Effects of phosphodefective FFAR4 C‐terminal mutants on COX‐2 expression in macrophages

Macrophages have critical roles in the initiating, maintaining, and resolving inflammation, and mediate this response partially through expression of cyclooxygenase‐2 (COX‐2), which generates prostaglandins (PG). Free‐fatty acid receptor‐4 (FFAR4), a G protein‐coupled receptor that was previously termed GPR120, has been shown to play important roles in regulating macrophage function to produce robust anti‐inflammatory effects. In this regard, agonism of FFAR4 with docosahexaenoic acid (DHA) has previously been shown to reduce expression of COX‐2 and subsequent PGE2 synthesis. Our previous work, as well as that of others, has shown that FFAR4 signaling is regulated by phosphorylation of the receptor on its C‐terminal tail and that mutation of these phosphorylation sites diminishes the receptors ability to interact with β‐arrestin partner proteins. The goal of the current study was to assess the role of the C‐terminal tail of FFAR4 in regulating COX2 expression and PGE2 synthesis. Here, we have created C‐terminal truncated mutants of both the short (FFAR4‐S) and long (FFAR4‐L) isoforms of FFAR4 and stably expressed them in murine RAW264.7 macrophages. Our results show that stable expression of wild‐type (WT) FFAR4‐S in RAW cells significantly reduces COX2 expression, while expression of the C‐terminal truncated mutant of FFAR4‐S does not affect COX2 expression compared to control RAW macrophages. Meanwhile, stable expression of WT‐FFAR4‐L, which has previously been shown to be intrinsically biased solely towards β‐arrestin signaling, also resulted in reduced COX‐2 expression, while expression of the truncated FFAR4‐L mutant reversed this effect. These results suggest that C‐terminal phosphorylation and subsequent β‐arrestin recruitment are integral in FFAR4‐mediated alteration of COX2 expression in macrophages. Support or Funding Information Supported by NIH/NIDDK grant DK098730 to N.H.M.