G Protein Preassembly Rescues Efficacy of W^6.48 Toggle Mutations in Neuropeptide Y₂ Receptor

Ligand binding and pathway-specific activation of G protein-coupled receptors is currently being studied with great effort. Individual answers may depend on the nature of the ligands and the effector pathway. Recently, we have presented a detailed model of neuropeptide Y bound to the Y₂R. Accordingly, the C-terminal part of the peptide binds deeply in the transmembrane bundle and brings the side chain of the most essential Y³⁶ in close proximity to W^6.48 Here, we investigate the role of this interaction for ligand binding and activation of this receptor. BRET sensors were used for detailed investigation of effector coupling and led to the identification of preassembly of the Y₂R-G_i complex. It further confirmed ligand-dependent recruitment of arrestin3. Using equally sensitive readouts for G_i activation and arrestin recruitment as well as quantification with operational models of agonism allowed us to identify a strong inherent bias for G_i activation over arrestin3 recruitment for the wild-type receptor. By systematic mutagenesis, we found that W^6.48 does not contribute to the binding affinity, but acts as an allosteric connector to couple ligand binding to G_i activation and arrestin3 recruitment. However, even mutagenesis to a small threonine did not lead to a complete loss of signaling. Interestingly, signaling was restored to wild-type levels by ligands that contain a naphthylalanine as the C-terminal residue instead of Y³⁶ Steric and polar contributions of W^6.48 for the activation of the receptor are discussed in the context of different mechanisms of G protein coupling and arrestin recruitment.