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(®) 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.
authors.affiliation: Department of Bio-Organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.|Department of Medicinal Chemistry, Leiden Academic Center for Drug Research, Leiden University, Leiden, The Netherlands.
authors.forename: Marjolein
authors.initials: M
authors.lastname: Soethoudt
authors.affiliation: Department of Medicinal Chemistry, Leiden Academic Center for Drug Research, Leiden University, Leiden, The Netherlands.
authors.forename: Noortje
authors.initials: N
authors.lastname: van Gils
authors.affiliation: Department of Bio-Organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Mario
authors.initials: M
authors.lastname: van der Stelt
authors.affiliation: Department of Medicinal Chemistry, Leiden Academic Center for Drug Research, Leiden University, Leiden, The Netherlands. l.h.heitman@lacdr.leidenuniv.nl.
authors.forename: Laura H
authors.initials: LH
authors.lastname: Heitman
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D016466:CHO Cells; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D017930:Genes, Reporter; D008024:Ligands; D011485:Protein Binding; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D043885:Receptor, Cannabinoid, CB2 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction; D000071557:beta-Arrestins / Q000378:metabolism*
chemical_list: D008024:Ligands; D043884:Receptor, Cannabinoid, CB1; D043885:Receptor, Cannabinoid, CB2; D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins
keywords: Biased signaling; Cannabinoid receptors; DiscoveRx Pathunter®; Functional selectivity; GPCRs; Internalization; Receptor signaling; β-Arrestin