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Direct visualisation of internalization of the adenosine A3 receptor and localization with arrestin3 using a fluorescent agonist

Stoddart, Leigh A.; Vernall, Andrea J.; Briddon, Stephen J.; Kellam, Barrie; Hill, Stephen J.

Authors

Leigh A. Stoddart

Andrea J. Vernall

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BARRIE KELLAM BARRIE.KELLAM@NOTTINGHAM.AC.UK
Professor of Medicinal Chemistry

STEPHEN HILL steve.hill@nottingham.ac.uk
Professor of Molecular Pharmacology



Abstract

Fluorescence based probes provide a novel way to study the dynamic internalization process of G protein-coupled receptors (GPCRs). Recent advances in the rational design of fluorescent ligands for GPCRs have been used here to generate new fluorescent agonists containing tripeptide linkers for the adenosine A3 receptor. The fluorescent agonist BY630-X-(D)-A-(D)-A-G-ABEA was found to be a highly potent agonist at the adenosine A3 receptor in both reporter gene (pEC50 = 8.48 ± 0.09) and internalization assays (pEC50 = 7.47 ± 0.11). Confocal imaging studies showed that BY630-X-(D)-A-(D)-A-G-ABEA was internalized with A3 linked to yellow fluorescent protein, which was blocked by the competitive antagonist MRS1220. Internalization of untagged adenosine A3 could also be visualized with BY630-X-(D)-A-(D)-A-G-ABEA treatment. Further, BY630-X-(D)-A-(D)-A-G-ABEA stimulated the formation of receptor–arrestin3 complexes and was found to localize with these intracellular complexes. This highly potent agonist with excellent imaging properties should be a valuable tool to study receptor internalization.

Citation

Stoddart, L. A., Vernall, A. J., Briddon, S. J., Kellam, B., & Hill, S. J. (2015). Direct visualisation of internalization of the adenosine A3 receptor and localization with arrestin3 using a fluorescent agonist. Neuropharmacology, 98, 68-77. https://doi.org/10.1016/j.neuropharm.2015.04.013

Journal Article Type Article
Acceptance Date Apr 1, 2015
Online Publication Date Apr 30, 2015
Publication Date 2015-11
Deposit Date Jul 18, 2016
Publicly Available Date Jul 18, 2016
Journal Neuropharmacology
Print ISSN 0028-3908
Electronic ISSN 1873-7064
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 98
Pages 68-77
DOI https://doi.org/10.1016/j.neuropharm.2015.04.013
Keywords Adenosine receptors; Adenosine A3 receptor; Fluorescent agonist; Internalization; Arrestin3
Public URL https://nottingham-repository.worktribe.com/output/981620
Publisher URL http://www.sciencedirect.com/science/article/pii/S0028390815001422

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