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 | |
| Publication status | Published - 2016 |
Cite this
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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 journal › Article › Academic › peer-review
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
VL - 1412
SP - 103
EP - 111
JO - Methods in Molecular Biology
JF - Methods in Molecular Biology
SN - 1064-3745
ER -