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

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

doi:10.1007/978-1-4939-3539-0_11

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

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

Hematology laboratory

Research output: Contribution to journal › Article › Academic › peer-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 language	English
Pages (from-to)	103-11
Number of pages	9
Journal	Methods in Molecular Biology
Volume	1412
DOIs	https://doi.org/10.1007/978-1-4939-3539-0_11
Publication status	Published - 2016

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10.1007/978-1-4939-3539-0_11

Cite this

@article{8a67c4d13cb34a74b929d35387ddc082,

title = "Protocol to Study β-Arrestin Recruitment by CB1 and CB2 Cannabinoid Receptors",

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({\textregistered}) assay. The latter is a cellular assay that can be performed in plates with 384-wells. The PathHunter({\textregistered}) 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.",

keywords = "Animals, Biological Assay/methods, CHO Cells, Cricetulus, Dose-Response Relationship, Drug, Gene Expression, Genes, Reporter, Ligands, Protein Binding, Receptor, Cannabinoid, CB1/agonists, Receptor, Cannabinoid, CB2/agonists, Receptors, G-Protein-Coupled/metabolism, Signal Transduction, beta-Arrestins/metabolism",

author = "Marjolein Soethoudt and {van Gils}, Noortje and {van der Stelt}, Mario and Heitman, {Laura H}",

year = "2016",

doi = "10.1007/978-1-4939-3539-0_11",

language = "English",

volume = "1412",

pages = "103--11",

journal = "Methods in Molecular Biology",

issn = "1064-3745",

publisher = "Humana Press",

}

TY - JOUR

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

Y1 - 2016

N2 - 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.

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.

KW - Animals

KW - Biological Assay/methods

KW - CHO Cells

KW - Cricetulus

KW - Dose-Response Relationship, Drug

KW - Gene Expression

KW - Genes, Reporter

KW - Ligands

KW - Protein Binding

KW - Receptor, Cannabinoid, CB1/agonists

KW - Receptor, Cannabinoid, CB2/agonists

KW - Receptors, G-Protein-Coupled/metabolism

KW - Signal Transduction

KW - beta-Arrestins/metabolism

U2 - 10.1007/978-1-4939-3539-0_11

DO - 10.1007/978-1-4939-3539-0_11

M3 - Article

C2 - 27245896

VL - 1412

SP - 103

EP - 111

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1064-3745

ER -