Role of Carboxyterminal Hypervariable Region in Rap1b Function

描述(由申请人提供)：Rap1b是一种普遍存在的膜相关低分子GTP酶，已被证明对整合素介导的血小板聚集和黏附具有重要作用。Rap1b的作用机制似乎是通过导致整合素激活的信号级联来实现的。缺乏Rap1b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，Rap1b的膜结合对其功能是重要的。这项工作的目的是更详细地探讨Rap1b的膜相互作用，以便更好地了解其作用机制。Rap1b与膜的结合是通过其C-末端高变区，该区域包含被认为与膜磷脂相互作用的多碱序列，翻译后添加的与膜相关的戊烯基基，以及疏水的C-末端羧甲基，也与膜相关。当升高cAMP水平的药物抑制血小板时，高变区也是cAMP(CAMP)依赖的蛋白激酶(PKA)磷酸化的部位。利用影响多碱基簇结构以及羧甲基化、苯基化和磷酸化位点的各种高变区突变，我们将研究这些不同的突变对Rap1b的亚细胞定位和功能的作用，高变区与特定光脂的相互作用，以及高变区与其他蛋白质的相互作用。这些研究的结果将有助于更好地理解GTP酶的一般工作原理，以及RAP1b如何在血小板中调节整合素的激活，并可能导致抑制血小板功能的新方法。公共卫生相关性：当血管损伤时，血小板是第一反应者。这个应用程序的总体目标是了解血小板对这种损伤的反应，以及与其反应有关的机制。这些发现将为人体如何治愈出血损伤以及血液凝结状况的根本原因提供洞察。

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.