Geijsen N, van Delft S, Raaijmakers J A, Lammers J W, Collard J G, Koenderman L, Coffer P J
Department of Pulmonary Diseases, University Hospital Utrecht, Utrecht, The Netherlands.
Blood. 1999 Aug 1;94(3):1121-30.
The small guanosine triphosphate (GTPase) p21rac is highly expressed in human neutrophils where it is thought to play a role in cytoskeletal reorganization and superoxide production. Using the p21rac binding domain of PAK (PAK-RBD) as an activation-specific probe, we have investigated agonist-stimulated activation of p21rac. Stimulation of neutrophils with the chemoattractants fMet-Leu-Phe (fMLP) or platelet-activating factor (PAF) induced an extremely rapid and transient p21rac activation, being optimal within 5 seconds. This activation correlates with the rapid changes of intracellular free Ca(2+) (Ca(2+)) stimulated by fMLP; however, changes in Ca(2+) were neither sufficient nor required for p21rac activation. Furthermore, fMLP-induced p21rac activation was not inhibited by broad tyrosine kinase inhibitors or specific inhibitors of ERK, p38 mitogen activated protein kinase, Src, or phosphatidylinositol 3-kinases. Surprisingly, the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha did not cause p21rac activation or modulate fMLP-induced p21rac activation. AlF(-), a potent activator of heterotrimeric G-protein alpha-subunits, however, was found to activate p21rac. Stimulation of neutrophils with phorbol myristate acetate (PMA) strongly activated the respiratory burst, but did not induce p21rac activation, suggesting that superoxide production per se can occur independently of p21rac activation. These data suggest that in human granulocytes, G-protein coupled receptors, but not cytokine receptors, activate p21rac via a rapid, novel exchange-mechanism independently of changes in Ca(2+), tyrosine phosphorylation, or PI3K.
小GTP酶（GTPase）p21rac在人类中性粒细胞中高表达，据认为它在细胞骨架重组和超氧化物生成中发挥作用。我们使用PAK的p21rac结合结构域（PAK-RBD）作为激活特异性探针，研究了激动剂刺激下p21rac的激活情况。用趋化因子N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）或血小板活化因子（PAF）刺激中性粒细胞，可诱导p21rac极其快速且短暂的激活，在5秒内达到最佳状态。这种激活与fMLP刺激引起的细胞内游离钙（Ca(2+)）的快速变化相关；然而，Ca(2+)的变化对于p21rac激活既非充分条件也非必要条件。此外，fMLP诱导的p21rac激活不受广泛的酪氨酸激酶抑制剂或ERK、p38丝裂原活化蛋白激酶、Src或磷脂酰肌醇3激酶的特异性抑制剂的抑制。令人惊讶的是，细胞因子粒细胞-巨噬细胞集落刺激因子（GM-CSF）和肿瘤坏死因子-α并未引起p21rac激活，也未调节fMLP诱导的p21rac激活。然而，发现异三聚体G蛋白α亚基的强效激活剂AlF(-)可激活p21rac。用佛波酯（PMA）刺激中性粒细胞可强烈激活呼吸爆发，但未诱导p21rac激活，这表明超氧化物生成本身可以独立于p21rac激活而发生。这些数据表明，在人类粒细胞中，G蛋白偶联受体而非细胞因子受体通过一种快速、新颖的交换机制激活p21rac，该机制独立于Ca(2+)的变化、酪氨酸磷酸化或PI3K。
