No Evidence for Endocannabinoid-Induced G Protein Subtype Selectivity at Human and Rodent Cannabinoid CB ₁ Receptors

<b>Introduction:</b> The endocannabinoid system (ECS) is a widespread neurotransmitter system. A key characteristic of the ECS is that there are multiple endogenous ligands (endocannabinoids). Of these, the most extensively studied are arachidonoyl ethanolamide (AEA) and 2-arachidonoyl-glycerol (2-AG), both act as agonists at the cannabinoid CB₁ receptor. In humans, three CB₁ variants have been identified: hCB₁, considered the most abundant G protein-coupled receptor in the brain, alongside the less abundant and studied variants, hCB_1a and hCB_1b. CB₁ exhibits a preference for coupling with inhibitory G_i/o proteins, although its interactions with specific members of the G_i/o family remain poorly characterized. This study aimed to compare the AEA and 2-AG-induced activation of various G protein subtypes at CB₁. Furthermore, we compared the response of human CB₁ (hCB₁, hCB_1a, hCB_1b) and explored species differences by examining rodent receptors (mCB₁, rCB₁). <b>Materials and Methods:</b> Activation of individual G protein subtypes in HEK293 cells transiently expressing CB₁ was measured with G protein dissociation assay utilizing TRUPATH biosensors. The performance of the TRUPATH biosensors was evaluated using Z-factor analysis. Pathway potencies and efficacies were analyzed using the operational analysis of bias to determine G protein subtype selectivity for AEA and 2-AG. <b>Results:</b> Initial screening of TRUPATH biosensors performance revealed variable sensitivities within our system. Based on the biosensor performance, the G protein subtypes pursued for further characterization were G_i1, G_i3, G_oA, G_oB, G_Z, G₁₂, and G₁₃. Across all pathways, AEA demonstrated partial agonism, whereas 2-AG exhibited full or high-efficacy agonism. Notably, we provide direct evidence that the hCB₁ receptor couples to G₁₂ and G₁₃ proteins. Our findings do not indicate any evidence of G protein subtype selectivity. Similar observations were made across the human receptor variants (hCB₁, hCB_1a, hCB_1b), as well as at mCB₁ and rCB₁. <b>Discussion:</b> There was no evidence suggesting G protein subtype selectivity for AEA and 2-AG at CB₁, and this finding remained consistent across human receptor variants and different species.