Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis

Biochim Biophys Acta Mol Cell Res. 2020 Dec;1867(12):118849. doi: 10.1016/j.bbamcr.2020.118849. Epub 2020 Sep 8.

Abstract

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.

Keywords: AP2; Actin cytoskeleton; Barbadin; Desensitization; Endocytosis; FPR2; G protein-coupled receptors; Neutrophils; Reactive oxygen species; Resensitization; β-Arrestin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Protein Complex 2 / chemistry
  • Adaptor Protein Complex 2 / genetics
  • Clathrin / chemistry
  • Endocytosis / drug effects
  • Endocytosis / genetics*
  • HEK293 Cells
  • Humans
  • NADPH Oxidases / genetics
  • Neutrophils / drug effects
  • Protein Binding / drug effects
  • Pyrimidines / chemistry
  • Pyrimidines / pharmacology*
  • Reactive Oxygen Species / metabolism
  • Receptors, Formyl Peptide / chemistry
  • Receptors, Formyl Peptide / genetics*
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, Lipoxin / chemistry
  • Receptors, Lipoxin / genetics*
  • Signal Transduction / drug effects
  • beta-Arrestin 1 / chemistry
  • beta-Arrestin 1 / genetics*

Substances

  • Adaptor Protein Complex 2
  • Clathrin
  • FPR2 protein, human
  • Pyrimidines
  • Reactive Oxygen Species
  • Receptors, Formyl Peptide
  • Receptors, G-Protein-Coupled
  • Receptors, Lipoxin
  • barbadin
  • beta-Arrestin 1
  • NADPH Oxidases