Conserved class B GPCR activation by a biased intracellular agonist.

Class B G protein-coupled receptors (GPCRs), including glucagon-like receptor 1 (GLP-1R) and parathyroid hormone receptor 1 (PTH1R), are important drug targets 1-5 . Injectable peptide drugs targeting these receptors have been developed but orally available small molecule drugs are hotly pursued 6,7 . Here we report the high-resolution structure of the 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 binding site of PCO371 is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. Residues that make up the PCO371 binding pocket are conserved in class B GPCRs and a single mutation in PTH type 2 receptor (PTH2R) and two residue mutations in GLP-1R convert these receptors to respond to PCO371 activation. Functional assays reveal that PCO371 is a G-protein biased agonist that is defective in promoting PTH1R-mediated arrestin signaling. Together, these results uncover a distinct binding site for designing small molecule agonists for PTH1R and possible other members of class B GPCRs and define a receptor conformation that is only specific for G protein activation but not arrestin signaling. These insights should facilitate the design of distinct types of class B GPCR small molecule agonists for various therapeutic indications.