The β-arrestin-biased negative allosteric modulator AP-7-168 functions as a molecular glue that stabilizes β2-adrenergic receptor homodimerization through a symmetric TM3-TM4-TM5 interface, selectively preventing β-arrestin and GRK engagement via steric exclusion while permitting G protein coupling, thereby achieving biased signaling through ligand-induced quaternary structure modulation rather than conventional conformational selection.

Family A G-protein coupled receptors (GPCRs) are typically described as monomers, yet growing evidence suggests they can form dimers with distinct signaling properties. The mechanisms and therapeutic potential of such dimerization, however, remain poorly understood. Here, we show that AP-7-168, an optimized derivative of a β-arrestin-biased negative allosteric modulator of the β2-adrenergic receptor (β2AR) that sustains bronchorelaxation in cell and tissue models, functions as a molecular glue to promote β2AR homodimerization. Cryo-EM structures reveal a unique binding mode in which two AP-7-168 molecules pack within a pocket formed by transmembrane helices 3, 4, and 5 of two protomers, stabilizing a dimeric conformation that selectively prevents β-arrestin coupling. In cells, AP-7-168 robustly induces β2AR dimerization and drives enlarged nanocluster formation. Combined with extensive functional studies, our findings unveil a novel allosteric mechanism by which a small molecule biases β2AR signaling through dimerization, highlighting ligand-induced dimerization as a strategy for GPCR modulation.