Modulation of integrin function by cytoplasmic signals

通过细胞质信号调节整合素功能

• 批准号: 6911709
• 负责人: ULHAS P NAIK
• 金额: $ 33.98万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6911709
• 关键词: CHO cells; calcium binding protein; cell differentiation; clinical research; enzyme activity; gene deletion mutation; gene targeting; genetically modified animals; human subject; immunoprecipitation; integrins; laboratory mouse; megakaryocytes; molecular biology; phosphorylation; platelet aggregation; platelets; polymerase chain reaction; protein structure function; serine threonine protein kinase; thrombopoiesis; western blottings

DESCRIPTION (provided by applicant): Blood platelets are anucleated cells with unique receptors that respond to blood vessel injury. One of such receptors is integrin alphaIIb-beta3, the fibrinogen receptor. Integrin alphaIIb-beta3, which is in a low-affinity state on resting platelets, changes its conformation upon platelet activation by an agonist and binds fibrinogen, thus initiating both physiological hemostasis and pathological thrombosis. The affinity of alphaIIb-beta3 to its ligand is modulated by cytoplasmic signals, through a process termed inside-out signaling which involves the propagation of signals from the cytoplasm to the extracellular domain in response to intracellular signaling. The binding of fibrinogen induces a cascade of signals inside the platelet termed outside-in signaling that includes tyrosine phosphorylation, cytoskeletal rearrangement and association of alphaIIb-beta3 with the cytoskeleton. As part of the original project, we have identified CIB (Calcium- and Integrin-Binding) protein that interacts specifically with the cytoplasmic domain of alphaIIb. CIB translocates to the cytoskeleton in an aggregation-dependent manner. CIB, which is present on the membranes of unactivated platelets, accumulates at the filopodia upon platelet activation. CIB associates with integrin alphaIIb-beta3and Plk3, a serine/threonine kinase belonging to the polo-like kinase family. Association of CIB with alphaIIb-beta3 is required for platelet spreading on fibrinogen. CIB overexpression in cells induces multinucleation (polyploidy). The proposed project is aimed at further understanding the role of CIB and Plk3 in the modulation of integrin alphaIIb-beta3 function, which may shed light on the mechanism of regulation of cellular processes such as megakaryocyte differentiation, platelet activation and cell migration. Specific aims of the project are to 1) Study the mechanism of regulation of integrin alphaIIb-beta3 (GPIIb/IIIa) by CIB; 2) Identify the role of Plk3 activity in platelet function; 3) Determine the role of CIB and Plk3 in megakaryocytopoiesis. We plan to use immunological studies in human platelets, a heterologous expression system in CHO cells, and platelets from CIB-null mice to determine the physiological function of CIB in platelet activation. To study the role of CIB and Plk3 in TPO-induced megakaryocytopoiesis, we will use real-time PCR and western blot analysis. We will also characterize the CIB promoter and use CIB-null mice to determine the role of CIB in this process. Since the integrin alphaIIb-beta3 links platelets to each other and to specific components of the vascular wall, it initiates both physiological hemostasis and pathological thrombosis. In addition, alphaIIb-beta3 has been shown to mediate contraction of fibrin clots. Information on the mechanism of activation of the platelet integrin alphaIIb-beta3 will help in the regulation of these processes in platelets and in other cells. Further, characterization of CIB and its interactions with integrin alphaIIb-beta3 and Plk3 may help in delineating the mechanisms by which cytoplasmic signals modulate integrin function.

描述（由申请人提供）：血小板是带有独特受体的无核细胞，对血管损伤有反应。这样的受体之一是纤维蛋白原受体的整联蛋白alphaiib-beta3。整合素α-beta3在静止血小板上处于低亲和力状态，改变了其对血小板激活的构象，并结合纤维蛋白原，从而启动生理止血和病理血栓形成。 Alphaiib-Beta3与其配体的亲和力通过胞质信号调节，该过程通过称为内外信号传导的过程，该过程涉及响应细胞内信号的信号从细胞质到细胞外域的传播。纤维蛋白原的结合诱导了称为外部信号传导的血小板内部的一系列信号，其中包括酪氨酸磷酸化，细胞骨架重排和alphaiib-Beta3与细胞骨架的结合。作为原始项目的一部分，我们已经确定了与Alphaiib的细胞质结构域特别相互作用的CIB（钙和整合素结合）蛋白。 CIB以聚集依赖性方式转移到细胞骨架上。存在于未激活血小板的膜上的CIB在血小板激活后积聚在丝状虫。 CIB与整合素alphaiib-Beta3and PLK3相关，这是一种属于类似马球的激酶家族的丝氨酸/苏氨酸激酶。 CIB与Alphaiib-Beta3的关联是血小板扩散在纤维蛋白原上所必需的。细胞中的CIB过表达诱导多核（多倍体）。所提出的项目旨在进一步了解CIB和PLK3在整合素Alphaiib-Beta3功能调节中的作用，该功能可能阐明了调节细胞过程的机制，例如巨核细胞分化，血小板激活和细胞迁移。该项目的具体目的是1）研究CIB的整合素alphaiib-Beta3（GPIIB/IIIA）调节的机理； 2）确定PLK3活性在血小板功能中的作用； 3）确定CIB和PLK3在巨核细胞的作用。我们计划在人血小板中使用免疫学研究，CHO细胞中的异源表达系统以及CIB无小鼠的血小板来确定CIB在血小板激活中的生理功能。为了研究CIB和PLK3在TPO诱导的巨核细胞的作用，我们将使用实时PCR和Western blot分析。我们还将表征CIB启动子，并使用CIB-NULL小鼠来确定CIB在此过程中的作用。由于整联蛋白alphaiib-Beta3将血小板彼此连接到血管壁的特定组成部分，因此它启动了生理止血和病理血栓形成。另外，已证明α-beta3可以介导纤维蛋白血块的收缩。有关血小板整合素α-beta3激活机制的信息将有助于调节血小板和其他细胞中的这些过程。此外，CIB及其与整合素alphaiib-Beta3和PLK3的相互作用的表征可能有助于描述细胞质信号调节整合素功能的机制。