Homodimerization of CB2 cannabinoid receptor triggered by a bivalent ligand enhances cellular signaling

G protein-coupled receptors (GPCRs) exist within a landscape of interconvertible conformational states and in dynamic equilibrium between monomers and higher-order oligomers, both influenced by ligand binding. Here, we show that a homobivalent ligand formed by equal chromenopyrazole moieties as pharmacophores, connected by 14 methylene units, can modulate the dynamics of the cannabinoid CB 2 receptor (CB 2 R) homodimerization by simultaneously binding both protomers of the CB 2 R-CB 2 R homodimer. Computational and pharmacological experiments showed that one of the ligand pharmacophores binds to the orthosteric site of one protomer, and the other pharmacophore to a membrane-oriented pocket between transmembranes 1 and 7 of the partner protomer. This results in unique pharmacological properties, including increased potency in G i -mediated signaling and enhanced recruitment of β-arrestin. Thus, by modulating dimerization dynamics, it may be possible to fine-tune CB 2 R activity, potentially leading to improved therapeutic outcomes. • A homobivalent ligand of CB 2 R (PM369) modulates the dynamics of receptor homodimerization. • PM369 binds to the orthosteric site of one protomer and to a complementary, membrane-facing, site of the other protomer. • PM369 triggers CB 2 R homodimerization via the TM 1/7 interface that provides unique pharmacological properties. • PM369 potentiates signaling, increases potency in G i binding and increases recruitment of β-arrestin. • These results disclose new approaches to modulating GPCR signaling.