LAUSR

Graphical abstract Figure. No Caption available. ABSTRACT Agonism of the G protein‐coupled free‐fatty acid receptor‐4 (FFA4) has been shown to promote numerous anti‐inflammatory effects in macrophages that arise due to interaction with &bgr;‐arrestin partner proteins. Humans express functionally distinct short and long FFA4 splice variants, such that FFA4‐S signals through G&agr;q/11 and &bgr;‐arrestin, while FFA4‐L is intrinsically biased solely towards &bgr;‐arrestin signaling. Recently, we and others have shown that phosphorylation of the FFA4 C‐terminal tail is responsible for &bgr;‐arrestin interactability and signaling. Given the significance of &bgr;‐arrestin in the anti‐inflammatory function of FFA4, the goal of this study was to examine the role of the C‐terminal &bgr;‐arrestin phosphosensor in FFA4 signaling induced by PMA and LPS in murine Raw 264.7 macrophages. Our data reveal for the first time that both FFA4 isoforms modulate PMA‐induced ROS generation, and that abolishment of the FFA4‐S, but not FFA4‐L C‐terminal phosphosensor, is detrimental to this effect. Furthermore, we show that while both isoforms reduce PMA‐induced expression of COX‐2, removal of the FFA4‐S phosphosensor significantly decreases this response, suggesting that these effects of FFA4‐S are &bgr;‐arrestin mediated. On the contrary, FFA4‐S, as well as the truncated C‐terminal congener lacking the &bgr;‐arrestin phosphosensor were both able to reduce LPS‐induced NF‐&kgr;B activity and ERK1/2 phosphorylation. However, FFA4‐L and its corresponding mutant were incapable of modulating either, suggesting that these responses are mediated by G protein coupling. Taken together, our data reveal important structure‐function and signaling differences between the two FFA4 isoforms, and for the first time link FFA4 to modulation of ROS in macrophages.