Functional selectivity of cannabinoids at CB₁ receptors: Comprehensive analysis of G proteins, β-arrestin and cAMP...

Therapeutic targeting of CB₁ has been limited by adverse effects of orthosteric ligands. Allosteric modulation and biased signalling have been proposed to achieve pathway-selective control, but the receptor's complex coupling complicates interpretation of ligand actions. In this study, we systematically compared orthosteric and allosteric CB₁ ligands in transfected HEK293T cells by measuring G-protein coupling and downstream cAMP regulation in real time using NanoBiT and GloSensor assays. Orthosteric agonists CP 55,940 and methanandamide activated both G_i and G_s with weak and transient β-arrestin2 recruitment, producing potent inhibition of forskolin-stimulated cAMP and modest increases in basal levels. The inverse agonist AM251 reduced basal G_i coupling and increased basal cAMP, consistent with inverse agonism at constitutively active CB₁ receptors. Under forskolin stimulation, AM251 attenuated agonist-induced inhibition of cAMP and produced concentration-dependent modulation of forskolin-stimulated responses. AM4113 marginally reduced G-protein coupling and increased basal, while modestly reducing forskolin-stimulated cAMP, consistent with very low inverse efficacy. Among allosteric ligands, the positive allosteric modulator GAT229 increased G_i coupling and, with orthosteric agonists, further enhanced G_i and G_s signalling, elevating basal cAMP, without affecting β-arrestin2. The negative allosteric modulators PSNCBAM-1 and ORG27569 each reduced G_i- and G_s-coupling, elevated basal cAMP, and attenuated agonist-induced inhibition of forskolin-stimulated cAMP. Cannabidiol showed no intrinsic activity at CB₁ and marginally reduced orthosteric G-protein signalling, consistent with indirect interaction with the receptor. In summary, this study shows that orthosteric and allosteric CB₁ ligands differ in G-protein coupling and cAMP regulation, clarifying key aspects of cannabinoid receptor signalling.