FPR2, a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to β-arrestin recruitment. β-Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of β-arrestin in FPR2 endocytosis and in NADPH-oxidase activation in neutrophils, Barbadin was used as a research tool in this study. Barbadin has been shown to bind the clathrin adaptor protein (AP2) and thereby prevent β-arrestin/AP2 interaction and β-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/β-arrestin interaction induced by an FPR2-specific agonist was inhibited by Barbadin. Unexpectedly, however, Barbadin did not inhibit FPR2 endocytosis, indicating that a mechanism independent of β-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in β-arrestin deficient cells, albeit at a diminished level as compared to wild type cells. Dissection of the Barbadin effects on FPR2-mediated neutrophil functions including NADPH-oxidase activation mediated release of reactive oxygen species (ROS) and chemotaxis revealed that Barbadin had no effect on chemotactic migration whereas the release of ROS was potentiated/primed. The effect of Barbadin on ROS production was reversible, independent of β-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of β-arrestin, while Barbadin selectively augments FPR2-mediated ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/β-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from neutrophils.

FPR2是G蛋白偶联受体（GPCR）家族的成员，它介导嗜中性粒细胞迁移，该反应与β-arrestin募集有关。β-arrestin调节GPCR的内吞作用，还可以引发非规范的受体信号传导。为了确定人们对β-抑制蛋白在FPR2内吞和嗜中性粒细胞中NADPH氧化酶激活中的作用了解甚少，巴巴丁被用作这项研究的研究工具。巴巴丁已显示与网格蛋白衔接蛋白（AP2）结合，从而阻止β-arrestin/ AP2相互作用和β-arrestin介导的GPCR内吞作用。与此一致，巴巴丁抑制了由FPR2特异性激动剂诱导的AP2 /β-arrestin相互作用。然而，出乎意料的是，巴巴丁并没有抑制FPR2的内吞作用，这表明独立于β-arrestin/ AP2相互作用的机制可能会维持FPR2的内吞作用。事实证实了这一点，即与野生型细胞相比，FPR2在β-arrestin缺陷型细胞中也经历了激动剂促进的内吞作用，尽管水平有所降低。剖析了巴巴丁对FPR2介导的中性粒细胞功能的影响，包括NADPH-氧化酶激活介导的活性氧（ROS）释放和趋化性表明，巴巴丁对趋化性迁移没有影响，而ROS的释放被增强/引发。Barbadin对ROS产生的作用是可逆的，与β-arrestin的募集无关，与拉古伦菌素A诱导的作用相似。综上，我们的数据表明FPR2的内吞摄取独立于β-arrestin，而Barbadin选择性地增加FPR2-介导的ROS产生，独立于受体内吞作用。