G protein-receptor kinases 5/6 are the key regulators of G protein-coupled receptor 35-arrestin interactions

Ganguly, A. et al. (2023) G protein-receptor kinases 5/6 are the key regulators of G protein-coupled receptor 35-arrestin interactions. Journal of Biological Chemistry, 299(10), 105218. (doi: 10.1016/j.jbc.2023.105218) (PMID:37660910) (PMCID:PMC10520886)

Abstract

Human G protein–coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phospho-acceptor amino acids within its C-terminal tail. Alteration of both Ser300 and Ser303 to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome edited to lack expression of combinations of G protein receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser300 and Ser303 (orce; the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues. Extensions of these studies demonstrated the importance of the GRK5/6-mediated phosphorylation of these amino acids for agonist-induced internalization of the receptor. Homology and predictive modeling of the interaction of human GPR35 with GRKs showed that the N terminus of GRK5 is likely to dock in the same methionine pocket on the intracellular face of GPR35 as the C terminus of the α5 helix of Gα13 and, that while this is also the case for GRK6, GRK2 and GRK3 are unable to do so effectively. These studies provide unique and wide-ranging insights into modes of regulation of GPR35, a receptor that is currently attracting considerable interest as a novel therapeutic target in diseases including ulcerative colitis.

• Item Type: Articles
• Additional Information: —These studies were supported in part by UKRI Biotechnology and Biological Sciences Research Council grants BB/P000649/1 and BB/P00069X/1 to G. M. and A. B. T., and the Luxembourg National Research Fund (INTER/FNRS grants 20/15084569) to A. C. A. G. thanks the Commonwealth Scholarship Commission for studentship support (reference BDCA2019-6).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Quon, Dr Tezz and Ganguly, Amlan and Milligan, Professor Graeme and Jenkins, Mrs Laura and Tobin, Andrew

Creator Roles

Milligan, G.: Conceptualization, Writing – original draft, Funding acquisition, Project administration
Tobin, A.: Conceptualization, Funding acquisition, Project administration
Jenkins, L.: Investigation, Formal analysis
Quon, T.: Investigation, Formal analysis
Ganguly, A.: Formal analysis

• Authors: Ganguly, A., Quon, T., Jenkins, L., Joseph, B., Al-awar, R., Chevigne, A., Tobin, A. B., Uehling, D. E., Hoffmann, C., Drube, J., and Milligan, G.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: Journal of Biological Chemistry
• Publisher: American Society for Biochemistry and Molecular Biology
• ISSN: 0021-9258
• ISSN (Online): 1083-351X
• Copyright Holders: Copyright: © 2023 The Authors
• First Published: First published in Journal of Biological Chemistry 299(10): 105218
• Publisher Policy: Reproduced under a Creative Commons licence

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
173305		Defining the functional roles of the enigmatic G protein coupled receptor GPR35	Graeme Milligan	Biotechnology and Biological Sciences Research Council (BBSRC)	BB/P000649/1	SMB - Molecular Biosciences
173689		Defining the functional role of the enigmatic G protein coupled receptor GPR35 - Leicester application - PART B	Andrew Tobin	Biotechnology and Biological Sciences Research Council (BBSRC)	BB/P00069X/1	SMB - Professional Services