1. Academic Validation
  2. Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor

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

  • J Biol Chem. 2022 Mar;298(3):101655. doi: 10.1016/j.jbc.2022.101655.
Nina Divorty 1 Laura Jenkins 2 Amlan Ganguly 2 Adrian J Butcher 3 Brian D Hudson 2 Stefan Schulz 4 Andrew B Tobin 2 Stuart A Nicklin 5 Graeme Milligan 6
Affiliations

Affiliations

  • 1 The Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom; Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
  • 2 The Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
  • 3 Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom.
  • 4 7TM Antibodies GmbH, Jena, Germany; Institute of Pharmacology and Toxicology, University Hospital Jena, Jena, Germany.
  • 5 Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
  • 6 The Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom. Electronic address: Graeme.Milligan@glasgow.ac.uk.
Abstract

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 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.

Keywords

GPCR; GPR35; arrestin; phospho-site–specific antisera; phosphorylation.

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