Abstact

Neuropeptide FF receptors 1 and 2 (NPFFR1 and NPFFR2) are RF-amide peptide receptors that couple to Gi/o proteins and regulate pain, opioid tolerance, and metabolism. Despite their physiological significance, their ligand selectivity and activation mechanisms remain unclear. Using cryoelectron microscopy, we resolved four NPFFR1 and NPFFR2 structures bound to NPFF or NPVF, revealing conserved C-terminal RF-amide interactions within the orthosteric pocket and N-terminal variations driving subtype specificity. Structural and mutagenesis analyses identified ECL2 and the receptor N terminus as key determinants of NPVF-NPFFR1 and NPFF-NPFFR2 selectivity. Additionally, the structures elucidate the activation mechanism and uncover distinct Gi-coupling features between NPFFR subtypes. These findings provide molecular insights into peptide recognition and receptor activation within the RF-amide family, offering a structural framework for designing selective NPFFR modulators to treat pain, addiction, and metabolic disorders with enhanced specificity and reduced off-target effects.