PXD031252-1

PXD031252 is an original dataset announced via ProteomeXchange.

Title	GRK2/3-regulated, but not constitutive, phosphorylation of the G protein-coupled receptor GPR84 controls arrestin interactions and subsequent function
Description	Although the GPR84 activators 2-HTP and 6-OAU promoted phosphorylation of human GPR84 and its interactions with arrestin-3, PSB-16671 and DL-175 did not. Replacement of all 21 serine and threonine residues within the third intracellular loop, but not the 2 serines in the C-terminal tail, eliminated incorporation of [32P] promoted by 2-HTP and greatly reduced receptor-arrestin-3 interactions. Mass spectrometry indicated that GPR84 was phosphorylated constitutively on residues Ser221 and Ser224 whilst a range of sites became phosphorylated in response to 2-HTP. An antiserum able to identify pSer221/pSer224 recognised GPR84 from cells treated both with and without 2-THP, whilst an antiserum able to identify pThr263/pThr264 recognised GPR84 only after exposure to 2-HTP. Treatment with neither PSB-16671 nor DL-175 was able to promote receptor recognition by this antiserum. 2-HTP-mediated phosphorylation of Thr263/Thr264 was prevented by two chemically distinct GPR84 antagonists but neither affected constitutive phosphorylation of Ser221/Ser224. 2-HTP-mediated phosphorylation of Thr263/Thr264 but not constitutive phosphorylation of Ser221/Ser224 was prevented by GRK2/3 inhibition. Mutation of residues Thr263 and Thr264 to alanine generated a variant of GPR84 as limited in 2-HTP-induced interactions with arrestin-3 as when all 21 serine and threonine were eliminated from the third intracellular loop. By contrast this mutant was unaffected in capacity to reduce cAMP levels in response to 2-HTP. Homology modelling and mutagenesis provided molecular insight into differences in agonist function. These studies define key residues, regulated by GRK2/3, that define effective interactions with arrestins and provide novel tools to monitor the phosphorylation and functional status of GPR84.
HostingRepository	PRIDE
AnnounceDate	2022-06-09
AnnouncementXML	Submission_2022-06-09_08:07:25.780.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD031252
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Adrian Butcher
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	LTQ Orbitrap Elite

Revision	Datetime	Status	ChangeLog Entry
0	2022-01-26 02:17:09	ID requested
⏵ 1	2022-06-09 08:07:26	announced

Marsango S, Ward RJ, Jenkins L, Butcher AJ, Al Mahmud Z, Dwomoh L, Nagel F, Schulz S, Tikhonova IG, Tobin AB, Milligan G, Selective phosphorylation of threonine residues defines GPR84-arrestin interactions of biased ligands. J Biol Chem, 298(5):101932(2022) [pubmed]

submitter keyword: GPCR, phosphorylation

Graeme Milligan
contact affiliation	The Centre for Translational Pharmacology, Institute of Molecular, Cellular and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom
contact email	ajb342@medschl.cam.ac.uk
lab head
Adrian Butcher
contact affiliation	University of Cambridge
contact email	ajb342@medschl.cam.ac.uk
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD031252
     1. Label: PRIDE project
     2. Name: GRK2/3-regulated, but not constitutive, phosphorylation of the G protein-coupled receptor GPR84 controls arrestin interactions and subsequent function