In response to three highly conserved neuropeptides, neuropeptide Y (NPY), peptide YY, and pancreatic polypeptide (PP), four G protein–coupled receptors mediate multiple essential physiological processes, such as food intake, vasoconstriction, sedation, and memory retention. Here, we report the structures of the human Y ₁ , Y ₂ , and Y ₄ receptors in complex with NPY or PP, and the G _i1 protein. These structures reveal distinct binding poses of the peptide upon coupling to different receptors, reflecting the importance of the conformational plasticity of the peptide in recognizing the NPY receptors. The N terminus of the peptide forms extensive interactions with the Y ₁ receptor, but not with the Y ₂ and Y ₄ receptors. Supported by mutagenesis and functional studies, subtype-specific interactions between the receptors and peptides were further observed. These findings provide insight into key factors that govern NPY signal recognition and transduction, and would enable development of selective drugs.

Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes.

Background: The neuropeptide Y system affects various processes, among others food intake, and is frequently discussed in the context of targeting obesity. Studies in model organisms are indispensable to enable molecular studies in a physiological context. Although the NPY system is evolutionarily conserved in all bilaterians, in the widely used modelCaenorhabditis elegansthere is controversy on the existence of NPY orthologous molecules. While the FMRFamide-like peptide (FLP)/Neuropeptide receptor-Resemblance (NPR) system in the nematode was initially suggested to be orthologous to the mammalian NPY system, later global phylogenetic studies indicate that FLP/NPR is protostome-specific.Methods: We performed a comprehensive pharmacological study of the FLP/NPR system in transfected cells in vitro, and tested for functional substitution inC. elegansknockout strains. Further, we phenotypically compared differentflploss-of-function strains. Differences between groups were compared by ANOVA and post-hoc testing (Dunnett, Bonferroni).Results: Our pharmacological analysis of the FLP/NPR system including formerly functionally uncharacterized NPY-like peptides fromC. elegansdemonstrates that G protein-coupling and ligand requirements for receptor activation are similar to the human NPY system. In vitro and in vivo analyses show cross-reactivity of NPY with the FLP/NPR system manifesting in the ability of the human GPCRs to functionally substitute FLP/NPR signaling in vivo. The high pharmacological/functional similarities enabled us to identifyC. elegansFLP-14 as a key molecule in avoidance behavior.Conclusions: Our data demonstrate the pharmacological and functional similarities of human NPY andC. elegansNPR systems. This adds a novel perspective to current phylogenetic reconstructions of the neuropeptide Y system. NPY and NPR receptors are pharmacologically so similar that the human receptors can functionally compensate for theC. elegansones, suggesting orthologous relationships. This is also underlined by the presence of NPY-like peptides and parallels in peptide requirements for receptor activation. Further, the results presented here highlight the potential of this knowledge for physiological as well as molecular studies on neuropeptide GPCRs such as the NPY system in the future.

Ligand binding and pathway-specific activation of G protein-coupled receptors (GPCRs) is currently study 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 NPY bound to the Y₂R (Kaiser et al., 2015). 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 pre-assembly 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 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 naphtylalanine 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.

Background: A sensitive balance between receptor activation and desensitization is crucial for cellular homeostasis. Like many other GPCR, the human neuropeptide Y₂ receptor (hY₂R) undergoes ligand dependent activation and internalization into intracellular compartments, followed by recycling to the plasma membrane. This receptor is involved in the pathophysiology of distinct diseases e.g. epilepsy and cancer progression and conveys anorexigenic signals which makes it an interesting and promising anti-obesity target. However, Y₂R desensitization was observed after daily treatment with a selective PYY_13–36 analog in vivo by a yet unknown mechanism. Materials: We studied the desensitization and activatability of recycled Y₂R in transiently transfected HEK293 cells as well as in endogenously Y₂R expressing SH-SY5Y and SMS-KAN cells. Results were evaluated by one-way ANOVA and Tukey post test. Results: We observed strong desensitization of the Y₂R in a second round of stimulation despite its reappearance at the membrane. Already the first activation of the Y₂R leads to depletion of the functional cellular Gα_i/o protein pool and consequently desensitizes the linked signal transduction pathways, independent of receptor internalization. This desensitization also extends to other Gα_i/o-coupled GPCR and can be detected in transfected HEK293 as well as in SH-SY5Y and SMS-KAN cell lines, both expressing the Y₂R endogenously. By overexpression of chimeric Gα_qi proteins in a model system, activation has been rescued, which identifies a critical role of the G protein status for cellular signaling. Furthermore, Y₂R displays strong allosteric coupling to inhibitory G proteins in radioligand binding assays, and loses 10-fold affinity in the G protein-depleted state observed after activation, which can be largely abrogated by overexpression of the Gα_i-subunit. Conclusion: The unusually persistent Gα_i-signaling of the Y₂R leads to a state of cellular desensitization of the inhibitory Gα_i-pathway. The strong allosteric effects of the Y₂R-Gα_i-interaction might be a mechanism that contributes to the burst of Gα_i-signaling, but also serves as a mechanism to limit the Y₂-mediated signaling after recycling. Thus, the cell is left in a refractory state, preventing further Gα_i-signaling of the Y₂R itself but also other Gα_i/o-coupled receptors by simply controlling the repertoire of downstream effectors. Graphical