Crosstalk between membrane traffic proteins and integrin activation

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)：血小板是止血的核心，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。血小板的分泌是一个多步骤的过程，包括胞浆细胞器的集中提供收缩力，膜运输蛋白控制的膜融合，以及颗粒内容物的释放。血小板整合素αIIbβ3与纤维蛋白原和纤维蛋白等黏附配体相互作用，介导血小板的黏附和聚集，在心脏病发作和中风等血栓性疾病的发生发展中发挥关键作用。这项建议的总体目标是在膜运输蛋白和整合素之间建立联系，这两个事件涉及到血小板功能的两个关键事件：血小板货物释放和整合素激活。VPS33B是Sec1/Munc18蛋白家族中的一员，在胞吐作用中具有多种作用。VPS33B在关节紊乱、肾功能不全、胆汁淤积症(ARC)综合征患者中存在缺陷。这些患者的血小板缺乏α颗粒。为了明确α颗粒在血小板功能和血栓形成中的作用，我们建立了α颗粒缺陷小鼠模型，在该模型中，VPS33B仅在巨核细胞和血小板中缺失。在特定的目标1中，我们将确定VPS33B在αIIbβ3依赖的内吞作用和αIIbβ3自外向内信号转导中的作用。α颗粒含量、vWF和纤维蛋白原在缺乏VPS33B的血小板中显著降低。令人惊讶的是，VPS33B-/-血小板无法收回凝块，并且在纤维蛋白原上的传播存在缺陷。利用CHO重组αIIbβ3激活模型，我们发现VPS33B的过表达显著增强了细胞在纤维蛋白原上的铺展和肌动蛋白的聚合。因此，我们假设，在血小板中，膜运输蛋白和整合素激活之间存在串扰，VPS33B是αIIbβ3自外向内信号的关键贡献者。这一假设将使用血小板特异性VPS33B条件性基因敲除小鼠和其他VPS33B基因敲除小鼠，如全身基因敲除小鼠和中国仓鼠卵巢(CHO)整合素激活模型进行验证。我们的初步数据表明，共聚焦显微镜检测到VPS33B与αIIbβ3共定位，并通过免疫共沉淀检测VPS33B与αIIbβ3形成复合体。在特定的目标2中，我们将使用多种方法来表征VPS33B和α胞质结构域的相互结合决定因素。在特定的目标3中，我们将确定依赖于VPS33B的αIIbβ3自外向内信号的分子机制。这些研究的完成不仅将确定一个新的αIIbβ3结合伙伴，它在αIIbβ3由外向内的信号转导中发挥重要作用，而且还将首次建立膜运输蛋白与血小板整合素激活之间的联系。

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.