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Growth factor receptor-bound protein 2 contributes to (hem)immunoreceptor tyrosine-based activation motif-mediated signaling in platelets.

ABSTRACT: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity but may also cause pathological vessel occlusion. One major pathway of platelet activation is triggered by 2 receptors that signal through an (hem)immunoreceptor tyrosine-based activation motif (ITAM), the activating collagen receptor glycoprotein (GP) VI and the C-type lectin-like receptor 2 (CLEC-2). Growth factor receptor-bound protein 2 (Grb2) is a ubiquitously expressed adapter molecule involved in signaling processes of numerous receptors in different cell types, but its function in platelets and MKs is unknown.We tested the hypothesis that Grb2 is a crucial adapter protein in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets.Here, we show that genetic ablation of Grb2 in MKs and platelets did not interfere with MK differentiation or platelet production. However, Grb2-deficiency severely impaired glycoprotein VI-mediated platelet activation because of defective stabilization of the linker of activated T-cell (LAT) signalosome and activation of downstream signaling proteins that resulted in reduced adhesion, aggregation, and coagulant activity on collagen in vitro. Similarly, CLEC-2-mediated signaling was impaired in Grb2-deficient platelets, whereas the cells responded normally to stimulation of G protein-coupled receptors. In vivo, this selective (hem)immunoreceptor tyrosine-based activation motif signaling defect resulted in prolonged bleeding times but affected arterial thrombus formation only after concomitant treatment with acetylsalicylic acid, indicating that defective glycoprotein VI signaling in the absence of Grb2 can be compensated through thromboxane A2-induced G protein-coupled receptor signaling pathways.These results reveal an important contribution of Grb2 in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets in hemostasis and thrombosis by stabilizing the LAT signalosome.

SUBMITTER: Dutting S

PROVIDER: S-EPMC4405766 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Growth factor receptor-bound protein 2 contributes to (hem)immunoreceptor tyrosine-based activation motif-mediated signaling in platelets.

Dütting Sebastian S Vögtle Timo T Morowski Martina M Schiessl Sarah S Schäfer Carmen M CM Watson Stephanie K SK Hughes Craig E CE Ackermann Jochen A JA Radtke Daniel D Hermanns Heike M HM Watson Steve P SP Nitschke Lars L Nieswandt Bernhard B

Circulation research 20131121 3

<h4>Rationale</h4>Platelets are anuclear cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity but may also cause pathological vessel occlusion. One major pathway of platelet activation is triggered by 2 receptors that signal through an (hem)immunoreceptor tyrosine-based activation motif (ITAM), the activating collagen receptor glycoprotein (GP) VI and the C-type lectin-like receptor 2 (CLEC-2). Growth factor receptor-bound protein 2 (Grb2) is a ubiquitou ...[more]

PMID: 24265393

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Project description:INTRODUCTION: The immunoreceptor tyrosine-based activation motif (ITAM) pathway provides osteoclast co-stimulatory signals and regulates proliferation, survival and differentiation of effector immune cells. In the osteoclast, the receptors Triggering Receptor Expressed on Myeloid cells 2 (TREM2) and Osteoclast Associated Receptor (OSCAR) and their respective adaptor proteins, DAP12 and FcR? mediate ITAM signals and induce calcium signaling and the crucial transcription factor, NFATc1. In rheumatoid arthritis (RA), OSCAR expression by monocytes is inversely correlated with disease activity. Additionally, serum levels of OSCAR are reduced in RA patients versus healthy controls suggesting that expression and secretion or cleavage of soluble (s) OSCAR is immune modulated. Recent data suggest that endothelial cells may also be a source of OSCAR. METHODS: ITAM receptors, their adaptor proteins, and NFATc1 and cathepsin K were detected in human synovial tissues by immunohistochemistry. Synovial tissues from patients with active RA were compared with tissue from patients in remission, osteoarthritis (OA) patients and healthy individuals. OSCAR was measured by immunoassay in synovial fluids recovered from active RA and OA patients. Endothelial cells were cultured with or without 5 ng/mL TNF-? or IL-1? over 72 hours. Temporal expression of OSCAR mRNA was assessed by qRT PCR and OSCAR protein in the supernatant was measured by ELISA. RESULTS: Significantly higher (P < 0.05) NFATc1-positive inflammatory cell aggregates were found in active RA tissues than in healthy synovial tissue. Similarly, the percentage of OSCAR, FcR?, DAP12 and TREM2 positive cells was significantly higher in active RA tissues compared to the healthy synovial tissue. Notably, OSCAR was strongly expressed in the microvasculature of the active RA tissues (9/9), inactive RA (8/9) weakly in OA (4/9) but only in the lumen of healthy synovial tissue (0/8). OSCAR levels were detected in synovial fluids from both RA (47 to 152 ng/mL) and OA (112 to 145 ng/mL) patients. Moreover, OSCAR mRNA expression and soluble OSCAR release was stimulated by TNF-? and IL1-? in cultured endothelial cells. CONCLUSIONS: Increased levels of ITAM related factors were present in synovial tissue from active RA joints compared to OA and healthy joints. OSCAR was strongly expressed by the vasculature of active RA patients and membrane bound and soluble OSCAR was stimulated by inflammatory mediators in endothelial cells in vitro.

Project description:The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing importance of mutant mouse models in establishing protein function in platelets. This approach identified all of the major classes of platelet transmembrane receptors, including multitransmembrane proteins. Strikingly 17 of the 25 most megakaryocyte-specific genes (relative to 30 other serial analysis of gene expression libraries) were transmembrane proteins, illustrating the unique nature of the megakaryocyte/platelet surface. The list of novel plasma membrane proteins identified using proteomics includes the immunoglobulin superfamily member G6b, which undergoes extensive alternate splicing. Specific antibodies were used to demonstrate expression of the G6b-B isoform, which contains an immunoreceptor tyrosine-based inhibition motif. G6b-B undergoes tyrosine phosphorylation and association with the SH2 domain-containing phosphatase, SHP-1, in stimulated platelets suggesting that it may play a novel role in limiting platelet activation.

Dataset Information

Growth factor receptor-bound protein 2 contributes to (hem)immunoreceptor tyrosine-based activation motif-mediated signaling in platelets.

Publications

Growth factor receptor-bound protein 2 contributes to (hem)immunoreceptor tyrosine-based activation motif-mediated signaling in platelets.

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