REGULATION OF GP 11B 111A BY THE PLATELET CYTOSKELETON

描述（改编自研究者摘要）：糖蛋白 (GP)IIb-IIIa复合物介导纤维蛋白原与血小板的结合 因此在诱导血小板形成中起主要作用 集料.在未受刺激的血小板上，GPIIb-IIIa不能结合 纤维蛋白原。血小板活化后，未鉴定的细胞内 事件引起GPIIb-IIIa的细胞外结构域的变化， 使其与纤维蛋白原结合。这被称为“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 如酪氨酸残基上特定蛋白质的磷酸化， 钙蛋白酶的活化和特定细胞骨架蛋白的水解。 这被称为“由外向内”信令。两个人的方式 跨GPIIb-IIIa的信令发生的方式尚不清楚。 GPIIb-IIIa是粘附受体的整合素家族的成员。在 在培养的细胞中，已有证据表明整合素可以与 这种相互作用调节了细胞的功能 整合素的活性。人们对这种可能性知之甚少， 细胞骨架调节整合素的功能活性， 其他细胞。在初步实验中，研究人员提供了 证据表明：1）GPIIb-IIIa与细胞膜骨架有关， 未受刺激的血小板，2）酪氨酸激酶，pp 60 c-src，似乎 与血小板的膜骨架有关， 有能力结合纤维蛋白原，和几种蛋白质， 血小板活化时酪氨酸残基迅速磷酸化 与去污剂裂解物中的膜骨架共分离; 3）结合 纤维蛋白原驱动GPIIb-IIIa和相关的膜骨架 与细胞质肌动蛋白丝结合的蛋白质，GPIIb-IIIa 簇，额外的细胞骨架蛋白成为磷酸化 酪氨酸残基，和另外的信号分子（例如，蛋白 激酶C和磷酸肌醇3-激酶）被募集到整合素- 丰富的骨架结构; 4）用细胞松弛素孵育血小板 抑制整合素的双向信号传导。这些发现 这与细胞骨架调节 血小板中GPIIb-IIIa的功能活性。在这方面的实验 建议旨在确定GPIIb-IIIa 与细胞骨架相关，并利用这些信息 直接检验假设，即相互作用对于 调节GPIIb-IIIa复合物的功能活性。的 拟议研究的具体目标是：1）表征 GPIIb-IIIa和细胞骨架蛋白之间的相互作用， 已知整联蛋白在体外相互作用; 2）鉴定另外的 与GPIIb-IIIa的细胞质结构域相互作用的蛋白质;和3） 为了阐明细胞骨架蛋白在调节两种途径中的作用， 通过GPIIb-IIIa进行信号传输。福克斯博士预计这些研究 将提供深入了解机制涉及信号通过一个 各种整合素，并可能导致理解的方式， 整联蛋白功能可能受到抑制。

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.