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

Marsango, S. et al. (2022) Selective phosphorylation of threonine residues defines GPR84-arrestin interactions of biased ligands. Journal of Biological Chemistry, 298(5), 101932. (doi: 10.1016/j.jbc.2022.101932) (PMID:35427647)

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Abstract

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.

Item Type:Articles
Additional Information:This work was funded by each of the Biotechnology and Biosciences Research Council (grant reference BB/T000562/1) to GM and ABT, and grant reference BB/R007101/1 to IGT, an ORBIT (Opportunities in Receptor Biology for Industrial Translation) grant from Sosei-Heptares to GM and ABT, and the Medical Research Council (Confidence in Concept, grant number MC_PC_17160 (to GM)).
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Milligan, Professor Graeme and Jenkins, Mrs Laura and Ward, Dr Richard and Marsango, Dr Sara and Tobin, Andrew and Dwomoh, Dr Louis
Creator Roles:
Marsango, S.Investigation, Writing – review and editing
Ward, R.Investigation
Jenkins, L.Investigation
Dwomoh, L.Investigation
Tobin, A.Conceptualization
Milligan, G.Conceptualization, Writing – original draft, Funding acquisition, Project administration
Authors: Marsango, S., Ward, R. J., Jenkins, L., Butcher, A. J., Al Mahmud, Z., Dwomoh, L., Nagel, F., Schulz, S., Tikhonova, I. G., Tobin, A. B., and Milligan, G.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Journal of Biological Chemistry
Publisher:Elsevier
ISSN:0021-9258
ISSN (Online):1083-351X
Published Online:15 April 2022
Copyright Holders:Copyright © 2022 The Authors
First Published:First published in Journal of Biological Chemistry 298(5): 101932
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
305987IPA Responsive Mode BBSRC Resubmission GPR84Graeme MilliganBiotechnology and Biological Sciences Research Council (BBSRC)BB/T000562/1Institute of Molecular, Cell & Systems Biology