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

Abstract

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 [32P] and greatly reduced receptor–arrestin-3 interactions promoted by 2-HTP. 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. 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 [32P] and greatly reduced receptor–arrestin-3 interactions promoted by 2-HTP. 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. GPR84 is a nominally orphan G protein–coupled receptor (GPCR) that can be activated by both medium-chain fatty acids (MCFAs) and a range of synthetic ligands that act as either orthosteric or allosteric activators (1Marsango S. Barki N. Jenkins L. Tobin A.B. Milligan G. Therapeutic validation of an orphan G protein-coupled receptor: The case of GPR84.Br. J. Pharmacol. 2020; https://doi.org/10.1111/bph.15248Crossref PubMed Scopus (7) Google immune response 2020; PubMed Scopus Google of the receptor by and 2020; PubMed Scopus Google The of a of both and immune cells J. N. L. fatty acids as ligands orphan G protein-coupled receptor PubMed Scopus Google S. fatty GPR84, is a proinflammatory PubMed Scopus Google L. L. L. of the receptor GPR84 and in PubMed Scopus Google Jenkins L. Tobin A.B. Milligan G. and of novel orthosteric and allosteric activators of PubMed Scopus Google and that and to the receptor are in response to proinflammatory L. L. L. of the receptor GPR84 and in PubMed Scopus Google Jenkins L. Tobin A.B. Milligan G. and of novel orthosteric and allosteric activators of PubMed Scopus Google promoted in GPR84 as a therapeutic target in a range of (1Marsango S. Barki N. Jenkins L. Tobin A.B. Milligan G. Therapeutic validation of an orphan G protein-coupled receptor: The case of GPR84.Br. J. Pharmacol. 2020; https://doi.org/10.1111/bph.15248Crossref PubMed Scopus (7) Google immune response 2020; PubMed Scopus Google both and fibrosis (1Marsango S. Barki N. Jenkins L. Tobin A.B. Milligan G. Therapeutic validation of an orphan G protein-coupled receptor: The case of GPR84.Br. J. Pharmacol. 2020; https://doi.org/10.1111/bph.15248Crossref PubMed Scopus (7) Google S. L. G. L. S. S. of a GPR84 allosteric in a 2020; PubMed Scopus Google Although both these on the and of ligands (1Marsango S. Barki N. Jenkins L. Tobin A.B. Milligan G. Therapeutic validation of an orphan G protein-coupled receptor: The case of GPR84.Br. J. Pharmacol. 2020; https://doi.org/10.1111/bph.15248Crossref PubMed Scopus (7) Google of the receptor by and 2020; PubMed Scopus Google S. L. G. L. S. S. of a GPR84 allosteric in a 2020; PubMed Scopus Google it that of GPR84 also the of J. S. 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S. of ligands orphan G protein-coupled PubMed Scopus Google show both 2-HTP and 6-OAU effective phosphorylation of GPR84 residues within the neither DL-175 of and DL-175 as orthosteric of GPR84, we and to provide a in and on we identify residues in the of GPR84 that are phosphorylated of that identify of both constitutive and phosphorylation to that phosphorylation amino acids Thr263 Thr264 is to effective interactions with arrestin-2 and and that these amino acids phosphorylated in a By contrast, neither Ser221 Ser224, are constitutively phosphorylated and not affected by inhibition of a in arrestin-2 or the phosphorylation status of neither of these of amino acids on the of the receptor to of as GPR84 is in a range of immune cells and in a of proinflammatory J. N. L. fatty acids as ligands orphan G protein-coupled receptor PubMed Scopus Google S. fatty GPR84, is a proinflammatory PubMed Scopus Google L. L. L. of the receptor GPR84 and in PubMed Scopus Google the status of GPR84 by the antiserum provide an in of GPR84 and of by of the proinflammatory GPR84 we a pair of orthosteric 2-HTP Jenkins L. Tobin A.B. Milligan G. and of novel orthosteric and allosteric activators of PubMed Scopus Google Jenkins L. S. Milligan G. of ligands to distinct on the orphan G protein-coupled receptor PubMed Scopus Google or and of and as GPR84 PubMed Scopus Google and 6-OAU S. fatty GPR84, is a proinflammatory PubMed Scopus Google G. with the orphan G protein-coupled receptor PubMed Scopus Google as well as the allosteric Jenkins L. Tobin A.B. Milligan G. and of novel orthosteric and allosteric activators of PubMed Scopus Google G. of the orphan G protein-coupled receptor PubMed Scopus Google we a a and to G with GPR84 L. agonist receptor GPR84 distinct functional in PubMed Scopus Google we in cells GPR84 with to the C-terminal of the receptor Jenkins L. S. Milligan G. of ligands to distinct on the orphan G protein-coupled receptor PubMed Scopus Google of the Milligan G. the to PubMed Scopus Google GPR84 is to be able to with of the (1Marsango S. Barki N. Jenkins L. Tobin A.B. Milligan G. Therapeutic validation of an orphan G protein-coupled receptor: The case of GPR84.Br. J. Pharmacol. 2020; https://doi.org/10.1111/bph.15248Crossref PubMed Scopus (7) Google J. N. L. fatty acids as ligands orphan G protein-coupled receptor PubMed Scopus Google from cells the of to of was promoted an in of in a with 2-HTP 6-OAU DL-175 and effective of GPR84 with but not ligands is to with L. agonist receptor GPR84 distinct functional in PubMed Scopus Google J. L. L. L. The two was in cells with to 2-HTP promoted and interactions the receptor and this 6-OAU was also able to interactions and 2-HTP was it was not to 6-OAU to to be both in the and contrast, DL-175 did not interactions and that was to and neither did as a allosteric GPR84 Jenkins L. Tobin A.B. Milligan G. and of novel orthosteric and allosteric activators of PubMed Scopus Google G. of the orphan G protein-coupled receptor PubMed Scopus Google the of 2-HTP in and this was also the case 6-OAU By contrast, of DL-175 did not the of 2-HTP or 6-OAU of a with an is phosphorylation of the receptor that to agonist of the receptor L. of G protein-coupled PubMed Scopus Google this we cells to with [32P] and treated the cells with or a of 2-HTP to an in the of these cells with an antiserum also and by to incorporation of [32P] was in incorporation of [32P] with to was with with to incorporation of [32P] within of agonist exposure 6-OAU also promoted incorporation of [32P] neither DL-175 effective interactions of with arrestin-3, and be we of these ligands that in on the as shown 2-HTP and 6-OAU to this in the in these incorporation of [32P] was in treated with either DL-175 or the of 2-HTP to phosphorylation of was prevented by with either of two distinct GPR84 S. L. G. L. S. 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