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Investigation of adenosine A1 receptor-mediated β-arrestin 2 recruitment using a split-luciferase assay
Background: Adenosine A1 receptor (A(1)AR) plays a prominent role in neurological and cardiac diseases and inflammatory processes. Its endogenous ligand adenosine is known to be one of the key players in the sleep–wake cycle. Like other G protein-coupled receptors (GPCRs), stimulation of A(1)AR lead...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10268005/ https://www.ncbi.nlm.nih.gov/pubmed/37324481 http://dx.doi.org/10.3389/fphar.2023.1172551 |
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author | Saecker, Luisa Häberlein, Hanns Franken, Sebastian |
author_facet | Saecker, Luisa Häberlein, Hanns Franken, Sebastian |
author_sort | Saecker, Luisa |
collection | PubMed |
description | Background: Adenosine A1 receptor (A(1)AR) plays a prominent role in neurological and cardiac diseases and inflammatory processes. Its endogenous ligand adenosine is known to be one of the key players in the sleep–wake cycle. Like other G protein-coupled receptors (GPCRs), stimulation of A(1)AR leads to the recruitment of arrestins in addition to the activation of G proteins. So far, little is known about the role of these proteins in signal transduction and regulation of A(1)AR compared to the activation of G proteins. In this work, we characterized a live cell assay for A(1)AR-mediated β-arrestin 2 recruitment. We have applied this assay to a set of different compounds that interact with this receptor. Methods: Based on NanoBit(®) technology, a protein complementation assay was developed in which the A(1)AR is coupled to the large part of the nanoluciferase (LgBiT), whereas its small part (SmBiT) is fused to the N-terminus of β-arrestin 2. Stimulation of A(1)AR results in the recruitment of β-arrestin 2 and subsequent complementation of a functional nanoluciferase. For comparison, corresponding data on the effect of receptor stimulation on intracellular cAMP levels were collected for some data sets using the GloSensor™ assay. Results: The assay gives highly reproducible results with a very good signal-to-noise ratio. Capadenoson, in contrast to adenosine, CPA, or NECA, shows only partial agonism in this assay with respect to the recruitment of β-arrestin 2, whereas it shows full agonism in the case of the inhibitory effect of A(1)AR on cAMP production. By using a GRK2 inhibitor, it becomes clear that the recruitment is at least partially dependent on the phosphorylation of the receptor by this kinase. Interestingly, this was also the first time that we demonstrate the A(1)AR-mediated recruitment of β-arrestin 2 by stimulation with a valerian extract. Conclusion: The presented assay is a useful tool for the quantitative study of A(1)AR-mediated β-arrestin 2 recruitment. It allows data collection for stimulatory, inhibitory, and modulatory substances and is also suitable for more complex substance mixtures such as valerian extract. |
format | Online Article Text |
id | pubmed-10268005 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-102680052023-06-15 Investigation of adenosine A1 receptor-mediated β-arrestin 2 recruitment using a split-luciferase assay Saecker, Luisa Häberlein, Hanns Franken, Sebastian Front Pharmacol Pharmacology Background: Adenosine A1 receptor (A(1)AR) plays a prominent role in neurological and cardiac diseases and inflammatory processes. Its endogenous ligand adenosine is known to be one of the key players in the sleep–wake cycle. Like other G protein-coupled receptors (GPCRs), stimulation of A(1)AR leads to the recruitment of arrestins in addition to the activation of G proteins. So far, little is known about the role of these proteins in signal transduction and regulation of A(1)AR compared to the activation of G proteins. In this work, we characterized a live cell assay for A(1)AR-mediated β-arrestin 2 recruitment. We have applied this assay to a set of different compounds that interact with this receptor. Methods: Based on NanoBit(®) technology, a protein complementation assay was developed in which the A(1)AR is coupled to the large part of the nanoluciferase (LgBiT), whereas its small part (SmBiT) is fused to the N-terminus of β-arrestin 2. Stimulation of A(1)AR results in the recruitment of β-arrestin 2 and subsequent complementation of a functional nanoluciferase. For comparison, corresponding data on the effect of receptor stimulation on intracellular cAMP levels were collected for some data sets using the GloSensor™ assay. Results: The assay gives highly reproducible results with a very good signal-to-noise ratio. Capadenoson, in contrast to adenosine, CPA, or NECA, shows only partial agonism in this assay with respect to the recruitment of β-arrestin 2, whereas it shows full agonism in the case of the inhibitory effect of A(1)AR on cAMP production. By using a GRK2 inhibitor, it becomes clear that the recruitment is at least partially dependent on the phosphorylation of the receptor by this kinase. Interestingly, this was also the first time that we demonstrate the A(1)AR-mediated recruitment of β-arrestin 2 by stimulation with a valerian extract. Conclusion: The presented assay is a useful tool for the quantitative study of A(1)AR-mediated β-arrestin 2 recruitment. It allows data collection for stimulatory, inhibitory, and modulatory substances and is also suitable for more complex substance mixtures such as valerian extract. Frontiers Media S.A. 2023-05-30 /pmc/articles/PMC10268005/ /pubmed/37324481 http://dx.doi.org/10.3389/fphar.2023.1172551 Text en Copyright © 2023 Saecker, Häberlein and Franken. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Pharmacology Saecker, Luisa Häberlein, Hanns Franken, Sebastian Investigation of adenosine A1 receptor-mediated β-arrestin 2 recruitment using a split-luciferase assay |
title | Investigation of adenosine A1 receptor-mediated β-arrestin 2 recruitment using a split-luciferase assay |
title_full | Investigation of adenosine A1 receptor-mediated β-arrestin 2 recruitment using a split-luciferase assay |
title_fullStr | Investigation of adenosine A1 receptor-mediated β-arrestin 2 recruitment using a split-luciferase assay |
title_full_unstemmed | Investigation of adenosine A1 receptor-mediated β-arrestin 2 recruitment using a split-luciferase assay |
title_short | Investigation of adenosine A1 receptor-mediated β-arrestin 2 recruitment using a split-luciferase assay |
title_sort | investigation of adenosine a1 receptor-mediated β-arrestin 2 recruitment using a split-luciferase assay |
topic | Pharmacology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10268005/ https://www.ncbi.nlm.nih.gov/pubmed/37324481 http://dx.doi.org/10.3389/fphar.2023.1172551 |
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