Cannabidiol biases A2A-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 A2A-cannabinoid CB2 receptor heteromer (A2AR-CB2R) is a functionally relevant complex implicated in diverse physiological processes. This study aimed to characterize the modulatory effects of CBD on A2AR-CB2R heteromers. A multi-technique approach was employed using HEK-293T cells co-transfected with A2AR 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 A2AR-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 CB2R agonist JWH-133-induced signaling than that induced by CGS 21680, an A2AR 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 A2AR-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 A2AR-CB2R heteromers as promising targets for biased signaling-based therapeutics.