Protocol to Study β-Arrestin Recruitment by CB1 and CB2 Cannabinoid Receptors

Marjolein Soethoudt, Noortje van Gils, Mario van der Stelt, Laura H Heitman

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Cannabinoid CB1 and CB2 receptors are G-protein-coupled receptors (GPCRs) that recruit β-arrestins upon activation by (partial) agonists. β-Arrestin recruitment is induced by phosphorylation of their C-terminal tails, and is associated with the termination of GPCR signaling; yet, it may also activate cellular signaling pathways independent of G-proteins. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB1 and CB2 receptors, by using the PathHunter(®) assay. The latter is a cellular assay that can be performed in plates with 384-wells. The PathHunter(®) assay makes use of β-galactosidase complementation, and has a chemiluminescent readout. We used this assay to characterize a set of reference ligands (both agonists and antagonists) on human CB1 and CB2 receptors.

Original languageEnglish
Pages (from-to)103-11
Number of pages9
JournalMethods in Molecular Biology
Volume1412
DOIs
Publication statusPublished - 2016

Cite this

Soethoudt, Marjolein ; van Gils, Noortje ; van der Stelt, Mario ; Heitman, Laura H. / Protocol to Study β-Arrestin Recruitment by CB1 and CB2 Cannabinoid Receptors. In: Methods in Molecular Biology. 2016 ; Vol. 1412. pp. 103-11.
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abstract = "Cannabinoid CB1 and CB2 receptors are G-protein-coupled receptors (GPCRs) that recruit β-arrestins upon activation by (partial) agonists. β-Arrestin recruitment is induced by phosphorylation of their C-terminal tails, and is associated with the termination of GPCR signaling; yet, it may also activate cellular signaling pathways independent of G-proteins. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB1 and CB2 receptors, by using the PathHunter({\circledR}) assay. The latter is a cellular assay that can be performed in plates with 384-wells. The PathHunter({\circledR}) assay makes use of β-galactosidase complementation, and has a chemiluminescent readout. We used this assay to characterize a set of reference ligands (both agonists and antagonists) on human CB1 and CB2 receptors.",
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author = "Marjolein Soethoudt and {van Gils}, Noortje and {van der Stelt}, Mario and Heitman, {Laura H}",
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Protocol to Study β-Arrestin Recruitment by CB1 and CB2 Cannabinoid Receptors. / Soethoudt, Marjolein; van Gils, Noortje; van der Stelt, Mario; Heitman, Laura H.

In: Methods in Molecular Biology, Vol. 1412, 2016, p. 103-11.

Research output: Contribution to journalArticleAcademicpeer-review

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T1 - Protocol to Study β-Arrestin Recruitment by CB1 and CB2 Cannabinoid Receptors

AU - Soethoudt, Marjolein

AU - van Gils, Noortje

AU - van der Stelt, Mario

AU - Heitman, Laura H

PY - 2016

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AB - Cannabinoid CB1 and CB2 receptors are G-protein-coupled receptors (GPCRs) that recruit β-arrestins upon activation by (partial) agonists. β-Arrestin recruitment is induced by phosphorylation of their C-terminal tails, and is associated with the termination of GPCR signaling; yet, it may also activate cellular signaling pathways independent of G-proteins. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB1 and CB2 receptors, by using the PathHunter(®) assay. The latter is a cellular assay that can be performed in plates with 384-wells. The PathHunter(®) assay makes use of β-galactosidase complementation, and has a chemiluminescent readout. We used this assay to characterize a set of reference ligands (both agonists and antagonists) on human CB1 and CB2 receptors.

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KW - Receptor, Cannabinoid, CB1/agonists

KW - Receptor, Cannabinoid, CB2/agonists

KW - Receptors, G-Protein-Coupled/metabolism

KW - Signal Transduction

KW - beta-Arrestins/metabolism

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