Cannabinoid CB1 and CB2 Receptor-Mediated Arrestin Translocation: Species, Subtype, and Agonist-Dependence

This study has utilized this assay (with receptors minimally modified with an extracellular epitope-tag to facilitate measurement of expression) to investigate the ability of CB1 and CB2 to induce β-arrestin-1 and -2 plasma membrane translocation in response to stimulation with a range of cannabinoid ligands. Furthermore, we have compared the CB1- and CB2-mediated activity profiles of human and bovine β-arrestins. Materials and Methods Plasmids and Cloning We used the human form of all receptor constructs and transiently expressed transgenes to ensure high expression levels. The pplss-3HA-hCB1 pEF4a construct has been described previously (Finlay et al., 2017). The use of the preprolactin signal sequence (pplss) chimera of CB1 was necessary to ensure sufficient expression levels. The 3HA-hCB2 pEF4a construct has been described previously (Grimsey et al., 2011). The 3HA-hV2R pcDNA3.1 construct and 3HA-hD2R pcDNA3.1 construct were both purchased from cDNA Resource Center (#AVR020TN00 and #DRD020TN01, respectively; www.cdna.org, Bloomsburg, PA, United States). The Rluc8-bβ-arrestin-2-Sp1 pcDNA3.1 (bovine β-arrestin-2) and mem-linker-Citrine-SH3 pcDNA3.1 were described in the original papers detailing the β-arrestin translocation assay (Clayton et al., 2014; Donthamsetti et al., 2015). Rluc8-bβ-arrestin-1-Sp1 was synthesized and cloned into pcDNA3.1 commercially, using the restriction sites HindIII and NotI (GenScript, Piscataway, NJ...
Source: Frontiers in Pharmacology - Category: Drugs & Pharmacology Source Type: research