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 CB receptor. In humans, three CB variants have been identified: hCB, considered the most abundant G protein-coupled receptor in the brain, alongside the less abundant and studied variants, hCB and hCB. CB exhibits a preference for coupling with inhibitory G proteins, although its interactions with specific members of the G family remain poorly characterized. This study aimed to compare the AEA and 2-AG-induced activation of various G protein subtypes at CB. Furthermore, we compared the response of human CB (hCB, hCB, hCB) and explored species differences by examining rodent receptors (mCB, rCB). Activation of individual G protein subtypes in HEK293 cells transiently expressing CB 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. 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, G, G, G, G, G, and G. Across all pathways, AEA demonstrated partial agonism, whereas 2-AG exhibited full or high-efficacy agonism. Notably, we provide direct evidence that the hCB receptor couples to G and G proteins. Our findings do not indicate any evidence of G protein subtype selectivity. Similar observations were made across the human receptor variants (hCB, hCB, hCB), as well as at mCB and rCB. There was no evidence suggesting G protein subtype selectivity for AEA and 2-AG at CB, and this finding remained consistent across human receptor variants and different species.
