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

GPR84 is an immune cell-expressed, pro-inflammatory 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 GRP84, but not the 2 serines in the C-terminal tail, eliminated incorporation of [32P] and greatly reduced receptor-arrestin-3 interactions promoted by 2-HTP. We demonstrate that GPR84 was phosphorylated constitutively on residues Ser221 and Ser224 while various other amino acids are phosphorylated in response to 2-HTP. Consistent with this, an antiserum able to identify pSer221/pSer224 recognized GPR84 from cells treated with and without activators, whereas an antiserum able to identify pThr263/pThr264 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 pThr263/pThr264, but neither strategy affected constitutive phosphorylation of Ser221/Ser224. Furthermore, mutation of residues Thr263 and Thr264 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