Human Y1 Receptor Research Articles

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.

Design, synthesis, and biological evaluation of a multifunctional neuropeptide-Y conjugate for selective nuclear delivery of radiolanthanides

BackgroundTargeting G protein-coupled receptors on the surface of cancer cells with peptide ligands is a promising concept for the selective tumor delivery of therapeutically active cargos, including radiometals for targeted radionuclide therapy (TRT). Recently, the radiolanthanide terbium-161 (161Tb) gained significant interest for TRT application, since it decays with medium-energy β-radiation but also emits a significant amount of conversion and Auger electrons with short tissue penetration range. The therapeutic efficiency of radiometals emitting Auger electrons, like 161Tb, can therefore be highly boosted by an additional subcellular delivery into the nucleus, in order to facilitate maximum dose deposition to the DNA. In this study, we describe the design of a multifunctional, radiolabeled neuropeptide-Y (NPY) conjugate, to address radiolanthanides to the nucleus of cells naturally overexpressing the human Y1 receptor (hY1R).By using solid-phase peptide synthesis, the hY1R-preferring [F7,P34]-NPY was modified with a fatty acid, a cathepsin B-cleavable linker, followed by a nuclear localization sequence (NLS), and a DOTA chelator (compound pb12). In this proof-of-concept study, labeling was performed with either native terbium-159 (natTb), as surrogate for 161Tb, or with indium-111 (111In).Results[natTb]Tb-pb12 showed a preserved high binding affinity to endogenous hY1R on MCF-7 cells and was able to induce receptor activation and internalization similar to the hY1R-preferring [F7,P34]-NPY. Specific internalization of the 111In-labeled conjugate into MCF-7 cells was observed, and importantly, time-dependent nuclear uptake of 111In was demonstrated. Study of metabolic stability showed that the peptide is insufficiently stable in human plasma. This was confirmed by injection of [111In]In-pb12 in nude mice bearing MCF-7 xenograft which showed specific uptake only at very early time point.ConclusionThe multifunctional NPY conjugate with a releasable DOTA-NLS unit represents a promising concept for enhanced TRT with Auger electron-emitting radiolanthanides. Our research is now focusing on improving the reported concept with respect to the poor plasmatic stability of this promising radiopeptide.

Dynamics of the Human Y1-Receptor in the Inactive and APO States Investigated by Solid-State Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulations

The Y1 receptor (Y1R) is part of the G protein-coupled receptor (GPCR) superfamily, which plays a crucial role in many biological signaling processes and is of high interest for medical applications. It is involved in many physiological and pathophysiological functions, related to obesity and cancer. Y1R is activated by neuropeptide Y (NPY), which is involved in several physiological processes including the stimulation of food intake. Here, we studied the receptor dynamics bound to UR-MK299, which is a small molecular weight antagonist, and in the apo form both embedded in POPC membranes at 37 °C. For investigation by nuclear magnetic resonance (NMR) spectroscopy the receptor was labeled uniformly with 13C. This allowed investigating its dynamics by measurement of order parameters. However, due to the uniform labeling the NMR order parameters are averaged over the whole receptor without any site resolution and therefore could only show general differences between the different states. To obtain information of individual segmental motions, we conducted molecular dynamics (MD) simulations of both states using the recently published crystal structure in presence of UR-MK299 (PDB entry code: 5ZBQ). For the apo state, the antagonist was removed and the receptor relaxed for several microseconds before analysis. For the UR-MK299 bound state the CHARMM force field of the antagonist was parametrized with Gaussian. The simulations were prepared following a newly developed general scheme for setup of GPCR MD simulations. The production runs were conducted for several microseconds and compared to the experimentally available data followed by a detailed analysis of the segmental motions. Aim of this analysis is to elucidate the complex structure function dynamics relationship of GPCRs combining state-of-the-art biophysical methods to obtain their time resolved motions.

Biased agonists at the human Y1 receptor lead to prolonged membrane residency and extended receptor G protein interaction.

Functionally selective ligands to address specific cellular responses downstream of G protein-coupled receptors (GPCR) open up new possibilities for therapeutics. We designed and characterized novel subtype- and pathway-selective ligands. Substitution of position Q34 of neuropeptide Y to glycine (G34-NPY) results in unprecedented selectivity over all other YR subtypes. Moreover, this ligand displays a significant bias towards activation of the Gi/o pathway over recruitment of arrestin-3. Notably, no bias is observed for an established Y1R versus Y2R selective ligand carrying a proline at position 34 (F7,P34-NPY). Next, we investigated the spatio-temporal signaling at the Y1R and demonstrated that G protein-biased ligands promote a prolonged localization at the cell membrane, which leads to enhanced G protein signaling, while endosomal receptors do not contribute to cAMP signaling. Thus, spatial components are critical for the signaling of the Y1R that can be modulated by tailored ligands and represent a novel mode for biased pathways.

Selective Neuropeptide Y Conjugates with Maximized Carborane Loading as Promising Boron Delivery Agents for Boron Neutron Capture Therapy.

G-protein-coupled receptors like the human Y1 receptor (hY1R) are promising targets in cancer therapy due to their high overexpression on cancer cells and their ability to internalize together with the bound ligand. This mechanism was exploited to shuttle boron atoms into cancer cells for the application of boron neutron capture therapy (BNCT), a noninvasive approach to eliminate cancer cells. A maximized number of carboranes was introduced to the hY1R-preferring ligand [F7,P34]-NPY by solid phase peptide synthesis. Branched conjugates loaded with up to 80 boron atoms per peptide molecule exhibited a maintained receptor activation profile, and the selective uptake into hY1R-expressing cells was demonstrated by internalization studies. In order to ensure appropriate solubility in aqueous solution, we proved the need for eight hydroxyl groups per carborane. Thus, we suggest the utilization of bis-deoxygalactosyl-carborane building blocks in solid phase peptide synthesis to produce selective boron delivery agents for BNCT.

Functional characterization in vitro of twelve naturally occurring variants of the human pancreatic polypeptide receptor NPY4R

Obesity has become a global health problem and therefore understanding of the mechanisms regulating hunger and satiety is of utmost importance for the development of new treatment strategies. The Y4 receptor, encoded by the NPY4R gene, and its ligand pancreatic polypeptide (PP) have been reported to mediate a satiety signal. Multiple genetic studies have reported an association between NPY4R copy number and body weight. The gene also displays several SNP variants, many of which lead to amino acid differences, making it interesting to study. We have investigated the functional properties of 12 naturally occurring amino acid sequence variants of the Y4 and interpret the results in relation to sequence conservation and our structural model of the human Y4 receptor protein. Three receptor variants, Cys201ECL2Tyr, Val2716.41Leu and Asn3187.49Asp, were found to completely lose functional response, measured as inositol phosphate turnover, while retaining membrane expression. They display high sequence conservation and have important roles in the receptor structure. For two receptor variants the potency of PP was significantly decreased, Cys34NTSer (EC50 = 2.9 nM, p < .001) and Val1353.46Met (EC50 = 3.0 nM, p < .01), compared to wild-type Y4 (EC50 = 0.68 nM). Cys34 forms a disulphide bond with Cys298, linking the N-terminal part to ECL3. The Val1353.46Met variant has an amino acid replacement located in the TM3 helix, one helix turn above the highly conserved ERH motif. This position has influence on the network of residues involved in receptor activation and subsequent inactivation. Sequence conservation and the structural model are consistent with these results. The remaining seven positions had no significant effect on the receptor's functional response compared to wild-type Y4. These positions display more variation during evolution. Understanding of the interactions between the Y4 receptor and its native PP agonist and the effects of amino acid variation on its functional response will hopefully lead to future therapeutic possibilities.

Carbaboranylation of Truncated C-Terminal Neuropeptide Y Analogue Leads to Full hY1 Receptor Agonism.

The human Y1 receptor is overexpressed in breast tumour cells and is, therefore, a valuable target for site-selective drug delivery. The well-established hY1 R-selective ligand [Phe7,Pro34]NPY has been used to couple to drugs but its length of 36 amino acids also implies complex synthesis and high production costs. Therefore, shorter ligands are desirable. However, truncated versions of neuropeptide Y (NPY) usually result in reduced binding, antagonists, or only partial G-protein-biased agonists. Herein, we report on a nonamer peptide derived from the C terminus of NPY that is modified by a carbaborane, which is able to activate hY1 R fully in terms of G-protein activation but also arrestin recruitment and internalisation. We provide evidence that this unique behaviour is due to the bulky nature of the carbaborane cluster, which might address a specific second binding pocket in hY1 R that is crucial for arrestin recruitment. Thus, this study helps in deciphering ligand-induced onset of different pathways in hY1 R mediated signalling.

Different mode of arrestin-3 binding at the human Y1 and Y2 receptor

GPCR internalization, which is induced by arrestin recruitment, is an important mechanism for the regulation of signaling and receptor quantity at the cell surface.In this study, differences in the mechanism of arrestin-3 (arr-3) recruitment to the neuropeptide Y1 and Y2 receptor were identified. These receptors play an essential role in the regulation of feeding, energy homeostasis and cancer. The Y1R displays high affinity to arr-3, which induces rapid internalization of the arrestin/receptor complex. In contrast, the Y2R has a lower affinity for arr-3. Internalization is induced by arrestin binding, but arr-3 is released from the receptor and remains at the membrane while the receptor internalizes. Moreover, the deletion of the finger loop region of arr-3 reduces its agonist-dependent recruitment to the Y2R significantly, but not to the Y1R suggesting different binding conformations.For the first time, the formation of a supercomplex consisting of Y receptor, Gα0 protein and arrestin was studied by BRET-assay. We demonstrated that the Y1R is able to bind Gα0 protein as well as arr-3 simultaneously and internalizes as a supercomplex. For the Y2R no supercomplex formation was observed.By substituting the C-terminus or specific residues within the intracellular loop 1 and 2 of the receptors, the arr-3 recruitment of the Y1R and Y2R can be switched. Thus, we shed light on the specific spatio-temporal distribution of Gα0 protein and arrestin in response to Y1 versus Y2 receptor activation and identified the molecular determinants.

Elucidation of the Binding Mode of the Carboxyterminal Region of Peptide YY to the Human Y2 Receptor.

Understanding the agonist-receptor interactions in the neuropeptide Y (NPY)/peptide YY (PYY) signaling system is fundamental for the design of novel modulators of appetite regulation. We report here the results of a multidisciplinary approach to elucidate the binding mode of the native peptide agonist PYY to the human Y2 receptor, based on computational modeling, peptide chemistry and in vitro pharmacological analyses. The preserved binding orientation proposed for full-length PYY and five analogs, truncated at the amino terminus, explains our pharmacological results where truncations of the N-terminal proline helix showed little effect on peptide affinity. This was followed by receptor mutagenesis to investigate the roles of several receptor positions suggested by the modeling. As a complement, PYY-(3-36) analogs were synthesized with modifications at different positions in the common PYY/NPY C-terminal fragment (32TRQRY36-amide). The results were assessed and interpreted by molecular dynamics and Free Energy Perturbation (FEP) simulations of selected mutants, providing a detailed map of the interactions of the PYY/NPY C-terminal fragment with the transmembrane cavity of the Y2 receptor. The amidated C-terminus would be stabilized by polar interactions with Gln2886.55 and Tyr2195.39, while Gln1303.32 contributes to interactions with Q34 in the peptide and T32 is close to the tip of TM7 in the receptor. This leaves the core, α-helix of the peptide exposed to make potential interactions with the extracellular loops. This model agrees with most experimental data available for the Y2 system and can be used as a basis for optimization of Y2 receptor agonists.

Structural insights in the binding mode of neuropeptide Y at G protein coupled receptors and consequences for drug development

Peptides hormones assume a significant job in the guideline of complex exercises in the body. A considerable lot of them transmit their movement through G-Protein Coupled Receptors (GPCR), which are among the most encouraging medication targets now-a-days. Likewise, explaining the coupling method of ligands is basic. While a few little particle frameworks have been all around portrayed, ligand official of enormous and adaptable peptides is still all the more testing. Notwithstanding ligand authoritative and receptor actuation, aberrant components have been appeared to assume a job for drugs tending to GPCRs. This incorporates desensitization, disguise and in like manner their expected use as medication transports, for example in tumor focusing on. As needs be, notwithstanding ligand official, disguise must be tended to and to be examined, including arrestin enrollment. As needs be, ligand authoritative, auxiliary elements and disguise must be tended to and to be concentrated to address G protein coupled receptors for medicate improvement. The neuropeptide Y/pancreatic polypeptide family contains 36 amino corrosive peptides that quandary in human to four unique purported Y-receptors. By a mix of X-beam investigation, NMR, sub-atomic demonstrating and cross connecting joined with mass spectrometry, we could as of late recognize the unmistakable restricting methods of NPY to the Y1-and the Y2 receptors. We could additionally exhibit that concoction adjustment of the ligand, including fluorescence marking, lipidization and PEGylation altogether changes the dealing of the ligand. By naming of the receptor with a novel format helped ligation technique, we can follow ligand/receptor buildings in living cells. Besides, we recognized an alternate method of arrestin authoritative and enrollment. Neuropeptide Y1 and Y2 receptors have been appeared to assume a significant job in various tumors. In bosom disease we showed that human Y1 receptors are addressable by peptide conjugates utilizing 99mTc or 18F PET-tracers. We presently planned Y1 receptor specific peptides connected to various toxophors. Moreover, we portrayed the system of direct and peptide-interceded take-up of tubulysin-related poisons. In the field of tumor treatment, peptide-tranquilize conjugates are now all around acknowledged. Be that as it may, the idea of receptor-intervened disguise and resulting tissue explicit intracellular application isn't restricted to the specific tending to of tumors. This may open up another field of focused treatment by medium sized medications.

Identification and Characterization of the First Selective Y4 Receptor Positive Allosteric Modulator.

The human Y4 receptor (Y4R) and its cognate ligand, pancreatic polypeptide (PP), are involved in the regulation of energy expenditure, satiety, and food intake. This system represents a potential target for the treatment of metabolic diseases and has been extensively investigated and validated in vivo. Here, we present the compound tBPC (tert-butylphenoxycyclohexanol), a novel and selective Y4R positive allosteric modulator that potentiates Y4R activation in G-protein signaling and arrestin3 recruitment experiments. The compound has no effect on the binding of the orthosteric ligands, implying its allosteric mode of action at the Y4R and evidence for a purely efficacy-driven positive allosteric modulation. Finally, the ability of tBPC to selectively potentiate Y4R agonism initiated by PP was confirmed in mouse descending colon mucosa preparations expressing native Y4R, demonstrating Y4R positive allosteric modulation in vitro.

C-terminal motif of human neuropeptide Y4 receptor determines internalization and arrestin recruitment

The human neuropeptide Y4 receptor is a rhodopsin-like G protein-coupled receptor (GPCR), which contributes to anorexigenic signals. Thus, this receptor is a highly interesting target for metabolic diseases. As GPCR internalization and trafficking affect receptor signaling and vice versa, we aimed to investigate the molecular mechanism of hY4R desensitization and endocytosis. The role of distinct segments of the hY4R carboxyl terminus was investigated by fluorescence microscopy, binding assays, inositol turnover experiments and bioluminescence resonance energy transfer assays to examine the internalization behavior of hY4R and its interaction with arrestin-3. Based on results of C-terminal deletion mutants and substitution of single amino acids, the motif 7.78EESEHLPLSTVHTEVSKGS7.96 was identified, with glutamate, threonine and serine residues playing key roles, based on site-directed mutagenesis. Thus, we identified the internalization motif for the human neuropeptide Y4 receptor, which regulates arrestin-3 recruitment and receptor endocytosis.

Single Cell Peptide Heterogeneity of Rat Islets of Langerhans.

Measuring the chemical composition of individual cells in mammalian organs can provide critical insights toward understanding the mechanisms leading to their normal and pathological function. In this work, single cell heterogeneity of islets of Langerhans is characterized with high throughput by microscopy-guided single cell matrix-assisted laser desorption/ionization mass spectrometry. Two levels of chemical heterogeneity were observed from the analysis of more than 3000 individual cells. Within a single islet, cellular heterogeneity was evident from the exclusive expression of the canonical biomarkers glucagon, insulin, pancreatic polypeptide (PP), and somatostatin within α-, β-, γ-, and δ-cells, respectively. We localized the neuropeptide WE-14, a known cell-to-cell signaling molecule, to individual δ-cells. Moreover, several unreported endogenous peptides generated by dibasic site cleavages of PP were detected within individual γ-cells. Of these, PP(27-36) was previously shown to activate the human Y4 receptor, suggesting it has a signaling role in vivo. Heterogeneity in cell composition was also observed between islets as evidenced by a 50-fold larger α-cell population in islets of the dorsal pancreas compared to the ventral-derived pancreatic islets. Finally, PP(27-36) was more abundant in γ-cells from the ventral region of the pancreas, indicating differences in the extent of PP-prohormone processing in the two regions of the pancreas.

Discovery of Small-Molecule Modulators of the Human Y4 Receptor.

The human neuropeptide Y4 receptor (Y4R) and its native ligand, pancreatic polypeptide, are critically involved in the regulation of human metabolism by signaling satiety and regulating food intake, as well as increasing energy expenditure. Thus, this receptor represents a putative target for treatment of obesity. With respect to new approaches to treat complex metabolic disorders, especially in multi-receptor systems, small molecule allosteric modulators have been in the focus of research in the last years. However, no positive allosteric modulators or agonists of the Y4R have been described so far. In this study, small molecule compounds derived from the Niclosamide scaffold were identified by high-throughput screening to increase Y4R activity. Compounds were characterized for their potency and their effects at the human Y4R and as well as their selectivity towards Y1R, Y2R and Y5R. These compounds provide a structure-activity relationship profile around this common scaffold and lay the groundwork for hit-to-lead optimization and characterization of positive allosteric modulators of the Y4R.

Double Methotrexate-Modified Neuropeptide Y Analogues Express Increased Toxicity and Overcome Drug Resistance in Breast Cancer Cells.

Bioconjugates containing the neuropeptide Y (NPY) analogue [F(7),P(34)]-NPY as targeting moiety are able to deliver toxic agents specifically to breast cancer cells that overexpress the human Y1-receptor (hY1R). To increase their activity, multiple toxophores can be attached to one peptide. Herein, synthesis and characterization of [F(7),P(34)]-NPY conjugates containing two methotrexate (MTX) molecules are presented. First, carboxytetramethylrhodamine was linked to [F(7),P(34)]-NPY by amide or enzymatic linkage. The conjugate containing the enzymatic cleavage site showed high extracellular stability and fast intracellular release. Then, MTX was introduced at positions four and 22 of [F(7),P(34)]-NPY, connected by enzymatic or amide linkage. The toxicity of the analogues on breast cancer cells was hY1R-mediated and dependent on the used linkage and amount of toxophores. Furthermore, conjugates revealed higher potency than MTX on MTX-resistant cells. These results emphasize that peptide-drug conjugates can overcome drug resistance and that the attachment of multiple cleavable toxophores enhances the efficiency of this smart delivery system.

Interactions of human NPY/PYY C-terminal pentapeptide with the human Y2 receptor

Interactions of human NPY/PYY C-terminal pentapeptide with the human Y2 receptor

Dynamic mass redistribution as a label-free method for investigations on human NPY Y2 and Y4 receptors

Dynamic mass redistribution as a label-free method for investigations on human NPY Y2 and Y4 receptors

Influence of distinct motifs within the carboxyl-terminusof the human neuropeptide Y4 receptor on internalization and arrestin3 recruitment

Influence of distinct motifs within the carboxyl-terminusof the human neuropeptide Y4 receptor on internalization and arrestin3 recruitment

Discovery of novel small-molecule modulators of the human Y4 receptor

Discovery of novel small-molecule modulators of the human Y4 receptor

Discovery of novel small-molecule modulators of the human Y4receptor

Discovery of novel small-molecule modulators of the human Y4receptor