M'Rabet L, Coffer P, Zwartkruis F, Franke B, Segal A W, Koenderman L, Bos J L
Laboratory for Physiological Chemistry and Department of Pulmonary Diseases, Utrecht University, Utrecht, The Netherlands; and the University of London Hospital, London, UK.
Blood. 1998 Sep 15;92(6):2133-40.
The small GTPase Rap1 is highly expressed in human neutrophils, but its function is largely unknown. Using the Rap1-binding domain of RalGDS (RalGDS-RBD) as an activation-specific probe for Rap1, we have investigated the regulation of Rap1 activity in primary human neutrophils. We found that a variety of stimuli involved in neutrophil activation, including fMet-Leu-Phe (fMLP), platelet-activating factor (PAF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IgG-coated particles, induce a rapid and transient Rap1 activation. In addition, we found that Rap1 is normally activated in neutrophils from chronic granulomatous disease patients that lack cytochrome b558 or p47phox and have a defective NADPH oxidase system. From these results we conclude that in neutrophils Rap1 is activated independently of respiratory burst induction. Finally, we found that Rap1 is activated by both the Ca2+ ionophore ionomycin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), indicating that phospholipase C (PLC) activation leading to elevated levels of intracellular free Ca2+ and diacylglycerol (DAG) can mediate Rap1 activation. However, inhibition of PLC and Ca2+ depletion only marginally affected fMLP-induced Rap1 activation, suggesting that additional pathways may control Rap1 activation.
小GTP酶Rap1在人中性粒细胞中高表达，但其功能在很大程度上尚不清楚。我们使用RalGDS的Rap1结合结构域（RalGDS-RBD）作为Rap1的激活特异性探针，研究了原代人中性粒细胞中Rap1活性的调节。我们发现，多种参与中性粒细胞激活的刺激因素，包括甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMet-Leu-Phe，fMLP）、血小板活化因子（PAF）、粒细胞-巨噬细胞集落刺激因子（GM-CSF）和IgG包被颗粒，均可诱导Rap1快速且短暂的激活。此外，我们发现，在缺乏细胞色素b558或p47phox且NADPH氧化酶系统存在缺陷的慢性肉芽肿病患者的中性粒细胞中，Rap1通常处于激活状态。从这些结果我们得出结论，在中性粒细胞中，Rap1的激活独立于呼吸爆发的诱导。最后，我们发现Rap1可被钙离子载体离子霉素和佛波酯12-O-十四酰佛波醇-13-乙酸酯（TPA）激活，这表明磷脂酶C（PLC）的激活导致细胞内游离钙离子和二酰甘油（DAG）水平升高可介导Rap1的激活。然而，抑制PLC和耗尽钙离子仅对fMLP诱导的Rap1激活产生轻微影响，这表明可能还有其他途径控制Rap1的激活。
