UDP-glucose and MRS2905 agonist-bound states of the purinergic P2Y₁₄ receptor

P2Y₁₄ receptors respond to uracil-diphosphate-hexose conjugates, yet how this receptor selectively recognizes both uracil-nucleotides and hexose moieties of diverse agonists remains unclear. Here we report the active agonist-bound G protein-bound states of the P2Y₁₄ G protein-coupled receptor (GPCR) bound to endogenous agonist uridine diphosphate glucose (UDP-glucose) and 2-thiouridine-5'-O-(α,β-methylene)diphosphate (MRS2905). The cryo-EM structures of the heterotrimeric complexes with 3.19 Å and 3.05 Å global resolution, respectively, with local refinements reaching 2.87 Å and 3.22 Å for the masked receptor region. Our structures reveal a pronounced extracellular facing electronegative vestibule connecting to a smaller nucleotide binding subpocket (~300ų volume) that is shielded by extracellular loop 2 (ECL2). A glucose-binding subpocket is spatially delimited by residue V93; mutation to Trp selectively blocks UDP-glucose while permitting MRS2905 and antagonist binding. These findings provide atomic insights into uracil recognition, reveal how the receptor accommodates diverse flexible UDP-sugars, and promise to enable rational drug discovery of therapeutic P2Y₁₄R modulators.