Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor.
View / Open Files
Authors
Divorty, Nina
Jenkins, Laura
Ganguly, Amlan
Butcher, Adrian J
Hudson, Brian D
Schulz, Stefan
Tobin, Andrew B
Nicklin, Stuart A
Milligan, Graeme
Publication Date
2022-03Journal Title
J Biol Chem
ISSN
0021-9258
Publisher
Elsevier BV
Volume
298
Issue
3
Number
101655
Pages
101655
Type
Article
This Version
VoR
Physical Medium
Print-Electronic
Metadata
Show full item recordCitation
Divorty, N., Jenkins, L., Ganguly, A., Butcher, A. J., Hudson, B. D., Schulz, S., Tobin, A. B., et al. (2022). Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor.. J Biol Chem, 298 (3. 101655), 101655. https://doi.org/10.1016/j.jbc.2022.101655
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
Identifiers
External DOI: https://doi.org/10.1016/j.jbc.2022.101655
This record's URL: https://www.repository.cam.ac.uk/handle/1810/335458
Statistics
Total file downloads (since January 2020). For more information on metrics see the
IRUS guide.
Recommended or similar items
The current recommendation prototype on the Apollo Repository will be turned off on 03 February 2023. Although the pilot has been fruitful for both parties, the service provider IKVA is focusing on horizon scanning products and so the recommender service can no longer be supported. We recognise the importance of recommender services in supporting research discovery and are evaluating offerings from other service providers. If you would like to offer feedback on this decision please contact us on: support@repository.cam.ac.uk