Tethered peptide activation mechanism of the adhesion GPCRs ADGRG2 and ADGRG4

Adhesion G protein-coupled receptors (aGPCRs) constitute an evolutionarily ancient family of receptors that often undergo autoproteolysis to produce α and β subunits 1 – 3 . A tethered agonism mediated by the ‘Stachel sequence’ of the β subunit has been proposed to have central roles in aGPCR activation 4 – 6 . Here we present three cryo-electron microscopy structures of aGPCRs coupled to the G s heterotrimer. Two of these aGPCRs are activated by tethered Stachel sequences—the ADGRG2-β–G s complex and the ADGRG4-β–G s complex (in which β indicates the β subunit of the aGPCR)—and the other is the full-length ADGRG2 in complex with the exogenous ADGRG2 Stachel-sequence-derived peptide agonist IP15 (ADGRG2(FL)–IP15–G s ). The Stachel sequences of both ADGRG2-β and ADGRG4-β assume a U shape and insert deeply into the seven-transmembrane bundles. Constituting the FXφφφXφ motif (in which φ represents a hydrophobic residue), five residues of ADGRG2-β or ADGRG4-β extend like fingers to mediate binding to the seven-transmembrane domain and activation of the receptor. The structure of the ADGRG2(FL)–IP15–G s complex reveals the structural basis for the improved binding affinity of IP15 compared with VPM–p15 and indicates that rational design of peptidic agonists could be achieved by exploiting aGPCR-β structures. By converting the ‘finger residues’ to acidic residues, we develop a method to generate peptidic antagonists towards several aGPCRs. Collectively, our study provides structural and biochemical insights into the tethered activation mechanism of aGPCRs. Cryo-electron microscopy structures of three adhesion G protein-coupled receptors (aGPCRs) complexes provide insight into the tethered activation mechanism of aGPCRs and show the potential for rational design of agonists.