An extracellular allosteric gate modulates the ligand efficacy of GPR119

GPR119 is a promising therapeutic target for metabolic diseases, yet the structural basis for its ligand-dependent efficacy remains unclear. Here, we report the cryo-EM structure of GPR119 bound to the partial agonist AS1269574 and Gs protein, revealing a non-canonical ligand-binding mode. Unlike full agonists, AS1269574 occupies a restricted pocket within the activation cavity and stabilizes a closed conformation of an extracellular allosteric "stacking gate" formed by F157^ECL2 and W265^7.39. Together with MD simulations, our structure shows that this confined binding mode attenuates the progression of activation switches, including the swing of W238^6.48 and outward movement of TM6, thereby shifting the receptor equilibrium toward a low-efficacy state. Gate-disrupting mutations enhance AS1269574-stimulated cAMP production and shift intracellular switch conformations to a full-agonist-like state. Our work elucidates a structural mechanism for partial agonism in G protein-coupled receptors (GPCRs), in which ligand-specific engagement of an extracellular gate allosterically tunes the transmembrane conformational landscape and signaling efficacy.