Selective phosphorylation of threonine residues defines GPR84–arrestin interactions of biased ligands

GPR84 is an immune cell–expressed, proinflammatory receptor currently being assessed as a therapeutic target in conditions including fibrosis and inflammatory bowel disease. Although it was previously shown that the orthosteric GPR84 activators 2-HTP and 6-OAU promoted its interactions with arrestin-3, a G protein–biased agonist DL-175 did not. Here, we show that replacement of all 21 serine and threonine residues within i-loop 3 of GPR84, but not the two serines in the C-terminal tail, eliminated the incorporation of [(32)P] and greatly reduced receptor–arrestin-3 interactions promoted by 2-HTP. GPR84 was phosphorylated constitutively on residues Ser(221) and Ser(224), while various other amino acids are phosphorylated in response to 2-HTP. Consistent with this, an antiserum able to identify pSer(221)/pSer(224) recognized GPR84 from cells treated with and without activators, whereas an antiserum able to identify pThr(263)/pThr(264) only recognized GPR84 after exposure to 2-HTP and not DL-175. Two distinct GPR84 antagonists as well as inhibition of G protein–coupled receptor kinase 2/3 prevented phosphorylation of pThr(263)/pThr(264), but neither strategy affected constitutive phosphorylation of Ser(221)/Ser(224). Furthermore, mutation of residues Thr(263) and Thr(264) to alanine generated a variant of GPR84 also limited in 2-HTP–induced interactions with arrestin-2 and -3. By contrast, this mutant was unaffected in its capacity to reduce cAMP levels. Taken together, these results define a key pair of threonine residues, regulated only by subsets of GPR84 small molecule activators and by GRK2/3 that define effective interactions with arrestins and provide novel tools to monitor the phosphorylation and functional status of GPR84.