Immunoreceptor Tyrosine-based Activation Motif Receptors Research Articles

The LAT Rheostat as a Regulator of Megakaryocyte Activation.

Specifically positioned negatively charged residues within the cytoplasmic domain of the adaptor protein, linker for the activation of T cells (LAT), have been shown to be important for efficient phosphorylation of tyrosine residues that function to recruit cytosolic proteins downstream of immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling. LAT tyrosine 132-the binding site for PLC-γ2-is a notable exception, preceded instead by a glycine, making it a relatively poor substrate for phosphorylation. Mutating Gly131 to an acidic residue has been shown in T cells to enhance ITAM-linked receptor-mediated signaling. Whether this is generally true in other cell types is not known. To examine whether LAT Gly131 restricts ITAM signaling in cells of the megakaryocyte lineage, we introduced an aspartic acid at this position in human induced pluripotent stem cells (iPSCs), differentiated them into megakaryocytes, and examined its functional consequences. iPSCs expressing G131D LAT differentiated and matured into megakaryocytes normally, but exhibited markedly enhanced reactivity to glycoprotein VI (GPVI)-agonist stimulation. The rate and extent of LAT Tyr132 and PLC-γ2 phosphorylation, and proplatelet formation on GPVI-reactive substrates, were also enhanced. These data demonstrate that a glycine residue at the -1 position of LAT Tyr132 functions as a kinetic bottleneck to restrain Tyr132 phosphorylation and signaling downstream of ITAM receptor engagement in the megakaryocyte lineage. These findings may have translational applications in the burgeoning field of in vitro platelet bioengineering.

Both G protein–coupled and immunoreceptor tyrosine-based activation motif receptors mediate venous thrombosis in mice

AbstractPlatelets are critical in hemostasis and a major contributor to arterial thrombosis (AT). (Pre)clinical studies suggest platelets also contribute to venous thrombosis (VT), but the mechanisms are largely unknown. We hypothesized that in VT, platelets use signaling machinery distinct from AT. Here we aimed to characterize the contributions of platelet G protein–coupled (GPCR) and immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling to VT. Wild-type (WT) and transgenic mice were treated with inhibitors to selectively inhibit platelet-signaling pathways: ITAM-CLEC2 (Clec2mKO), glycoprotein VI (JAQ1 antibody), and Bruton's tyrosine kinase (ibrutinib); GPCR-cyclooxygenase 1 (aspirin); and P2Y12 (clopidogrel). VT was induced by inferior vena cava stenosis. Thrombin generation in platelet-rich plasma and whole-blood clot formation were studied ex vivo. Intravital microscopy was used to study platelet–leukocyte interactions after flow restriction. Thrombus weights were reduced in WT mice treated with high-dose aspirin + clopidogrel (dual antiplatelet therapy [DAPT]) but not in mice treated with either inhibitor alone or low-dose DAPT. Similarly, thrombus weights were reduced in mice with impaired ITAM signaling (Clec2mKO + JAQ1; WT + ibrutinib) but not in Clec2mKO or WT + JAQ1 mice. Both aspirin and clopidogrel, but not ibrutinib, protected mice from FeCl3-induced AT. Thrombin generation and clot formation were normal in blood from high-dose DAPT- or ibrutinib-treated mice; however, platelet adhesion and platelet–neutrophil aggregate formation at the vein wall were reduced in mice treated with high-dose DAPT or ibrutinib. In summary, VT initiation requires platelet activation via GPCRs and ITAM receptors. Strong inhibition of either signaling pathway reduces VT in mice.

Confocal Real-Time Analysis of Cutaneous Platelet Recruitment during Immune Complex‒Mediated Inflammation

Platelets preserve vascular integrity during immune complex‒mediated skin inflammation by preventing neutrophil-provoked hemorrhage. However, the single-cell dynamics of this hemostatic process have never been studied in real-time. To monitor the onset of thrombocytopenia-associated hemorrhages and analyze platelet recruitment, we developed a confocal microscopy‒based video-imaging platform for the dorsal skinfold chamber in living mice. For ultrastructural analysis of recruited platelets, we correlated our imaging approach with serial block-face scanning electron microscopy. We found that bleeding events were transient and occurred preferentially at vascular sites, which were repeatedly penetrated by extravasating neutrophils. Hemorrhage only resumed when previously affected sites were again breached by yet another neutrophil. In non-thrombocytopenic mice, we observed that neutrophil extravasation provoked the recruitment of single platelets to the vessel wall, which required platelet immunoreceptor tyrosine-based activation motif receptors glycoprotein VI and C-type-lectin-like receptor 2. Recruited platelets were found to spread across the endothelial barrier and some even across the basement membrane while retaining their granules. Thus, by visualizing the spatiotemporal dynamics of thrombocytopenia-associated bleeding and platelet recruitment on a single-cell level and in real-time, we provide further insights into how platelets preserve vascular integrity during immune complex‒mediated skin inflammation.

Targeting of C‐type lectin‐like receptor 2 or P2Y12 for the prevention of platelet activation by immunotherapeutic CpG oligodeoxynucleotides

Background Synthetic phosphorothioate-modified CpG oligodeoxynucleotides (ODNs) show potent immunostimulatory properties that are widely exploited in clinical trials of anticancer treatment. Unexpectedly, a recent study indicated that CpG ODNs activate human platelets via the immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor glycoproteinVI. Objective To further analyze the mechanisms of CpG ODN-induced platelet activation and identify potential inhibitory strategies. Methods Invitro analyses were performed on human and mouse platelets, and on cell lines expressing platelet ITAM receptors. CpG ODN platelet-activating effects were evaluated in a mouse model of thrombosis. Results We demonstrated platelet uptake of CpG ODNs, resulting in platelet activation and aggregation. C-type lectin-like receptor2 (CLEC-2) expressed in DT40 cells bound CpG ODNs. CpG ODN uptake did not occur in CLEC-2-deficient mouse platelets. Inhibition of human CLEC-2 with a blocking antibody inhibited CpG ODN-induced platelet aggregation. CpG ODNs caused CLEC-2 dimerization, and provoked its internalization. They induced dense granule release before the onset of aggregation. Accordingly, pretreating platelets with apyrase, or inhibiting P2Y12 with cangrelor or clopidogrel, prevented CpG ODN platelet-activating effect. Invivo, intravenously injected CpG ODN interacted with platelets adhered to mouse injured endothelium, and promoted thrombus growth, which was inhibited by CLEC-2 deficiency or by clopidogrel. Conclusions CLEC-2 and P2Y12 are required for CpG ODN-induced platelet activation and thrombosis, and might be targeted to prevent adverse events in patients at risk.

Platelet immunoreceptor tyrosine‐based activation motif (ITAM) and hemITAM signaling and vascular integrity in inflammation and development

Platelets are essential for maintaining hemostasis following mechanical injury to the vasculature. Besides this established function, novel roles of platelets are becoming increasingly recognized, which are critical in non-injury settings to maintain vascular barrier integrity. For example, during embryogenesis platelets act to support the proper separation of blood and lymphatic vessels. This role continues beyond birth, where platelets prevent leakage of blood into the lymphatic vessel network. During the course of inflammation, platelets are necessary to prevent local hemorrhage due to neutrophil diapedesis and disruption of endothelial cell-cell junctions. Surprisingly, platelets also work to secure tumor-associated blood vessels, inhibiting excessive vessel permeability and intra-tumor hemorrhaging. Interestingly, many of these novel platelet functions depend on immunoreceptor tyrosine-based activation motif (ITAM) signaling but not on signaling via G protein-coupled receptors, which plays a crucial role in platelet plug formation at sites of mechanical injury. Murine platelets express two ITAM-containing receptors: the Fc receptor γ-chain (FcRγ), which functionally associates with the collagen receptor GPVI, and the C-type lectin-like 2 (CLEC-2) receptor, a hemITAM receptor for the mucin-type glycoprotein podoplanin. Human platelets express an additional ITAM receptor, FcγRIIA. These receptors share common downstream effectors, including Syk, SLP-76 and PLCγ2. Here we will review the recent literature that highlights a critical role for platelet GPVI/FcRγ and CLEC-2 in vascular integrity during development and inflammation in mice and discuss the relevance to human disease.

Ibrutinib-Treated Platelets Secure Vascular Integrity in Inflammation

Background: Ibrutinib is a potent and irreversible inhibitor of Bruton's tyrosine kinase (Btk), and has been approved for treatment of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In recent clinical trials, Ibrutinib has shown high efficacy with an excellent safety profile. However, bleeding events have been observed in a subset of patients in several of these trials. While these bleeding events are generally low grade and have not led to interruption or dose reduction of treatment, the origin of bleeding remains incompletely understood. In the setting of thrombocytopenia, inflammation can induce robust vascular permeability and hemorrhage, and our lab has previously shown that immunoreceptor tyrosine-based activation motif (ITAM) signaling is necessary for the protective effects of platelets against hemorrhage. In platelets Btk acts downstream of ITAM receptors, therefore we hypothesized that bleeding in Ibrutinib-treated patients may be, at least in part, the result of hemorrhage at sites of inflammation.Methods: To investigate the possibility that bleeding events in Ibrutinib-treated patients are due to inflammatory hemorrhage, we used two inflammatory models - the dermal reverse passive Arthus reaction (rpA) and P. aeruginosa LPS-induced lung inflammation, both of which have been shown to induce hemorrhage in the absence of platelet ITAM signaling. To selectively inhibit Btk in platelets, thrombocytopenic mice were reconstituted with Ibrutinib- or vehicle-treated wild-type platelets and challenged in rpA or LPS model. Bleeding in the dermis or broncho-alveolar lavage (BAL) fluid was assessed by analysis of hemoglobin. In the clinic, patients generally receive once-a-day doses of 420-560 mg of the Btk inhibitor Ibrutinib, and plasma concentrations can transiently reach 0.5-1 µM. Thus, during their lifetime, circulating platelets are exposed to compounding doses of Ibrutinib, which may have greater inhibitory effects than a single dose, a phenomenon which we have observed in mice (see results below). Therefore, we treated mouse platelets in vitro with 0.5 µM Ibrutinib (low dose, equal to a single dose) or 5 µM Ibrutinib (high dose, comparable to multiple dose exposure) before adoptive transfer into mice.Results: While low dose Ibrutinib did not prevent αIIbβ3 integrin activation induced by collagen or convulxin (Cvx), high dose Ibrutinib abolished collagen-induced aggregation. This dose-dependent inhibition of aggregation was also observed in platelets isolated from mice which had received a single vs. multiple doses of Ibrutinib in vivo. αIIbβ3 activation induced by the stronger GPVI agonist, Cvx, however was only partially impaired, suggesting that Ibrutinib may not completely inhibit ITAM-dependent platelet activation. We next tested whether platelets treated ex vivo with Ibrutinib and transfused into thrombocytopenic mice were able to secure vascular integrity at sites of inflammation. In both models, platelet depletion without transfusion of wild-type platelets induced substantial bleeding. However, transfusion of Ibrutinib-treated platelets was able to rescue bleeding to the same extent as vehicle-treated platelets.Summary: Our findings demonstrate that Ibrutinib-treated platelets are capable of securing vascular integrity at sites of inflammation in mice. Our studies suggest that bleeding events in patients treated with Ibrutinib are not due to inflammation-associated hemorrhage. DisclosuresBergmeier:AHA: Research Funding; NIH: Research Funding.

Abstract 2075: Signaling thresholds and negative B cell selection in acute lymphoblastic leukemia

Abstract Introduction: Unlike other cell types, B cells are selected for an intermediate level of signaling strength. Critical survival and proliferation signals emanate from the (pre-) B cell receptor (BCR): Both attenuation below minimum (e.g. non-functional pre-BCR) and hyperactivation above maximum (e.g. autoreactive pre-BCR) thresholds of signaling strength trigger negative selection and cell death. The oncogenic BCR-ABL1 tyrosine kinase mimics active pre-BCR signaling in Ph+ acute lymphoblastic leukemia (ALL) which defines the ALL subgroup with the worst clinical outcome. Current therapy approaches are largely focused on the development of more potent tyrosine kinase inhibitors (TKI) to suppress oncogenic signaling. However resistance to TKI is developed invariably. Here, we test the hypothesis that targeting hyperactivation above a maximum threshold will selectively kill Ph+ ALL cells, similar to removal of self-reactive B cells. Results: The Ph+ ALL cells don not express ITAM (immunoreceptor tyrosine-based activation motif) receptor Igα or Igβ on the cell surface, indicating defects for a functional pre-BCR. Reconstitution of ITAM receptor was sufficient to induce cell death through increasing pre-BCR signaling strength indicated by phosphorylation of SYK, SRC, BTK and PLCγ2. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ ALL cells in this experimental setting. Surprisingly, patient-derived Ph+ ALL cells express the ITIM (immunoreceptor tyrosine-based inhibitory motif) receptors PECAM1, CD300A and LAIR1 at high levels compared to normal pre-B cells. Importantly, high expression levels of ITIM-receptors are predictive of poor outcome in two clinical trials, including both pediatric and adult ALL patients. Genetic studies revealed that Pecam1, Cd300a and Lair1 were critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases Ptpn6 (Shp1) and Inpp5d (Ship1). Genetic deletion of Lair1, Ptpn6 or Inpp5d in BCR-ABL1 ALL caused cell death in vitro and in vivo through hyperactivation of pre-BCR signaling. Testing various components of proximal pre-BCR signaling, we found that an incremental increase of SYK tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive SYK was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Using chimeric PECAM1, CD300A and LAIR1 receptor decoys and a novel small molecule inhibitor of INPP5D, we demonstrated that pharmacological hyperactivation of pre-BCR signaling and engagement of negative B cell selection represents a promising new strategy to overcome drug-resistance in human Ph+ ALL. Conclusion: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph+ ALL, and identified targeting hyperactivation of pre-BCR signaling as a potential novel class of therapeutic strategy. Note: This abstract was not presented at the meeting. Citation Format: Zhengshan Chen, Seyedmehdi Shojaee, Maike Buchner, Huimin Geng, Jae Woong Lee, Lars Klemm, Eugene Park, Ying Xim Tan, Anne Satterthwaite, Elisabeth Paietta, Stephen P. Hunger, Mignon L. Loh, Jae U. Jung, John E. Coligan, Silvia Bolland, Tak W. Mak, Andre Limnander, Hassan Jumaa, Michael Reth, Arthur Weiss, Clifford A. Lowell, Markus Müschen. Signaling thresholds and negative B cell selection in acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2075. doi:10.1158/1538-7445.AM2015-2075

Impact of the PI3-kinase/Akt pathway on ITAM and hemITAM receptors: Haemostasis, platelet activation and antithrombotic therapy

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated in response to various stimulants, and they regulate many processes including inflammation; the stress response; gene transcription; and cell proliferation, differentiation, and death. Increasing reports have shown that the PI3Ks and their downstream effector Akt are activated by several platelet receptors that regulate platelet activation and haemostasis. Platelets express two immunoreceptor tyrosine based activation motif (ITAM) receptors, collagen receptor glycoprotein VI (GPVI) and Fcγ receptor IIA (FcγRIIA), which are characterized by two YxxL sequences separated by 6–12 amino acids. Activation of an ITAM receptor initiates a reaction cascade via its YxxL sequence in which signaling molecules such as spleen tyrosine kinase (Syk), linker for activation of T cells (LAT) and phospholipase C γ2 (PLCγ2) become activated, leading to platelet activation. Platelets also express another receptor, C-type lectin 2 (CLEC-2), which has a single YxxL sequence, so it is appropriately called a hemITAM receptor. ITAM receptors and the hemITAM receptor share many signaling features. Here we will summarize our current knowledge about how the PI3K/Akt pathway regulates (hem)ITAM receptor-mediated platelet activation and haemostasis and discuss the possible benefits of targeting PI3K/Akt as an antithrombotic therapy.

Platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling and vascular integrity.

Platelets are well-known for their critical role in hemostasis, that is, the prevention of blood loss at sites of mechanical vessel injury. Inappropriate platelet activation and adhesion, however, can lead to thrombotic complications, such as myocardial infarction and stroke. To fulfill its role in hemostasis, the platelet is equipped with various G protein-coupled receptors that mediate the response to soluble agonists such as thrombin, ADP, and thromboxane A2. In addition to G protein-coupled receptors, platelets express 3 glycoproteins that belong to the family of immunoreceptor tyrosine-based activation motif receptors: Fc receptor γ chain, which is noncovalently associated with the glycoprotein VI collagen receptor, C-type lectin 2, the receptor for podoplanin, and Fc receptor γII A, a low-affinity receptor for immune complexes. Although both genetic and chemical approaches have documented a critical role for platelet G protein-coupled receptors in hemostasis, the contribution of immunoreceptor tyrosine-based activation motif receptors to this process is less defined. Studies performed during the past decade, however, have identified new roles for platelet immunoreceptor tyrosine-based activation motif signaling in vascular integrity in utero and at sites of inflammation. The purpose of this review is to summarize recent findings on how platelet immunoreceptor tyrosine-based activation motif signaling controls vascular integrity, both in the presence and absence of mechanical injury.

Loss of the ITAM receptor CD200R1 attenuates HSV-1 infection and encephalitis. (68.15)

Abstract We have discovered a novel role for the Immunoreceptor tyrosine-based activation motif (ITAM) containing receptor CD200R1 in virus infection. The host response to viral pathogens is driven by the activation of innate immune receptors that are triggered by pathogen-associated molecular pattern (PAMPs). These receptors include the transmembrane Toll-like receptors as well as the intracellular nucleic acid sensors RIG-I, Mda5, AIM2 and IFI16. ITAM receptors are known to both attenuate and activate signaling by innate receptors like TLRs but little is known about the potential impact of ITAMs on the outcome of viral infection. We generated CD200R1 knockout (KO) mice and infected with herpes simplex virus (HSV)-1. CD200R1 KO mice were protected from lethal HSV-1 encephalitis with >75% of the CD200R1 KOs surviving compared to 25% of the wild type (WT) mice, i.e., loss of CD200R1 conferred a survival advantage. This was not due to any obvious effects on the inflammatory response to infection. CD200R1 KO and WT mice had equivalent cytokine responses and leukocyte infiltration during the initial infection. Remarkably, HSV-1 virus replication was attenuated in CD200R1 KOs. Thus 50% of the CD200R1 KOs had cleared the virus from their brain by day 3-4 post-infection. In contrast, HSV-1 continued to replicate in the brains of 100% of the WT mice. Infection of cultured cells confirmed that CD200R1 expressing WT cells were more permissive for HSV-1 infection than CD200R1 KO cells.

Multiple ITAM-coupled NK-cell receptors engage the Bcl10/Malt1 complex via Carma1 for NF-κB and MAPK activation to selectively control cytokine production

AbstractNatural killer (NK) cells are innate immune cells that mediate resistance against viruses and tumors. They express multiple activating receptors that couple to immunoreceptor tyrosine-based activation motif (ITAM)–containing signaling chains for downstream cell activation. Ligation of activating NK-cell receptors induces NK-cell cytotoxicity and cytokine release. How these distinct events are selectively controlled is not well defined. Here we report the identification of a specific signaling pathway that operates downstream of the ITAM-coupled NK-cell receptors NK1.1, Ly49D, Ly49H, and NKG2D. Using primary NK cells from Bcl10−/−, Malt1−/−, Carma1−/−, and Card9−/− mice, we demonstrate a key role for Bcl10 signalosomes in the activation of canonical NF-κB signaling as well as JNK and p38 MAPK upon NK-cell triggering. Bcl10 directly cooperates with Malt1 and depends on Carma1 (Card11) but not on Card9 for NK-cell activation. These Bcl10-dependent cascades selectively control cytokine and chemokine production but do not affect NK-cell differentiation or killing. Thus, we identify a molecular basis for the segregation of NK-cell receptor–induced signals for cytokine release and target cell killing and extend the previously recognized roles for CARD-protein/Bcl10/Malt1 complexes in ITAM receptor signaling in innate and adaptive immune cells.

Platelet integrins and immunoreceptors

Stable platelet adhesion to extracellular matrices and the formation of a hemostatic or pathological thrombus are dependent on integrin alphaIIbbeta3, also known as GPIIb-IIIa. However, maximal platelet responses to vascular injury may involve the participation of other integrins expressed in platelets (alphaVbeta3, alpha2beta1, alpha5beta1, and alpha6beta1). Platelet membrane 'immunoreceptors' contain at least one subunit with an extracellular immunoglobulin superfamily domain and/or an intracellular stimulatory immunoreceptor tyrosine-based activation motif (ITAM) or immunoreceptor tyrosine-based inhibitory motif (ITIM). Platelet ITAM receptors, such as FcgammaRIIA and the GPVI-FcRgamma complex, promote activation of integrins, while ITIM receptors, such as platelet-endothelial cell adhesion molecule-1, may promote their inhibition. This review summarizes the structure and function of platelet integrins and immunoreceptors, the emerging functional relationships between these receptor classes, and the consequences of their interaction for platelet function in hemostasis and thrombosis.

The C-type Lectin Receptors CLEC-2 and Dectin-1, but Not DC-SIGN, Signal via a Novel YXXL-dependent Signaling Cascade

The two lectin receptors, CLEC-2 and Dectin-1, have been shown to signal through a Syk-dependent pathway, despite the presence of only a single YXXL in their cytosolic tails. In this study, we show that stimulation of CLEC-2 in platelets and in two mutant cell lines is dependent on the YXXL motif and on proteins that participate in signaling by immunoreceptor tyrosine-based activation motif receptors, including Src, Syk, and Tec family kinases, and on phospholipase Cgamma. Strikingly, mutation of either Src homology (SH) 2 domain of Syk blocks signaling by CLEC-2 despite the fact that it has only a single YXXL motif. Furthermore, signaling by CLEC-2 is only partially dependent on the BLNK/SLP-76 family of adapter proteins in contrast to that of immunoreceptor tyrosine-based activation motif receptors. The C-type lectin receptor, Dectin-1, which contains a YXXL motif preceded by the same four amino acids as for CLEC-2 (DEDG), signals like CLEC-2 and also requires the two SH2 domains of Syk and is only partially dependent on the BLNK/SLP-76 family of adapters. In marked contrast, the C-type lectin receptor, DC-SIGN, which has a distinct series of amino acids preceding a single YXXL, signals independent of this motif. A mutational analysis of the DEDG sequence of CLEC-2 revealed that the glycine residue directly upstream of the YXXL tyrosine is important for CLEC-2 signaling. These results demonstrate that CLEC-2 and Dectin-1 signal through a single YXXL motif that requires the tandem SH2 domains of Syk but is only partially dependent on the SLP-76/BLNK family of adapters.

Vav family proteins are required for optimal regulation of PLCγ2 by integrin αIIbβ3

Vav proteins belong to the family of guanine-nucleotide-exchange factors for the Rho/Rac family of small G-proteins. In addition, they serve as important adapter proteins for the activation of PLCgamma (phospholipase Cgamma) isoforms by ITAM (immunoreceptor tyrosine-based activation motif) receptors, including the platelet collagen receptor GPVI (glycoprotein VI). Vav proteins are also regulated downstream of integrins, including the major platelet integrin alphaIIbbeta3, which has recently been shown to regulate PLCgamma2. In the present study, we have investigated the role of Vav family proteins in filopodia and lamellipodia formation on fibrinogen using platelets deficient in Vav1 and Vav3. Wild-type mouse platelets undergo a limited degree of spreading on fibrinogen, characterized by the formation of numerous filopodia and limited lamellipodia structures. Platelets deficient in Vav1 and Vav3 exhibit reduced filopodia and lamellipodia formation during spreading on fibrinogen. This is accompanied by reduced alphaIIbbeta3-mediated PLCgamma2 tyrosine phosphorylation and reduced Ca(2+) mobilization. In contrast, the G-protein agonist thrombin stimulates full spreading of control and Vav1/3-deficient platelets. Consistent with this, stimulation of F-actin (filamentous actin) formation and Rac activation by thrombin is not altered in Vav-deficient cells. These results demonstrate that Vav1 and Vav3 are required for optimal spreading and regulation of PLCgamma2 by integrin alphaIIbbeta3, but that their requirement is by-passed upon G-protein receptor activation.

Osteopetroses and immunodeficiencies in humans

This review focuses on human and murine pathologies involving both osteoclasts and immune cells. These diseases have been relevant to the discovery of novel interactions and pathways shared between these two types of cells. Interactions between immune cells and osteoclasts were originally shown in murine models by gene targeting of molecules involved in the early steps of osteoclast differentiation, since receptor activator of nuclear factor kappa-B ligand (RANKL), RANK and TNFR-associated factor 6 knockout mice bore abnormalities of both bone resorption and immune system. Subsequently, osteoclast stimulation by RANKL secreted by lymphocytes in autoimmune diseases, such as rheumatoid arthritis, was found. More recently, the identification of immunoreceptor tyrosine-based activation motif receptors and adaptors important for both dendritic cells and osteoclast function has established a link between innate and adaptive immunity and bone. Finally, osteoclasts are also important for hematopoietic stem-cell mobilization, providing a further level of regulation of lymphoid cells. These findings open up a new field of research, osteoimmunology, which will unravel previously unsuspected links between bone remodelling and the immune response.

Differential Interaction of Crkl with Cbl or C3G, Hef-1, and γ Subunit Immunoreceptor Tyrosine-Based Activation Motif in Signaling of Myeloid High Affinity Fc Receptor for IgG (FcγRI)

AbstractCbl-Crkl and Crkl-C3G interactions have been implicated in T cell and B cell receptor signaling and in the regulation of the small GTPase, Rap1. Recent evidence suggests that Rap1 plays a prominent role in the regulation of immunoreceptor tyrosine-based activation motif (ITAM) signaling. To gain insight into the role of Crkl in myeloid ITAM signaling, we investigated Cbl-Crkl and Crkl-C3G interactions following FcγRI aggregation in U937IF cells. FcγRI cross-linking of U937IF cells results in the tyrosine phosphorylation of Cbl, Crkl, and Hef-1, an increase in the association of Crkl with Cbl via direct SH2 domain interaction and increased Crkl-Hef-1 binding. Crkl constitutively binds to the guanine nucleotide-releasing protein, C3G, via direct SH3 domain binding. Our data show that distinct Cbl-Crkl and Crkl-C3G complexes exist in myeloid cells, suggesting that these complexes may modulate distinct signaling events. Anti-Crkl immunoprecipitations demonstrate that the ITAM-containing γ subunit of FcγRI is induced to form a complex with the Crkl protein, and Crkl binds to the cytoskeletal protein, Hef-1. The induced association of Crkl with Cbl, Hef-1, and FcγRIγ after FcγRI activation and the constitutive association between C3G and Crkl provide the first evidence that a FcγRIγ-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells.