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

Sundqvist, M.; Holdfeldt, A.; Wright, S.; Moller, T.; Siaw, E.; Jennbacken, K.; Franzyk, H.; Bouvier, M.; Dahlgren, C.; forsman, h.

doi:10.1101/2020.04.30.070011

bioRxiv · 10.1101/2020.04.30.070011

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

Sundqvist, M.·Holdfeldt, A.·Wright, S.·Moller, T.·Siaw, E.·Jennbacken, K.·Franzyk, H.·Bouvier, M.·Dahlgren, C.·forsman, h.

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Abstract

Formyl peptide receptor 2 (FPR2), a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to {beta}-arrestin recruitment. {beta}-Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of {beta}-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 {beta}- arrestin/AP2 interaction and {beta}-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/{beta}-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 {beta}-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in {beta}-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 reveled 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 {beta}-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of {beta}-arrestin, while Barbadin selectively augments FPR2-mediated neutrophil ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/{beta}-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from human neutrophils.

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Sundqvist, M., Holdfeldt, A., Wright, S., Moller, T., Siaw, E., Jennbacken, K., Franzyk, H., Bouvier, M., Dahlgren, C., forsman, h.. 2020-05-01. Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis. https://doi.org/10.1101/2020.04.30.070011

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