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

Methods Mol Biol. 2016:1412:103-11. doi: 10.1007/978-1-4939-3539-0_11.

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.

Keywords: Biased signaling; Cannabinoid receptors; DiscoveRx Pathunter®; Functional selectivity; GPCRs; Internalization; Receptor signaling; β-Arrestin.

MeSH terms

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

Substances

  • Ligands
  • Receptor, Cannabinoid, CB1
  • Receptor, Cannabinoid, CB2
  • Receptors, G-Protein-Coupled
  • beta-Arrestins