Cloning and characterization of the guinea pig neuropeptide Y receptor Y5

The Y5 receptor has been postulated to be the main receptor mediating NPY-induced food intake in rats, based on its pharmacological profile and mRNA distribution. To further characterize this important receptor subtype, we isolated the Y5 gene in the guinea pig, a widely used laboratory animal in which all other known NPY receptors (Y1, Y2, Y4, y6) [2,13,33,37] have recently been cloned by our group. Our results show that the Y5 receptor is well conserved between species; guinea pig Y5 displays 96% overall amino acid sequence identity to human Y5, the highest identity reported for any non-primate NPY receptor orthologue, regardless of subtype. Thirteen of the twenty substitutions occur in the large third cytoplasmic loop. The identities between the guinea pig Y5 receptor and the dog, rat, and mouse Y5 receptors are 93%, 89%, and 89% respectively. When transiently expressed in EBNA cells, the guinea pig Y5 receptor showed a high binding affinity to iodinated porcine PYY with a dissociation constant of 0.41 nM. Competition experiments showed that the rank order of potency for NPY-analogues was PYY = NPY = NPY2-36 > gpPP > rPP > NPY 22-36. Thus the pharmacological profile of the guinea pig Y5 receptor agrees well with that reported for the Y5 receptor from other cloned species.     

Characterization of neuropeptide Y Y1-like and Y2-like receptor subtypes in the mouse brain

To characterize receptor subtypes in the mouse, we performed autoradiographic localization and pharmacological characterization studies using the selective radiolabeled agonists, [(125)I]-Leu(31), Pro(34)-PYY and [(125)I]-PYY 3-36. The pharmacology of [(125)I]-Leu(31), Pro(34)-PYY and [(125)I]-PYY 3-36 binding to mouse brain homogenates were consistent with Y1-like and Y2-like receptors, respectively. Using receptor autoradiography, high Y1-like binding was observed in the islands of Calleja and dentate gyrus. [(125)I]-PYY 3-36 binding was highest in the hippocampus, lateral septum, stria terminalis of the thalamus, and compacta and lateralis of the substantia nigra. In addition, there are differences in receptor distribution in mouse brain compared to other species that may translate into different functional roles for the NPY receptors within each species.     

Mutations in arrestin-3 differentially affect binding to neuropeptide Y receptor subtypes

Based on the identification of residues that determine receptor selectivity in arrestins and the phylogenetic analysis of the arrestin (arr) family, we introduced fifteen mutations of receptor-discriminator residues in arr-3, which were identified previously using mutagenesis, in vitro binding, and BRET-based recruitment assay in intact cells. The effects of these mutations were tested using neuropeptide Y receptors Y1R and Y2R. NPY-elicited arr-3 recruitment to Y1R was not affected by these mutations, or even alanine substitution of all ten residues (arr-3-NCA), which prevented arr-3 binding to other receptors tested so far. However, NCA and two other mutations prevented agonist-independent arr-3 pre-docking to Y1R. In contrast, eight out of 15 mutations significantly reduced agonist-dependent arr-3 recruitment to Y2R. NCA eliminated arr-3 binding to active Y2R, whereas Tyr239Thr reduced it ~ 7-fold. Thus, manipulation of key residues on the receptor-binding surface generates arr-3 with high preference for Y1R over Y2R. Several mutations differentially affect arr-3 pre-docking and agonist-induced recruitment. Thus, arr-3 recruitment to the receptor involves several mechanistically distinct steps. Targeted mutagenesis can fine-tune arrestins directing them to specific receptors and particular activation states of the same receptor.     

Agonist induced receptor internalization of neuropeptide Y receptor subtypes depends on third intracellular loop and C-terminus

Agonist stimulation of G-protein coupled receptors (GPCRs) results in the redistribution of the receptor from the cell surface into intracellular compartments through the process of endocytosis. Monitoring ligand-mediated internalization of GPCRs in living cells has become experimentally accessible by applying fluorescent reagents and fluorescence microscopy. By using cell lines that transiently, stably or endogenously express the human Y receptor (hYR) subtypes hY(1)R, hY(2)R, hY(4)R and hY(5)R and differently fluorescently tagged receptor proteins we were able to unravel further details concerning the internalization behavior of this multi-receptor/multi-ligand system. For the first time we could show that also the hY(2)R is internalized with a rate which is comparable to the hY(1)R and the hY(4)R. In contrast, the hY(5)R was internalized much slower and the rate remained unaffected by co-expression with other hYR subtypes. Furthermore receptor subtype co-expressing cells and selectively binding peptides revealed a receptor subtype selective internalization. By using novel hY(5)/hY(2) receptor chimera the receptor subtype dependent differences in hY receptor internalization could be identified on a molecular level.     

Cloning and characterization of a zebrafish Y2 receptor

The NPY receptors belong to the superfamily of G-protein coupled receptors and in mammals this family has five members, named Y1, Y2, Y4, Y5, and Y6. In bony fish, four receptors have been identified, named Ya, Yb, Yc and Y7. Yb and Y7 arose prior to the split between ray-fined fishes and tetrapods and have been lost in mammals. Yc appeared as a copy of Yb in teleost fishes. Ya may be an ortholog of Y4, but surprisingly no unambiguous receptor ortholog to any of the mammalian subtypes has yet been identified in bony fishes. Here we present the cloning and pharmacological characterization of a Y2 receptor in zebrafish, Danio rerio. To date, this is the first Y2 receptor outside mammals and birds that has been characterized pharmacologically. Phylogenetic analysis and synteny confirmed that this receptor is orthologous to mammalian Y2. We show that the receptor is pharmacologically most similar to chicken Y2 which leads to the conclusion that Y2 has acquired several novel characteristics in mammals. Y2 from zebrafish binds very poorly to the Y2-specific antagonist BIIE0246. Our pharmacological characterization supports our previous conclusions regarding the binding pocket of BIIE0246 in the human Y2 receptor.     

Ligands of the neuropeptide Y Y2 receptor

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and exerts a variety of physiological processes in humans via four different receptor subtypes Y1, Y2, Y4 and Y5. Y2 receptor is the most abundant Y subtype receptor in the central nervous system and implicated with food intake, bone formation, affective disorders, alcohol and drugs of abuse, epilepsy, pain, and cancer. The lack of small molecule non-peptidic Y2 receptor modulators suitable as in vivo pharmacological tools hampered the progress to uncover the precise pharmacological role of Y2. Only in recent years, several potent, selective and non-peptidic Y2 antagonists have been discovered providing the tools to validate Y2 receptor as a therapeutic target. This Letter reviews Y2 receptor modulators mainly non-peptidic antagonists and their structure–activity relationships.     

Novel non-peptidic neuropeptide Y Y2 receptor antagonists

Through SAR studies of a piperidinylindoline cinnamide HTS lead, the first potent, non-peptide, low molecular weight selective Neuropeptide Y Y2 (NPY Y2) antagonists have been synthesized. The SAR studies around the piperidinyl, the indolinyl, and the cinnamyl moieties are discussed.     

Pharmacological characterization of cloned chicken neuropeptide Y receptors Y1 and Y5

The neuropeptide Y (NPY) receptor subtypes Y1 and Y5 are involved in the regulation of feeding and several other physiological functions in mammals. To increase our understanding of the origin and mechanisms of the complex NPY system, we report here the cloning and pharmacological characterization of receptors Y1 and Y5 in the first non-mammal, chicken (Gallus gallus). The receptors display 80-83% and 64-72% amino acid sequence identity, respectively, with their mammalian orthologues. The three endogenous ligands NPY, peptide YY (PYY) and pancreatic polypeptide (PP) have similar affinities as in mammals, i.e. NPY and PYY have subnanomolar affinity for both receptors whereas chicken PP bound with nanomolar affinity to Y5 but not to Y1. A notable difference to mammalian receptor subtypes is that the Y1 antagonist SR120819A does not bind chicken Y1, whereas BIBP3226 does. The Y5 antagonist CGP71863A binds to the chicken Y5 receptor. Anatomically, both Y1 and Y5 have high mRNA expression levels in the infundibular nucleus which is the homologous structure of the hypothalamic arcuate nucleus in mammals. These results suggest that some of the selective Y1 and Y5 antagonists developed in mammals can be used to study appetite regulation in chicken.     

Dihydropyridine neuropeptide Y Y(1) receptor antagonists

Dihydropyridine 5a was found to be an inhibitor of neuropeptide Y(1) binding in a high throughput (125)I-PYY screening assay. Structure-activity studies around certain portions of the dihydropyridine chemotype identified BMS-193885 (6e) as a potent and selective Y(1) receptor antagonist. In a forskolin-stimulated c-AMP production assay using CHO cells expressing the human Y(1) receptor, 6e demonstrated full functional antagonism (K(b)=4.5 nM). Compound 6e inhibited NPY-induced feeding in satiated rats when dosed at 3.0 and 10.0 mg/kg (ip), and also decreased spontaneous overnight food consumption in rats at doses of 10 and 20 mg/kg (ip).     

Molecular characterization of the ligand–receptor interaction of the neuropeptide Y family

Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) belong to the NPY hormone family and activate a class of receptors called the Y‐receptors, and also belong to the large superfamily of the G‐protein coupled receptors. Structure–affinity and structure–activity relationship studies of peptide analogs, combined with studies based on site‐directed mutagenesis and anti‐receptor antibodies, have given insight into the individual characterization of each receptor subtype relative to its interaction with the ligand, as well as to its biological function. A number of selective antagonists at the Y₁‐receptor are available whose structures resemble that of the C‐terminus of NPY. Some of these compounds, like BIBP3226, BIBO3304 and GW1229, have recently been used for in vivo investigations of the NPY‐induced increase in food intake. Y₂‐receptor selective agonists are the analog cyclo‐(28/32)‐Ac‐[Lys²⁸‐Glu³²]‐(25–36)‐pNPY and the TASP molecule containing two units of the NPY segment 21–36. Now the first antagonist with nanomolar affinity for the Y₂‐receptor is also known, BIIE0246. So far, the native peptide PP has been shown to be the most potent ligand at the Y₄‐receptor. However, by the design of PP/NPY chimera, some analogs have been found that bind not only to the Y₄‐, but also to the Y₅‐receptor with subnanomolar affinities, and are as potent as NPY at the Y₁‐receptor. For the characterization of the Y₅‐receptor in vitro and in vivo, a new class of highly selective agonists is now available. This consists of analogs of NPY and of PP/NPY chimera which all contain the motif Ala³¹‐Aib³². This motif has been shown to induce a 3₁₀‐helical turn in the region 28–31 of NPY and is suggested to be the key motif for high Y₅‐receptor selectivity. The results of feeding experiments in rats treated with the first highly specific Y₅‐receptor agonists support the hypothesis that this receptor plays a role in the NPY‐induced stimulation of food intake. In conclusion, the selective compounds for the different Y‐receptor subtypes known so far are promising tools for a better understanding of the physiological properties of the hormones of the NPY family and related receptors. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.     

Isolation and characterization of neuropeptide Y from porcine intestine

The isolation and primary structure of intestinal neuropeptide Y (NPY) is described. The peptide was purified from porcine intestinal extracts using a chemical assay and radioimmunoassay for NPY. The amino acid sequence of this peptide is: Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala- Arg-Tyr-Tyr- Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2. This the structure of intestinal NPY is identical to the NPY of brain origin.     

Molecular characterization of the human neuropeptide Y Y2-receptor.

Five neuropeptide Y receptors, the Y1-, Y2-, Y4-, Y5- and y6-subtypes, have been cloned, which belong to the rhodopsin-like G-protein-coupled, 7-transmembrane helix-spanning receptors and bind the 36-mer neuromodulator NPY (neuropeptide Y) with nanomolar affinity. In this study, the Y2-receptor subtype expressed in a human neuroblastoma cell line (SMS-KAN) and in transfected Chinese hamster ovary cells (CHO-hY2) was characterized on the protein level by using photoaffinity labeling and antireceptor antibodies. Two photoactivatable analogues of NPY were synthesized, in which a Tyr residue was substituted by the photoreactive amino acid 4-(3-trifluoromethyl)-3H-diazirin-3-ylphenylalanine ((Tmd)Phe), [Nalpha-biotinyl-Ahx2,(Tmd)Phe36]NPY (Tmd36), and the Y2-receptor subtype selective [Nalpha-biotinyl-Ahx2,Ahx5-24,(Tmd)Phe27]N PY (Tmd27). Both analogues were labeled with [3H]succinimidyl-propionate at Lys4 and bind to the Y2-receptor with affinity similar to that of the native ligand. A synthetic fragment of the second (E2) extracellular loop was used to generate subtype selective antireceptor antibodies against the Y2-receptor. Photoaffinity labeling of the receptor followed by SDS-PAGE and detection of bound radioactivity and SDS-PAGE of solubilized receptors and subsequent Western blotting revealed the same molecular masses. Two proteins correspondingly have been detected for each cell line with molecular masses of 58 +/- 4 and 50 +/- 4 kDa, respectively.     

Cloning and characterization of the pheromone biosynthesis activating neuropeptide receptor gene in Spodoptera littoralis larvae

In noctuid moths cuticular pigmentation is regulated by the pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family, which also mediates a variety of other functions in moths and other insects. Numerous studies have shown that these neuropeptides exert their functions through activation of the PBAN receptor (PBAN-R), with subsequent Ca(2+) influx, followed by either activation of cAMP or direct activation of downstream kinases. Recently, several PBAN-Rs have been identified, all of which are from the pheromone gland of adult female moths, but evidence shows that functional PK/PBAN-Rs can also be expressed in insect larvae, where they mediate melanization and possibly other functions (e.g., diapause). Here, we identified a gene encoding a G-protein-coupled receptor from the 5th instar larval tissue of the moth Spodoptera littoralis. The cDNA of this gene contains an open reading frame with a length of 1050 nucleotides, which translates to a 350-amino acid, 42-kDa protein that shares 92% amino acid identity with Helicoverpa zea and Helicoverpa armigera PBAN-R, 81% with Bombyx mori PBAN-R and 72% with Plutella xylostella PBAN-R. The S. littoralis PBAN-R gene was stably expressed in NIH3T3 cells and transiently in HEK293 cells. We show that it mediates the dose-dependent PBAN-induced intracellular Ca(2+) response and activation of the MAP kinase via a PKC-dependent but Galphai-independent signaling mechanism. Other PK/PBAN family peptides (pheromonotropin and a C-terminally PBAN-derived peptide PBAN(28-33)NH(2)) also triggered MAP kinase activation. This receptor, together with the previously cloned PBAN-R, may facilitate our understanding of the cell-specific responses and functional diversities of this diverse neuropeptide family.     

Neuropeptide Y and neuropeptide Y18-36. Structural and biological characterization

Neuropeptide Y (NPY), a 36-residue peptide amide, has been shown by numerous studies to be a potent vasoconstrictor. In order to gain an appreciation of the structural requirements for this action, we have previously synthesized a number of fragments of NPY. It had been shown that sequential deletions from the N-terminus resulted in peptides with decreasing hypertensive activity. In the present study we present data supporting the unexpected finding of two fragments, NPY17-36 and NPT18-36 with substantial hypotensive action in vivo. This action was dose dependent (data not shown) and was also observed to a lesser extent with NPY19-36 but not NPY16-36 or NPY20-36. It was, however, slower in onset and of longer duration than the hypertensive action of NPY. These differing kinetics of action may suggest that NPY and NPY18-36 act through different mechanisms. Structural studies using circular dichroism were performed. While NPY was found to assume an ordered helical structure in both aqueous buffer and trifluoroethanol (TFE), 30% TFE in aqueous buffer was required to induce substantial helicity for NPY18-36. This structural investigation suggests that both NPY and NPY18-36 assume an ordered conformation upon reaching the lipid rich receptor environment.     

Synthesis and SAR of selective small molecule neuropeptide Y Y2 receptor antagonists

Highly potent and selective small molecule neuropeptide Y Y2 receptor antagonists are reported. The systematic SAR exploration of a hit molecule N-(4-ethoxyphenyl)-4-[hydroxy(diphenyl)methyl]piperidine-1-carbothioamide, identified from HTS, led to the discovery of highly potent NPY Y2 antagonists 16 (CYM 9484) and 54 (CYM 9552) with IC(50) values of 19 nM and 12 nM respectively.     

A Y2 receptor mimetic aptamer directed against neuropeptide Y.

Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that exerts its activity by at least five different receptor subtypes that belong to the family of G-protein-coupled receptors. We isolated an aptamer directed against NPY from a nuclease-resistant RNA library. Mapping experiments with N-terminally, C-terminally, and centrally truncated analogues of NPY revealed that the aptamer recognizes the C terminus of NPY. Individual replacement of the four arginine residues at positions 19, 25, 33, and 35 by l-alanine showed that arginine 33 is essential for binding. The aptamer does not recognize pancreatic polypeptide, a highly homologous Y4 receptor-specific peptide of the gut. Furthermore, the affinity of the aptamer to the Y5 receptor-selective agonist [Ala(31),Aib(32)]NPY and the Y1/Y5 receptor-binding peptide [Leu(31),Pro(34)]NPY was considerably reduced, whereas Y2 receptor-specific NPY mutants were bound well by the aptamer. Accordingly, the NPY epitope was recognized by the Y2 receptor, and the aptamer was highly similar. This Y2 receptor mimicking effect was further confirmed by competition binding studies. Whereas the aptamer competed with the Y2 receptor for binding of [(3)H]NPY with high affinity, a low affinity displacement of [(3)H]NPY was observed at the Y1 and the Y5 receptors. Consequently, competition at the Y2 receptor occurred with a considerably lower K(i) value compared with the Y1 and Y5 receptors. These results indicate that the aptamer mimics the binding of NPY to the Y2 receptor more closely than to the Y1 and Y5 receptors.     

1,3-Disubstituted benzazepines as neuropeptide Y Y1 receptor antagonists.

A novel class of potent and selective non-peptide neuropeptide Y (NPY) Y1 receptor antagonists, having benzazepine nuclei, have been designed, synthesized, and evaluated for activity. Through a blind screening we found the compound 1-N-(3-(N'-(tert-butoxycarbonyl)amino)benzyl)-7-methoxy-(3-(3)-methyl ureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (9: IC50 = 1.6 microM). Chemical modifications of 9 gave a potent NPY Y1 antagonist 3-(N-(4-hydroxyphenyl)-N'-methylguanidino)-1-N-(3-(N'-(tert-butoxy carbonyl)amino)benzyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (14c: IC5(0=43 nM), which had no affinity for NPY Y2 and Y5 receptors.