Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor

Divorty, N., Jenkins, L. , Ganguly, A., Butcher, A. J., Hudson, B. D. , Schulz, S., Tobin, A. B. , Nicklin, S. A. and Milligan, G. (2022) Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor. Journal of Biological Chemistry, 298(3), 101655. (doi: 10.1016/j.jbc.2022.101655) (PMID:35101446) (PMCID:PMC8892012)

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G 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 labelling 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 GPCR 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) GPR35 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.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Jenkins, Mrs Laura and Hudson, Dr Brian and Nicklin, Professor Stuart and Tobin, Andrew and Divorty, Nina and Ganguly, Amlan and Milligan, Professor Graeme
Creator Roles:
Milligan, G.Conceptualization, Funding acquisition, Project administration, Writing – original draft
Nicklin, S.Conceptualization, Project administration
Tobin, A.Conceptualization, Funding acquisition
Divorty, N.Investigation
Ganguly, A.Investigation
Hudson, B.Resources
Authors: Divorty, N., Jenkins, L., Ganguly, A., Butcher, A. J., Hudson, B. D., Schulz, S., Tobin, A. B., Nicklin, S. A., and Milligan, G.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Journal of Biological Chemistry
ISSN (Online):1083-351X
Published Online:29 January 2022
Copyright Holders:Copyright © 2022 The Authors
First Published:First published in Journal of Biological Chemistry 298(3):101655
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
173305Defining the functional roles of the enigmatic G protein coupled receptor GPR35Graeme MilliganBiotechnology and Biological Sciences Research Council (BBSRC)BB/P000649/1MCSB - Molecular Pharmacology
173689Defining the functional role of the enigmatic G protein coupled receptor GPR35 - Leicester application - PART BAndrew TobinBiotechnology and Biological Sciences Research Council (BBSRC)BB/P00069X/1MCSB - Administration
190814BHF centre of excellenceRhian TouyzBritish Heart Foundation (BHF)RE/13/5/30177Institute of Cardiovascular & Medical Sciences
169494Molecular Functions in Disease (PhD Studentship 2012-2016)Graeme MilliganWellcome Trust (WELLCOTR)099787/Z/12/ZMCSB - Molecular Pharmacology