Cannabidiol biases A_2A-CB₂ receptor heteromer function by decoupling β-arrestin signaling from complex formation

Cannabidiol (CBD), a phytocannabinoid with pleiotropic effects, exhibits complex mechanisms of action that remain incompletely understood, particularly its role as an allosteric modulator of G protein-coupled receptor (GPCR) heteromers. Among these, the adenosine A_2A-cannabinoid CB₂ receptor heteromer (A_2AR-CB₂R) is a functionally relevant complex implicated in diverse physiological processes. This study aimed to characterize the modulatory effects of CBD on A_2AR-CB₂R heteromers. A multi-technique approach was employed using HEK-293T cells co-transfected with A_2AR and CB₂R. Real-time NanoBRET assays at 37 °C were used to assess receptor interaction kinetics. Functional consequences were evaluated via β-arrestin II recruitment assays, and complex formation was visualized and quantified using proximity ligation assays (PLA). CBD acted as a potent allosteric modulator, enhancing the kinetics of agonist-induced A_2AR-CB₂R interaction. Notably, this effect was accompanied by functional dissociation: CBD inhibited β-arrestin II recruitment in a probe-dependent manner, with significant modulatory effects at concentrations of 100 nM, and showed a greater inhibition of the selective CB₂R agonist JWH-133-induced signaling than that induced by CGS 21680, a selective A_2AR agonist. PLA data confirmed that this functional modulation occurred without altering the extent of heteromer complex formation. These findings reveal that CBD operates as a biased allosteric modulator of the A_2AR-CB₂R heteromer. Rather than disrupting heteromer formation, CBD selectively induces a conformational state that uncouples physical receptor interaction from β-arrestin II signaling. This defines a distinct mechanism of action for CBD and highlights A_2AR-CB₂R heteromers as promising targets for biased signaling-based therapeutics.