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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

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.

Journal Article Type Article
Publication Date 2015-11
Journal Neuropharmacology
Print ISSN 0028-3908
Electronic ISSN 1873-7064
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 98
Pages 68-77
APA6 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
DOI https://doi.org/10.1016/j.neuropharm.2015.04.013
Keywords Adenosine receptors; Adenosine A3 receptor; Fluorescent agonist; Internalization; Arrestin3
Publisher URL http://www.sciencedirect.com/science/article/pii/S0028390815001422
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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