Conserved class B GPCR activation by a biased intracellular agonist

Class B G-protein-coupled receptors (GPCRs), including glucagon-like peptide 1 receptor (GLP1R) and parathyroid hormone 1 receptor (PTH1R), are important drug targets 1 – 5 . Injectable peptide drugs targeting these receptors have been developed, but orally available small-molecule drugs remain under development 6 , 7 . Here we report the high-resolution structure of human PTH1R in complex with the stimulatory G protein (G s ) and a small-molecule agonist, PCO371, which reveals an unexpected binding mode of PCO371 at the cytoplasmic interface of PTH1R with G s . The PCO371-binding site is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. The residues that make up the PCO371-binding pocket are conserved in class B GPCRs, and a single alteration in PTH2R and two residue alterations in GLP1R convert these receptors to respond to PCO371. Functional assays reveal that PCO371 is a G-protein-biased agonist that is defective in promoting PTH1R-mediated arrestin signalling. Together, these results uncover a distinct binding site for designing small-molecule agonists for PTH1R and possibly other members of the class B GPCRs and define a receptor conformation that is specific only for G-protein activation but not arrestin signalling. These insights should facilitate the design of distinct types of class B GPCR small-molecule agonist for various therapeutic indications.