REGULATION OF GP 11B 111A BY THE PLATELET CYTOSKELETON

描述(改编自研究人员摘要)：糖蛋白 (GP)IIb-IIIa复合体介导纤维蛋白原与血小板的结合 因此在诱导血小板形成方面起着重要作用。 集合体。在未经刺激的血小板上，GPIIb-IIIa不能结合 纤维蛋白原。当血小板被激活时，细胞内不明原因 这些事件导致GPIIb-IIIa的胞外结构域发生变化 使其与纤维蛋白原结合。这就是所谓的“由内而外”的信号。 纤维蛋白原的结合导致细胞内事件的诱导 例如酪氨酸残基上特定蛋白质的磷酸化， 钙蛋白酶的激活和特定细胞骨架蛋白的水解性。 这就是所谓的“自外而内”信令。两个人的方式 通过GPIIb-IIIa发送信号的方式尚不清楚。 GPIIb-IIIa是黏附受体整合素家族的一员。在……里面 培养的细胞，有证据表明整合素可以与 与细胞骨架相互作用，这种相互作用调节功能 整合素的活性。人们对这种可能性知之甚少 细胞骨架调控整合素的功能活性 其他细胞。在初步实验中，调查人员提供了 1)GPIIb-IIIa与膜骨架有关的证据 未刺激的血小板，2)酪氨酸激酶，pp60c-src，似乎 与血小板的膜骨架有关，目前还没有 有能力结合纤维蛋白原，以及几种成为 当血小板被激活时，酪氨酸残基被快速磷酸化 与洗涤剂裂解液中的膜骨架共隔离；3)结合 纤维蛋白原驱动GPIIb-IIIa和相关的膜骨架 与细胞质肌动蛋白细丝相关的蛋白质，GPIIb-IIIa 簇，额外的细胞骨架蛋白被磷酸化 酪氨酸残基和额外的信号分子(例如蛋白质 激酶C和磷脂酰肌醇3-激酶)被招募到整合素- 丰富的骨骼结构；4)血小板与细胞松弛素的孵育 抑制整合素的双向信号传递。这些发现是 与细胞骨架调节的假设一致 血小板内GPIIb-IIIa的功能活性。在这方面的实验 建议的目的是确定GPIIb-IIIa 与细胞骨架相关联，并使用此信息 直接检验互动(S)是必不可少的假设 调节GPIIb-IIIa复合体的功能活动。这个 拟议研究的具体目的是：1)描述 GPIIb-IIIa与细胞骨架蛋白的相互作用 已知整合素在体外相互作用；2)识别其他 与GPIIb-IIIa细胞质结构域相互作用的蛋白质；以及3) 阐明细胞骨架蛋白在双向调节中的作用 通过GPIIb-IIIa发送信号。福克斯博士预计，这些研究 将深入了解跨 整合素的多样性，并可能导致理解 整合素功能可能受到抑制。

DESCRIPTION (Adapted from investigator's abstract): The glycoprotein (GP) IIb-IIIa complex mediates the binding of fibrinogen to platelets and thus plays a major role in inducing the formation of platelet aggregates. On unstimulated platelets, GPIIb-IIIa cannot bind fibrinogen. Upon activation of platelets, unidentified intracellular events cause a change in the extracellular domain of GPIIb-IIIa that allows it to bind fibrinogen. This is known as "inside-out" signaling. Binding of fibrinogen leads to the induction of intracellular events such as phosphorylation of specific proteins on tyrosine residues, activation of calpain and hydrolysis of specific cytoskeletal proteins. This is referred to as "outside-in" signaling. The way in which the two way signaling across GPIIb-IIIa occurs is not understood. GPIIb-IIIa is a member of the integrin family of adhesion receptors. In cultured cells, evidence has been provided that integrins can associate with the cytoskeleton and that this interaction regulates functional activities of the integrins. Little is known about the possibility that the cytoskeleton regulates the functional activities of integrins in other cells. In preliminary experiments, the investigators have provided evidence that 1) GPIIb-IIIa is associated with the membrane skeleton in unstimulated platelets, 2) the tyrosine kinase, pp60c-src, appears to be associated with the membrane skeleton of platelets that are not yet competent to bind fibrinogen, and several of the proteins that become rapidly phosphorylated on tyrosine residues when platelets are activated co-isolate with the membrane skeleton in detergent lysates; 3) binding of fibrinogen drives GPIIb-IIIa and associated membrane skeleton proteins into association with cytoplasmic actin filaments, GPIIb-IIIa clusters, additional cytoskeletal proteins become phosphorylated on tyrosine residues, and additional signaling molecules (e.g., protein kinase C and phosphoinositide 3-kinase) are recruited to the integrin- rich skeletal structure; 4) incubation of platelets with cytochalasins inhibits the two way signaling across the integrin. These findings are consistent with the hypothesis that the cytoskeleton regulates functional activities of GPIIb-IIIa in platelets. Experiments in this proposal are designed to identify the mechanism(s) by which GPIIb-IIIa associates with the cytoskeleton, and to use this information to directly test the hypothesis that the interaction(s) is essential for regulating functional activities of the GPIIb-IIIa complex. The Specific Aims of the proposed research are 1) to characterize interactions between GPIIb-IIIa and cytoskeletal proteins with which the integrin is known to interact in vitro; 2) to identify additional proteins that interact with the cytoplasm domains of GPIIb-IIIa; and 3) to elucidate the role of cytoskeletal proteins in regulating two way signaling across GPIIb-IIIa. Dr. Fox anticipates that these studies will provide insight into mechanisms involved in signaling across a variety of integrins, and may lead to understanding of ways in which integrin functions could be inhibited.