Crosstalk between membrane traffic proteins and integrin activation

膜运输蛋白和整合素激活之间的串扰

基本信息

批准号：
8837170
负责人：
ZHENYU Li
金额：
$ 37.5万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-25 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8837170
关键词：
Actins Acute Adhesions Adhesives Anabolism Arthrogryposis Atherosclerosis Binding Binding Proteins Biochemical Blood Platelets Blood Vessels Cause of Death Cell Adhesion Cell surface Cells Chinese Hamster Chinese Hamster Ovary Cell Cholestasis Co-Immunoprecipitations Coagulation Process Complex Confocal Microscopy Cytomegalovirus Cytoplasmic Granules Cytoplasmic Organelle Cytoplasmic Tail Data Defect Development Disease Endocytosis Epitopes Event Exocytosis Extracellular Matrix Fibrin Fibrinogen Functional disorder Goals Health Hemostatic function Host Defense Human Inflammation Integrin Binding Integrins Kidney Knockout Mice Ligands Link MAP Kinase Gene Malignant Neoplasms Maps Mediating Megakaryocytes Membrane Fusion Membrane Protein Traffic Membrane Proteins Modeling Molecular Mouse Strains Mus Myocardial Infarction Ovary P-Selectin Pathway interactions Patients Phosphorylation Plasma Platelet Activation Play Process Protein Family Proteins Recombinants Research Project Grants Role SRC gene Signal Pathway Signal Transduction Stroke Syndrome Testing Thrombosis Time United States VWF gene Work Wound Healing angiogenesis antimicrobial base extracellular in vivo insight member mouse model mutant novel overexpression polymerization prevent promoter receptor receptor mediated endocytosis recombinase research study rho GTP-Binding Proteins stem syntaxin binding protein 1

项目摘要

DESCRIPTION (provided by applicant): Crosstalk between membrane traffic proteins and integrin activation DESCRIPTION (provided by applicant): Platelets are central to hemostasis because they respond to vascular damage and secrete a variety of granule cargo molecules, which are critical to thrombosis and its sequellae. Platelet secretion is a multistep process involving centralization of cytoplasmic organelles that provides a contractile force, membrane fusion controlled by membrane traffic proteins, and release of granule contents. The platelet integrin, αIIbβ3 interacts with adhesive ligands such as fibrinogen and fibrin and mediates platelet adhesion and aggregation and thus plays a critical role in the development of thrombotic diseases such as heart attack and stroke. The overall goal of this proposal is to establish a link between the membrane traffic proteins and integrins that involve the two key events to platelet function, platelet cargo release and integrin activation. VPS33B is a member of the Sec1/Munc18 protein family that has multiple roles in exocytosis. VPS33B is defective in patients with arthrogryposis, renal dysfunction, cholestasis (ARC) syndrome. Platelets from these patients lack α granules. In an attempt to define the roles of α granules in platelet functio and thrombosis, we produced a mouse model of α granule deficiency, in which VPS33B was deleted only in megakaryocytes and platelets. In Specific Aim 1, we will determine the roles of VPS33B in αIIbβ3 dependent endocytosis and αIIbβ3 outside-in signaling. α granule contents vWF and fibrinogen are significantly reduced in the platelets lacking VPS33B. Surprisingly, VPS33B-/- platelets fail to retract a clot and are defective in spreading on fibrinogen. Using the CHO recombinant αIIbβ3activation model, we showed that overexpression of VPS33B markedly potentiates cell spreading on fibrinogen and actin polymerization. Thus, we hypothesize that in platelets, there exists a crosstalk between the membrane traffic proteins and integrin activation and that VPS33B is a key contributor to αIIbβ3 outside-in signaling. This hypothesis will be tested using the platelet- specific VPS33B conditional knockout mice and other VPS33B knockout mice such as whole-body knockout mice and a Chinese Hamster Ovary (CHO) integrin activation model. Our preliminary data demonstrated that VPS33B co-localizes with αIIbβ3 as detected by confocal microscopy and forms a complex with αIIbβ3 as detected by co-immunoprecipitation. In Specific Aim 2 we will use multiple approaches to characterize the binding determinants in VPS33B and theα cytoplasmic domains for each other. In Specific Aim 3 we will identify the molecular mechanisms of VPS33B-dependent αIIbβ3 outside-in signaling. Completion of these studies will not only identify a novel αIIbβ3 binding partner that plays an important role in αIIbβ3 outside-in signaling, but also for the first time establish a lin between membrane traffic proteins and integrin activation in platelets.

描述（由申请人提供）：膜运输蛋白和整合素激活之间的串扰描述（由申请人提供）：血小板对于止血至关重要，因为它们对血管损伤做出反应并分泌各种颗粒货物分子，这对血栓形成及其后遗症至关重要血小板分泌是细胞质细胞器集中的多步骤过程，涉及收缩力、膜运输蛋白控制的膜融合以及释放血小板整合素αIIbβ3与纤维蛋白原和纤维蛋白等粘附配体相互作用，介导血小板粘附和聚集，因此在心脏病和中风等血栓性疾病的发展中发挥着关键作用。在膜运输蛋白和整合素之间建立联系，涉及血小板功能的两个关键事件，即血小板货物释放和整合素激活。在患有关节弯曲、肾功能障碍、胆汁淤积 (ARC) 综合征的患者中，具有多种胞吐作用的 Sec1/Munc18 蛋白家族存在缺陷。为了研究血小板功能和血栓形成，我们制作了α颗粒缺陷小鼠模型，其中仅在巨核细胞和血小板中删除了VPS33B。具体目标 1，我们将确定 VPS33B 在 αIIbβ3 依赖性内吞作用和 αIIbβ3 由外向内信号传导中的作用，在缺乏 VPS33B 的血小板中，vWF 和纤维蛋白原的含量显着降低。使用 CHO 重组 αIIbβ3 激活模型，我们证明了它们在纤维蛋白原上的扩散存在缺陷。 VPS33B 的过度表达显着增强细胞在纤维蛋白原和肌动蛋白聚合上的扩散，因此，我们发现在血小板中，膜运输蛋白和整合素激活之间存在串扰，并且 VPS33B 是 αIIbβ3 由外向内信号传导的关键贡献者。将使用血小板特异性VPS33B条件敲除小鼠和其他VPS33B敲除小鼠（例如全身敲除小鼠和中国仓鼠）进行测试卵巢 (CHO) 整合素激活模型。我们的初步数据表明，通过共聚焦显微镜检测，VPS33B 与 αIIbβ3 共定位，并通过共免疫沉淀检测，与 αIIbβ3 形成复合物。在特定目标 2 中，我们将使用多种方法来表征结合。在特定目标 3 中，我们将确定 VPS33B 和 α 细胞质结构域的分子机制。 VPS33B依赖性αIIbβ3外向内信号传导的完成不仅将鉴定出在αIIbβ3外向内信号传导中发挥重要作用的新型αIIbβ3结合配偶体，而且将首次在膜交通蛋白和整合素激活之间建立联系。在血小板中。