Negative regulation of mast cell signaling and function by the adaptor LAB/NTAL

Engagement of the Fcepsilon receptor I (FcepsilonRI) on mast cells and basophils initiates signaling pathways leading to degranulation. Early activation events include tyrosine phosphorylation of two transmembrane adaptor proteins, linker for activation of T cells (LAT) and non-T cell activation linker (NTAL; also called LAB; a product of Wbscr5 gene). Previous studies showed that the secretory response was partially inhibited in bone marrow-derived mast cells (BMMCs) from LAT-deficient mice. To clarify the role of NTAL in mast cell degranulation, we compared FcepsilonRI-mediated signaling events in BMMCs from NTAL-deficient and wild-type mice. Although NTAL is structurally similar to LAT, antigen-mediated degranulation responses were unexpectedly increased in NTAL-deficient mast cells. The earliest event affected was enhanced tyrosine phosphorylation of LAT in antigen-activated cells. This was accompanied by enhanced tyrosine phosphorylation and enzymatic activity of phospholipase C gamma1 and phospholipase C gamma2, resulting in elevated levels of inositol 1,4,5-trisphosphate and free intracellular Ca2+. NTAL-deficient BMMCs also exhibited an enhanced activity of phosphatidylinositol 3-OH kinase and Src homology 2 domain-containing protein tyrosine phosphatase-2. Although both LAT and NTAL are considered to be localized in membrane rafts, immunogold electron microscopy on isolated membrane sheets demonstrated their independent clustering. The combined data show that NTAL is functionally and topographically different from LAT.     

Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells

Aggregation of the high affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells results in rapid tyrosine phosphorylation and activation of Syk, a cytoplasmic protein tyrosine kinase. To examine the role of Syk in the Fc epsilon RI signaling pathway, we identified a variant of RBL-2H3 cells that has no detectable Syk by immunoblotting and by in vitro kinase reactions. In these Syk-deficient TB1A2 cells, aggregation of Fc epsilon RI induced no histamine release and no detectable increase in total cellular protein tyrosine phosphorylation. However, stimulation of these cells with the calcium ionophore did induce degranulation. Fc epsilon RI aggregation induced tyrosine phosphorylation of the beta and gamma subunits of the receptor, but no increase in the tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2 and no detectable increase in intracellular free Ca2+ concentration. By transfection, cloned lines were established with stable expression of Syk. In these reconstituted cells, Fc epsilon RI aggregation induced tyrosine phosphorylation of phospholipase C- gamma 1 and phospholipase C-gamma 2, an increase in intracellular free Ca2+ and histamine release. These results demonstrate that Syk plays a critical role in the early Fc epsilon RI-mediated signaling events. It further demonstrates that Syk activation occurs downstream of receptor phosphorylation, but upstream of most of the Fc epsilon RI-mediated protein tyrosine phosphorylations.     

Regulation of high-affinity IgE receptor-mediated mast cell activation by murine low-affinity IgG receptors.

Allergic symptoms result from the release of granular and lipidic mediators and of cytokines by inflammatory cells. The whole process is initiated by the aggregation of mast cell and basophil high-affinity IgE receptors (Fc epsilon RI) by IgE and antigen. We report here that IgE-induced release of mediator and cytokine can be inhibited by cross-linking Fc epsilon RI to low-affinity IgG receptors (Fc gamma RII) which are constitutively expressed on mast cells and basophils. Using a model of stable transfectants in RBL-2H3 cells expressing endogeneous rat Fc epsilon RI and recombinant murine Fc gamma RII, we showed that inhibition requires that Fc epsilon RI be crosslinked to Fc gamma RII by the same multivalent ligand. Inhibition of cross-linked receptors left non-cross-linked Fc epsilon RI capable of triggering mediator release and was reversible upon disengagement. Both isoforms of wild-type Fc gamma RII were equally capable of inhibiting Fc epsilon RI-mediated mast cell activation provided they had an intact intracytoplasmic domain. Our results demonstrate that mast cell secretory responses triggered by high-affinity receptors for IgE may be controlled by low-affinity receptors for IgG. This regulation of Fc epsilon RI-mediated mast cell activation is of potential interest in mast cell physiology and in allergic pathology.     

Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non-T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcgamma- and Fc epsilon -receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non-T cells.     

Antigen-independent induction of histamine synthesis by immunoglobulin E in mouse bone marrow-derived mast cells

Immunoglobulin (Ig)E-mediated activation of mast cells has long been thought to occur only when Fc(epsilon)RI receptor-bound IgE is cross-linked via multivalent antigens. However, recent studies have raised the possibility that mast cells may be activated by the binding of IgE to the Fc(epsilon)RI receptor in the absence of antigen. Here we demonstrate that IgE binding without antigen induces the expression of histidine decarboxylase (HDC) in mouse interleukin (IL)-3-dependent bone marrow-derived mast cells (BMMCs). The induction of HDC by the binding of IgE was found to require an influx of extracellular calcium ions, which was attenuated by pretreatment with U73122, a phospholipase C inhibitor. Furthermore, the increase in HDC activity upon sensitization with IgE was completely suppressed by pretreatment of BMMCs with protein kinase C inhibitors, such as H7, staurosporine, and G?6976. In addition, immediate activation of the tyrosine kinase Lyn was not detectable upon treatment with IgE. These results suggest that the binding of IgE to its receptor in the absence of antigen results in de novo synthesis of HDC in BMMCs through a signaling pathway distinct to that operating during antigen-stimulated Fc(epsilon)RI activation.     

Knock-in mutation of the distal four tyrosines of linker for activation of T cells blocks murine T cell development.

The integral membrane adapter protein linker for activation of T cells (LAT) performs a critical function in T cell antigen receptor (TCR) signal transduction by coupling the TCR to downstream signaling pathways. After TCR engagement, LAT is tyrosine phosphorylated by ZAP-70 creating docking sites for multiple src homology 2-containing effector proteins. In the Jurkat T cell line, the distal four tyrosines of LAT bind PLCgamma-1, Grb2, and Gads. Mutation of these four tyrosine residues to phenylalanine (4YF) blocked TCR-mediated calcium mobilization, Erk activation, and nuclear factor (NF)-AT activation. In this study, we examined whether these four tyrosine residues were essential for T cell development by generating LAT "knock-in" mutant mice that express the 4YF mutant protein under the control of endogenous LAT regulatory sequences. Significantly, the phenotype of 4YF knock-in mice was identical to LAT(-/)- (null) mice; thymocyte development was arrested at the immature CD4(-)CD8(-) stage and no mature T cells were present. Knock-in mice expressing wild-type LAT protein, generated by a similar strategy, displayed a normal T cell developmental profile. These results demonstrate that the distal four tyrosine residues of LAT are essential for preTCR signaling and T cell development in vivo.     

Positive and negative regulation of FcepsilonRI-mediated signaling by the adaptor protein LAB/NTAL

Linker for activation of B cells (LAB, also called NTAL; a product of wbscr5 gene) is a newly identified transmembrane adaptor protein that is expressed in B cells, NK cells, and mast cells. Upon BCR activation, LAB is phosphorylated and interacts with Grb2. LAB is capable of rescuing thymocyte development in LAT-deficient mice. To study the in vivo function of LAB, LAB-deficient mice were generated. Although disruption of the Lab gene did not affect lymphocyte development, it caused mast cells to be hyperresponsive to stimulation via the FcepsilonRI, evidenced by enhanced Erk activation, calcium mobilization, degranulation, and cytokine production. These data suggested that LAB negatively regulates mast cell function. However, mast cells that lacked both linker for activation of T cells (LAT) and LAB proteins had a more severe block in FcepsilonRI-mediated signaling than LAT(-/-) mast cells, demonstrating that LAB also shares a redundant function with LAT to play a positive role in FcepsilonRI-mediated signaling.     

Mast cells lacking the high affinity immunoglobulin E receptor are deficient in Fc epsilon RI gamma messenger RNA

A population of cells that express mast cell markers, including the membrane protein p161, but that lack expression of the high affinity IgE receptor, Fc epsilon RI, can be routinely grown from bone marrow. Ionomycin, but not IgE immune complexes, causes these cells to release serotonin and to express IL-3 and IL-13 mRNA, consistent with their being FC epsilon RI-deficient mast cells. These p161+/Fc epsilon RI- mast cells expressed normal amounts of Fc epsilon RI alpha and beta chain mRNA, but extremely low levels of Fc epsilon RI gamma chain mRNA. In addition, this novel mast cell population expressed CD3 zeta chain mRNA, which p161+/Fc epsilon RI+ mast cells did not. CD3 zeta stable transfectants of Abelson-murine leukemia virus-transformed p161+/Fc epsilon RI+ mast cells continued to express Fc epsilon RI. This strongly suggests that the failure of p161+/Fc epsilon RI- mast cells to express IgE receptors was not caused by the presence of CD3 zeta chain. Transfection of human Fc epsilon RI gamma cDNA into p161+/Fc epsilon RI- mast cells rescued IgE binding. These stable transfectants released serotonin in response to cross-linkage of Fc epsilon RI, demonstrating that the molecular defect of p161+/Fc epsilon RI- mast cells is indeed the loss of Fc epsilon RI gamma expression.     

Recruitment of SLP-76 to the membrane and glycolipid-enriched membrane microdomains replaces the requirement for linker for activation of T cells in T cell receptor signaling

Two hematopoietic-specific adapters, src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) and linker for activation of T cells (LAT), are critical for T cell development and T cell receptor (TCR) signaling. Several studies have suggested that SLP-76 and LAT function coordinately to promote downstream signaling. In support of this hypothesis, we find that a fraction of SLP-76 localizes to glycolipid-enriched membrane microdomains (GEMs) after TCR stimulation. This recruitment of SLP-76 requires amino acids 224-244. The functional consequences of targeting SLP-76 to GEMs for TCR signaling are demonstrated using a LAT/SLP-76 chimeric protein. Expression of this construct reconstitutes TCR-inducted phospholipase Cgamma1 phosphorylation, extracellular signal-regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2). Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling. Expression of a chimera that lacks SLP-76 amino acids 224-244 restores NFAT promoter activity, suggesting that if localized, SLP-76 does not require an association with Gads to promote T cell activation. In contrast, mutation of the protein tyrosine kinase phosphorylation sites of SLP-76 in the context of the LAT/SLP-76 chimera abolishes reconstitution of TCR function. Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane.     

Impaired degranulation but enhanced cytokine production after Fc epsilonRI stimulation of diacylglycerol kinase zeta-deficient mast cells

Calcium and diacylglycerol are critical second messengers that together effect mast cell degranulation after allergen cross-linking of immunoglobulin (Ig)E-bound FcepsilonRI. Diacylglycerol kinase (DGK)zeta is a negative regulator of diacylglycerol-dependent signaling that acts by converting diacylglycerol to phosphatidic acid. We reported previously that DGKzeta-/- mice have enhanced in vivo T cell function. Here, we demonstrate that these mice have diminished in vivo mast cell function, as revealed by impaired local anaphylactic responses. Concordantly, DGKzeta-/- bone marrow-derived mast cells (BMMCs) demonstrate impaired degranulation after Fc epsilonRI cross-linking, associated with diminished phospholipase Cgamma activity, calcium flux, and protein kinase C-betaII membrane recruitment. In contrast, Ras-Erk signals and interleukin-6 production are enhanced, both during IgE sensitization and after antigen cross-linking of Fc epsilonRI. Our data demonstrate dissociation between cytokine production and degranulation in mast cells and reveal the importance of DGK activity during IgE sensitization for proper attenuation of Fc epsilonRI signals.     

Preferential signaling and induction of allergy-promoting lymphokines upon weak stimulation of the high affinity IgE receptor on mast cells

Mast cell degranulation and de novo cytokine production is a consequence of antigen-aggregation of the immunoglobulin E (IgE)-occupied high affinity receptor for IgE (Fc epsilon RI). Herein, we report that lymphokines that promote allergic inflammation, like MCP-1, were potently induced at low antigen (Ag) concentrations or at low receptor occupancy with IgE whereas some that down-regulate this response, like interleukin (IL)-10, required high receptor occupancy. Weak stimulation of mast cells caused minimal degranulation whereas a half-maximal secretory response was observed for chemokines and, with the exception of TNF-alpha, a weaker cytokine secretory response was observed. The medium from weakly stimulated mast cells elicited a monocyte/macrophage chemotactic response similar to that observed at high receptor occupancy. Weak stimulation also favored the phosphorylation of Gab2 and p38MAPK, while LAT and ERK2 phosphorylation was induced by a stronger stimulus. Gab2-deficient mast cells were severely impaired in chemokine mRNA induction whereas LAT-deficient mast cells showed a more pronounced defect in cytokines. These findings demonstrate that perturbation of small numbers of IgE receptors on mast cells favors certain signals that contribute to a lymphokine response that can mediate allergic inflammation.     

Quercetin inhibits collagen-stimulated platelet activation through inhibition of multiple components of the glycoprotein VI signaling pathway

Summary.  Background : The regulation of platelet function by pharmacological agents that modulate platelet signaling has proven a successful approach to the prevention of thrombosis. A variety of molecules present in the diet have been shown to inhibit platelet activation, including the antioxidant quercetin. Objectives : In this report we investigate the molecular mechanisms through which quercetin inhibits collagen-stimulated platelet aggregation. Methods : The effect of quercetin on platelet aggregation, intracellular calcium release, whole cell tyrosine phosphorylation and intracellular signaling events including tyrosine phosphorylation and kinase activity of proteins involved in the collagen-stimulated glycoprotein (GP) signaling pathway were investigated. Results : We report that quercetin inhibits collagen-stimulated whole cell protein tyrosine phosphorylation and intracellular mobilization of calcium, in a concentration-dependent manner. Quercetin was also found to inhibit various events in signaling generated by the collagen receptor GPVI. This includes collagen-stimulated tyrosine phosphorylation of the Fc receptor γ-chain, Syk, LAT and phospholipase Cγ2. Inhibition of phosphorylation of the Fc receptor γ-chain suggests that quercetin inhibits early signaling events following stimulation of platelets with collagen. The activity of the kinases that phosphorylate the Fc receptor γ-chain, Fyn and Lyn, as well as the tyrosine kinase Syk and phosphoinositide 3-kinase was also inhibited by quercetin in a concentration-dependent manner, both in whole cells and in isolation. Conclusions : The present results provide a molecular basis for the inhibition by quercetin of collagen-stimulated platelet activation, through inhibition of multiple components of the GPVI signaling pathway, and may begin to explain the proposed health benefits of high quercetin intake.     

Identification of the low affinity receptor for immunoglobulin E on mouse mast cells and macrophages as Fc gamma RII and Fc gamma RIII

In addition to their well characterized high affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) mast cells have long been suspected to express undefined Fc receptors capable of binding IgE with low affinity. In this paper, we show that Fc gamma RII and Fc gamma RIII, but not Mac-2, on mouse mast cells and macrophages bind IgE-immune complexes. This binding is efficiently competed by 2.4G2, a monoclonal antibody against the extracellular homologous region of both Fc gamma RII and Fc gamma RIII. Furthermore, IgE-immune complexes bind specifically to Fc gamma RII or Fc gamma RIII transfected into COS-7 cells. The association constants of IgE binding estimated from competition experiments are about 3.1 x 10(5) M-1 for Fc gamma RII, and 4.8 x 10(5) M-1 for Fc gamma RIII. Engagement of Fc gamma RII and Fc gamma RIII with IgE-immune complexes (after blocking access to Fc epsilon RI) or with IgG-immune complexes triggers C57.1 mouse mast cells to release serotonin. This release is inhibited by 2.4G2, and at maximum, reaches 30-40% of the intracellular content, about half of the maximal release (60-80%) obtained after Fc epsilon RI engagement. These data demonstrate that mouse Fc gamma RII and Fc gamma RIII are not isotype specific, and that the binding of IgE-immune complexes to these receptors induces cell activation.     

Different roles for the Fc epsilon RI gamma chain as a function of the receptor context

The high affinity immunoglobulin E receptor (Fc epsilon RI) and the B and T cell antigen receptors (TCR) are multimeric complexes containing subunits with cytoplasmic antigen recognition activation motifs (ARAMs). The presence of multiple motifs may be a way to amplify a single signal or provide independent activation modules. Here we have compared the signaling capacity of the same Fc epsilon RI gamma motif in the context of two different receptors, Fc epsilon RI and TCR/CD3, simultaneously reconstituted on the surface of the same zeta-deficient T cell line. Both reconstituted receptors mediate early (phosphorylation) and late (interleukin [IL]-2 release) signals. Mutation of the two tyrosine residues of ARAM gamma alters early signaling by both receptors, but the set of substrates phosphorylated via ARAM gamma is different for each receptor and is thus dependent on the receptor context. Furthermore, the mutations prevent Fc epsilon RI- but not TCR/CD3-mediated IL-2 release. These data demonstrate that ARAM gamma is necessary for allowing both receptors to phosphorylate the complete set of substrates, and that the CD3 complex, unlike the Fc epsilon RI beta chain, contains activation modules capable of compensating for the absence of a functional ARAM gamma in generating late signals such as IL-2 release.     

Enhanced mast cell activation in mice deficient in the A2b adenosine receptor

Antigen-mediated cross-linking of IgE bound to mast cells via the high affinity receptor for IgE triggers a signaling cascade that results in the release of intracellular calcium stores, followed by an influx of extracellular calcium. The collective increase in intracellular calcium is critical to the release of the granular contents of the mast cell, which include the mediators of acute anaphylaxis. We show that the sensitivity of the mast cell to antigen-mediated degranulation through this pathway can be dramatically influenced by the A2b adenosine receptor. Loss of this Gs-coupled receptor on mouse bone marrow-derived mast cells results in decreased basal levels of cyclic AMP and an excessive influx of extracellular calcium through store-operated calcium channels following antigen activation. Mice lacking the A2b receptor display increased sensitivity to IgE-mediated anaphylaxis. Collectively, these findings show that the A2b adenosine receptor functions as a critical regulator of signaling pathways within the mast cell, which act in concert to limit the magnitude of mast cell responsiveness when antigen is encountered.     

Characterization of Cbl tyrosine phosphorylation and a Cbl-Syk complex in RBL-2H3 cells

Tyrosine phosphorylation of the Cbl protooncogene has been shown to occur after engagement of a number of different receptors on hematopoietic cells. However, the mechanisms by which these receptors induce Cbl tyrosine phosphorylation are poorly understood. Here we demonstrate that engagement of the high affinity IgE receptor (Fc epsilon R1) leads to the tyrosine phosphorylation of Cbl and analyze how this occurs. We show that at least part of Fc epsilon R1-induced Cbl tyrosine phosphorylation is mediated by the Syk tyrosine kinase, and that the Syk-dependent tyrosine phosphorylation of Cbl occurs mainly distal to the Cbl proline-rich region within the COOH-terminal 250 amino acids. Furthermore, we show by coprecipitation that Cbl is present in a complex with Syk before receptor engagement, that the proline-rich region of Cbl and a region of Syk comprised of the two SH2 domains and intradomain linker are required for formation of the complex, and that little or no tyrosine-phosphorylated Cbl is detected in complex with Syk. Overexpression of truncation mutants of Cbl capable of binding Syk has the effect of blocking tyrosine phosphorylation of endogenous Cbl. These results define a potentially important intramolecular interaction in mast cells and suggest a complex function for Cbl in intracellular signaling pathways.     

Expression of functional high affinity immunoglobulin E receptors (Fc epsilon RI) on monocytes of atopic individuals

Suggestive evidence indicates that immunoglobulin E (IgE)-dependent activation of mononuclear phagocytes plays an important pathogenic role in allergic tissue inflammation. Prevailing opinion holds that low affinity IgE receptors are the relevant IgE-binding structures on monocytes/macrophages and that functional events occurring after cross- linking of membrane-bound IgE on these cells are mediated by these receptors. Here we demonstrate that peripheral blood monocytes can bind monomeric IgE via the high affinity IgE receptor (Fc epsilon RI) and that Fc epsilon RI expression on these cells is upregulated in atopic persons. Further, we demonstrate that, upon monocyte adherence to substrate, bridging of monocyte Fc epsilon RI is followed by cell activation. We propose that direct interaction of multivalent allergen with Fc epsilon RI(+)-bound IgE on mononuclear phagocytes results in cell signaling via Fc epsilon RI and that the biological consequences of this event may critically influence the outcome of allergic reactions.     

WIP regulates signaling via the high affinity receptor for immunoglobulin E in mast cells

Wiskott-Aldrich syndrome protein-interacting protein (WIP) stabilizes actin filaments and is important for immunoreceptor-mediated signal transduction leading to actin cytoskeleton rearrangement in T and B cells. Here we report a role for WIP in signaling pathways downstream of the high affinity receptor for immunoglobulin (Ig)E (FcepsilonRI) in mast cells. WIP-deficient bone marrow-derived mast cells (BMMCs) were impaired in their capacity to degranulate and secrete interleukin 6 after FcepsilonRI ligation. Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs. WIP was found to associate with Syk after FcepsilonRI ligation and to inhibit Syk degradation as evidenced by markedly diminished Syk levels in WIP-deficient BMMCs. WIP-deficient BMMCs exhibited no apparent defect in their subcortical actin network and were normal in their ability to form protrusions when exposed to an IgE-coated surface. However, the kinetics of actin changes and the cell shape changes that follow FcepsilonRI signaling were altered in WIP-deficient BMMCs. These results suggest that WIP regulates FcepsilonRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement.     

Platelet endothelial cell adhesion molecule‐1 regulates collagen‐stimulated platelet function by modulating the association of phosphatidylinositol 3‐kinase with Grb‐2‐associated binding protein‐1 and linker for activation of T cells

Summary. Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)γ‐chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3‐kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb‐2‐associated binding protein‐1 (Gab1), which is regulated by binding of the Src homology 2 domain‐containing protein tyrosine phosphatase‐2 (SHP‐2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule‐1 (PECAM‐1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcRγ‐chain signaling via recruitment of SHP‐2 to phosphorylated immunoreceptor tyrosine‐based inhibitory motifs in PECAM‐1. Objective: To investigate the possibility that PECAM‐1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI‐mediated platelet activation in platelets. Methods: The ability of PECAM‐1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM‐1‐associated SHP‐2 in collagen‐stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen‐stimulated PI3K signaling. We therefore propose that PECAM‐1‐mediated inhibition of GPVI‐dependent platelet responses result, at least in part, from recruitment of SHP‐2–p85 complexes to tyrosine‐phosphorylated PECAM‐1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.