G Protein Preassembly Rescues Efficacy of W6.48 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 W6.48 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-Gi complex. It further confirmed ligand-dependent recruitment of arrestin3. Using equally sensitive readouts for Gi activation and arrestin recruitment as well as quantification with operational models of agonism allowed us to identify a strong inherent bias for Gi activation over arrestin3 recruitment for the wild-type receptor. By systematic mutagenesis, we found that W6.48 does not contribute to the binding affinity, but acts as an allosteric connector to couple ligand binding to Gi 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 W6.48 for the activation of the receptor are discussed in the context of different mechanisms of G protein coupling and arrestin recruitment.