Characterization of different G protein coupling properties of CB₁ and CB₂ cannabinoid receptors and GPR55 receptor using BRET

Background

CB₁ and CB₂ cannabinoid receptors are G protein-coupled receptors which have been described to couple mainly to the G_i/o subfamily of G proteins. However, in some cell types and upon stimulation with certain cannabinoid agonists, activation of other G protein subtypes has also been observed. GPR55 is an orphan G protein-coupled receptor which has been suggested to be a novel member of the cannabinoid receptor family.

Methods

In this study we wanted to characterize the G protein activation properties of the two known cannabinoid receptors and GPR55 following stimulation with different cannabinoid ligands, using bioluminescence resonance energy transfer (BRET). We monitored the activation of different G protein subtypes (G_o, G_q, G_s or G₁₂) using Renilla luciferase-tagged wild type or chimeric Gα_o subunits (i.e. Gα_o with the C-terminal 5 amino acids replaced with those of Gα_q, Gα_s or Gα₁₂, respectively) co-expressed with EYFP-tagged α₁α₁₁ subunit and the receptor in CHO cells.

Results

We found that CB₁ was able to activate all four subtypes of G proteins, with different pharmacokinetic properties, following stimulation by non-selective (WIN55 and 2-AG) or CB₁-selective (ACEA) cannabinoid agonists. Basal activity of CB₁ could also be detected with G_o and G₁₂ subtypes, as the CB₁ inverse agonist AM251 caused significant BRET increase (i.e. G protein subunit association) when tested with these G proteins. In contrast, CB₂ showed no G protein activation other than G_o, upon either WIN55 or 2-AG stimuli. Stimulation of GPR55 with WIN55, 2-AG or AM251 did not alter the activity of the tested G proteins even at considerably high ligand concentrations.