Structural insight into the self-activation and G-protein coupling of P2Y2 receptor

Abstract Purinergic P2Y2 receptor (P2Y2R) represents a typically extracellular ATP and UTP sensor for mediating purinergic signaling. Despite its importance as a pharmacological target, the molecular mechanisms underlying ligand recognition and G-protein coupling have remained elusive due to lack of structural information. In this study, we determined the cryo-electron microscopy (cryo-EM) structures of the apo P2Y2R in complex with G q , ATP-bound P2Y2R in complex with G q or G o , and UTP-bound P2Y4R in complex with G q . These structures reveal the similarities and distinctions of ligand recognition within the P2Y receptor family. Furthermore, a comprehensive analysis of G-protein coupling reveals that P2Y2R exhibits promiscuity in coupling with both G q and G o proteins. Combining molecular dynamics simulations and signaling assays, we elucidate the molecular mechanisms by which P2Y2R differentiates pathway-specific G q or G o coupling through distinct structural components on the intracellular side. Strikingly, we identify a helix-like segment within the N-terminus that occupies the orthosteric ligand-binding pocket of P2Y2R, accounting for its self-activation. Taken together, these findings provide a molecular framework for understanding the activation mechanism of P2Y2R, encompassing ligand recognition, G-protein coupling, and a novel N-terminus-mediated self-activation mechanism.