Orthogonal Peptide-Templated Labeling Elucidates Lateral ETA R/ETB R Proximity and Reveals Altered Downstream Signaling.

Fine-tuning of G protein-coupled receptor (GPCR) signaling is important to maintain cellular homeostasis. Recent studies demonstrated that lateral GPCR interactions in the cell membrane can impact signaling profiles. Here, we report on a one-step labeling method of multiple membrane-embedded GPCRs. Based on short peptide tags, complementary probes transfer the cargo (e. g. a fluorescent dye) by an acyl transfer reaction with high spatial and temporal resolution within 5 min. We applied this approach to four receptors of the cardiovascular system: the endothelin receptor A and B (ETA R and ETB R), angiotensin II receptor type 1, and apelin. Wild type-like G protein activation after N-terminal modification was demonstrated for all receptor species. Using FRET-competent dyes, a constitutive proximity between hetero-receptors was limited to ETA R/ETB R. Further, we demonstrate, that ETA R expression regulates the signaling of co-expressed ETB R. Our orthogonal peptide-templated labeling of different GPCRs provides novel insight into the regulation of GPCR signaling.

Arrestin-dependent internalization of rhodopsin-like G protein-coupled receptors.

The internalization of G protein-coupled receptors (GPCRs) is an important mechanism regulating the signal strength and limiting the opportunity of receptor activation. Based on the importance of GPCRs, the detailed knowledge about the regulation of signal transduction is crucial. Here, current knowledge about the agonist-induced, arrestin-dependent internalization process of rhodopsin-like GPCRs is reviewed. Arrestins are conserved molecules that act as key players within the internalization process of many GPCRs. Based on highly conserved structural characteristics within the rhodopsin-like GPCRs, the identification of arrestin interaction sites in model systems can be compared and used for the investigation of internalization processes of other receptors. The increasing understanding of this essential regulation mechanism of receptors can be used for drug development targeting rhodopsin-like GPCRs. Here, we focus on the neuropeptide Y receptor family, as these receptors transmit various physiological processes such as food intake, energy homeostasis, and regulation of emotional behavior, and are further involved in pathophysiological processes like cancer, obesity and mood disorders. Hence, this receptor family represents an interesting target for the development of novel therapeutics requiring the understanding of the regulatory mechanisms influencing receptor mediated signaling.

Illuminating the neuropeptide Y4 receptor and its ligand pancreatic polypeptide from a structural, functional, and therapeutic perspective.

The neuropeptide Y4 receptor (Y4R), a rhodopsin-like G protein-coupled receptor (GPCR) and the hormone pancreatic polypeptide (PP) are members of the neuropeptide Y family consisting of four receptors (Y1R, Y2R, Y4R, Y5R) and three highly homologous peptide ligands (neuropeptide Y, peptide YY, PP). In this family, the Y4R is of particular interest as it is the only subtype with high affinity to PP over NPY. The Y4R, as a mediator of PP signaling, has a pivotal role in appetite regulation and energy homeostasis, offering potential avenues for the treatment of metabolic disorders such as obesity. PP as anorexigenic peptide is released postprandial from the pancreas in response to food intake, induces satiety signals and contributes to hamper excessive food intake. Moreover, this system was also described to be associated with different types of cancer: overexpression of Y4R have been found in human adenocarcinoma cells, while elevated levels of PP are related to the development of pancreatic endocrine tumors. The pharmacological relevance of the Y4R advanced the search for potent and selective ligands for this receptor subtype, which will be significantly progressed through the elucidation of the active state PP-Y4R cryo-EM structure. This review summarizes the development of novel PP-derived ligands, like Obinepitide as dual Y2R/Y4R agonist in clinical trials or UR-AK86c as small hexapeptide agonist with picomolar affinity, as well as the first allosteric modulators that selectively target the Y4R, e.g. VU0506013 as potent Y4R positive allosteric modulator or (S)-VU0637120 as allosteric antagonist. Here, we provide valuable insights into the complex physiological functions of the Y4R and PP and the pharmacological relevance of the system in appetite regulation to open up new avenues for the development of tool compounds for targeted therapies with potential applications in metabolic disorders.

Receptor-specific recognition of NPY peptides revealed by structures of NPY receptors.

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 Y1, Y2, and Y4 receptors in complex with NPY or PP, and the Gi1 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 Y1 receptor, but not with the Y2 and Y4 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.