conversely, the Y-2 isoleucine that determines the in vitro binding of SHP-1 and SHP-2 affected neither the binding nor the recruitment of SHIP1 or SHIP2. One hydrophobic residue, in the ITIM of FcgammaRIIB therefore determines the affinity for SHIPs. This residue is symmetrical to the hydrophobic residue that determines the affinity of all ITIMs for SHPs. It defines a SHIP-binding site, distinct from a SHP-binding site, that enables FcgammaRIIB to recruit SHIP1 and SHIP2 and that is preferentially used in vivo.     

Novel binding site for Src homology 2-containing protein-tyrosine phosphatase-1 in CD22 activated by B lymphocyte stimulation with antigen

CD22, a B lymphocyte membrane glycoprotein, contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic region and recruits Src homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) to the phosphorylated ITIMs upon ligation of B lymphocyte antigen receptor (BCR), thereby negatively regulating BCR signaling. Among the three previously identified ITIMs, both ITIMs containing tyrosine residues at position 843 (Tyr(843)) and 863 (Tyr(863)), respectively, are shown to be required for CD22 to recruit SHP-1 and regulate BCR signaling upon BCR ligation by anti-Ig antibody (Ab), indicating that CD22 has the SHP-1-binding domain at the region containing Tyr(843) and Tyr(863). Here we address the requirement of CD22 for SHP-1 recruitment and BCR regulation upon BCR ligation by antigen, which induces much stronger CD22 phosphorylation than anti-Ig Ab does. We demonstrate that the CD22 mutant in which both Tyr(843) and Tyr(863) are replaced by phenylalanine (CD22F5/6) recruits SHP-1 and regulates BCR signaling upon stimulation with antigen but not anti-Ig Ab. This result strongly suggests that CD22 contains another SHP-1 binding domain that is specifically activated upon stimulation with antigen. Both of the flanking sequences of Tyr(783) and Tyr(817) fit the consensus sequence of ITIM, and the CD22F5/6 mutant requires these tyrosine residues for SHP-1 binding and BCR regulation. Thus, these ITIMs constitute a novel conditional SHP-1-binding site of CD22 that is activated upon BCR ligation by antigen but not by anti-Ig Ab.     

CD72 down-modulates BCR-induced signal transduction and diminishes survival in primary mature B lymphocytes

CD72, a 45-kDa type II transmembrane glycoprotein carrying an ITIM motif, is believed to be an inhibitory coreceptor of the BCR. Mature B cells lacking CD72 show enhanced Ca(2+) mobilization and are hyperproliferative in response to BCR ligation. However, the signal transduction pathways downstream of BCR signaling that transmit the inhibitory effect of CD72 in mature B cells remain unknown. To address this question, we used hen egg lysozyme-specific BCR transgenic mice to elucidate the differential cell signaling between wild-type and CD72-deficient B cells in response to hen egg lysozyme Ag stimulation. Our results demonstrate that CD72 predominantly down-regulates the major signal transduction pathways downstream of the BCR, including NF-AT, NF-kappaB, ERK, JNK, p38-MAPK, and PI3K/Akt in mature B cells. CD72 ligation with anti-CD72 Ab (K10.6), which mimics the binding of CD100 (a natural ligand for CD72) to release the inhibitory function of CD72, augments cell proliferation, Ca(2+) flux, IkappaBalpha activation, and ERK MAPK activity upon Ag stimulation in wild-type B cells. In addition, we show direct evidence that CD72 promotes cell cycle arrest and apoptosis after Ag stimulation in mature B cells. Taken together, our findings conclude that CD72 plays a dominant role as a negative regulator of BCR signaling in primary mature B lymphocytes.