Role of Carboxyterminal Hypervariable Region in Rap1b Function

描述（由申请方提供）：Rap 1b是一种普遍存在的膜相关低分子量GTdR，已证明其对整合素介导的血小板聚集和粘附很重要。rap 1b的作用机制似乎是通过一个信号级联，导致整合素的激活。rap 1b缺陷的小鼠对所有测试的激动剂都有异常的血小板反应。与所有低分子量GTP酶一样，rap 1b的膜结合对其功能很重要。这项工作的目的是更详细地探索rap 1b的膜相互作用，以更好地了解其作用机制。rap 1b通过其C-末端高变区连接到膜上，该区域包含被认为与膜磷脂相互作用的多元序列，与膜相关的后-分离添加的异戊二烯基，以及也与膜相关的疏水C-末端羧甲基。当血小板被增加cAMP水平的药物抑制时，高变区也是环AMP（cAMP）依赖性蛋白激酶（PKA）磷酸化的位点。使用各种高变区突变，影响的结构的多元簇和羧甲基化，异戊烯化和磷酸化的网站，我们将研究这些不同的moeities的作用，rap 1b的亚细胞定位和功能，高变区与特定的磷脂的相互作用，和高变区与其他蛋白质的相互作用。这些研究的结果将导致更好地了解GTP酶一般如何工作，rap 1b如何在血小板中调节整合素活化，并可能导致抑制血小板功能的新方法。公共卫生相关性：当血管受伤时，血小板是第一反应者。本申请的总体目标是了解血小板如何对这种损伤作出反应，以及与其反应有关的机制。这些发现将提供深入了解身体如何愈合出血损伤，以及血液凝固条件的根本原因。

DESCRIPTION (provided by applicant): Rap1b is a ubiquitous, membrane associated low molecular weight GTPase that has been shown to be important for integrin-mediated platelet aggregation and adhesion. The mechanism of rap1b action appears to be through a signaling cascade that leads to activation of integrins. Mice deficient in rap1b have abnormal platelet responses to all agonists tested. As with all low molecular weight GTPases, the membrane association of rap1b is important for its function. The purpose of the work in this proposal is to explore the membrane interactions of rap1b in more detail in order to better understand its mechanism of action. The attachment of rap1b to the membrane is through its C-terminal hypervariable region, a region that contains polybasic sequences believed to interact with membrane phospholipids, a post-translationally added prenyl group that associates with membranes, and a hydrophobic C-terminal carboxymethyl group that also associates with the membrane. The hypervariable region is also the site of phosphorylation by cyclic AMP (cAMP)-dependent protein kinase (PKA) when platelets are inhibited by agents that increase cAMP levels. Using various hypervariable region mutations that affect the structure of the polybasic cluster and the sites of carboxymethylation, prenylation, and phosphorylation, we will examine the role of these various moeities on the subcellular localization and function of rap1b, the interaction of the hypervariable region with specific phoshoplipids, and the interaction of the hypervariable region with other proteins. The results of these studies will lead to an improved understand of how GTPases work in general, how rap1b functions in platelets to regulate integrin activation, and may lead to new methods for inhibiting platelet function. PUBLIC HEALTH RELEVANCE: Platelets are the first responders when there is an injury to a blood vessel. The overall goal of this application is to understand how platelets respond to such an injury, as well as the mechanisms involved with their response. These findings will provide insight into how the body heals bleeding injuries, as well as the underlying cause of blood clotting conditions.