N-terminally modified GLP1R agonists drive G protein bias via extracellular loop 3 displacement.

The glucagon-like peptide-1 receptor (GLP1R), a class B G protein-coupled receptor (GPCR), is a pivotal therapeutic target for type 2 diabetes mellitus and obesity. GLP1R agonists signal through both G protein and β-arrestin pathways, associated with distinct receptor conformational and trafficking states. Here, we develop G protein-biased GLP1R agonists through N-terminal modifications, including acetylation or amino acid substitutions, which exhibit preferential G protein signaling relative to the reference ligand. Cryo-EM structure of GLP1-Y11-GLP1R-G complex at 2.64 Å resolution reveals that outward displacement of extracellular loop 3 (ECL3) is the key structural feature associated with signaling bias, distinguishing the complex from β-arrestin-biased agonist-bound state. Ac-GLP1 and Ac-semaglutide display altered receptor trafficking, attenuated β-arrestin recruitment, and prolonged cAMP signaling. In diet-induced obese mice, Ac-semaglutide maintains glucose-lowering activity three days post-administration. These findings elucidate the structural basis of GLP1R biased agonism and provide mechanistic insights into class B GPCR signaling modulation.