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NanoBiT Complementation to Monitor Agonist-Induced Adenosine A1 Receptor Internalization

Soave, Mark; Kellam, Barrie; Woolard, Jeanette; Briddon, Stephen J.; Hill, Stephen J.


Mark Soave

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Professor of Medicinal Chemistry

Professor of Cardiovascular Physiology and Pharmacology

Professor of Molecular Pharmacology


Receptor internalization in response to prolonged agonist treatment is an important regulator of G protein–coupled receptor (GPCR) function. The adenosine A1 receptor (A1AR) is one of the adenosine receptor family of GPCRs, and evidence for its agonist-induced internalization is equivocal. The recently developed NanoBiT technology uses split NanoLuc Luciferase to monitor changes in protein interactions. We have modified the human A1AR on the N-terminus with the small high-affinity HiBiT tag. In the presence of the large NanoLuc subunit (LgBiT), complementation occurs, reconstituting a full-length functional NanoLuc Luciferase. Here, we have used complemented luminescence to monitor the internalization of the A1AR in living HEK293 cells. Agonist treatment resulted in a robust decrease in cell-surface luminescence, indicating an increase in A1AR internalization. These responses were inhibited by the A1AR-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), with an antagonist affinity that closely matched that measured using ligand binding with a fluorescent A1 receptor antagonist (CA200645). The agonist potencies for inducing A1AR internalization were very similar to the affinities previously determined by ligand binding, suggesting little or no amplification of the internalization response. By complementing the HiBiT tag to exogenous purified LgBiT, it was also possible to perform NanoBRET ligand-binding experiments using HiBiT–A1AR. This study demonstrates the use of NanoBiT technology to monitor internalization of the A1AR and offers the potential to combine these experiments with NanoBRET ligand-binding assays.


Soave, M., Kellam, B., Woolard, J., Briddon, S. J., & Hill, S. J. (2019). NanoBiT Complementation to Monitor Agonist-Induced Adenosine A1 Receptor Internalization. Slas Discovery,

Journal Article Type Article
Acceptance Date Sep 12, 2019
Online Publication Date Oct 4, 2019
Publication Date Oct 4, 2019
Deposit Date Oct 4, 2019
Publicly Available Date Oct 4, 2019
Journal SLAS DISCOVERY: Advancing Life Sciences R&D
Electronic ISSN 2472-5560
Publisher SAGE Publications
Peer Reviewed Peer Reviewed
Keywords GPCR; Adenosine; Receptor internalization; NanoBiT; Nanoluciferase complementation
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