SMART MODE:

NORMAL GENOMIC

• Berglund MM, Lundell I, Cabrele C, Serradeil-Le Gal C, Beck-Sickinger AG, Larhammar D
• Binding properties of three neuropeptide Y receptor subtypes from zebrafish: comparison with mammalian Y1 receptors.
• Biochem Pharmacol. 2000; 60: 1815-22
• Display abstract

• Neuropeptide Y (NPY) and peptide YY (PYY) are two related 36-amino-acid peptides found in all vertebrates and are involved in many physiological processes. Five receptor subtypes have been cloned in mammals (Y1, Y2, Y4, Y5, and y6). We have recently cloned three NPY/PYY receptor subtypes in zebrafish, called Ya, Yb, and Yc. Here we report on a direct comparison of the pharmacological properties of these three receptors in vitro using porcine NPY with alanine substitutions in positions 33-36 as ligands and three analogues with internal deletions: [Ahx(8-20)]NPY, [Ahx(8-20), Pro(34)]NPY, and [Ahx(5-24)]NPY. In all cases, the zYc receptor was the most sensitive to the modifications of the NPY molecule and zYa was the least sensitive (except for the Arg --> Ala replacement at position 33). Our data identified zYa as a receptor that can bind ligands specific for Y1, Y2, and Y4 receptors, while zYb and zYc were more Y1-like. All peptides with internal deletions bound to the zYa receptor with affinities similar to that of intact pNPY. Neither the Y1-selective antagonists BIBP3226 and SR120819A nor the Y2-selective BIIE0246 bound to any of the zebrafish receptors, although the amino acids identified as important for BIBP3226 binding were almost completely conserved. These results may prove helpful in molecular modeling of the three-dimensional receptor structure.

• Wraith A et al.
• Evolution of the neuropeptide Y receptor family: gene and chromosome duplications deduced from the cloning and mapping of the five receptor subtype genes in pig.
• Genome Res. 2000; 10: 302-10
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• Neuropeptide Y (NPY) receptors mediate a variety of physiological responses including feeding and vasoconstriction. To investigate the evolutionary events that have generated this receptor family, we have sequenced and determined the chromosomal localizations of all five presently known mammalian NPY receptor subtype genes in the domestic pig, Sus scrofa (SSC). The orthologs of the Y(1) and Y(2) subtypes display high amino acid sequence identities between pig, human, and mouse (92%-94%), whereas the Y(4), Y(5), and y(6) subtypes display lower identities (76%-87%). The lower identity of Y(5) is due to high sequence divergence in the large third intracellular loop. The NPY1R, NPY2R, and NPY5R receptor genes were localized to SSC8, the NPY4R to SSC14, and NPY6R to SSC2. Our comparisons strongly suggest that the tight cluster of NPY1R, NPY2R, and NPY5R on human chromosome 4 (HSA4) represents the ancestral configuration, whereas the porcine cluster has been split by two inversions on SSC8. These 3 genes, along with adjacent genes from 14 other gene families, form a cluster on HSA4 with extensive similarities to a cluster on HSA5, where NPY6R and >13 other paralogs reside, as well as another large cluster on HSA10 that includes NPY4R. Thus, these gene families have expanded through large-scale duplications. The sequence comparisons show that the NPY receptor triplet NPY1R-NPY2R-NPY5R existed before these large-scale duplications.

• St-Pierre JA, Nouel D, Dumont Y, Beaudet A, Quirion R
• Sub-population of cultured hippocampal astrocytes expresses neuropeptide Y Y(1) receptors.
• Glia. 2000; 30: 82-91
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• The expression and pharmacological characterization of neuropeptide Y (NPY) receptors of the Y(1) subtype on cultured hippocampal neurons was reported using radioreceptor assays and immunohistochemical approaches (St-Pierre et al., 1998). The present study aimed to establish the presence of NPY Y(1) receptors on cultured hippocampal astrocytes using similar strategies. Immunocytochemical experiments were carried out using three antisera directed against distinct domains (amino acids sequence 185-203, 198-213 and 355-382) of the Y(1) receptor. Double-labeling experiments and confocal microscopy with these Y(1) receptor antisera demonstrated their recognition of the same sub-population (20%) of GFAP-positive astrocytes in culture. The immunostaining seen with all three Y(1) receptor antisera took the form of large irregular clusters distributed throughout cell bodies and processes. Further experiments using radioactive ligands confirmed the presence of NPY receptors on cultured hippocampal astrocytes. Emulsion receptor autoradiography using a newly developed ligand, [(125)I]GR231118 in the presence of PYY, hPP or BIBP3226 (1 microM), pharmacologically established the Y(1) nature of these receptors. Specific [(125)I]GR231118 binding was competed by PYY and the selective Y(1) antagonist BIBP3226 but not by hPP (a Y(4)/Y(5) agonist). Similar autoradiographic labeling patterns were obtained using [(125)I][Leu(31).Pro(34)]PYY (a Y(1)/Y(4)/Y(5) agonist) whereas [(125)I]PYY(3-36) (a Y(2)/Y(5) agonist) failed to generate any specific signal. Hence, rat cultured hippocampal astrocytes express a typical Y(1) receptor without evidence for the presence of Y(2), Y(4) or Y(5) subtypes. These data suggest a preferential regulation by NPY, acting via the Y(1) receptors, of astrocytic function.

• Soderberg C et al.
• Zebrafish genes for neuropeptide Y and peptide YY reveal origin by chromosome duplication from an ancestral gene linked to the homeobox cluster.
• J Neurochem. 2000; 75: 908-18
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• Neuropeptide Y (NPY) and peptide YY (PYY) are related 36-amino acid peptides. NPY is widely distributed in the nervous system and has several physiological roles. PYY serves as an intestinal hormone as well as a neuropeptide. We report here cloning of the npy and pyy genes in zebrafish (Danio rerio). NPY differs at only one to four amino acid positions from NPY in other jawed vertebrates. Zebrafish PYY differs at three positions from PYY from other fishes and at 10 positions from mammals. In situ hybridization showed that neurons containing NPY mRNA have a widespread distribution in the brain, particularly in the telencephalon, optic tectum, and rhombencephalon. PYY mRNA was found mainly in brainstem neurons, as reported previously for vertebrates as divergent as the rat and the lamprey, suggesting an essential role for PYY in these neurons. PYY mRNA was observed also in the telencephalon. These results were confirmed by immunocytochemistry. As in the human, the npy gene is located adjacent to homeobox (hox) gene cluster A (copy a in zebrafish), whereas the pyy gene is located close to hoxBa. This suggests that npy and pyy arose from a common ancestral gene in a chromosomal duplication event that also involved the hox gene clusters. As zebrafish has seven hox clusters, it is possible that additional NPY family genes exist or have existed. Also, the NPY receptor system seems to be more complex in zebrafish than in mammals, with at least two receptor genes without known mammalian orthologues.

• Parker SL, Parker MS
• FMRFamides exert a unique modulation of rodent pancreatic polypeptide sensitive neuropeptide Y (NPY) receptors.
• Can J Physiol Pharmacol. 2000; 78: 150-61
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• FMRFamide and related peptides (RFamides) were found to inhibit the association binding of iodinated human pancreatic polypeptide ([125I]hPP) to Y5-like neuropeptide Y (NPY) receptor in rodent tissues. An allosteric regulation of the activity of the rodent kidney PP-sensitive neuropeptide Y (NPY) receptor by RFamides was indicated by potency decrease with particle concentration in the inhibition of the association binding of 125I-labeled human pancreatic polypeptide (hPP) by RFamides at rabbit kidney membranes. The competition by C-terminal hexapeptide of hPP (LTRPRY.NH2) did not show such affinity change. The steady-state binding of hPP showed little sensitivity to any of the RFamides tested. The Y1-selective binding of [125I][Leu31,Pro34]hPYY (at 2 nM hPP) was much less sensitive to RFamides than the binding of [125I]hPP, albeit with some differences across tissue or cell types. The binding of Y2-selective agonist 125I-labeled human peptide YY (3-36) was quite insensitive to RFamides. The presence of a unique component in the inhibition of hPP binding by RFamides was further indicated by a degree of antagonism with phospholipase C inhibitor U-73122, and by an only limited cooperation with a N5-amiloride compound, and with alkylator chloroethylclonidine. Change of the chirality of individual residues in the FMRFamide molecule produced a significant reduction of inhibitory potency only with D-Phe in the C-terminal position. Substitution of the (C-3) L-Met by L-Leu greatly increased the inhibitory potency of RFamides relative to otherwise identical congeners. RFamides could act both as ligands of membrane neighbors of the PP receptor, and as competitors of Y5-like NPY receptor epitopes that accommodate the C-terminal aspects of agonist peptides.

• Lundell I, Berglund MM, Larhammar D
• Y4 receptor in different species. Functional expression and binding.
• Methods Mol Biol. 2000; 153: 45-51

• Mullins DE, Guzzi M, Xia L, Parker EM
• Pharmacological characterization of the cloned neuropeptide Y y(6) receptor.
• Eur J Pharmacol. 2000; 395: 87-93
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• Neuropeptide Y has potent appetite stimulating effects which are mediated by hypothalamic receptors believed to be of the neuropeptide Y Y(1) and/or neuropeptide Y Y(5) subtype. In mice, the neuropeptide Y y(6) receptor is also expressed in the hypothalamus, suggesting that it too may function as a feeding receptor in this species. Several laboratories have studied the pharmacology of the neuropeptide Y y(6) receptor, but their results are not in agreement. Using neuropeptide Y and a variety of peptide analogs and small molecule antagonists, we have determined that the pharmacology of the cloned mouse neuropeptide Y y(6) receptor is distinct from that of the other known neuropeptide Y receptors. The rank order of binding affinity for the mouse neuropeptide Y y(6) receptor is [(Ile, Glu,Pro,Dpr,Tyr,Arg,Leu,Arg,Tyr-NH(2))(2)human peptide YY=human, rat neuropeptide Y=human, rat neuropeptide Y-(2-36)=human, rat [Leu(31), Pro(34)porcine (Cys(2))-neuropeptide Y-(1-4)-8-aminooctanoyl-(D-Cys(27)porcine [D-Trp(32)rat pancreatic polypeptide=human pancreatic polypeptide. A similar rank order of potency is seen for inhibition of forskolin-stimulated cyclic AMP. The neuropeptide Y Y(5) receptor antagonist trans-naphthalene-1-sulfonic acid ?4-[4-amino-quinazolin-2-ylamino)-methyl]-cyclohexylmethy l?-amide hydrochloride (CGP 71683A) and the neuropeptide Y Y(1) receptor antagonist ((R)-N(2)-diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-argininam ide) (BIBP3226) bind weakly to the neuropeptide Y y(6) receptor (K(i)10, 000 nM, respectively). Although the function of the neuropeptide Y y(6) receptor remains to be elucidated, its pharmacology is not consistent with a role in appetite regulation.

• Salaneck E, Holmberg SK, Berglund MM, Boswell T, Larhammar D
• Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y2(1).
• FEBS Lett. 2000; 484: 229-34
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• Here we report the molecular cloning of the chicken (Gallus gallus) neuropeptide Y (NPY) receptor Y2, the first non-mammalian Y2 receptor. It displays 75-80% identity to mammalian Y2 and has a surprisingly divergent cytoplasmic tail. Expression of the receptor protein in a cell line showed that the receptor did not bind the mammalian Y2 selective antagonist BIIE0246. Furthermore, porcine [Leu(31), Pro(34)]NPY, which binds poorly to mammalian Y2, exhibited an unexpectedly high affinity for chicken Y2. In situ hybridisation revealed expression in the hippocampus. Thus, the chicken Y2 receptor exhibits substantial differences with regard to sequence and pharmacological profile in comparison to mammalian Y2 receptors, while the expression pattern in the central nervous system resembles that observed in mammals.

• Cerda-Reverter JM, Martinez-Rodriguez G, Zanuy S, Carrillo M, Larhammar D
• Molecular evolution of the neuropeptide Y (NPY) family of peptides: cloning of three NPY-related peptides from the sea bass (Dicentrarchus labrax).
• Regul Pept. 2000; 95: 25-34
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• Neuropeptide Y (NPY) is a 36-amino-acid peptide that is widely and abundantly expressed in the central nervous system of all vertebrates investigated. Related peptides have been found in various vertebrate groups: peptide YY (PYY) is present in gut endocrine cells of many species and pancreatic polypeptide (PP) is made in the pancreas of all tetrapods. In addition, a fish pancreatic peptide called PY has been reported in three species of fishes. The evolutionary relationships of fish PY have been unclear and it has been proposed to be the orthologue (species homologue) of each of the three tetrapod peptides. We demonstrate here with molecular cloning techniques that the sea bass (Dicentrarchus labrax), an acanthomorph fish, has orthologues of both NPY and PYY as well as a separate PY peptide. Sequence comparisons suggest that PY arose as a copy of the PYY gene, presumably in a duplication event separate from the one that generated PP from PYY in tetrapods. PY sequences from four species of fish indicate that, similar to PP, PY evolves much more rapidly than NPY and PYY. The physiological role of PY is unknown, but we demonstrate here that sea bass PY, like NPY and PYY but in contrast to the tetrapod PP, is expressed in brain.

• Starback P, Wraith A, Eriksson H, Larhammar D
• Neuropeptide Y receptor gene y6: multiple deaths or resurrections?
• Biochem Biophys Res Commun. 2000; 277: 264-9
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• The neuropeptide Y family of G-protein-coupled receptors consists of five cloned members in mammals. Four genes give rise to functional receptors in all mammals investigated. The y6 gene is a pseudogene in human and pig and is absent in rat, but generates a functional receptor in rabbit and mouse and probably in the collared peccary (Pecari tajacu), a distant relative of the pig family. We report here that the guinea pig y6 gene has a highly distorted nucleotide sequence with multiple frame-shift mutations. One evolutionary scenario may suggest that y6 was inactivated before the divergence of the mammalian orders and subsequently resurrected in some lineages. However, the pseudogene mutations seem to be distinct in human, pig, and guinea pig, arguing for separate inactivation events. In either case, the y6 gene has a quite unusual evolutionary history with multiple independent deaths or resurrections.

• Pheng LH, Regoli D
• Receptors for NPY in peripheral tissues bioassays.
• Life Sci. 2000; 67: 847-62
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• Neuropeptide Y (NPY) and its congeners, peptide YY (PYY) and the pancreatic polypeptide (PP), have a large spectrum of peripheral actions. NPY is found in peripheral neurons, co-localized or not with noradrenaline; PYY and PP are expressed in endocrine cells of the pancreas and in the intestine of vertebrates. NPY is the most abundant peptide in the brain and is involved in the regulation of food intake and of circadian rhythm. It intervenes also in the process of anxiety and memory. NPY is a potent vasoconstrictor, a cardiac stimulant, and may affect the gut through enteric neurons. PYY and PP act mainly on the gastrointestinal system; however, when in blood, they can cross-react with functional sites elsewhere and replace NPY in some parts of the brain (e.g. regions involved in feeding behavior). These peptides act through G protein coupled receptors (GPCR) of which five different types are known and have been cloned (1,2); functional sites (receptors) for NPY have been found in vessels, the gut, and in vasa deferentia (3-6).

• Couzens M et al.
• Peptide YY-2 (PYY2) and pancreatic polypeptide-2 (PPY2): species-specific evolution of novel members of the neuropeptide Y gene family.
• Genomics. 2000; 64: 318-23
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• Several gene duplication events have led to the creation of at least five distinct members of the neuropeptide Y gene family. We now reveal that the most recent of these events, involving the PYY-PPY gene cluster on chromosome 17q21.1, has led to the creation of novel PYY- and PP-like genes on chromosome 17q11 in the human genome. Sequence analysis of the novel human PYY2 and PPY2 genes shows an extensive homology to the peptide YY-pancreatic polypeptide genes, at the level of gene structure, nucleotide sequence, and primary amino acid sequence. The extremely high degree of homology between the PYY-PPY and the PYY2-PPY2 gene clusters, in both coding regions and especially noncoding regions, suggests that the PYY2 and PPY2 genes have arisen by a very recent gene duplication. Similar gene duplication events of the PYY-PPY gene cluster have also occurred in other species, including cow and baboon, but have not been confirmed in the rat and mouse genomes. Interestingly, despite the greater than 92% nucleotide sequence identity between these new genes, a few specific mutations have resulted in significantly altered peptide sequences. These altered sequences are accompanied by acquisition of new functions apparently unrelated to the neurotransmitter/endocrine role of PYY and PPY, as demonstrated by the major involvement of bovine PYY2, also known as seminal plasmin, in the fertilization process.

• Mannon PJ, Kanungo A, Mannon RB, Ludwig KA
• Peptide YY/neuropeptide Y Y1 receptor expression in the epithelium and mucosal nerves of the human colon.
• Regul Pept. 1999; 83: 11-9
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• OBJECTIVES: Peptide YY is an abundant distal gut hormone which regulates secretion, motility, and possibly epithelial proliferation in the gut. Though messenger RNA for the peptide YY Y1 receptor subtype occurs in the basal colonic crypts of humans, peptide YY receptors themselves have not been clearly localized within the adult human gastrointestinal tract. Using an antiserum directed against the C-terminus of the Y1 receptor we determined the actual extent of Y1 receptor protein expression in the human colon in order to identify areas targeted for peptide YY effects and suggest additional physiological roles for PYY in the human gut. RESULTS: Y1 receptor protein expression was seen throughout the colonic epithelium along its basolateral aspect. There was an unexpected dense distribution of Y1 receptor immunoreactivity in varicose fibers within the mucosa. Staining was also noted in nerve fibers of the muscularis mucosae, in the submucous and myenteric plexuses, and in nerves in the muscularis propria. CONCLUSIONS: Widespread distribution of Y1 receptors in the colonic epithelium and mucosal nerve fibers suggests diverse regulatory roles for peptide YY in modulating epithelial function as well as secretomotor reflexes in response to lumenal peptide YY-release signals.

• Starback P et al.
• Neuropeptide Y receptor subtype with unique properties cloned in the zebrafish: the zYa receptor.
• Brain Res Mol Brain Res. 1999; 70: 242-52
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• Neuropeptide Y (NPY) belongs to a family of structurally related neuroendocrine peptides for which five different G-protein-coupled receptor subtypes have been cloned in mammals. To identify additional subtypes we have performed PCR with degenerate primers in different species. We describe here the cloning and pharmacological profile of a unique NPY receptor subtype in the zebrafish that has tentatively been called the zYa receptor. It has 46-50% amino acid identity to the mammalian Y1, Y4 and y6 receptors and the previously cloned zebrafish receptors zYb and zYc, and only about 27% to Y2 and Y5. The zYa receptor binds NPY and PYY from mammals as well as zebrafish with high affinities and has a K(d) of 28 pM for porcine (125)I-PYY. It has a unique binding profile displaying some features in common with each of the mammalian Y1, Y2 and Y5 receptors. In a microphysiometer assay the receptor responds with extracellular acidification. Chromosomal mapping in the zebrafish genome of zYa, zYb and zYc receptor genes indicates a possible orthologous relationship between zYc and mammalian y6, but identifies no obvious mammalian ortholog for zYa (zYb is a recent copy of zYc in the fish lineage). These results imply that previous studies of NPY in fishes, which have striven to interpret the effects within the framework of mammalian Y1, Y2, and Y5 receptors, need to be reevaluated. Thus, the sequence comparisons, pharmacological properties, and chromosomal localization suggest that the zYa receptor is a novel NPY receptor subtype which is likely to be present also in mammals.

• Ingenhoven N, Eckard CP, Gehlert DR, Beck-Sickinger AG
• Molecular characterization of the human neuropeptide Y Y2-receptor.
• Biochemistry. 1999; 38: 6897-902
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• 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.

• Ingenhoven N, Beck-Sickinger AG
• Molecular characterization of the ligand-receptor interaction of neuropeptide Y.
• Curr Med Chem. 1999; 6: 1055-66
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• Neuropeptide Y (NPY) consists of 36 amino acids and is one of the most abundant peptides in the peripheral and central nervous system. Several subtypes of NPY receptors have been described (Y1- y6) using segments and analogues of NPY. The Y1-, Y2- and the Y5-receptor, which have been cloned, belong to the G-protein coupled hormone receptor family and will be specially addressed, because they are the endogenous binding sites of neuropeptide Y in human. In contrast, Y4-receptors recognize endogenous PP, Y3 receptors are discussed controversially and the y6-receptor is truncated in human. In this review, we summarize the data of neuropeptide Y with respect to ligand binding, selectivity, receptor structures and ligand-receptor complexes by using ligand analogues, site directed mutagenesis and photoaffinity labeling.

• Le MT, Vanderheyden PM, De Backer JP, Vanquelin G, Broeck JV
• High affinity displacement of [(3)H]NPY binding to the crude venom of conus anemone by insect neuropeptides.
• Biochem Biophys Res Commun. 1999; 262: 180-6
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• The venom from Conus anemone contains a protein, named ANPY toxin, which displayed high affinity (IC(50) in nanomolar range) to neuropeptide Y (NPY), [Leu(31), Pro(34)]NPY, peptide YY, pancreatic polypeptide, the Y(1) antagonist 1229U91, and C-terminal NPY fragments. N-terminal fragments and the free acid form of NPY did not bind to ANPY. The truncated NPY fragments displayed very low affinity to Y(1) receptors and partially inhibited [(3)H]NPY binding to anti-NPY antiserum. Several insect neuropeptides, the sequences of which related to the C-terminal fragments of NPY, were observed to bind with similar affinity or even 20 times higher (Lom-MS and Scg-NPF) affinity than NPY. In contrast, no significant binding of these insect peptides was observed for Y(1) receptors and anti-NPY antiserum. Therefore, ANPY can be viewed as an acceptor that binds with very high affinity to a broad spectrum of vertebrate and invertebrate neuropeptides that share a similar C-terminal amino acid sequence.

• Berglund MM et al.
• The cloned guinea pig neuropeptide Y receptor Y1 conforms to other mammalian Y1 receptors.
• Peptides. 1999; 20: 1043-53
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• We have cloned the guinea pig neuropeptide Y (NPY) Y1 receptor and found it to be 92-93% identical to other cloned mammalian Y1 receptors. Porcine NPY and peptide YY (PYY) displayed affinities of 43 pM and 48 pM, respectively. NPY2-36 and NPY3-36 had 6- and 46-fold lower affinity, respectively, than intact NPY. Functional coupling was measured by using a microphysiometer. Human NPY and PYY were equipotent in causing extracellular acidification with EC50 values of 0.59 nM and 0.69 nM, respectively, whereas NPY2-36 and NPY3-36 were about 15-fold and 500-fold less potent, respectively, than NPY. The present study shows that the cloned guinea pig Y1 receptor is very similar to its orthologues in other mammals, both with respect to sequence and pharmacology. Thus, results from previous studies on guinea pig NPY receptors might imply the existence of an additional Y1-like receptor sensitive to B1BP3226.

• Pheng LH et al.
• Neuropeptide Y-induced contraction is mediated by neuropeptide Y Y2 and Y4 receptors in the rat colon.
• Eur J Pharmacol. 1999; 374: 85-91
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• Ascending and descending segments of the rat colon were studied to analyze their contractile responses to neuropeptide Y and related peptides. These responses are (a) completely eliminated by tetrodotoxin (1 microM), (b) reduced to a variable extent (20 to 60%) by atropine (1 microM) and (c) not modified by indomethacin, diphenhydramine or methysergide. The order of potency of agonists for peptides related to neuropeptide Y was as follows: human pancreatic polypeptide = rat pancreatic polypeptide > peptide YY = peptide YY-(3-36) = [Leu31,Pro34]neuropeptide Y > neuropeptide Y-(2-36) > C2-neuropeptide Y = neuropeptide Y > neuropeptide Y-(13-36), with minor differences observed between the two parts of the colon. This selectivity pattern does not correspond to the profile of any known cloned neuropeptide Y receptors. BIBP3226, a selective antagonist for the neuropeptide Y Y1 receptor sub-type, was found to be inactive, while a neuropeptide Y Y2 receptor antagonist, T4-[NPY-(33-36)]4, reduced the effects of neuropeptide Y, peptide YY, peptide YY-(3-36) and C2-neuropeptide Y without affecting those of human pancreatic polypeptide, rat pancreatic polypeptide and [Leu31,Pro34]neuropeptide Y. JCF 104 (compound 28), a putative neuropeptide Y Y5 receptor antagonist, showed no effect or a weak inhibition of human pancreatic polypeptide or [Leu31,Pro34]neuropeptide Y-induced contraction. Taken together, these data suggest that: (1) at least two neuropeptide Y receptor types are present in the rat colon autonomic nerve terminals and modulate the release of acetylcholine and possibly other transmitters; (2) a proportion of the receptors mediating the contractile response of the rat colon (especially descending part) to neuropeptide Y and related peptides appears to be of the Y2 type and (3) the significant portion of the response is mediated by a receptor which is insensitive to neuropeptide Y Y1, Y2 and to neuropeptide Y Y5 receptor antagonists. This receptor behaves as a neuropeptide Y Y4 receptor sub-type and appears to be located on enteric nerves.

• Eckard CP, Beck-Sickinger AG, Wieland HA
• Comparison of antibodies directed against receptor segments of NPY-receptors.
• J Recept Signal Transduct Res. 1999; 19: 379-94
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• The Y1-, Y2-, Y4- and Y5-receptor, which belong to the rhodopsin-like G-protein coupled, 7 transmembrane helix spanning receptors, bind the 36-mer neuromodulator NPY (neuropeptide Y) with nanomolar affinity. Synthetic fragments of the second (E2) and third (E3) extracellular loop were used to generate subtype selective anti-receptor antibodies against the Y-receptors. As investigated on intact receptors by ELISA and on solubilized receptors by SDS-PAGE and subsequent Western blotting, subtype selectivity was only partly achieved. Nevertheless, selectivity can be obtained by using several antisera in combination. These antibodies represent tools for molecular mass determination, receptor purification by affinity chromatography with antibody-columns and receptor localization studies.

• Goumain M, Voisin T, Lorinet AM, Balasubramaniam A, Laburthe M
• Pharmacological profile of the rat intestinal crypt peptide YY receptor vs. the recombinant rat Y5 receptor.
• Eur J Pharmacol. 1998; 362: 245-9
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• Peptide YY and neuropeptide Y have potent antisecretory effects in rat small intestine. Scatchard analysis of [125I]peptide YY binding revealed a 10-fold higher concentration of receptors in rat jejunal crypt cells than in villus cells and no detectable receptors in colonic epithelium. Reverse transcription polymerase chain reaction analysis of neuropeptide Y Y5 receptor mRNA indicated that they are mainly expressed in rat jejunal crypts with very few or no expression in villus cells and colon epithelium, respectively. In order to determine whether neuropeptide Y Y5 receptors could represent the intestinal crypt receptor for peptide YY and neuropeptide Y, the ability of peptide YY, neuropeptide Y, pancreatic polypeptide and analogues to inhibit [125I]peptide YY binding to membrane prepared from rat crypt cells and COS-7 cells (African green monkey kidney cells) transfected with the rat neuropeptide Y Y5 receptor cDNA was tested. It appeared that several analogues displayed different inhibition constants (Ki) in the two binding assays, more especially N-alpha-acetyl-peptide YY-(22-36) which was 1200 x more potent in the crypt cell binding assay than in the recombinant neuropeptide Y Y5 receptor binding assay. These data support that the intestinal crypt peptide YY receptor is not a Y5 receptor. reserved.

• Marsh DJ, Hollopeter G, Kafer KE, Palmiter RD
• Role of the Y5 neuropeptide Y receptor in feeding and obesity.
• Nat Med. 1998; 4: 718-21
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• Neuropeptide Y (NPY), a 36-amino-acid neuromodulator abundantly expressed in the brain, has been implicated in the regulation of food intake and body weight. Pharmacological data suggest that NPY's stimulatory effect on appetite is transduced by the G-protein-coupled NPY Y5 receptor (Y5R). We have inactivated the Y5R gene in mice and report that younger Y5R-null mice feed and grow normally; however, they develop mild late-onset obesity characterized by increased body weight, food intake and adiposity. Fasting-induced refeeding is unchanged in younger Y5R-null mice and they exhibit normal sensitivity to leptin. Their response to intracerebroventricular (i.c.v.) administration of NPY and related peptides is either reduced or absent. NPY deficiency attenuates the obesity syndrome of mice deficient for leptin (ob/ob), but these effects are not mediated by NPY signaling through the Y5R because Y5R-null ob/ob mice are equally obese. These results demonstrate that the Y5R contributes to feeding induced by centrally administered NPY and its analogs, but is not a critical physiological feeding receptor in mice.

• Pooga M et al.
• Novel galanin receptor ligands.
• J Pept Res. 1998; 51: 65-74
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• Galanin is a neuroendocrine peptide which is 29/30 amino acids in length and is recognised by G-protein-coupled central nervous system receptors via its N-terminus. We synthesised several galanin receptor ligands and fragments around C-terminal extensions of galanin(1-13) to yield chimeric peptides with C-terminals corresponding to bioactive peptides like bradykinin(2-9), mastoparan, neuropeptide Y(25-36) or substance P(5-11), respectively. We also synthesised short galanin analogs in which galanin(1-13) was C-terminally elongated with Lys14; different pharmacologically active small molecules were then attached to the epsilon-amino group of Lys14. Several cysteine-substituted linear and ring closed analogs of galanin(1-9) and galanin(1-16) were also synthesised. The equilibrium binding constants for these peptides at hypothalamic galanin receptors were determined and found in the subnanomolar to micromolar range. The large number of peptides and their binding affinities presented here permit structure-activity relationship analysis of peptide-type ligands to galanin receptors.

• Shetzline MA, Zipf WB, Nishikawara MT
• Pancreatic polypeptide: identification of target tissues using an in vivo radioreceptor assay.
• Peptides. 1998; 19: 279-89
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• The definitive function of pancreatic polypeptide in mammalian physiology remains unknown. The identification of specific PP target tissues should be helpful to further investigations into the possible regulatory actions of this peptide. An in vivo radioreceptor assay was used in the rat to locate potential binding sites of I(125) bovine PP. In vitro, high concentrations of unlabeled hormone competitively inhibit binding to receptors by low concentrations of labeled hormone. In vivo studies showed that, in the presence of concentrated unlabeled pancreatic polypeptide, labeled PP distributes between the plasma and interstitial fluid. When excess unlabeled PP is replaced with saline in the companion animals, the labeled peptide appears to distribute in a volume that exceeds the combined plasma volume and interstitial fluid volume of the tissue. Using this in vivo receptor assay, the distribution volume that exceeds the anatomic extracellular volume has been identified as the receptor compartment. With this assay we demonstrated in the rat specific and displaceable PP binding to the ductus choledochus, duodenum, ileum, and adrenal gland. The NVV determined in the adrenal gland of experimental animals was 3.9 times greater than that found in the control group. Binding was rapid and was displaced only by excess unlabeled pancreatic polypeptide. Neither excess insulin nor excess neuropeptide Y significantly reduced this binding.

• Larhammar D, Soderberg C, Lundell I
• Evolution of the neuropeptide Y family and its receptors.
• Ann N Y Acad Sci. 1998; 839: 35-40

• Smith-White M, Moriarty MJ, Potter EK
• A comparison of actions of neuropeptide Y (NPY) agonists and antagonists at NPY Y1 and Y2 receptors in anaesthetized rats.
• Neuropeptides. 1998; 32: 109-18
• Display abstract

• The pancreatic polypeptide family includes three members, neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), with sequence homology between members and species varying from approximately 50 to 80%. Some of these peptides were compared in the mammalian cardiovascular system for activity mediated by actions on pre- (Y2) and post-junctional (Y1) NPY receptors. NPY and PYY, with sequence homology of 67% have similar actions on Y1 and Y2 receptors. Rat pancreatic polypeptide (rPP) with sequence homology of approximately 50% is inactive at both. This study reports that the chimeric peptide, hPP1-11/NPY12-36 and the truncated peptide NPY2-36 show similar activity to NPY mediated through both receptor types in vivo, while salmon PYY (sPYY), with 81% homology to NPY, has improved potency at both receptor subtypes. NPY3-36 has equal activity with NPY on actions mediated through Y2 receptors, but significantly reduced activity mediated through Y1 receptors. Two NPY antagonists were also examined: PYX2 was inactive in vivo and 1229U91 showed potent, long-lasting activity on Y1 receptor-mediated effects.

• Zimanyi IA, Fathi Z, Poindexter GS
• Central control of feeding behavior by neuropeptide Y.
• Curr Pharm Des. 1998; 4: 349-66
• Display abstract

• Obesity is a serious health problem in the Western societies, therefore its treatment has become the subject of intense interest in the scientific community. A significant number of recent publications enlist different central and peripheral factors which play important roles in the regulation of food intake, body weight and energy expenditure. Neuropeptide Y, a 36 amino acid peptide, which is quite abundant in the brain, seems to be one of the more important players in these regulations. Recently five NPY receptors have been cloned and pharmacological evidence strongly supports the existence of a sixth receptor. There are many contradictory findings regarding which NPY receptor mediates the effect of NPY on food intake. This article will review the effects of NPY on the regulation of food intake and energy expenditure and will discuss the pharmacological and molecular evidence as to which NPY receptor(s) mediate this effect. The review will also summarize the progress which has been made in the design of novel NPY-ergic ligands, especially NPY receptor antagonists, for potential use in the treatment of obesity.

• Cerda-Reverter JM, Martinez Rodriguez G, Zanuy S, Carrillo M, Larhammar D
• Cloning of neuropeptide Y, peptide YY, and peptide Y from sea bass (Dicentrarchus labrax), a marine teleost.
• Ann N Y Acad Sci. 1998; 839: 493-5

• Wieland HA, Eckard CP, Doods HN, Beck-Sickinger AG
• Probing of the neuropeptide Y-Y1-receptors interaction with anti-receptor antibodies.
• Eur J Biochem. 1998; 255: 595-603
• Display abstract

• The Y1 receptor, which belongs to the family of rhodopsin-like GTP-binding protein-coupled, seven-transmembrane helix-spanning receptors, binds the 36-mer neuromodulator neuropeptide Y (NPY) with nanomolar affinity. Synthetic fragments of the N-terminus, extracellular loops and C-terminus of the Y1 receptor were used to generate 18 anti-receptor antibodies; ten of them recognize the receptor expressed on intact cells as well as on membranes that have been prepared (with the exception of one antibody raised against the intracellular C-terminus) as investigated by ELISA. SDS/PAGE of solubilized membranes, subsequent Western blotting and staining with the antibodies revealed two proteins of 73 kDa and 51 kDa for both, the rat and the human receptor. Competition with neuropeptide Y showed that the binding of seven antibodies is strongly inhibited in the presence of the native ligand. Using photoactivatible analogues, it could be demonstrated that the competition efficiency strongly depends on the position of the crosslinker within the ligand. Based on these studies, a model for the ligand-receptor interaction is suggested. These antibodies represent novel tools for the structural characterization of the Y1 receptor and its interaction with NPY and antagonists as well as for localization studies.

• Parker EM et al.
• GR231118 (1229U91) and other analogues of the C-terminus of neuropeptide Y are potent neuropeptide Y Y1 receptor antagonists and neuropeptide Y Y4 receptor agonists.
• Eur J Pharmacol. 1998; 349: 97-105
• Display abstract

• GR231118, BW1911U90, Bis(31/31')[[Cys31, Trp32, Nva34] neuropeptide Y(31-36)] (T-190) and [Trp-Arg-Nva-Arg-Tyr]2-NH2 (T-241) are peptide analogs of the C-terminus of neuropeptide Y that have recently been shown to be antagonists of the neuropeptide Y Y1 receptor. In this study, the activity of these peptides at each of the cloned neuropeptide Y receptor subtypes is determined in radioligand binding assays and in functional assays (inhibition of forskolin-stimulated cAMP formation). GR231118 is a potent antagonist at the human and rat neuropeptide Y Y1 receptors (pA2 = 10.5 and 10.0, respectively; pKi = 10.2 and 10.4, respectively), a potent agonist at the human neuropeptide Y Y4 receptor (pEC50 = 8.6; pKi = 9.6) and a weak agonist at the human and rat neuropeptide Y Y2 and Y5 receptors. GR231118 also has high affinity for the mouse neuropeptide Y Y6 receptor (pKi = 8.8). Therefore, GR231118 is a relatively selective neuropeptide Y Y1 receptor antagonist, but has appreciable activity at the neuropeptide Y Y4 and Y6 receptors as well. BW1911U90, T-190 and T-241 are moderately potent neuropeptide Y Y1 receptor antagonists (pA2 = 7.1, 5.8 and 6.5, respectively; pKi = 8.3, 6.5 and 6.8, respectively) and neuropeptide Y Y4 receptor agonists (pEC50 = 6.8, 6.3 and 6.6, respectively; pKi; 8.3, 7.7 and 8.3, respectively). These data suggest that the C-terminus of neuropeptide Y and related peptides is sufficient for activation of the neuropeptide Y Y4 receptor, but is not sufficient for activation of the neuropeptide Y Y1 receptor. Because BW1911U90, T-190 and T-241 are significantly less potent at the cloned human neuropeptide Y Y1 receptor than at the neuropeptide Y receptor in human erythroleukemia cells, these cells may express a novel neuropeptide Y receptor with high affinity for these peptides.

• Uegaki K, Murase S, Nemoto N, Kobayashi Y, Yoshikawa S, Yumoto N
• Effects of covalent dimerization on the structure and function of the carboxy-terminal fragment of neuropeptide Y.
• Biochem Biophys Res Commun. 1997; 241: 737-43
• Display abstract

• To determine whether or not the dimeric structure of neuropeptide Y (NPY) that is found in solution is necessary for its function, we investigated the effects of covalent dimerization on the structure and function of NPY using the carboxy-terminal fragment, NPY(12-36), in which residues 12 and 31 (located at both ends of alpha-helical region) were replaced by Cys residues. Among the three species (the parallel dimer, the anti-parallel dimer, and the intramolecularly cross-linked monomer) obtained by oxidation of the fragment, the anti-parallel dimer was predominant. NMR analysis showed that both parallel and anti-parallel dimers had alpha-helices similar to that of intact NPY, suggesting that covalent dimerization might have little effect on the helical structure. A binding assay with Y2 receptors on porcine hippocampal membranes revealed that the IC50 value of the anti-parallel dimer was almost the same as that of NPY (13-36), which is known as a Y2-specific ligand. By contrast, the binding by the parallel dimer was weaker by more than one order of magnitude. Our results suggest that the formation of dimers of NPY is not essential for binding to the receptor.

• Statnick MA, Schober DA, Gehlert DR
• Identification of multiple neuropeptide Y receptor subtypes in the human frontal cortex.
• Eur J Pharmacol. 1997; 332: 299-305
• Display abstract

• Recently, we found abundant mRNA and binding sites for neuropeptide Y Y1-like receptors in the human cerebral cortex. However, an earlier study using indirect labeling methods failed to detect substantial neuropeptide Y1-like receptor binding in numerous areas of the human brain, including the cerebral cortex. To resolve the disparity in these findings, we characterized the neuropeptide Y receptor subtypes labeled with [125I]peptide YY in homogenates of human frontal cortex. Competition experiments using 100 pM [125I]peptide YY binding to human frontal cortex homogenates indicated predominantly neuropeptide Y Y2 receptors are labeled with this concentration of ligand. However, saturation analysis of [125I]peptide YY binding to frontal cortex membranes resulted in isotherms best characterized by a two-site fit. Binding of [125I]peptide YY to the high affinity (Kd = 40 pM) binding site was prevented using a 100 nM concentration of the neuropeptide Y Y2 receptor agonist peptide YY-(3-36). By masking the higher affinity site, we found a low affinity [125I]peptide YY binding site (Kd = 1.4 nM) exhibiting a pharmacology consistent with a neuropeptide Y Y1-like receptor. It appears that neuropeptide Y Y2 receptors are the predominant subtype labeled with low concentrations of[125I]peptide YY and that the neuropeptide Y Y1 receptor is a low affinity [125I]peptide YY binding site in the human frontal cortex.

• Blomqvist AG, Herzog H
• Y-receptor subtypes--how many more?
• Trends Neurosci. 1997; 20: 294-8
• Display abstract

• The Y-receptors belong to the G protein-coupled receptor superfamily and mediate a wide variety of physiological effects, such as regulation of blood pressure, anxiety, memory retention, hormone release and food intake. Since the first human Y-receptor was cloned in 1992, the search for additional subtypes has been an area of intense study. Recently four new NPY-receptor subtypes have been isolated, revealing surprisingly limited sequence identity with values as low as 30%. Several reports indicate further heterogeneity of this receptor family, for example a peripheral Y2 receptor. However, since many studies have been carried out with different peptide analogs and radioligands in different species, there is substantial confusion regarding the pharmacological profile of the receptors. This may have led to an exaggeration of the potential number of discrete receptors.

• Roche C et al.
• Genetic studies of neuropeptide Y and neuropeptide Y receptors Y1 and Y5 regions in morbid obesity.
• Diabetologia. 1997; 40: 671-5
• Display abstract

• Synthesis and release of neuropeptide Y (NPY) are both regulated by leptin binding to its hypothalamic receptor mediating some of the effects of leptin on food intake. Moreover, NPY administration is a powerful stimulant of feeding behaviour. Thus, we investigated the potential implication of NPY, NPY-Y1 and -Y5 subtype receptors [rNPY-Y1/-Y5] in the development of human obesity. Two complementary genetic approaches were used: 1) linkage analyses between obesity and polymorphic markers located nearby NPY and rNPY-Y1/-Y5 genes (respectively on chromosomes 7p15.1 and 4q[31.3-32]) in 93 French Caucasian morbidly obese families; 2) single strand conformation polymorphism (SSCP) scanning of the coding region of the NPY and rNPY-Y1 genes performed in 50 unrelated obese patients ascertained on the basis of a body mass index of 27 kg/m2 or more and a family history of obesity. No evidence of linkage between morbid obesity or obesity-related quantitative traits and NPY and rNPY-Y1/ Y5 regions was found in this population. Moreover, SSCP scanning revealed no mutation in the coding region of NPY and rNPY-Y1 genes among obese subjects. These results suggest that NPY and NPY-Y1/ Y5 receptors are unlikely to be implicated in the development of human morbid obesity, at least in the French Caucasian population.

• Jacques D, Dumont Y, Fournier A, Quirion R
• Characterization of neuropeptide Y receptor subtypes in the normal human brain, including the hypothalamus.
• Neuroscience. 1997; 79: 129-48
• Display abstract

• The aim of the present study was to investigate the existence and distribution of neuropeptide Y receptor subtypes in various regions of the normal human brain using the peptide YY derivative receptor probes, [125I][Leu31,Pro34]polypeptide YY/Y1 and [125I]polypeptide YY(3-36)/Y2, in addition to the non-selective ligand [125I]polypeptide YY. Membrane binding assays performed with post mortem frontal cortex homogenates revealed that [125I]polypeptide YY and [125I]polypeptide YY(3-36) bound in a time- and protein concentration-dependent manner. Very low amounts of specific [125I][Leu31,Pro34]polypeptide YY binding could be detected even in the presence of high amounts of protein, contrasting with results obtained with [125I]polypeptide YY and [125I]polypeptide YY(3-36), a preferential Y2 receptor probe. Analysis of saturation isotherms revealed that [125I]polypeptide YY(3-36) bound to a single class of high-affinity sites (0.5-2 nM). Significantly higher binding capacities were evident for [125I]polypeptide YY(3-36) as compared to [125I][Leu31,Pro34]polypeptide YY, suggesting that the human frontal cortex, in contrast to the rat, is mostly enriched with Y2 receptors. Ligand selectivity profile confirmed the hypothesis that polypeptide YY(3-36), neuropeptide Y and polypeptide YY but not the [Leu31,Pro34] derivatives are potent competitors of [125I]polypeptide YY and [125I]polypeptide YY(3-36) binding sites. Autoradiographic studies demonstrated further that cortical areas, as well as most other regions of the human brain, are particularly enriched with Y2/[125I]polypeptide YY(3-36) sites, while only low to very low amounts of Y1 binding were detected except in the dentate gyrus of the hippocampal formation. In the human hypothalamus, a preponderance of Y2 binding sites was also noted. Taken together, these results clearly establish that the distribution of the Y1 and Y2 receptor subtypes in human is different from the rodent brain, the Y2 subtype being most abundant in the human brain.

• Ringvall M, Berglund MM, Larhammar D
• Multiplicity of neuropeptide Y receptors: cloning of a third distinct subtype in the zebrafish.
• Biochem Biophys Res Commun. 1997; 241: 749-55
• Display abstract

• Five different receptor subtypes for neuropeptide Y (NPY) have recently been cloned in mammals. We have discovered three distinct subtypes by PCR in the zebrafish, Danio rerio, and describe here one of these called zYc. The protein sequence identity is 46-51% to mammalian subtypes Y1, Y4 and Y6 and to zebrafish Ya, i.e., the same degree of identity as these subtypes display to one another. The identity to zYb is higher, 75%, indicating that zYb and zYc share a more recent ancestor. The zYc receptor binds NPY and PYY (peptide YY) from mammals as well as zebrafish with high affinities and has a Kd of 16 pM for 125I-pPYY. The pharmacological profile is similar to, but distinct-from, mammalian Y1. zYc inhibits cAMP synthesis. This work suggests that NPY has more receptor subtypes than any other peptide that binds to G protein-coupled receptors. Work is in progress to see if the zebrafish receptors are present in mammals.

• Muller M, Knieps S, Gessele K, Dove S, Bernhardt G, Buschauer A
• Synthesis and neuropeptide Y Y1 receptor antagonistic activity of N,N-disubstituted omega-guanidino- and omega-aminoalkanoic acid amides.
• Arch Pharm (Weinheim). 1997; 330: 333-42
• Display abstract

• Potent arpromidine-type histamine H2 receptor agonists such as BU-E-76 (He 90481) were among the first non-peptides reported to display weak neuropeptide Y (NPY) Y1 receptor antagonist activity. In search of new chemical leads for the development of more potent NPY antagonists, a series of N,N-disubstituted omega-guanidino and omega-aminoalkanoic acid amides were synthesized on the basis of structure-activity relationships and molecular modeling studies of arpromidine and related imidazolylpropylguanidines. In one group of compounds the imidazole ring was retained whereas in the second group it was replaced with a phenol group representing a putative mimic of Tyr36 in NPY. Although the substitution patterns have not yet been optimized, the title compounds are NPY Y1 antagonists in human erythroleukemia (HEL) cells (Ca2+ assay) achieving pKB values in the range of 6.3-6.6. For representative new substances tested in the isolated guinea pig right atrium histamine H2 receptor agonism could not be found. In the N-(diphenylalkyl)amide series, compounds with a trimethylene chain were more active Y1 antagonists than the ethylene homologs. Concerning the spacer in the omega-amino or omega-guanidinoalkanoyl portion, the best activity was found in compounds with a four- or five-membered alkyl chain or a 1,4-cyclohexylene group. Surprisingly, in contrast to the phenol series, in the imidazole series the compounds with a side chain amino group turned out to be considerably more potent than the correspondence strongly basic guanidines. Thus, the structure-activity relationships appear to be different for the diphenylalkylamide NPY antagonists with one or two basic groups.

• Niebergall-Roth E, Teyssen S, Rippel K, Singer MV
• [Effects of peptide YY on functions of the gastrointestinal tract]
• Dtsch Tierarztl Wochenschr. 1997; 104: 108-13
• Display abstract

• The present report gives a review about the localization, release and gastrointestinal actions of peptide YY in different animal species and in humans. Possible mechanisms of action, the physiological and pathophysiological significance of peptide YY and the role of peptide YY 3-36 are discussed. Finally, unanswered questions are specified.

• Pheng LH, Quirion R, Iyengar S, Fournier A, Regoli D
• The rabbit ileum: a sensitive and selective preparation for the neuropeptide Y Y5 receptor.
• Eur J Pharmacol. 1997; 333: 35-35
• Display abstract

• The rabbit ileum shows high sensitivity to neuropeptide Y. Relaxations are obtained in this tissue with human pancreatic polypeptide > peptide YY > > [Leu31,Pro34]neuropeptide Y > rat pancreatic polypeptide > human neuropeptide Y in this order of potency that is indicative of a Y5 receptor. Effects of neuropeptide Y and congeners are not affected by neuropeptide Y Y1 receptor antagonist (BIBP 3226), but are reduced by the neuropeptide Y Y5 receptor antagonist JCF 104 (2-(naphtalen-1-yl)-3-phenylpropane-1,2-diamine). Rabbit ilea provide sensitive and selective neuropeptide Y Y5 receptor preparations.

• Lundell I, Berglund MM, Starback P, Salaneck E, Gehlert DR, Larhammar D
• Cloning and characterization of a novel neuropeptide Y receptor subtype in the zebrafish.
• DNA Cell Biol. 1997; 16: 1357-63
• Display abstract

• Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) form a family of structurally related peptides. As we have previously isolated clones for NPY and PYY from the zebrafish (Danio rerio), we wished to clone the receptors for these peptides to allow correlation of ligand and receptor distribution. We describe here the cloning and functional expression of a receptor with equally high identity to the NPY-Y1 receptor as to the recently cloned Y4/PP1 and Y6 receptors with an overall amino acid sequence identity of approximately 50%. Furthermore, the zebrafish receptor gene lacks the intron present in the coding region in vertebrate Y1 genes. These features strongly suggest that the zebrafish receptor represents a separate subtype. Hence, we have named it zYb for zebrafish Y-receptor b. (We have also discovered a unique receptor called zYa.) The zYb receptor has a binding profile that is reminiscent of Y1 with affinities for NPY and PYY in the low picomolar range, whereas affinities for Y2-selective ligands are considerably lower. It couples to adenylyl cyclase by inhibiting cAMP synthesis. Receptor mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) in brain, eye, and intestine. The binding profile and amino acid identity show that the zebrafish zYb receptor is related to Y1 but represents a distinct subtype that is likely to be present also in mammals.

• Betancur C, Azzi M, Rostene W
• Nonpeptide antagonists of neuropeptide receptors: tools for research and therapy.
• Trends Pharmacol Sci. 1997; 18: 372-86
• Display abstract

• The recent development of selective and highly potent nonpeptide antagonists for peptide receptors has constituted a major breakthrough in the field of neuropeptide research. Following the discovery of the first nonpeptide antagonists for peptide receptors ten years ago, numerous other antagonists have been developed for most neuropeptide families. These new, metabolically stable compounds, orally active and capable of crossing the blood-brain barrier, offer clear advantages over the previously available peptide antagonists. Nonpeptide antagonists have provided valuable tools to investigate peptide receptors at the molecular, pharmacological and anatomical levels, and have considerably advanced our understanding of the pathophysiological roles of peptides in the CNS and periphery. Evidence from animal and clinical studies suggests that nonpeptide antagonists binding to peptide receptors could be useful for the treatment of disease states associated with high levels of neuropeptides. In this article Catalina Batancur, Mounia Azzi and William Rostene will address the recent developments in nonpeptide antagonists for neuropeptide receptors, with a particular focus on their CNS actions.

• Walker MW et al.
• A structure-activity analysis of the cloned rat and human Y4 receptors for pancreatic polypeptide.
• Peptides. 1997; 18: 609-12
• Display abstract

• We cloned and expressed the rat Y4 receptor for pancreatic polypeptide (PP). Structure-activity profiles derived from 125I-PP binding assays and [cAMP] radioimmunoassays reveal a selective receptor interaction with rat PP vs. neuropeptide Y (NPY) or peptide YY (PYY). Rat and human Y4 receptor clones share 75% amino acid identity. Based on [cAMP] radioimmunoassay, the human Y4 receptor exhibits a less selective interaction with rat PP vs. NPY or PYY and a greater dependence on N-terminal PP residues, relative to rat Y4. Differences in sequence and structure-activity profiles suggest the rat be used with caution to model human Y4 receptor function.

• Sanchez-Margalet V, Santos-Alvarez J
• Solubilization and molecular characterization of active pancreastatin receptors from rat liver membranes.
• Endocrinology. 1997; 138: 1712-8
• Display abstract

• Pancreastatin receptors were solubilized from rat liver membranes with the nonionic detergent Triton X-100. Binding of a iodinated analog of rat pancreastatin ([125I-Tyr0]pancreastatin) to the soluble fraction was time dependent, saturable, and reversible. Scatchard analysis of binding under equilibrium conditions indicated that the soluble extracts contained a single class of pancreastatin-binding sites, with a binding capacity of 14 fmol/mg protein and a Kd of 0.3 nM. As observed with membrane-bound receptors, binding of [125I]pancreastatin to soluble extracts was inhibited by guanine nucleotides with the following rank order of potency: guanyl-5'-yl-imidodiphosphate > GTP > GDP > GMP, indicating that the soluble receptors are functionally linked to G proteins. Molecular analysis of the soluble pancreastatin receptor by covalent cross-linking to [125I]pancreastatin using disuccinimidyl suberate and further identification on SDS-PAGE indicated a single band of 85,000 Mr. Gel filtration of soluble extracts on Sephacryl S-300 revealed two molecular components with binding abilities (Mr 80,000 and 170,000). The higher molecular mass component was more sensitive to guanine nucleotides, and covalent cross-linking of both components to [125I]pancreastatin and further SDS-PAGE analysis revealed again a single band of 85,000 Mr, suggesting an association of the receptor with a G protein. Moreover, direct evidence that a Gq was present in the same chromatographic fraction was obtained by specific immunodetection. The soluble receptor is a glycoprotein that can be specifically bound to the wheat-germ agglutinin lectin. We conclude that we solubilized active pancreastatin receptors from rat liver membranes, and these results support the conclusion that the liver pancreastatin receptor consists of a 80,000 Mr glycoprotein associated with G proteins.

• Davison JS, Pearce CM
• Actions of intrahypothalamic NPY on gastric function.
• Proc West Pharmacol Soc. 1997; 40: 135-6

• Lutz CM et al.
• Neuropeptide Y receptor genes mapped in human and mouse: receptors with high affinity for pancreatic polypeptide are not clustered with receptors specific for neuropeptide Y and peptide YY.
• Genomics. 1997; 46: 287-90
• Display abstract

• Ppyr1, Npy5r, and Npy6r, the genes encoding mouse type 4, type 5, and type 6 members of the neuropeptide Y receptor family, have been mapped by interspecific backcross analysis to conserved linkage groups on mouse Chr 14, Chr 8, and Chr 18, respectively. The human genes, PPYR1 and NPY5R, have been localized to chromosomes 10q and 4q, respectively, by analysis of a panel of rodent-human somatic cell hybrids and yeast artificial chromosomes. These studies complete the mapping of the cloned NPY receptor subtypes in human and mouse and, together with previous studies, establish that the genes encoding receptors with high affinity for pancreatic polypeptide are not clustered with the genes encoding receptors specific for neuropeptide Y and peptide YY. The physical association of these receptor genes correlates with ligand-binding properties, rather than sequence identity, and suggests a complex evolutionary relationship.

• Larhammar D
• Evolution of neuropeptide Y, peptide YY and pancreatic polypeptide.
• Regul Pept. 1996; 62: 1-11
• Display abstract

• The neuropeptide Y family of peptides consists of neuropeptide Y (NPY), which is expressed in the central and peripheral nervous systems, and peptide YY (PYY) and pancreatic polypeptide (PP) which are gut endocrine peptides. All three peptides are 36 amino acids long and act on G-protein-coupled receptors. NPY and PYY are present in all vertebrates, whereas PP probably arose as a copy of PYY in an early tetrapod ancestor. NPY is one of the most conserved peptides during evolution and no gnathostome (jawed) species differs from the ancestral gnathostome sequence at more than five positions. PYY is more variable, particularly in mammals which have nine differences to the gnathostome ancestor. PP may be the most rapidly evolving neuroendocrine peptide among tetrapods with only 50% identity between mammals, birds, and amphibians. Ancestral gnathostome NPY and PYY seem to have differed at only four positions, suggesting that the gene duplication occurred shortly before the appearance of the gnathostomes. The two peptides differ from one another at 9-12 positions in tetrapod species and share at least two receptor subtypes in mammals. In bony and cartilaginous fishes, NPY and PYY have only 5-6 differences which, together with more extensive neuronal localization of PYY, indicate an even greater functional overlap between the two peptides in these animal groups. The emergence of sequence information for several receptor subtypes from various species will shed additional light on the evolution of the functions of the NPY-family peptides.

• Larhammar D
• Structural diversity of receptors for neuropeptide Y, peptide YY and pancreatic polypeptide.
• Regul Pept. 1996; 65: 165-74
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• The NPY (neuropeptide Y) family of neuroendocrine peptides consists of NPY, PYY (peptide YY) and PP (pancreatic polypeptide). Several receptors have been characterized pharmacologically of which three have now been cloned. All three belong to the superfamily of receptors that couple to G proteins and all three cause inhibition of cAMP accumulation. Receptor subtypes Y1 and Y2 bind both NPY and PYY. Surprisingly, Y1 and Y2 share only 31% overall sequence identity, the lowest percentage reported for receptors that bind the same peptide ligand. Nevertheless, each subtype is 94% identical between human and rat, suggesting a slow rate of change. These observations suggest that Y1 and Y2 started to diverge from one another very long ago, possibly before the origin of vertebrates. The PP receptor, called PP1 or Y4, is 42% identical to the Y1 receptor (57% in the transmembrane regions) and is one of the most rapidly evolving receptors with only 75% overall identity between man and rat. Interestingly, this receptor's preferred ligand, PP, also evolves extremely rapidly. The PP receptor also differs between man and rat in tissue distribution and binding properties. The Y1 and PP receptors bind to both termini of their ligands whereas Y2 mainly interacts with the C-terminal part. Thus, within the same family there are highly conserved receptors and peptide ligands as well as one rapidly evolving receptor and ligand.

• Satoh M, Itoh Z
• [Motilin and motilin receptor]
• Nippon Rinsho. 1996; 54: 1092-6
• Display abstract

• Motilin, a 22 amino acid polypeptide, is known to play an important role in the initiation of phase III activity of the interdigestive migrating contractions (IMC) in the dog and man. The precursor of human motilin consists of 115 amino acids including a 25 amino acid signal peptide in direct linkage with the 22 amino acid motilin sequence and a 66 amino acid carboxy-terminal motilin-associated peptide (MAP). Northern blot analysis revealed that motilin mRNA is abundant in the duodenum. In vitro studies suggested that motilin acts directly on motilin receptors located on gastrointestinal smooth muscle cells in the rabbit, cat and man. On the contrary, in vivo studies suggested that motilin receptors are likely to be present in the nervous system. The cloning of motilin receptors is needed to clarify the detailed mechanism(s) of motilin's action.

• Gehlert DR et al.
• Characterization of the peptide binding requirements for the cloned human pancreatic polypeptide-preferring receptor.
• Mol Pharmacol. 1996; 50: 112-8
• Display abstract

• Traditionally, neuropeptide Y (NPY) receptors have been divided into Y1 and Y2 subtypes based on peptide pharmacology and synaptic localization. Other receptor subtypes have been proposed based on preferences for NPY, peptide YY (PYY), or pancreatic polypeptide (PP). Recently, we discovered a novel human member of this receptor family exhibiting high affinity for PP and PYY. In the current study, we expressed a DNA clone encoding this human PP-preferring receptor [hPP1 (or Y4)] in Chinese hamster ovary cells and performed a peptide structure-activity study. [125I]pPYY bound to homogenates of hPP1-Chinese hamster ovary cells with a Kd of 0.064 +/- 0.006 nM and a Bmax of 244 +/- 12 fmol/mg protein. Human PP inhibited binding with a Ki of 0.023 nM, whereas human PYY (Ki = 0.31 nM) and human NPY Ki = 12 nM) were significantly less potent. Rat, porcine, and bovine PP inhibited binding with similar affinities to human PP, whereas avian PP was substantially less potent (Ki = 1 nM). Deletion of the first four amino acids reduced the affinity of bovine PP to 1 nM. Carboxyl-terminal fragments of NPY and PYY also had reduced potency compared with the native peptides. In addition, deletion of Tyr36-amide produced a substantial reduction in affinity. Pro34-substituted NPY and PYY had modestly increased affinity compared with the native peptides, although Gln34-bPP had similar affinity compared with bovine PP. The carboxyl-terminally derived Y1 antagonist 1229U91 was a very potent (Ki = 0.042 nM) inhibitor of binding to hPP1. Thus, the carboxyl-terminal region of PP seems to be the most important part of the peptide for high affinity binding to hPP1. A few key residues (amino acids 2 and 3) in the amino-terminal region of PP contribute to the high affinity of the native peptide. Thus, features required for peptide recognition by the hPP1 receptor seem to be distinct from the Y1 and Y2 receptor.

• Matsumoto M et al.
• Inactivation of a novel neuropeptide Y/peptide YY receptor gene in primate species.
• J Biol Chem. 1996; 271: 27217-20
• Display abstract

• Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to a family of structurally related peptides which have numerous functions in both neural and endocrine signaling. By homology screening, we cloned a novel gene sharing the highest homology with the NPY Y1 receptor gene from humans, rabbits, and several other species. This novel gene of rabbit encodes a functional NPY/PYY receptor, designated Y2b, which prefers NPY13-36 rather than [Leu31,Pro34]NPY despite its higher identity with the Y1 receptor. Although, at low levels, mRNA was detected in the tissues and brain regions, including hypothalamus. Further, sequence data revealed that this gene is the orthologue of the recently cloned mouse novel NPY receptor, Y5. However, our study demonstrates that the receptor function of this gene has been inactivated in primates by a frameshift mutation occurring early in primate evolution. This novel NPY receptor represents the first neurotransmitter receptor identified that has universally lost its receptor function in primate species. Interestingly, despite its inactivation in humans, the transcripts were abundantly detected in the heart and skeletal muscle, suggesting a novel function of the human gene.

• Gregor P, Millham ML, Feng Y, DeCarr LB, McCaleb ML, Cornfield LJ
• Cloning and characterization of a novel receptor to pancreatic polypeptide, a member of the neuropeptide Y receptor family.
• FEBS Lett. 1996; 381: 58-62
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• We report isolation of a murine gene, NPYR-D, which predicts an intronless novel G protein-coupled receptor of 375 amino acids. Percent identities of NPYR-D to the clone Y1, Y2, rat Y4/PP1 and human Y4/PP1 receptors are 45, 32, 92 and 76, respectively. Southern blots indicate that NPYR-D and human Y4/PP1 receptor genes are species homologues. Rat [125I]pancreatic polypeptide ([125I]rPP) bound to NPYR-D transfected COS-7 cell membranes with a high affinity, i.e. IC50=90 pM. Pharmacological characterization of [125I]rPP binding showed a rank order of potency of P >> PYY > or = NPY, such that PYY and NPY were at least 5000-fold weaker than PP. Interestingly, [125I]rPYY binding produced the same rank order, but PYY and NPY were only 25-fold weaker than PP, which had an IC50 value of approximately 120 pM. Tissue distribution studies in mouse and humans suggest potential roles of this novel receptor in the gastrointestinal tract, heart, prostate, as well as in neural and endocrine signalling.

• Gehlert DR et al.
• The neuropeptide Y Y1 receptor selective radioligand, [125I][Leu31,Pro34]peptide YY, is also a high affinity radioligand for human pancreatic polypeptide 1 receptors.
• Eur J Pharmacol. 1996; 318: 485-90
• Display abstract

• A number of receptors for the pancreatic polypeptide-fold peptides are proposed based on findings from pharmacology and molecular biology studies. Neuropeptide Y and peptide YY have similar affinity for neuropeptide Y Y1 and neuropeptide Y Y2 while pancreatic polypeptide has highest affinity for pancreatic polypeptide 1. Pro34-substituted analogs of neuropeptide Y and peptide YY have selectivity for neuropeptide Y Y1 over neuropeptide Y Y2 receptors. In the present study, we found that one such 'neuropeptide Y Y1-selective' radioligand, [125I][Leu31,Pro34]peptide YY, also binds with high affinity to the pancreatic polypeptide 1 receptor. Therefore, caution needs to be exercised when using Pro34-analogs to define the neuropeptide Y Y1 receptor in vivo and using tissue preparations.

• Uesaka T, Yano K, Sugimoto S, Ando M
• Effects of eel neuropeptide Y on ion transport across the seawater eel intestine.
• Zoolog Sci. 1996; 13: 341-6
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• A neuropeptide Y (eNPY) was isolated from the intestinal extract of eels. This peptide enhanced significantly the serosa-negative transepithelial potential difference (PD) and short-circuit current (Isc) across the intestine of the seawater eel after pretreatment with isobutylmethylxanthine, serotonin and methacholine. The effects of eNPY on the Isc were concentration-dependent with a threshold concentration of 3 x 10(-9) M and a maximal effect at 3 x 10(-7) M. Similar concentration-response curve was obtained by porcine peptide YY (pPYY). Since 9 amino acid residues are replaced in the pPYY, this result indicates that these substitutions do not change the potency and the efficacy. These stimulatory actions of eNPY were not blocked by tetrodotoxin, an inhibitor of neural firing, or yohimbine, an alpha 2-adrenoceptor antagonist, indicating that eNPY acts without enteric neural firing or catecholamine release. When eNPY and adrenaline (AD) were applied simultaneously, the effects were additive only at lower dosage (3 x 10(-8) M for eNPY, 3 x 10(-8) M for AD), but not at high dosage (10(-6) M eNPY, 10(-7) M AD). The ceiling effect at high dosage suggests that these two regulators act through common signal transduction systems and affect the Na(+)-K(+)-Cl- cotransport system, since both effects were completely blocked by bumetanide, a specific inhibitor of Na(+)-K(+)-Cl- cotransporter.

• Nakamura M, Aoki Y, Hirano D
• Cloning and functional expression of a cDNA encoding a mouse type 2 neuropeptide Y receptor.
• Biochim Biophys Acta. 1996; 1284: 134-7
• Display abstract

• A cDNA clone homologous with the human neuropeptide Y (NPY)-Y2 receptor has been isolated from a mouse brain cDNA library. Analysis of the predicted amino-acid sequence indicates that the polypeptide encoded by this cDNA is 94% homologous to the human NPY-Y2 receptor. In Chinese hamster ovary (CHO) cells expressing the mouse NPY-Y2 receptor, an increase in intracellular Ca2+ and inhibition of forskolin-induced cAMP accumulation were observed due to stimulation with NPY, NPY-(13-36) and peptide YY, but not with pancreatic polypeptide or [Leu31, Pro34]NPY. The fact that the NPY-induced increase in intracellular Ca2+ and inhibition of forskolin-induced cAMP accumulation were eliminated by pretreatment with pertussis toxin suggests that the NPY-Y2 receptor couples to PTX-sensitive G-protein(s), probably Gi/Go, in CHO cells.

• Lundell I et al.
• The cloned rat pancreatic polypeptide receptor exhibits profound differences to the orthologous receptor.
• Proc Natl Acad Sci U S A. 1996; 93: 5111-5
• Display abstract

• Pancreatic polypeptide (PP) is produced in the islets of Langerhans and released in response to meals. It belongs to a family of peptides that also includes neuropeptide Y and peptide YY. In the present communication, we describe a rat receptor with high affinity for PP, therefore named PP1. Clones for the PP1 receptor were obtained by PCR using sequence information for the neuropeptide Y receptor Y1 from several species. The PP1 receptor has 46% overall amino acid sequence identity to the rat Y1 receptor and 56% identity in the transmembrane regions. The PP1 receptor displays a pharmacological profile that is distinct from previously described neuropeptide Y-family receptors. In competition with iodinated bovine PP, it binds rat PP with an affinity (K(i)) of 0.017 nM, while the affinities for peptide YY and neuropeptide Y are substantially lower with K(i) values of 162 and 192 nM, respectively. In stably transfected CHO cells, the PP1 receptor inhibits forskolin-stimulated cAMP synthesis. Northern blot hybridizations to a panel of mRNAs detected transcripts in testis and lung. A faint band was seen in colon and total brain. In contrast, the human receptor is expressed primarily in colon and small intestine. Whereas rat and human PP1 bind PP with the same affinity, the rat receptor has much lower affinity than its human ortholog for peptide YY and neuropeptide Y. Interestingly, the amino acid sequence identity between rat and human PP1 is only 75%. Thus, the sequence, the tissue distribution, and the binding profile of the PP1 receptor differ considerably between rat and human.

• Trinh T, van Dumont Y, Quirion R
• High levels of specific neuropeptide Y/pancreatic polypeptide receptors in the rat hypothalamus and brainstem.
• Eur J Pharmacol. 1996; 318: 13-13
• Display abstract

• It is known that the paraventricular hypothalamic nucleus is responsible for some of the stimulatory effects of neuropeptide Y, peptide YY and the pancreatic polypeptides on food intake. However, specific neuropeptide Y Y1 and Y2 receptors were not abundantly expressed in the hypothalamus. In contrast, specific [125I]human pancreatic polypeptide binding sites were detected in this hypothalamic nucleus as well as the medial preoptic area, interpeduncular nucleus, nucleus tractus solitarius, area postrema and dorsal vagal nucleus while cortical areas and the hippocampus contained negligible levels of labeling. The ligand binding profile of the various competitors suggests that the binding sites labeled by [125I]human pancreatic polypeptide are predominantly of the neuropeptide Y Y4 and/or Y5 subtypes. These newly cloned receptors may play a key role in the modulatory effects of neuropeptide Y and related peptides on appetite.

• Yan H et al.
• Cloning and functional expression of cDNAs encoding human and rat pancreatic polypeptide receptors.
• Proc Natl Acad Sci U S A. 1996; 93: 4661-5
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• PCR was used to isolate nucleotide sequences that may encode novel members of the neuropeptide Y receptor family. By use of a PCR product as a hybridization probe, a full-length human cDNA was isolated that encodes a 375-aa protein with a predicted membrane topology identifying it as a member of the G-protein-coupled receptor superfamily. After stable transfection of the cDNA into human embryonic kidney 293 cells, the receptor exhibited high affinity (Kd = 2.8 nM) for 125I-labeled human pancreatic polypeptide (PP). Competition binding studies in whole cells indicated the following rank order of potency: human PP = bovine PP > or = human [Pro34]peptide YY > rat PP > human peptide YY = human neuropeptide Y. Northern blot analysis revealed that human PP receptor mRNA is most abundantly expressed in skeletal muscle and, to a lesser extent, in lung and brain tissue. A rat cDNA clone encoding a high-affinity PP receptor that is 74% identical to the human PP receptor at the amino acid level was also isolated. These receptor clones will be useful in elucidating the functional role of PP and designing selective PP receptor agonists and antagonists.

• Mao YK, Wang YF, Ward G, Cipris S, Daniel EE, McDonald TJ
• Peptide YY receptor in submucosal and myenteric plexus synaptosomes of canine small intestine.
• Am J Physiol. 1996; 271: 3641-3641
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• PYY receptors were characterized and their loci determined in canine small intestine. The density of 125I-labeled peptide tyrosine tyrosine (PYY) binding was highest in myenteric (MY) and submucosal (SUB) plexus fractions enriched in synaptosomes. Two binding sites [high affinity (H) and low affinity (L)] were found in the submucosal synaptosome-enriched membrane: dissociation constant (Kd)H = 7.6 pM, maximal binding capacity (Bmax)H = 28 fmol/mg; KdL = 0.18 nM, BmaxL = 120 fmol/mg protein. The binding of 125I-PYY reached a maximum within 30 min; dissociation was incomplete in the presence of unlabeled PYY. The rate of dissociation was enhanced after exposure of synaptosomes to guanosine 5'-O-(3-thiotriphosphate). Binding of 125I-PYY was completely inhibited by neuropeptide Y (NPY)-(13-36) (in SUB and MY) and by [Leu31,Pro34]NPY (in MY) but only partially by [Leu31,Pro34]NPY in SUB, suggesting the presence of Y2 receptor in SUB and the presence of Y1 and Y2 receptors in MY. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PYY receptor complex revealed a radioactive band at 70 kDa. The PYY receptors in the canine small intestinal myenteric and submucosal plexus correspond in location to that of PYY in synaptosomes and are coupled with G proteins. Different subtypes are present in different loci.

• Playford RJ, Cox HM
• Peptide YY and neuropeptide Y: two peptides intimately involved in electrolyte homeostasis.
• Trends Pharmacol Sci. 1996; 17: 436-8

• Gregor P, Feng Y, DeCarr LB, Cornfield LJ, McCaleb ML
• Molecular characterization of a second mouse pancreatic polypeptide receptor and its inactivated human homologue.
• J Biol Chem. 1996; 271: 27776-81
• Display abstract

• The family of mammalian neuropeptide Y (NPY)/peptide YY (PYY)/pancreatic polypeptide (PP) receptors comprises several G protein-coupled receptors, i.e. Y1, Y2, and Y4/PP1. We now report cloning of a novel member of this family named PP2. The coding region of the mouse PP2 gene reveals no introns and predicts a seven transmembrane domain (TM) receptor of 371 amino acids. Percent identities of the mouse PP2 to mouse Y1, mouse Y4/PP1 and human Y2 receptors are 53, 42, and 31, respectively. The mouse PP2 receptor expressed in COS cells binds rat 125I-PP with high affinity, i.e. IC50 = 65 pM. Pharmacological characterization of 125I-PP binding shows a rank order of potency of PP >> PYY >/= NPY, which is similar to that of the mouse Y4/PP1 receptor. Mouse PP2 transcripts were not detectable by Northern analysis in adult tissues and in 11-, 15-, and 17-day-old embryos. However, a 9.8-kb PP2 transcript was detectable in 7-day-old mouse embryo, i.e. prior to the organogenesis of pancreas and the onset of PP production. We have also cloned the human homologue of PP2, which is a single copy gene and maps to human chromosome 5q31. Surprisingly, the human PP2 cDNAs and gene sequences display a single base deletion in the coding region. This frameshifting mutation predicts a truncated receptor of 290 amino acids without TM7. Transfection of COS-7 cells with several different human PP2 expression constructs failed to confirm any specific binding of 125I-PP, 125I-PYY, or 125I-NPY to cell membranes. These data suggest that in mouse there are at least two PP receptors, Y4/PP1 and PP2, whereas in humans, PP2 is either functionally inactive or it has acquired a PP-independent function.

• Herzog H, Hort Y, Schneider R, Shine J
• Seminalplasmin: recent evolution of another member of the neuropeptide Y gene family.
• Proc Natl Acad Sci U S A. 1995; 92: 594-8
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• Seminalplasmin, the major basic protein of bull semen, an important regulator of calcium transport in bovine sperm and a positive modulator of the zona pellucida-induced acrosome reaction, is shown to be a recently created member of the neuropeptide Y gene family. Sequence analysis of the bovine peptide YY-pancreatic polypeptide gene cluster reveals an unexpected and extensive homology between seminalplasmin and the neuropeptide Y gene family, at the level of both gene structure and primary amino acid and nucleotide sequences. The extremely high degree of homology to the peptide YY gene, in both coding and especially noncoding regions, suggests that the seminalplasmin gene has arisen by a very recent gene duplication of the bovine peptide YY gene. Despite the more than 95% nucleotide sequence identity, a few specific mutations in the seminalplasmin gene have resulted in both the loss of the amino- and carboxyl-terminal cleavage sites characteristic of all other members of the neuropeptide Y family and the acquisition of a function apparently unrelated to the neurotransmitter/endocrine role of peptide YY.

• Hort Y, Baker E, Sutherland GR, Shine J, Herzog H
• Gene duplication of the human peptide YY gene (PYY) generated the pancreatic polypeptide gene (PPY) on chromosome 17q21.1.
• Genomics. 1995; 26: 77-83
• Display abstract

• Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) are structurally related but functionally diverse peptides, encoded by separate genes and expressed in different tissues. Although the human NPY gene has been mapped to chromosome 7, we demonstrate here that the genes for human PYY and PP (PPY) are localized only 10 kb apart from each other on chromosome 17q21.1. The high degree of homology between the members of this gene family, both in primary sequence and exon/intron structure, suggests that the NPY and the PYY genes arose from an initial gene duplication event, with a subsequent tandem duplication of the PYY gene being responsible for duplication of the PYY gene being responsible for the creation of the PPY gene. A second weaker hybridization signal also found on chromosome 17q11 and results obtained by Southern blot analysis suggest that the entire PYY-PPY region has undergone a further duplication event.

• Blomqvist AG, Roubos EW, Larhammar D, Martens GJ
• Cloning and sequence analysis of a neuropeptide Y/peptide YY receptor Y1 cDNA from Xenopus laevis.
• Biochim Biophys Acta. 1995; 1261: 439-41
• Display abstract

• Neuropeptide Y (NPY) and peptide YY (PYY) are structurally related peptides that share at least two distinct receptors denoted Y1 and Y2. The Y1 receptor has previously been cloned in man, rat and mouse. We describe here the cloning and sequence of a Xenopus laevis Y1 receptor that shares 81% amino acid sequence identity with the human receptor in the region spanning transmembrane (TM) regions I to VII. The extracellular amino-terminal part, TM IV and the second extracellular loop contain several replacements suggesting that these portions have no or limited direct interactions with the peptide ligands. The intracellular regions including the carboxy-terminal tail are nearly identical between Xenopus and mammals, suggesting strong structural constraints on the portions that may interact with G proteins.

• Freitag C, Svendsen AB, Feldthus N, Lossl K, Sheikh SP
• Coupling of the human Y2 receptor for neuropeptide Y and peptide YY to guanine nucleotide inhibitory proteins in permeabilized SMS-KAN cells.
• J Neurochem. 1995; 64: 643-50
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• Using guanine nucleotides, pertussis toxin, and specific antisera against the COOH-terminals of the alpha-subunits of Gi1/2, Gi3, and G(o), the binding and biological response of the Y2 receptor (Y2R) for peptide YY (PYY) was probed in SMS-KAN neuroblastoma cells. The specific binding of radiolabeled PYY exhibited a single apparent dissociation constant, KD = 76 pM for intact cells and KD = 906 pM for permeabilized cells. However, other data suggested existence of multiple receptor affinity states. A shift in KD and a decrease in apparent number of binding sites (Bmax) was observed in permeabilized cells when incubated with guanine nucleotides. By contrast, in membrane preparations guanine nucleotides induced only a decrease in Bmax. In intact cells, agonist exposure inhibited the intracellular accumulation of forskolin-stimulated cyclic AMP by 80% (IC50 = 420 nM) compared with 94% inhibition (IC50 = 380 nM) in permeabilized cells. In permeabilized cells, preincubation with antisera against alpha i1/2 and alpha i3 blocked the functional response of PYY, with anti-alpha i3 being the most potent; whereas anti-alpha o failed to affect the cyclic AMP levels. These results suggest that permeabilized SMS-KAN cells serve as a good model system for analysis of Y2R binding kinetics and functional response and that the Y2R interacts directly with several different GiS (but not G(o)).

• Gerald C, Walker MW, Vaysse PJ, He C, Branchek TA, Weinshank RL
• Expression cloning and pharmacological characterization of a human hippocampal neuropeptide Y/peptide YY Y2 receptor subtype.
• J Biol Chem. 1995; 270: 26758-61
• Display abstract

• The pancreatic polypeptide family includes neuropeptide Y (NPY), one of the most abundant neuropeptides in the mammalian nervous system, as well as peptide YY (PYY) and pancreatic polypeptide (PP). This peptide family is involved in numerous physiological processes such as memory, pain, blood pressure, appetite, anxiety, and circadian rhythm. Of the multiple Y-type receptors proposed for PP family members, only the Y1 subtype was cloned previously. We now report the isolation of a human Y2 (hhY2) receptor cDNA by expression cloning from a human hippocampal cDNA library, using a 125I-PYY binding assay. hhY2 cDNA encodes a predicted protein of 381 amino acids with low amino acid identity to the human Y1 receptor (31% overall; 41% transmembrane). 125I-PYY binding to transiently expressed hY2 receptors was saturable (pKd = 10.17) and displaceable by human PP family members in rank order: PYY (pKi = 9.47) approximately NPY (pKi = 9.27) >> PP (pKi < 6) and by peptide analogs: NPY2-36 (pKi = 8.80) approximately NPY13-36 (pKi = 8.55) approximately C2-NPY (pKi = 8.54) > NPY26-36 (pKi = 6.51) approximately [Leu31,Pro34]NPY (pKi = 6.23). Human PYY decreased [cAMP] and increased intracellular [Ca2+] in hY2-transfected 293 cells.

• Bard JA, Walker MW, Branchek TA, Weinshank RL
• Cloning and functional expression of a human Y4 subtype receptor for pancreatic polypeptide, neuropeptide Y, and peptide YY.
• J Biol Chem. 1995; 270: 26762-5
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• The pancreatic polypeptide family includes pancreatic polypeptide (PP), neuropeptide Y (NPY), and peptide YY (PYY). Members of the PP family regulate numerous physiological processes, including appetite, gastrointestinal transit, anxiety, and blood pressure. Of the multiple Y-type receptors proposed for PP family members, only the Y1 subtype has been cloned previously. We now report the cloning of an additional Y-type receptor, designated Y4, by homology screening of a human placental genomic library with transmembrane (TM) probes derived from the rat Y1 gene. The Y4 genomic clone encodes a predicted protein of 375 amino acids that is most homologous to Y1 receptors from human, rat, and mouse (42% overall; 55% in TM). 125I-PYY binding to transiently expressed Y4 receptors was saturable (pKd = 9.89) and displaceable by human PP family derivatives: PP (pKi = 10.25) approximately PP2-36 (pKi = 10.06) > PYY (pKi = 9.06) approximately [Leu31,Pro34]NPY (pKi = 8.95) > NPY (pKi = 8.68) > PP13-36 (pKi = 7.13) > PP31-36 (pKi = 6.46) > PP31-36 free acid (pKi < 5). Human PP decreased [cAMP] and increased intracellular [Ca2+] in Y4-transfected LMTK- cells. Y4 mRNA was detected by reverse transcriptase-polymerase chain reaction in human brain, coronary artery, and ileum, suggesting potential roles for Y4 receptors in central nervous system, cardiovascular, and gastrointestinal function.

• Nguyen TD, Wolfe MS, Heintz GG
• Solubilization of receptors for pancreatic polypeptide from rat liver membranes.
• Am J Physiol. 1995; 268: 21523-21523
• Display abstract

• We have previously identified, on rat liver microsomes and plasma membranes, proteins that bind pancreatic polypeptide (PP) with high affinity and specificity and that may serve as receptors for a hepatic effect of PP (J. Biol. Chem. 267: 9416-9421, 1992). Further characterization of these proteins requires the solubilization of receptors with conserved ability to bind PP selectively and efficiently. In this report, using 6 mM of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), we solubilized, from liver microsomes, receptors that bound PP with high affinity (dissociation constant 6.15 +/- 1.6 nM) and specificity (no interaction with the homologous peptides neuropeptide Y and peptide YY). Gel filtration chromatography showed different degrees of receptor aggregation related to different concentrations of CHAPS in the eluent. To characterize the structure of these solubilized receptors, the chemical cross-linker N-(5-azido-2-nitrobenzoyloxy)succinimide was used to covalently bind these receptors to radiolabeled PP, and the resulting PP-receptor complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A radioactive band with an apparent molecular weight (M(r)) of 46,000 was detected that was inhibited by unlabeled PP with a half-maximal inhibitory concentration of approximately 10(-8) M. It most likely reflected a PP receptor with an estimated M(r) of 42,000, excluding the molecular weight of PP. The migration of this complex was not affected by the reducing agent dithiothreitol, suggesting the absence of disulfide bonding. The solubilization and identification of a bioactive hepatic PP receptor will allow further characterization and purification of this receptor and will lead to the clarification of the interaction between PP and the digestive system.

• Beck-Sickinger AG, Wieland HA, Brunner J
• Synthesis, receptor binding, and crosslinking of photoactive analogues of neuropeptide Y.
• J Recept Signal Transduct Res. 1995; 15: 473-85
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• Five photoactive analogues of porcine neuropeptide Y (NPY), a 36 amino acid hormone of the pancreatic polypeptide family, have been synthesized by solid phase peptide synthesis method, Fmoc/tBu strategy and carefully characterized. The analogues contain the photoactivatable amino acid 4'-(3-trifluoromethyl)-3H-diazirine-3-yl-phenyl-alanine ((Tmd)Phe) individually at different positions (1, 20, 21, 27 or 36) instead of tyrosine in the wildtype sequence. Affinity to membranes prepared from SMS-KAN cells, which stably express the Y2 receptor has been investigated by measuring the displacement of 125I-Bolton Hunter-NPY. After incubation of the membranes with different concentrations of the crosslinker and subsequent photolysis, the specific binding of 125I-Bolton Hunter-NPY at those membranes was tested in order to quantify the crosslinking efficiency. Whereas [(Tmd)Phe20] NPY, [(Tmd)Phe21] NPY and [(Tmd)Phe27] NPY revealed highest affinity to the Y2 receptor, crosslinking was most efficient when Tyr36 was replaced by (Tmd)Phe. This is in good agreement with the previously suggested C-terminal binding site of neuropeptide Y.

• Nakajima M et al.
• Effects of pancreatic polypeptide family peptides on feeding and learning behavior in mice.
• J Pharmacol Exp Ther. 1994; 268: 1010-4
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• We studied the effects of intra-third cerebroventricular administration of neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) on the locomotor activity and the feeding and learning behavior of mice. NPY (0.3-10 micrograms), PYY (0.1-10 micrograms) and PP (3.0-10 micrograms) produced significant increases in locomotor activity. A significant decrease was then observed 15 min after administration of 10 micrograms of PYY. NPY, PYY and PP significantly increased food intake at 20 min and this effect continued for 2 to 4 hr at the high doses. The feeding response to PP family peptides were quite similar to that in locomotor activity with respect to dose-response, time course and peptide specificity. Learning behaviors were evaluated at three different stages of memory processing, acquisition, consolidation and retrieval, in a battery of step-down type passive avoidance tests. NPY and PYY had no effect on acquisition, but significantly improved consolidation at a dose of 0.03 and 0.3 microgram, respectively. NPY also improved retrieval at a dose of 0.03 microgram. The ranking order of potency in stimulating feeding and locomotor activity was PYY > NPY > PP, and in improving memory consolidation NPY > PYY >> PP. These observations suggest that NPY and PYY influence different neural substrates in the brain involved in feeding and learning.

• Medeiros MS, Turner AJ
• Post-secretory processing of regulatory peptides: the pancreatic polypeptide family as a model example.
• Biochimie. 1994; 76: 283-7
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• Post-secretory metabolism is an important event in the overall homeostasis of regulatory peptides and the enzymes involved in these processes may be suitable targets for pharmacological intervention. Some examples are reviewed here. Peptide YY and neuropeptide Y, both members of the pancreatic polypeptide family, can be processed by dipeptidyl peptidase IV to their (3-36) fragments by removal of the N-terminal Tyr-Pro dipeptide, which generates a metabolite of different receptor selectivity. Aminopeptidase P and endopeptidase-24.11 also metabolize these peptides and the relative levels of these three cell-surface enzymes may regulate their interconversion between receptor-selective forms and inactive metabolites.

• Grandt D et al.
• [Pro34]peptide YY is a Y1-selective agonist at peptide YY/neuropeptide Y receptors.
• Eur J Pharmacol. 1994; 269: 127-32
• Display abstract

• We have investigated binding and functional effects of a new peptide YY analogue, [Pro34]peptide YY, at Y1 and Y2-like subtypes of receptors for peptide YY and neuropeptide Y. In binding studies [Pro34]peptide YY had a similarly high affinity as peptide YY to human Y1-like receptors in SK-N-MC cells, a human neuroblastoma cell line of presumed neurogenic origin, and HEL cells, a human cell line derived from a patient with Hodgkin's disease. In functional studies [Pro34]peptide YY stimulated Ca2+ elevations in both Y1-like receptor cell lines with similar potency and efficacy as peptide YY. In contrast to peptide YY [Pro34]peptide YY was 1000-fold less potent in binding to Y2-like receptors in porcine splenic membranes and lacked agonistic effects in another Y2-like receptor-mediated model system, i.e. inhibition of [3H]serotonin release from rat cerebral cortical slices. Thus, [Pro34]peptide YY is a highly Y1-selective full agonist of peptide YY/neuropeptide Y receptors. [Pro34]peptide YY could be useful for studying the importance of Y receptor subtypes in mediating peptide YY physiological actions.

• Mannon PJ, Mervin SJ, Sheriff-Carter KD
• Characterization of a Y1-preferring NPY/PYY receptor in HT-29 cells.
• Am J Physiol. 1994; 267: 9017-9017
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• Equilibrium binding studies showed that butyrate-treated HT-29 cells express a high-affinity 125I-labeled peptide YY (125I-PYY) binding site with a dissociation constant of 0.32 +/- 0.12 nM (mean +/- SE, n = 4). This site was Y1 preferring because neuropeptide Y (NPY) and the Y1-selective agonist [Leu31,Pro34]NPY were equipotent to PYY at displacing 125I-PYY; PYY-(13-36) and pancreatic polypeptide were > 1,000- and 10,000-fold less potent at displacing the radioligand. PYY and [Leu31,Pro34]NPY inhibited forskolin-stimulated adenosine 3',5'-cyclic monophosphate production 63% and 48%, respectively, with a half-maximal inhibitory concentration between 0.1 and 1.0 nM. PYY and [Leu31,Pro34]NPY had no effect on release of intracellular calcium alone or on the increase in intracellular calcium concentration caused by carbachol or neurotensin. Northern blot analysis of poly(A)+ RNA from HT-29 cells demonstrated a single transcript of 2.5 kb that hybridized to a human Y1-receptor cDNA probe. Sequence analysis of a reverse transcription-polymerase chain reaction product amplified with primers based on human Y1-receptor cDNA confirmed that these cells contained mRNA encoding the human Y1 receptor. These studies show that butyrate-treated HT-29 cells constitutively express the Y1-preferring NPY/PYY receptor and Y1 mRNA and provide a new model for studies of PYY-regulated epithelial cell function and tissue-specific expression of the human Y1-receptor gene.

• Miura M et al.
• Dynorphin binds to neuropeptide Y and peptide YY receptors in human neuroblastoma cell lines.
• Am J Physiol. 1994; 267: 7029-7029
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• The modulation of neuropeptide Y (NPY) and peptide YY (PYY) receptors by dynorphin, luteinizing hormone-releasing hormone (LHRH), corticotropin-releasing factor (CRF), and cholecystokinin octapeptide has been studied in human neuroblastoma cell lines SK-N-MC and SMS-MSN, which express Y1 and Y2 receptors for NPY/PYY. Dynorphin A and LHRH inhibited the binding of NPY/PYY to SK-N-MC cell membranes at concentrations ranging from 10(-7) to 10(-5) M, whereas dynorphin A and CRF were effective in SMS-MSN cells. The inhibitory effect of dynorphin A on NPY/PYY binding was observed in the presence of guanosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable GTP analogue, as well as H-7 and H-8, novel inhibitors of protein kinases C and A. However, U-50488, the most potent kappa-selective compound did not mimic the dynorphin action. Dynorphin A showed neither effect on the dissociation of NPY/PYY from their receptors nor inhibition on the basal as well as forskolin-stimulated adenosine 3',5'-cyclic monophosphate response. These results indicate that the interaction of dynorphin A with Y1 and Y2 receptors is not mediated by changes in receptor-G protein interaction, receptor phosphorylation, and allosteric binding to NPY/PYY receptors but that dynorphin A binds to NPY/PYY receptors at high concentrations, probably in an antagonistic manner.

• Huang SC, Tsai MF
• Receptors for peptide YY and neuropeptide Y on guinea pig pancreatic acini.
• Peptides. 1994; 15: 405-10
• Display abstract

• We examined the effects of peptide YY (PYY), neuropeptide Y (NPY), and their analogues on dispersed pancreatic acini. Binding of [125I]PYY to acini was saturable, reversible, and specific, and PYY binding was best fit with a two-site model. The relative potencies for inhibiting [125I]PYY binding were PYY > or = NPY > NPY(13-36). There was no inhibition of binding with [Leu31,Pro34]NPY, PYX-2, or pancreatic polypeptide. Both PYY and NPY (0.1 microM) inhibited amylase release stimulated by vasoactive intestinal polypeptide (VIP) (0.3 nM) and forskolin (1 microM) by about 30%, but not that stimulated by cholecystokinin-8 or bombesin. The relative potencies for inhibiting VIP-stimulated amylase release were PYY > or = NPY > NPY(13-36), the same as those for inhibiting VIP-stimulated cAMP increase in acini. No inhibition was detected with [Leu31,Pro34]NPY. This work demonstrates Y2 receptors on guinea pig pancreatic acini mediating inhibitory actions of PYY and NPY on pancreatic enzyme secretion.

• Gehlert DR
• Subtypes of receptors for neuropeptide Y: implications for the targeting of therapeutics.
• Life Sci. 1994; 55: 551-62
• Display abstract

• Neuropeptide Y is a 36 amino acid peptide that was originally discovered in extracts of porcine brain. The peptide has a broad distribution in the central or peripheral nervous system. Receptors for this peptide were originally subdivided into postsynaptic Y-1 receptors and presynaptic Y-2 receptors. The Y-1 receptor has recently been cloned and appears to mediate several effects of NPY including vasoconstriction and an anxiolytic effect in animal models of anxiety. The Y-2 receptor inhibits the release of neurotransmitters in the CNS by the inhibition of the mobilization of intracellular calcium. Additional receptors have been proposed including a Y-3 receptor that recognizes NPY but not the related endocrine peptide, PYY. The functional importance of these newer receptors remains to be established. The absence of useful antagonists has made the study of NPY a challenge for investigators in the field. The potential utility of such molecules is discussed.

• Mentlein R, Dahms P, Grandt D, Kruger R
• Proteolytic processing of neuropeptide Y and peptide YY by dipeptidyl peptidase IV.
• Regul Pept. 1993; 49: 133-44
• Display abstract

• Neuropeptide Y, peptide YY and pancreatic polypeptide share an evolutionary conserved proline-rich N-terminal sequence, a structure generally known to be inert to the attack of common proteinases, but a potential target for specialized proline-specific aminopeptidases. Purified human dipeptidyl peptidase IV (also termed CD 26) liberated N-terminal Tyr-Pro from both, neuropeptide Y and peptide YY, with very high specific activities and Km values in the micromolar range, but almost no Ala-Pro from pancreatic polypeptide. Other proline-specific aminopeptidases exhibited low (aminopeptidase P, liberation of N-terminal Tyr) or totally no activity (dipeptidyl peptidase II), as was also observed with less-specific aminopeptidases (aminopeptidase M, leucine aminopeptidase). When human serum was incubated with neuropeptide Y or peptide YY at micro- and nanomolar concentrations, Tyr-Pro was detected as a metabolite of both peptides. Formation of Tyr-Pro in serum was blocked in the presence of Lys-pyrrolidine and diprotin A (Ile-Pro-Ile), specific, competitive inhibitors of dipeptidyl peptidase IV. Incubation of neuropeptide Y or peptide YY with immunocytochemically defined, cultivated endothelial cells from human umbilical cord also yielded Tyr-Pro. Dipeptidyl peptidase IV could be immunostained on most endothelial cells by a specific antibody. We suggest that dipeptidyl peptidase IV might be involved in the degradation of neuropeptide Y and peptide YY to N-terminal truncated neuropeptide Y(3-36) and peptide YY(3-36). Since specific binding to Y1, but not to Y2 subtype of neuropeptide Y/peptide YY receptors requires intact N- as well as C-termini of neuropeptide Y and peptide YY, removal of their amino-terminal dipeptides by dipeptidyl peptidase IV inactivates them for binding to one receptor subtype.

• Bjornholm B, Jorgensen FS, Schwartz TW
• Conservation of a helix-stabilizing dipole moment in the PP-fold family of regulatory peptides.
• Biochemistry. 1993; 32: 2954-9
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• Investigation of the charge distribution for all known members of the PP-fold family of peptides reveals a common pattern characterized by a cluster of negative charges in the beta-turn region and a cluster of positive charges in the receptor-binding region of the peptide. Detailed analysis of the electrostatic properties of five representative members of the PP-fold family of peptides (human neuropeptide Y, human peptide YY, human pancreatic polypeptide, avian PP, and lamprey peptide methionine tyrosine) shows that this characteristic charge clustering gives rise to a common dipole moment of 325-450 D directed from the beta-turn region toward the receptor-binding region. This overall dipole moment is antiparallel to the dipole moment of the alpha-helix caused by alignment of the peptide dipoles parallel to the helix. Calculations of the stabilization energy for this antiparallel dipole moment arrangement were performed in two ways: (1) by the use of a Poisson-Boltzmann approach which allows for an estimate of the screening effect, and (2) by the use of a uniform dielectric model (Coulomb's law). It is found that the alpha-helix is stabilized by approximately 5-10 kcal/mol due to electrostatic forces alone when the screening effect is considered. This energy is of the same order of magnitude as the enthalpy change for the unfolding of avian PP (approximately 30 kcal/mol), strongly indicating that the charge-dipole interactions are of significant importance for the stability of the three-dimensional structure of the PP-fold peptides.

• Arvidsson K, Land T, Langel U, Bartfai T, Ehrenberg A
• Solution structure by 2D 1H-NMR of a chimeric peptide recognized by galanin and neuropeptide Y receptors.
• Biochemistry. 1993; 32: 7787-98
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• The 25 amino acid residue chimeric peptide M32, galanin(1-13)-neuropeptide Y(25-36)-amide, was synthesized. The peptide was found to be recognized by both galanin and NPY receptors. The solution structure in 30% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol was examined by 2-D 1H-NMR and by CD. Proton resonance assignments were made, and structures were calculated using DIANA and refined by restrained energy minimization and molecular dynamics. The obtained structures contain an alpha-helical part in the NPY portion of the peptide including residues 13-20, and in some structures it continues to the C-terminal Tyr25. The more flexible N-terminal portion of the peptide has the freedom to approach the C-terminal alpha-helix, via a reverse turn or a nascent alpha-helix, which permits the N-terminus with Trp2 to come into close contact with the C-terminus with Tyr25. Among the ten NMR structures with lowest energy, there are structures reminiscent of the horseshoe shape of aPP, a close relative of NPY with known crystal structure. It appears that the strong alpha-helical character of the NPY (25-36) amide fragment of M32 helps to stabilize structural features in the galanin-derived part of the peptide. It is noteworthy that this rigid NPY portion of M32 does not prevent the recognition of the peptide by galanin receptors; rather, the peptide has unusually high affinity: IC50 = 0.1 nM at galanin receptors. The chimeric peptide M32 is also recognized by NPY receptors with submicromolar affinity (IC50 = 0.25 microM). The availability of a solution structure for peptide M32, which is recognized by two peptide receptors that are both members of the family of G-protein-coupled receptors, may be useful in understanding peptide receptor-ligand interactions and in designing new galanin and NPY receptor ligands.

• Hazelwood RL
• The pancreatic polypeptide (PP-fold) family: gastrointestinal, vascular, and feeding behavioral implications.
• Proc Soc Exp Biol Med. 1993; 202: 44-63

• Marks NJ et al.
• Isolation and primary structure of a novel avian pancreatic polypeptide from five species of Eurasian crow.
• Regul Pept. 1993; 47: 187-94
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• Chicken pancreatic polypeptide is the prototype of the neuropeptide Y (NPY)/PP superfamily of regulatory peptides. This polypeptide was appended the descriptive term avian, despite the presence of some 8600 extant species of bird. Additional primary structures from other avian species, including turkey, goose and ostrich, would suggest that the primary structure of this polypeptide has been highly-conserved during avian evolution. Avian pancreatic polypeptides structurally-characterised to date have distinctive primary structural features unique to this vertebrate group including an N-terminal glycyl residue and a histidyl residue at position 34. The crow family, Corvidae, is representative of the order Passeriformes, generally regarded as the most evolutionarily recent and diverse avian taxon. Pancreatic polypeptide has been isolated from pancreatic tissues from five representative Eurasian species (the magpie, Pica pica; the jay, Garrulus glandarius; the hooded crow, Corvus corone; the rook, Corvus frugilegus; the jackdaw, Corvus monedula) and subjected to structural analyses. Mass spectroscopy estimated the molecular mass of each peptide as 4166 +/- 2 Da. The entire primary structures of 36 amino acid residue peptides were established in single gas-phase sequencing runs. The primary structures of pancreatic polypeptides from all species investigated were identical: APAQPAYPGDDAPVEDLLRFYNDLQQYLNVVTRPRY. The peptides were deemed to be amidated due to their full molar cross-reactivity with the amide-requiring PP antiserum employed. The molecular mass (4165.6 Da), calculated from the sequences, was in close agreement with mass spectroscopy estimates. The presence of an N-terminal alanyl residue and a prolyl residue at position 34 differentiates crow PP from counterparts in other avian species.(ABSTRACT TRUNCATED AT 250 WORDS)

• Balasubramaniam A, Cox HM, Voisin T, Laburthe M, Stein M, Fischer JE
• Structure-activity studies of peptide YY(22-36): N-alpha-Ac-[Phe27]PYY(22-36), a potent antisecretory peptide in rat jejunum.
• Peptides. 1993; 14: 1011-6
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• Peptide YY (PYY) and its homologous peptide, neuropeptide Y (NPY), are known to exhibit potent antisecretory effects in the intestine. To determine the structural requirements to elicit antisecretory effects, we have synthesized several analogs of the PYY active site, PYY(22-36), and compared their binding affinities and antisecretory potencies in rat jejunum. These investigations revealed that the hydroxyl groups of Ser23 and Thr32, as well as the imidazole group of His26, are important for activity in the intestine. N-alpha-acetylation of PYY(22-36) increased both the binding affinity and antisecretory potency. Structure-activity studies with N-alpha-Ac-PYY(22-36) showed that substitution of His26 with parachlorophenylalanine (pCl-Phe) or Tyr36 with N-Me-Tyr reduced receptor affinity, while replacement of Tyr27 with Phe increased the activity substantially. Furthermore, acylation of the alpha-NH2 group with hydrophobic groups, myristic and naphthaleneacetic acids, substantially reduced the antisecretory potencies but not the binding affinities. Further modification of N-alpha-Ac-[Phe27]PYY(22-36) may lead to the development of more potent agonist compounds, which may provide a framework for the design of a new class of antidiarrheal drugs.

• Jensen J, Conlon JM
• Characterization of peptides related to neuropeptide tyrosine and peptide tyrosine-tyrosine from the brain and gastrointestinal tract of teleost fish.
• Eur J Biochem. 1992; 210: 405-10
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• Neuropeptide Y was isolated from the brain of the Atlantic cod, Gadus morhua and its primary structure established as Tyr-Pro-Ile*-Lys-Pro-Glu*-Asn-Pro-Gly-Glu10-Asp-Ala-Pro-Ala-Asp*-G lu*-Leu*-Ala- Lys*-Tyr20-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Ile-Thr -Arg-Gln-Arg-Tyr- CONH2. Residues denoted by an asterisk are different from the corresponding sequence of human neuropeptide Y. A structurally similar peptide was isolated from the brain of the trout, Oncorhynchus mykiss. Trout neuropeptide Y contains four substitutions (Ile3-->Val, Ala14-->Thr, Asp15-->Glu and Ser22-->Thr) compared with cod neuropeptide Y. A second peptide of the neuropeptide Y family was identified in the trout brain and this component was structurally similar to peptide tyrosine-tyrosine previously isolated from frog intestine (six amino acid substitutions) and identical to a peptide isolated from the pancreas of the closely related species, Oncorhynchus kisutch (Coho salmon). Peptide tyrosine-tyrosine, with the same primary structure as the brain peptide, was also isolated from an extract of the trout stomach. The data indicate that a peptide analogous to mammalian neuropeptide Y is present in the brain of teleost fish and a peptide analogous to mammalian peptide tyrosine-tyrosine is present in brain, gastrointestinal tissue and pancreas. We speculate, therefore, that the putative gene duplication that led to pancreatic polypeptide in the higher vertebrates took place after the time of divergence of fish and tetrapods.

• Li XJ, Wu YN, North RA, Forte M
• Cloning, functional expression, and developmental regulation of a neuropeptide Y receptor from Drosophila melanogaster.
• J Biol Chem. 1992; 267: 9-12
• Display abstract

• Neuropeptide Y, peptide YY, and pancreatic polypeptide are homologous 36-amino acid peptides that differ from most other peptide transmitters by having a relatively rigid conformation in aqueous solutions, defined as the pancreatic polypeptide fold, and a critical C-terminal tyrosine amide. These peptides serve as gastrointestinal hormones and neurotransmitters. A cDNA encoding a novel G protein-coupled receptor activated by neuropeptide Y was cloned from Drosophila by use of degenerate oligonucleotide primers and polymerase chain reaction amplification of cDNA prepared from transcripts expressed early in embryogenesis. The cDNA encodes a protein of 449 amino acids with the characteristics of a G protein-coupled receptor and shares significant amino acid identity with mammalian tachykinin receptors. When expressed in Xenopus oocytes, the PR4 protein is activated by mammalian neuropeptides in the order: peptide YY greater than neuropeptide Y much greater than pancreatic polypeptide. Northern analysis showed that PR4 receptor is expressed at equivalent levels in adult Drosophila head and body and that the expression of the PR4 receptor is regulated during development. The molecular characterization of this receptor should lead to a better understanding of the functional role of this important family of hormone receptors in adult organisms and during development.

• Conlon JM, Bjenning C, Moon TW, Youson JH, Thim L
• Neuropeptide Y-related peptides from the pancreas of a teleostean (eel), holostean (bowfin) and elasmobranch (skate) fish.
• Peptides. 1991; 12: 221-6
• Display abstract

• Homologous peptides belonging to the pancreatic polypeptide (PP) family were isolated from the pancreas of a teleostean fish, the American eel (Anguilla rostrata), an holostean fish, the bowfin (Amia calva) and an elasmobranch fish, the skate (Raja rhina), and their primary structures were determined. The peptides show stronger homology to neuropeptide Y, particularly in their COOH-terminal regions, than to peptide YY or pancreatic polypeptide and contain an alpha-amidated COOH-terminal tyrosine residue. The skate peptide Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Asp10-Asp-Ala-Ala-Pro-Glu-Glu- Leu-Ala-Lys- Tyr20-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Ile-Thr-Arg- Gln-Arg-Tyr-NH2 represents the first member of the PP family to be isolated from a cartilaginous fish. The primary structure of the pancreatic PP family peptide has been more strongly conserved among the phylogenetically more ancient holostean and elasmobranch fishes than among the teleosts. A comparison of the primary structures of all PP family peptides supports the hypothesis and evolution has acted to conserve features of tertiiary structure in the molecules (e.g., the polyproline- and alpha-helices) rather than individual amino acid residues.

• Conlon JM, Bjornholm B, Jorgensen FS, Youson JH, Schwartz TW
• Primary structure and conformational analysis of peptide methionine-tyrosine, a peptide related to neuropeptide Y and peptide YY isolated from lamprey intestine.
• Eur J Biochem. 1991; 199: 293-8
• Display abstract

• A peptide belonging to the pancreatic-polypeptide-fold family of regulatory peptides has been isolated from the intestine of an Agnathan, the sea lamprey (Petromyzon marinus). The primary structure of the peptide (termed peptide methionine-tyrosine) was established as Met-Pro-Pro-Lys-Pro-Asp-Asn- Pro-Ser-Pro10-Asp-Ala-Ser-Pro-Glu-Leu-Ser-Lys-Tyr20-Met-Leu- Ala-Val-Arg-Asn- Tyr-Ile-Asn-Leu30-Ile-Thr-Arg-Gln-Arg-Tyr CONH2. This sequence shows stronger structural similarity with pig neuropeptide Y (64%), particularly in the COOH-terminal region, than with pig peptide tyrosine--tyrosine (61%) or with pig pancreatic polypeptide (42%). Molecular modelling and dynamic simulation, based upon sequence similarity with turkey pancreatic polypeptide, indicates that the conformations of the polyproline-helix-like region (residues 1-8) and the alpha-helical region (residues 15-30) in turkey pancreatic polypeptide are conserved in peptide methionine-tyrosine, and that non-bonded interactions between these domains have preserved the overall polypeptide fold in the molecule. The substitution of the otherwise totally conserved Gly9 residue by serine in lamprey peptide methionine-tyrosine, however, results in a preferred structure in which the conformation of the beta-turn between the two helical domains (residues 9-14) is appreciably different.

• Gingerich RL, Akpan JO, Gilbert WR, Leith KM, Hoffmann JA, Chance RE
• Structural requirements of pancreatic polypeptide receptor binding.
• Am J Physiol. 1991; 261: 31924-31924
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• Pancreatic polypeptide (PP) receptors have been identified and characterized on the basolateral membranes (BLM) of canine intestinal mucosa. The present study was designed to ascertain the structural requirements of the PP molecule for binding to its receptor. A radioreceptor assay using purified BLM was employed to elucidate receptors specific to PPs of various mammalian species and to modified bovine PP (bPP) fragments. Receptor cross-reactivities (CR) to various PPs and bPP fragments were established. Results show that percent receptor CR by PPs of various species was as follows: bPP (100%) greater than human PP (68%) greater than porcine PP (50%) greater than canine PP (45%) greater than ovine PP (36%) greater than rat PP (3%). The fragments bPP-(1-15), bPP-(1-17), bPP-(1-26), bPP-(16-23), bPP-(18-30), bPP-(24-36), bPP-(27-35), and bPP-(31-36) at 500 nM did not significantly displace tracer from receptor (less than 0.1% CR). Des-COOH-terminal tyrosinamide [bPP-(1-35)] produced less than 0.1% CR. Oxidation of bPP methionine-30 residue to methionine sulfoxide decreased displacement to 67%. Modification of native amidated tyrosinamide to the free acid abolished receptor binding, whereas esterification to the methyl ester of COOH-terminal tyrosine restored binding to 60%. Additionally, percent CR decreased progressively as amino acid residues were deleted from the NH2-terminal region. We conclude that the molecular homologue of PP primary structure is necessary for full receptor binding. Both the NH2- and COOH-terminal residues are required for recognition, and the COOH-terminal tyrosinamide must be intact for PP binding to its receptor.

• Jorgensen JC, Fuhlendorff J, Schwartz TW
• Structure-function studies on neuropeptide Y and pancreatic polypeptide--evidence for two PP-fold receptors in vas deferens.
• Eur J Pharmacol. 1990; 186: 105-14
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• The biological effects of neuropeptide Y (NPY), rat pancreatic polypeptide (rPP), hybrid analogs of NPY and PP, and C-terminal fragments of NPY were studied in the field-stimulated rat vas deferens model. The results were correlated with peptide binding experiments in Y1 and PP receptor assays on rat PC-12 cells and Y2 receptors on porcine hippocampal membranes. NPY and rPP inhibited the electrically induced contractions in the vas deferens with an IC50 of 25 and 22 nM respectively. However, in contrast to NPY, rPP could not totally block muscle activity. The inhibitory action of the long C-terminal fragment of NPY, NPY-(19-36) and NPY-(11-36), indicated that NPY acts through a Y2 receptor in the vas deferens. The structural basis for the differential recognition of NPY and PP by Y2 receptors and partly also by PP receptors, could be defined with hybrid analogs of PP and NPY. The analogs, [Ile31,Gln34]PP and [Leu31,Pro33]NPY reacted in the vas deferens preparation in accordance with their relative potency in the Y2 and PP receptor assays. [Ile31,Gln34]PP, which bound to the Y2 receptor like NPY, was also able to block the part of the contractile response which was resistant to rPP. It is concluded that in the vas deferens, PP-fold peptides act through two types of receptors: Y2 and PP, and that residues in the C-terminal part of the molecules determine the differential recognition of the peptides by these receptor types.

• Cloarec D, Rigaud D
• [Pancreatic polypeptide, peptide YY and neuropeptide Y. A new family of regulatory peptides]
• Gastroenterol Clin Biol. 1987; 11: 29-36

• Ganeshan K, Perlman MO, Perlman JM, Adamo ML, Hazelwood RL, Dyckes DF
• Recognition of neuropeptides FMRFamide and LPLRFamide by chicken cerebellum avian pancreatic polypeptide binding sites.
• Proc Soc Exp Biol Med. 1987; 185: 409-12
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• Binding isotherms were constructed for the binding of synthetic tetrapeptide and pentapeptide fragments to membranes prepared from chicken cerebellar tissue. Both the tetrapeptide (FMRFamide), which was originally isolated from ganglia of mollusks, and the pentapeptide (LPLRFamide) previously isolated from chicken brain are known to increase blood pressure and modulate brain neurons in rats. The C-terminal dipeptide sequences of the two peptides are identical and both show similarity to the dipeptide sequence established for the pancreatic polypeptide (PP) family. Specific high-affinity binding sites exist for the latter peptide, sites which are competed for (though with less affinity) by neuropeptide Y (NPY). Affinity for cerebellar membranes was virtually equivalent for the synthetic peptide LPLRFamide and FRMFamide; the binding affinities (IC50) of all fragments tested (C-terminal pentapeptides of avian PP and NPY, and FMRFamide and LPLRFamide) fell in the same approximate range. Since the N-terminal residues of FMRFamide and LPLRFamide are not homologous with equivalent residues of APP or NPY, our results indicate that only Arg-Tyr-NH2 or Arg-Phe-NH2 sequences are necessary for binding of the carboxy terminus peptides of the PP family. In this respect, these sequences are functionally equivalent.

• Leiter AB et al.
• Peptide YY. Structure of the precursor and expression in exocrine pancreas.
• J Biol Chem. 1987; 262: 12984-8
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• Peptide YY is a 36-residue gastrointestinal hormone which inhibits both pancreatic and gastric secretion. We have isolated a cDNA encoding the peptide YY precursor by screening a rat intestinal lambda gt11 cDNA library with an antiserum directed against the porcine hormone. The nucleotide sequence of the cDNA encodes a 98-residue protein (molecular weight, 11, 121) which has an amino acid sequence identical to that of porcine peptide YY. Rat peptide YY is preceded immediately by a signal sequence and followed by a cleavage-amidation sequence Gly-Lys-Arg plus 31 additional amino acids. Thus the peptide YY precursor is similar in structure to that of two related peptides, pancreatic polypeptide and neuropeptide Y. RNA blot hybridizations reveal that the peptide YY gene is much more actively expressed in pancreas than previously realized. In situ hybridizations localized peptide YY cells exclusively to the exocrine pancreas. The abundance of peptide YY in one of its target organs, the pancreas, suggests a paracrine mechanism for peptide YY in regulating pancreatic enzyme secretion.

• Solomon TE
• Pancreatic polypeptide, peptide YY, and neuropeptide Y family of regulatory peptides.
• Gastroenterology. 1985; 88: 838-41

• Hoffman JA, Chance RE
• Crystallization of bovine pancreatic polypeptide.
• Biochem Biophys Res Commun. 1983; 116: 830-5
• Display abstract

• Bovine pancreatic polypeptide, a 36-residue polypeptide hormone, was successfully crystallized for the first time. During purification by extensive dialysis against distilled water a surprisingly insoluble form of the peptide was obtained which led to its crystallization as orthorhombic needles up to 2 mm in length.

• Tatemoto K
• Neuropeptide Y: complete amino acid sequence of the brain peptide.
• Proc Natl Acad Sci U S A. 1982; 79: 5485-9
• Display abstract

• The amino acid sequence of neuropeptide Y, a 36-residue peptide recently isolated from porcine brain, has been determined by using high performance liquid chromatography for separation of its tryptic and chymotryptic fragments and subsequent sequence analysis of the isolated fragments by an improved dansyl Edman subtractive technique. The amino acid sequence of neuropeptide Y has been found to be: Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Ar g-Tyr -Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2. Neuropeptide Y has a high degree of sequence homology with peptide YY (70%), the newly isolated porcine intestinal peptide, and pancreatic polypeptide (50%). It is therefore proposed that neuropeptide Y, peptide YY, and pancreatic polypeptide are members of a newly recognized peptide family.

• Diemel JM, Lamers CB
• [Pancreatic polypeptide: a peptide with hormonal properties?]
• Ned Tijdschr Geneeskd. 1980; 124: 975-7

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