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dc.contributor.authorDivorty, Nina
dc.contributor.authorJenkins, Laura
dc.contributor.authorGanguly, Amlan
dc.contributor.authorButcher, Adrian J
dc.contributor.authorHudson, Brian D
dc.contributor.authorSchulz, Stefan
dc.contributor.authorTobin, Andrew B
dc.contributor.authorNicklin, Stuart A
dc.contributor.authorMilligan, Graeme
dc.date.accessioned2022-03-28T23:30:55Z
dc.date.available2022-03-28T23:30:55Z
dc.date.issued2022-03
dc.identifier.issn0021-9258
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/335458
dc.description.abstractG protein-coupled receptor 35 (GPR35) is poorly characterized but nevertheless has been revealed to have diverse roles in areas including lower gut inflammation and pain. The development of novel reagents and tools will greatly enhance analysis of GPR35 functions in health and disease. Here, we used mass spectrometry, mutagenesis, and [32P] orthophosphate labeling to identify that all five hydroxy-amino acids in the C-terminal tail of human GPR35a became phosphorylated in response to agonist occupancy of the receptor and that, apart from Ser294, each of these contributed to interactions with arretin-3, which inhibits further G protein-coupled receptor signaling. We found that Ser303 was key to such interactions; the serine corresponding to human GPR35a residue 303 also played a dominant role in arrestin-3 interactions for both mouse and rat GPR35. We also demonstrated that fully phospho-site-deficient mutants of human GPR35a and mouse GPR35 failed to interact effectively with arrestin-3, and the human phospho-deficient variant was not internalized from the surface of cells in response to agonist treatment. Even in cells stably expressing species orthologues of GPR35, a substantial proportion of the expressed protein(s) was determined to be immature. Finally, phospho-site-specific antisera targeting the region encompassing Ser303 in human (Ser301 in mouse) GPR35a identified only the mature forms of GPR35 and provided effective sensors of the activation status of the receptors both in immunoblotting and immunocytochemical studies. Such antisera may be useful tools to evaluate target engagement in drug discovery and target validation programs.
dc.format.mediumPrint-Electronic
dc.publisherElsevier BV
dc.rightsAttribution 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectGPCR
dc.subjectGPR35
dc.subjectarrestin
dc.subjectphospho-site–specific antisera
dc.subjectphosphorylation
dc.titleAgonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor.
dc.typeArticle
dc.publisher.departmentDivision of Neurology
dc.date.updated2022-03-28T07:51:23Z
prism.issueIdentifier3
prism.number101655
prism.publicationDate2022
prism.publicationNameJ Biol Chem
prism.startingPage101655
prism.volume298
dc.identifier.doi10.17863/CAM.82887
dcterms.dateAccepted2022-01-11
rioxxterms.versionofrecord10.1016/j.jbc.2022.101655
rioxxterms.versionVoR
dc.contributor.orcidButcher, Adrian [0000-0001-5723-8720]
dc.identifier.eissn1083-351X
rioxxterms.typeJournal Article/Review
cam.issuedOnline2022-01-29
cam.depositDate2022-03-28
pubs.licence-identifierapollo-deposit-licence-2-1
pubs.licence-display-nameApollo Repository Deposit Licence Agreement


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International