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1.
Structure ; 31(1): 44-57.e6, 2023 01 05.
Article in English | MEDLINE | ID: mdl-36525977

ABSTRACT

Neuropeptide Y (NPY) and its receptors are expressed in various human tissues including the brain where they regulate appetite and emotion. Upon NPY stimulation, the neuropeptide Y1 and Y2 receptors (Y1R and Y2R, respectively) activate GI signaling, but their physiological responses to food intake are different. In addition, deletion of the two N-terminal amino acids of peptide YY (PYY(3-36)), the endogenous form found in circulation, can stimulate Y2R but not Y1R, suggesting that Y1R and Y2R may have distinct ligand-binding modes. Here, we report the cryo-electron microscopy structures of the PYY(3-36)‒Y2R‒Gi and NPY‒Y2R‒Gi complexes. Using cell-based assays, molecular dynamics simulations, and structural analysis, we revealed the molecular basis of the exclusive binding of PYY(3-36) to Y2R. Furthermore, we demonstrated that Y2R favors G protein signaling over ß-arrestin signaling upon activation, whereas Y1R does not show a preference between these two pathways.


Subject(s)
Neuropeptide Y , Peptide YY , Humans , Neuropeptide Y/metabolism , Peptide YY/metabolism , Receptors, Neuropeptide Y/genetics , Receptors, Neuropeptide Y/chemistry , Receptors, Neuropeptide Y/metabolism , Cryoelectron Microscopy , Signal Transduction , Receptors, G-Protein-Coupled
2.
Molecules ; 27(12)2022 Jun 09.
Article in English | MEDLINE | ID: mdl-35744852

ABSTRACT

Neuropeptide Y (NPY) is a vastly studied biological peptide with numerous physiological functions that activate the NPY receptor family (Y1, Y2, Y4 and Y5). Moreover, these receptors are correlated with the pathophysiology of several diseases such as feeding disorders, anxiety, metabolic diseases, neurodegenerative diseases, some types of cancers and others. In order to deepen the knowledge of NPY receptors' functions and molecular mechanisms, neuroimaging techniques such as positron emission tomography (PET) have been used. The development of new radiotracers for the different NPY receptors and their subsequent PET studies have led to significant insights into molecular mechanisms involving NPY receptors. This article provides a systematic review of the imaging biomarkers that have been developed as PET tracers in order to study the NPY receptor family.


Subject(s)
Neuropeptide Y , Receptors, Neuropeptide Y , Neuropeptide Y/metabolism , Positron-Emission Tomography , Receptors, Neuropeptide Y/chemistry
3.
J Med Chem ; 64(22): 16746-16769, 2021 11 25.
Article in English | MEDLINE | ID: mdl-34748345

ABSTRACT

The family of neuropeptide Y (NPY) receptors comprises four subtypes (Y1R, Y2R, Y4R, Y5R), which are addressed by at least three endogenous peptides, i.e., NPY, peptide YY, and pancreatic polypeptide (PP), the latter showing a preference for Y4R. A series of cyclic oligopeptidic Y4R ligands were prepared by applying a novel approach, i.e., N-terminus to arginine side-chain cyclization. Most peptides acted as Y4R partial agonists, showing up to 60-fold higher Y4R affinity compared to the linear precursor peptides. Two cyclic hexapeptides (18, 24) showed higher Y4R potency (Ca2+ aequorin assay) and, with pKi values >10, also higher Y4R affinity compared to human pancreatic polypeptide (hPP). Compounds such as 18 and 24, exhibiting considerably lower molecular weight and considerably more pronounced Y4R selectivity than PP and previously described dimeric peptidic ligands with high Y4R affinity, represent promising leads for the preparation of labeled tool compounds and might support the development of drug-like Y4R ligands.


Subject(s)
Arginine/chemistry , Neuropeptide Y/metabolism , Receptors, Neuropeptide Y/metabolism , Amino Acid Sequence , Cyclization , HEK293 Cells , Humans , Ligands , Molecular Docking Simulation , Protein Binding , Receptors, Neuropeptide Y/chemistry
4.
J Biol Chem ; 297(5): 101289, 2021 11.
Article in English | MEDLINE | ID: mdl-34634305

ABSTRACT

Scribble, a member of the LAP protein family, contributes to the apicobasal polarity (ABP) of epithelial cells. The LAP-unique region of these proteins, which is essential and sufficient for ABP, includes a conserved Leucine-Rich Repeat (LRR) domain. The major binding partners of this region that could regulate ABP remain unknown. Here, using proteomics, native gel electrophoresis, and site-directed mutagenesis, we show that the concave surface of LRR domain in Scribble participates in three types of mutually exclusive interactions-(i) homodimerization, serving as an auto-inhibitory mechanism; (ii) interactions with a diverse set of polarity proteins, such as Llgl1, Llgl2, EPB41L2, and EPB41L5, which produce distinct multiprotein complexes; and (iii) a direct interaction with the protein phosphatase, PP1. Analogy with the complex between PP1 and LRR domain of SDS22, a well-studied PP1 regulator, suggests that the Scibble-PP1 complex stores a latent form of PP1 in the basolateral cell cortex. Such organization may generate a dynamic signaling network wherein PP1 could be dispatched from the complex with Scribble to particular protein ligands, achieving fast dephosphorylation kinetics.


Subject(s)
Cell Polarity , Epithelial Cells/metabolism , Membrane Proteins/metabolism , Protein Multimerization , Receptors, Neuropeptide Y/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolism , Cell Line , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Protein Binding , Protein Domains , Receptors, Neuropeptide Y/chemistry , Receptors, Neuropeptide Y/genetics , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics
5.
J Mol Biol ; 433(13): 166992, 2021 06 25.
Article in English | MEDLINE | ID: mdl-33865871

ABSTRACT

The neuropeptide Y (NPY) family is a peptide-activated G protein-coupled receptor system conserved across all bilaterians, and is involved in food intake, learning, and behavior. We hypothesized that comparing the NPY system in evolutionarily ancient organisms can reveal structural determinants of peptide recognition and receptor activation conserved in evolution. To test this hypothesis, we investigated the homologous FLP/NPR system of the protostome C.elegans. For three prototypic peptide-receptor complexes representing different ligand types, we integrate extensive functional data into structural models of the receptors. Common features include acidic patches in the extracellular loops (ECLs) of the receptors that cooperatively 'draw' the peptide into the binding pocket, which was functionally validated in vivo. A structurally conserved glutamate in the ECL2 anchors the peptides by a conserved salt bridge to the arginine of the RFamide motif. Beyond this conserved interaction, peptide binding show variability enabled by receptor-specific interactions. The family-conserved residue Q3.32 is a key player for peptide binding and receptor activation. Altered interaction patterns at Q3.32 may drastically increase the efficacy to activate the receptor.


Subject(s)
Caenorhabditis elegans/metabolism , Neuropeptide Y/chemistry , Neuropeptide Y/metabolism , Receptors, Neuropeptide Y/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/metabolism , Humans , Models, Molecular , Mutation/genetics , Protein Binding , Receptors, Neuropeptide Y/chemistry
6.
ChemMedChem ; 16(1): 164-178, 2021 01 08.
Article in English | MEDLINE | ID: mdl-32700391

ABSTRACT

G protein-coupled receptors (GPCRs) can be used to shuttle peptide-drug conjugates into cells. But, for efficient therapy, a high concentration of cargo needs to be delivered. To explore this, we studied the pharmacologically interesting neuropeptide Y1 receptor (Y1 R) in one recombinant and three oncogenic cell systems that endogenously express the receptor. We demonstrate that recycled receptors behave identically to newly synthesized receptors with respect to ligand binding and internalization pathways. Depending on the cell system, biosynthesis, recycling efficiency, and peptide uptake differ partially, but shuttling was efficient in all systems. However, by comparing continuous application of the ligand for four hours to four cycles of internalization and recycling in between, a significantly higher amount of peptide uptake was achieved in the pulsed application (150-250 % to 300-400 %). Accordingly, in this well-suited drug shuttle system pulsed application is superior under all investigated conditions and should be considered for innovative, targeted drug delivery in general.


Subject(s)
Neuropeptide Y/chemistry , Pharmaceutical Preparations/chemistry , Receptors, Neuropeptide Y/metabolism , Arrestin/chemistry , Arrestin/metabolism , Cell Line, Tumor , Drug Carriers/chemistry , Drug Carriers/metabolism , Fluorescent Dyes/chemistry , HEK293 Cells , Humans , Ligands , Microscopy, Confocal , Neuropeptide Y/metabolism , Protein Binding , Receptors, Neuropeptide Y/chemistry
7.
Molecules ; 25(23)2020 Nov 24.
Article in English | MEDLINE | ID: mdl-33255213

ABSTRACT

We report data on the structural dynamics of the neuropeptide Y (NPY) G-protein-coupled receptor (GPCR) type 1 (Y1R), a typical representative of class A peptide ligand GPCRs, using a combination of solid-state NMR and molecular dynamics (MD) simulation. First, the equilibrium dynamics of Y1R were studied using 15N-NMR and quantitative determination of 1H-13C order parameters through the measurement of dipolar couplings in separated-local-field NMR experiments. Order parameters reporting the amplitudes of the molecular motions of the C-H bond vectors of Y1R in DMPC membranes are 0.57 for the Cα sites and lower in the side chains (0.37 for the CH2 and 0.18 for the CH3 groups). Different NMR excitation schemes identify relatively rigid and also dynamic segments of the molecule. In monounsaturated membranes composed of longer lipid chains, Y1R is more rigid, attributed to a higher hydrophobic thickness of the lipid membrane. The presence of an antagonist or NPY has little influence on the amplitude of motions, whereas the addition of agonist and arrestin led to a pronounced rigidization. To investigate Y1R dynamics with site resolution, we conducted extensive all-atom MD simulations of the apo and antagonist-bound state. In each state, three replicas with a length of 20 µs (with one exception, where the trajectory length was 10 µs) were conducted. In these simulations, order parameters of each residue were determined and showed high values in the transmembrane helices, whereas the loops and termini exhibit much lower order. The extracellular helix segments undergo larger amplitude motions than their intracellular counterparts, whereas the opposite is observed for the loops, Helix 8, and termini. Only minor differences in order were observed between the apo and antagonist-bound state, whereas the time scale of the motions is shorter for the apo state. Although these relatively fast motions occurring with correlation times of ns up to a few µs have no direct relevance for receptor activation, it is believed that they represent the prerequisite for larger conformational transitions in proteins.


Subject(s)
Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Protein Conformation , Receptors, Neuropeptide Y/chemistry , Drug Discovery , Magnetic Resonance Spectroscopy/methods , Nuclear Magnetic Resonance, Biomolecular , Receptors, Neuropeptide Y/antagonists & inhibitors , Recombinant Proteins/chemistry , Structure-Activity Relationship
8.
Molecules ; 25(18)2020 Sep 10.
Article in English | MEDLINE | ID: mdl-32927734

ABSTRACT

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron-electron resonance (DEER) pulsed EPR technique on the receptor.


Subject(s)
Electron Spin Resonance Spectroscopy , Receptors, Neuropeptide Y/chemistry , Amino Acid Sequence , Biomarkers , Cell Membrane , Cysteine/chemistry , Cysteine/genetics , Electron Spin Resonance Spectroscopy/methods , Gene Expression , HEK293 Cells , Humans , Intracellular Space , Models, Molecular , Mutagenesis, Site-Directed , Promoter Regions, Genetic , Protein Conformation , Receptors, Neuropeptide Y/genetics , Receptors, Neuropeptide Y/metabolism , Structure-Activity Relationship , Transcriptional Activation
9.
Angew Chem Int Ed Engl ; 59(52): 23854-23861, 2020 12 21.
Article in English | MEDLINE | ID: mdl-32790043

ABSTRACT

Dynamic structural transitions within the seven-transmembrane bundle represent the mechanism by which G-protein-coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist-, and arrestin-bound states of Y2R were prepared by cell-free expression, functional refolding, and reconstitution into lipid membranes. To study conformational transitions between these states, all six tryptophans of Y2R were 13 C-labeled. NMR-signal assignment was achieved by dynamic-nuclear-polarization enhancement and the individual functional states of the receptor were characterized by monitoring 13 C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp2816.48 of the highly conserved SWLP motif and Trp3277.55 adjacent to the NPxxY motif. Furthermore, a conformationally preserved "cysteine lock"-Trp11623.50 was identified.


Subject(s)
Receptors, Neuropeptide Y/chemistry , Humans , Models, Molecular , Molecular Conformation
10.
Elife ; 82019 12 06.
Article in English | MEDLINE | ID: mdl-31808746

ABSTRACT

Microtubules segregate chromosomes by attaching to macromolecular kinetochores. Only microtubule-end attached kinetochores can be pulled apart; how these end-on attachments are selectively recognised and stabilised is not known. Using the kinetochore and microtubule-associated protein, Astrin, as a molecular probe, we show that end-on attachments are rapidly stabilised by spatially-restricted delivery of PP1 near the C-terminus of Ndc80, a core kinetochore-microtubule linker. PP1 is delivered by the evolutionarily conserved tail of Astrin and this promotes Astrin's own enrichment creating a highly-responsive positive feedback, independent of biorientation. Abrogating Astrin:PP1-delivery disrupts attachment stability, which is not rescued by inhibiting Aurora-B, an attachment destabiliser, but is reversed by artificially tethering PP1 near the C-terminus of Ndc80. Constitutive Astrin:PP1-delivery disrupts chromosome congression and segregation, revealing a dynamic mechanism for stabilising attachments. Thus, Astrin-PP1 mediates a dynamic 'lock' that selectively and rapidly stabilises end-on attachments, independent of biorientation, and ensures proper chromosome segregation.


Subject(s)
Alcian Blue/metabolism , Chromosome Segregation , Kinetochores/metabolism , Microtubules/metabolism , Phenazines/metabolism , Phenothiazines/metabolism , Receptors, Neuropeptide Y/metabolism , Resorcinols/metabolism , Alcian Blue/chemistry , Aurora Kinase B , Chromosomal Proteins, Non-Histone , Cytoskeletal Proteins/metabolism , HeLa Cells , Humans , Kinetochores/chemistry , Metaphase , Microtubule-Associated Proteins/metabolism , Molecular Docking Simulation , Phenazines/chemistry , Phenothiazines/chemistry , Protein Conformation , Protein Interaction Domains and Motifs , Receptors, Neuropeptide Y/chemistry , Receptors, Neuropeptide Y/genetics , Resorcinols/chemistry
11.
J Chem Theory Comput ; 15(10): 5461-5473, 2019 Oct 08.
Article in English | MEDLINE | ID: mdl-31436990

ABSTRACT

Predicting the effect of single-point mutations on protein stability or protein-ligand binding is a major challenge in computational biology. Free energy calculations constitute the most rigorous approach to this problem, though the estimation of converged values for amino acid mutations remains challenging. To overcome this limitation, we developed tailored protocols to calculate free energy shifts associated with single-point mutations. We herein describe the QresFEP protocol, which includes an extension of our recent protocols to cover all amino acids mutations, based on the latest versions of the OPLS-AA force field. QresFEP is implemented in an application programming interface framework and the graphic interface QGui, for the molecular dynamics software Q. The complete protocol is benchmarked in several model systems, optimizing a number of sampling parameters and the implementation of Zwanzig's exponential formula and Bennet's acceptance ratio methods. QresFEP shows an excellent performance on estimating the hydration free energies of amino acid side-chain mimics, including their charged analogues. We also examined its performance on a protein-ligand binding problem of pharmaceutical relevance, the antagonism of neuropeptide Y1 G protein-coupled receptor. Here, the calculations show very good agreement with the experimental effect of 16 mutations on the binding of antagonists BIBP3226, in line with our recent applications in this field. Finally, the characterization of 43 mutations of T4-lysozyme reveals the capacity of our protocol to assess variations of the thermal stability of proteins, achieving a similar performance to alternative free energy perturbation (FEP) approaches. In summary, QresFEP is a robust, versatile, and user-friendly computational FEP protocol to examine biochemical effects of single-point mutations with high accuracy.


Subject(s)
Arginine/analogs & derivatives , Automation , Molecular Dynamics Simulation , Mutant Proteins/chemistry , Receptors, Neuropeptide Y/chemistry , Software , Thermodynamics , Arginine/chemistry , Arginine/pharmacology , Ligands , Mutant Proteins/antagonists & inhibitors , Protein Stability , Receptors, Neuropeptide Y/antagonists & inhibitors
12.
Cell Rep ; 28(8): 2206-2219.e8, 2019 08 20.
Article in English | MEDLINE | ID: mdl-31433993

ABSTRACT

PP1 and PP2A-B56 are major serine/threonine phosphatase families that achieve specificity by colocalizing with substrates. At the kinetochore, however, both phosphatases localize to an almost identical molecular space and yet they still manage to regulate unique pathways and processes. By switching or modulating the positions of PP1/PP2A-B56 at kinetochores, we show that their unique downstream effects are not due to either the identity of the phosphatase or its precise location. Instead, these phosphatases signal differently because their kinetochore recruitment can be either inhibited (PP1) or enhanced (PP2A) by phosphorylation inputs. Mathematical modeling explains how these inverse phospho-dependencies elicit unique forms of cross-regulation and feedback, which allows otherwise indistinguishable phosphatases to produce distinct network behaviors and control different mitotic processes. Furthermore, our genome-wide analysis suggests that these major phosphatase families may have evolved to respond to phosphorylation inputs in opposite ways because many other PP1 and PP2A-B56-binding motifs are also phospho-regulated.


Subject(s)
Kinetochores/metabolism , Protein Phosphatase 2/metabolism , Receptors, Neuropeptide Y/metabolism , Amino Acid Motifs , Amino Acid Sequence , HeLa Cells , Humans , Microtubule-Associated Proteins/metabolism , Models, Biological , Phenotype , Phosphorylation , Protein Phosphatase 2/chemistry , Receptors, Neuropeptide Y/chemistry , Signal Transduction
13.
J Biol Chem ; 293(43): 16751-16760, 2018 10 26.
Article in English | MEDLINE | ID: mdl-30185622

ABSTRACT

Splicing generates many mRNA strands from a single precursor mRNA, expanding the proteome and enhancing intracellular diversity. Both initial assembly and activation of the spliceosome require an essential family of splicing factors called serine-arginine (SR) proteins. Protein phosphatase 1 (PP1) regulates the SR proteins by controlling phosphorylation of a C-terminal arginine-serine-rich (RS) domain. These modifications are vital for the subcellular localization and mRNA splicing function of the SR protein. Although PP1 has been shown to dephosphorylate the prototype SR protein splicing factor 1 (SRSF1), the molecular nature of this interaction is not understood. Here, using NMR spectroscopy, we identified two electrostatic residues in helix α2 and a hydrophobic residue in helix α1 in the RNA recognition motif 1 (RRM1) of SRSF1 that constitute a binding surface for PP1. Substitution of these residues dissociated SRSF1 from PP1 and enhanced phosphatase activity, reducing phosphorylation in the RS domain. These effects lead to shifts in alternative splicing patterns that parallel increases in SRSF1 diffusion from speckles to the nucleoplasm brought on by regiospecific decreases in RS domain phosphorylation. Overall, these findings establish a molecular and biological connection between PP1-targeted amino acids in an RRM with the phosphorylation state and mRNA-processing function of an SR protein.


Subject(s)
Arginine/metabolism , Receptors, Neuropeptide Y/metabolism , Serine-Arginine Splicing Factors/metabolism , Serine/metabolism , Tumor Suppressor Proteins/metabolism , Alternative Splicing , Amino Acid Sequence , Arginine/chemistry , Arginine/genetics , Crystallography, X-Ray , Humans , Phosphorylation , Protein Binding , Protein Conformation , Protein Processing, Post-Translational , Receptors, Neuropeptide Y/chemistry , Receptors, Neuropeptide Y/genetics , Ribonucleoside Diphosphate Reductase , Sequence Homology , Serine/chemistry , Serine/genetics , Serine-Arginine Splicing Factors/chemistry , Serine-Arginine Splicing Factors/genetics , Spliceosomes , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics
14.
J Biol Chem ; 293(39): 15152-15162, 2018 09 28.
Article in English | MEDLINE | ID: mdl-30115685

ABSTRACT

The protein Ser/Thr phosphatase PP1 catalyzes an important fraction of protein dephosphorylation events and forms highly specific holoenzymes through an association with regulatory interactors of protein phosphatase one (RIPPOs). The functional characterization of individual PP1 holoenzymes is hampered by the lack of straightforward strategies for substrate mapping. Because efficient substrate recruitment often involves binding to both PP1 and its associated RIPPO, here we examined whether PP1-RIPPO fusions can be used to trap substrates for further analysis. Fusions of an hypoactive point mutant of PP1 and either of four tested RIPPOs accumulated in HEK293T cells with their associated substrates and were co-immunoprecipitated for subsequent identification of the substrates by immunoblotting or MS analysis. Hypoactive fusions were also used to study RIPPOs themselves as substrates for associated PP1. In contrast, substrate trapping was barely detected with active PP1-RIPPO fusions or with nonfused PP1 or RIPPO subunits. Our results suggest that hypoactive fusions of PP1 subunits represent an easy-to-use tool for substrate identification of individual holoenzymes.


Subject(s)
Cell Nucleus/chemistry , Holoenzymes/chemistry , Protein Phosphatase 1/chemistry , Receptors, Neuropeptide Y/chemistry , Animals , Binding Sites , COS Cells , Cell Nucleus/genetics , Chlorocebus aethiops/genetics , HEK293 Cells , Holoenzymes/genetics , Humans , Immunoprecipitation , Phosphorylation , Protein Binding , Protein Phosphatase 1/genetics , Receptors, Neuropeptide Y/genetics , Substrate Specificity
15.
Nature ; 556(7702): 520-524, 2018 04.
Article in English | MEDLINE | ID: mdl-29670288

ABSTRACT

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology 1,2 . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity 3 . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y1 receptor (Y1R) 4 . A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity 4 , tumour 1 and bone loss 5 . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability 6 . Here we report crystal structures of the human Y1R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y1R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery that targets NPY receptors.


Subject(s)
Arginine/analogs & derivatives , Dihydropyridines/chemistry , Dihydropyridines/metabolism , Diphenylacetic Acids/chemistry , Diphenylacetic Acids/metabolism , Neuropeptide Y/metabolism , Phenylurea Compounds/chemistry , Phenylurea Compounds/metabolism , Receptors, Neuropeptide Y/antagonists & inhibitors , Receptors, Neuropeptide Y/chemistry , Arginine/chemistry , Arginine/metabolism , Arginine/pharmacology , Binding Sites , Crystallography, X-Ray , Dihydropyridines/pharmacology , Diphenylacetic Acids/pharmacology , Humans , Inositol Phosphates/metabolism , Ligands , Molecular Docking Simulation , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation , Neuropeptide Y/chemistry , Neuropeptide Y/pharmacology , Nuclear Magnetic Resonance, Biomolecular , Phenylurea Compounds/pharmacology , Protein Binding , Receptors, Neuropeptide Y/agonists , Receptors, Neuropeptide Y/metabolism , Structure-Activity Relationship , Substrate Specificity
16.
Mol Pharmacol ; 93(4): 387-401, 2018 04.
Article in English | MEDLINE | ID: mdl-29436493

ABSTRACT

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.


Subject(s)
Mutation/genetics , Neuropeptide Y/genetics , Neuropeptide Y/metabolism , Receptors, Neuropeptide Y/genetics , Receptors, Neuropeptide Y/metabolism , Amino Acid Sequence , Binding Sites/physiology , Dose-Response Relationship, Drug , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , HEK293 Cells , Humans , Neuropeptide Y/chemistry , Protein Structure, Secondary , Receptors, Neuropeptide Y/chemistry
17.
Cell Physiol Biochem ; 45(1): 88-107, 2018.
Article in English | MEDLINE | ID: mdl-29310113

ABSTRACT

Human neuropeptide Y (hNPY) is one of the most widely expressed neurotransmitters in the human central and peripheral nervous systems. It consists of 36 highly conserved amino acid residues, and was first isolated from the porcine hypothalamus in 1982. While it is the most recently discovered member of the pancreatic polypeptide family (which includes neuropeptide Y, gut-derived hormone peptide YY, and pancreatic polypeptide), NPY is the most abundant peptide found in the mammalian brain. In order to exert particular functions, NPY needs to bind to the NPY receptor to activate specific signaling pathways. NPY receptors belong to the class A or rhodopsin-like G-protein coupled receptor (GPCR) family and signal via cell-surface receptors. By binding to GPCRs, NPY plays a crucial role in various biological processes, including cortical excitability, stress response, food intake, circadian rhythms, and cardiovascular function. Abnormal regulation of NPY is involved in the development of a wide range of diseases, including obesity, hypertension, atherosclerosis, epilepsy, metabolic disorders, and many cancers. Thus far, five receptors have been cloned from mammals (Y1, Y2, Y4, Y5, and y6), but only four of these (hY1, hY2, hY4, and hY5) are functional in humans. In this review, we summarize the structural characteristics of human NPY receptors and their role in metabolic diseases.


Subject(s)
Metabolic Diseases/etiology , Receptors, Neuropeptide Y/metabolism , Diabetes Mellitus/etiology , Diabetes Mellitus/metabolism , Diabetes Mellitus/therapy , Dyslipidemias/etiology , Dyslipidemias/metabolism , Dyslipidemias/therapy , Humans , Hypertension/etiology , Hypertension/metabolism , Hypertension/therapy , Metabolic Diseases/metabolism , Metabolic Diseases/therapy , Obesity/etiology , Obesity/metabolism , Obesity/therapy , Receptors, Neuropeptide Y/chemistry , Receptors, Neuropeptide Y/genetics , Signal Transduction
18.
Mol Pharmacol ; 93(4): 323-334, 2018 04.
Article in English | MEDLINE | ID: mdl-29367257

ABSTRACT

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.


Subject(s)
Peptide YY/genetics , Peptide YY/metabolism , Receptors, Neuropeptide Y/genetics , Receptors, Neuropeptide Y/metabolism , Amino Acid Sequence , Animals , Binding Sites/physiology , HEK293 Cells , Humans , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide YY/chemistry , Protein Structure, Secondary , Receptors, Neuropeptide Y/chemistry , Swine
19.
Biochem Biophys Res Commun ; 484(4): 864-870, 2017 03 18.
Article in English | MEDLINE | ID: mdl-28188792

ABSTRACT

Centrosomal protein of 192 kDa (CEP192) is a scaffolding protein that recruits the mitotic protein kinases Aurora A and PLK1 to the centrosome. Here we demonstrate that CEP192 also recruits the type one protein phosphatase (PP1) via a highly conserved KHVTF docking motif. The threonine of the KHVTF motif is phosphorylated during mitosis and protein kinase inhibition studies suggest this to be a PLK1-dependent process.


Subject(s)
Centrosome/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Mitosis/physiology , Receptors, Neuropeptide Y/chemistry , Receptors, Neuropeptide Y/metabolism , Binding Sites , Centrosome/ultrastructure , Enzyme Activation , HeLa Cells , Humans , Phosphorylation , Protein Binding
20.
Cell Signal ; 29: 233-239, 2017 01.
Article in English | MEDLINE | ID: mdl-27818291

ABSTRACT

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.


Subject(s)
Endocytosis , Receptors, Neuropeptide Y/chemistry , Receptors, Neuropeptide Y/metabolism , beta-Arrestin 2/metabolism , Amino Acid Motifs , Amino Acid Sequence , Amino Acids/metabolism , Animals , COS Cells , Chlorocebus aethiops , HEK293 Cells , Humans , Mutant Proteins/metabolism , Reproducibility of Results , Sequence Alignment , Sequence Deletion , Structure-Activity Relationship
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