RHO GTPASES IN ADHESION/CHEMOATTRACTANT RECEPTOR CROSST

在生理条件下，白细胞在细胞外基质中爬行的氧化反应与分离的白细胞的氧化反应显著不同。 在非粘附细胞中，适度程度的氧化反应被化学引诱物和/或细胞因子迅速刺激。 与此相反，粘附白细胞的反应表现出延长的延迟，随后是显着增强和延长生成的氧代谢产物。 粘附受体（整合素）和启动氧化反应的化学引诱物受体之间的这种串扰的基础尚未确定。来自我们小组的大量数据表明了这样的假设，即这种串扰发生在Rho GTP酶的水平上，Rho GTP酶控制整联蛋白向细胞骨架的信号传导和吞噬细胞NADPH氧化酶的化学引诱物诱导的活化。为了系统地研究粘附调节的氧化反应，我们将确定粘附白细胞中控制Rho GTdR功能和NADPH氧化酶激活的信号通路。 我们将确定是否NADPH氧化酶组装和/或活化在贴壁细胞中被修改。 将使用完整细胞和破碎细胞系统检查单个氧化酶调节蛋白的功能。 在粘附调节的氧化反应期间，将使用新的生物化学测定法测量Rho家族GTP酶的活化。 由粘附引发的信号转导事件将被定量，并与GT酶活性和NADPH氧化酶活化的变化相关。 将使用生物化学手段和遗传方法来检查特定信号传导途径的作用，其中使用病毒表达系统将显性活性或显性阴性突变体引入细胞中。 最后，将使用缺乏特定信号蛋白的转基因小鼠来确认这些途径在调节粘附调节的氧化反应组分中的作用。

The oxidative responses of leukocytes crawling through an extracellular matrix under physiological conditions differ dramatically from those of isolated leukocytes. In nonadherent cells, an oxidative response of modest extent is rapidly stimulated by chemoattractants and/or cytokines. In contrast, the response of adherent leukocytes exhibits a prolonged delay, followed by a dramatically enhanced and prolonged generation of oxygen metabolites. The basis for this crosstalk between adhesion receptors (integrins) and chemoattractant receptors that initiate the oxidative response has not been determined. Substantial data from our group suggests the hypothesis that this crosstalk occurs at the level of Rho GTPases which control both integrin signaling to the cytoskeleton and chemoattractant- induced activation of the phagocyte NADPH oxidase. In order to systematically investigate the adhesion-regulated oxidative response, we will determine the signaling pathways controlling Rho GTPase function and NADPH oxidase activation in adherent leukocytes. We will determine whether NADPH oxidase assembly and/or activation is modified in adherent cells. The function of individual oxidase regulatory proteins will be examined using intact cell and broken cell systems. The activation of Rho family GTPases will be measured during the adhesion-regulated oxidative response using novel biochemical assays. Signal transduction events initiated by adhesion will be quantified and correlated pharmacologically with changes in GTPase activity and NADPH oxidase activation. The role of specific signaling pathways will be examined using biochemical means and genetic approaches in which dominant active or dominant negative mutants will be introduced into cells using a viral expression system. Finally, transgenic mice lacking specific signaling proteins will be used to confirm the roles of these pathways in regulating components of the adhesion-regulated oxidative response.