The antiparallel pancreatic polypeptide fold in the binding of neuropeptide Y to Y1 and Y2 receptors

Neuropeptide Y (NPY) belongs to the pancreatic polypeptide fold (PP-fold) family of regulatory peptides. Analysis of circular dicroic spectra of NPY showed that it has a high degree of secondary structure in aqueous solution which is in agreement with the globular, folded crystal structure of PP. Using three different approaches with synthetic peptides, we have probed the importance of the PP-fold structure in the interaction of NPY with two types of binding sites, Y1 and Y2 receptors. First, stepwise construction of the NPY molecule from the C-terminal amidated end, showed that although C-terminal fragments encompassing most of the long alpha-helix reacted reasonably well with the Y2 receptor, both Y1 and Y2 receptors required the presence of both ends of the PP-fold for full activity. Second, perturbation of the PP-fold by substitution with a helix-breaking proline residue, resulted in the loss of recognition of the N-terminal segment of the molecule by both types of receptors. Finally, a hybrid analog was constructed in which the essential, but by itself inactive, C-terminal segment of NPY was joined with the PP-fold motif of PP. This segment of PP is only 43% homologous to the similar motif in NPY, and most of the common residues cluster in the hydrophobic core of the fold. Nevertheless, the hybrid analog reacted with almost full potency on the Y2 receptors. It is concluded that the antiparallel PP-fold is of structural importance for the receptor binding of NPY, and that its main function is to present the combined C- and N-terminal segments of the molecule to the receptors.     

Y-receptor affinity modulation by the design of pancreatic polypeptide/neuropeptide Y chimera led to Y(5)-receptor ligands with picomolar affinity

Neuropeptide Y (NPY) and pancreatic polypeptide (PP) bind to the Y-receptors with very different affinities: NPY has high affinity for the receptors Y(1), Y(2) and Y(5), while PP binds only to Y(4)-receptor with picomolar affinity. By exchanging of specific amino acid positions between the two peptides, we developed 38 full-length PP/NPY chimeras with binding properties that are completely different from those of the two native ligands. Pig NPY (pNPY) analogs containing the segment 19-23 from human PP (hPP) bound to the Y-receptors with much lower affinity than NPY itself. The affinity of the hPP analog containing the pNPY segments 1-7 and 19-23 was comparable to that of pNPY at the Y(1)- and Y(5)-receptor subtypes, and to that of hPP at the Y(4)-receptor. Furthermore, the presence of the segments 1-7 from chicken PP (cPP) and 19-23 from pNPY within the hPP sequence led to a ligand with IC(50) of 40 pM at the Y(5)-receptor. This is the most potent Y(5)-receptor ligand known so far, with 15-fold higher affinity than NPY.     

Structural similarities of micelle-bound peptide YY (PYY) and neuropeptide Y (NPY) are related to their affinity profiles at the Y receptors

Here, we investigate the structure of porcine peptide YY (pPYY) both when unligated in solution at pH 4.2 and when bound to dodecylphosphocholine (DPC) micelles at pH 5.5. pPYY in solution displays the PP-fold, with the N-terminal segment being back-folded onto the C-terminal alpha-helix, which extends from residue 17 to 31. In contrast to the solution structure of Keire et al. published in the year 2000 the C-terminal helix does not display a kink around residue 23-25. The root mean square deviation (RMSD) for backbone atoms of the NMR ensemble of conformers to the mean structure is 0.99(+/-0.35) Angstrom for residues 14-31. The back-fold is supported by values of 0.60+/-0.1 for the (15)N(1)H-NOE and by generalized order parameters S(2) of 0.74+/-0.1 for residues 5-31 which indicate that the peptide is folded in that segment. We have additionally used DPC micelles as a membrane model and determined the structure of pPYY when bound to it. Therein, an alpha-helix occurs in the segment comprising residues 17-31 and the N terminus freely diffuses in solution. The hydrophobic side of the amphipathic helix forms the micelle-binding interface and hydrophobic side-chains extend into the micelle interior. A significant stabilization of helical conformation occurs in the C-terminal pentapeptide, which is important for receptor binding. The latter is supported by positive values of the heteronuclear NOE in that segment (0.52+/-0.1 compared to 0.08+/-0.4 for the unligated form) and by values of S(2) of 0.6+/-0.2 (versus 0.38+/-0.2 for the unligated form). The structures of micelle-bound pPYY and pNPY are much more similar than those of pPYY and bPP with pairwise RMSDs of 1.23(+/-0.21)A or 3.21(+/-0.39) Angstrom, respectively. In contrast to the conformational similarities in the DPC-bound state their structures in solution are very different. In fact pPYY is more similar to bPP, which with its strong preference for the Y(4) receptor displays a completely different binding profile. Considering the high degree of sequence homology of pNPY and pPYY (>80%) and the fact, that their binding affinities at all receptor subtypes are high and, more importantly, rather similar, it is much more likely that PYY and NPY are recognized by the Y receptors from the membrane-bound state. As a consequence of the latter the PP-fold is not important for recognition of PYY or NPY at the Y receptors. To our knowledge this work provides for the first time strong arguments derived from structural data that support a membrane-bound receptor recognition pathway.     

Receptors on phaeochromocytoma cells for two members of the PP-fold family--NPY and PP

Pancreatic polypeptide (PP) and neuropeptide Y (NPY) belong to a family of regulatory peptides which hold a distinct tertiary structure, the PP-fold, even in dilute aqueous solution. High-affinity receptors, specific for both PP and NPY, are described on the rat phaeochromocytoma cell line, PC-12. The binding of [125I-Tyr36]PP to PC-12 cells was inhibited by concentrations of unlabeled PP which correspond to physiological concentrations of the hormone, 10(-11)-10(-9) mol/l. The affinity of the receptor for the neuropeptide, NPY, was 10(2)-times lower than that of the PP receptor. C-terminal fragments of both PP (PP24-36) and NPY (NPY13-36) were between 10(2)- and 10(3)-times less potent in displacing the radiolabeled 36-amino-acid peptides from their respective receptors. It is concluded that PC-12 cells are suited for structure-function studies of the PP-fold peptides and studies on the cellular events following cellular binding of PP-fold peptides.     

2-36[K4,RYYSA(19-23)]PP a novel Y5-receptor preferring ligand with strong stimulatory effect on food intake

Members of the neuropeptide Y (NPY) family regulate many physiological processes via interaction with at least four functional, pharmacologically distinct Y-receptors. However, selective antagonists developed for several subtypes have not been useful in defining particular Y-receptor functions in vivo. To identify critical residues within members of the NPY family required for Y-receptor subtype-selectivity we have determined the contribution of each residue within NPY to receptor binding by replacing them with L-alanine. In a second study, chimeric peptides where single or stretches of residues were interchanged between members of the NPY family were generated and tested in radioligand binding studies. Overall, substituted alanine analogues exhibited similar orders of affinities at each Y-receptor subtype with no obvious subtype-selectivity. Residues of particular interest are Leu30 which exhibited selectivity for the Y4-receptor, whereas Asp16 does not appear to play any role in ligand binding. Several chimeric peptides, e.g., [K4]pancreatic polypeptide ([K4]PP) and [RYYSA(19-23)]PP clearly showed higher affinity at the Y4 and Y5 subtypes compared to the Y1 and Y2 subtypes. In addition, the transfer of a proline residue from position 14 to 13 in peptide YY decreases its affinity at the Y1-, Y4- and Y5-receptors but is unchanged at the Y2 subtype. Combining these results, and with the help of molecular modelling, second generation chimeras were designed. The most significant improvement was achieved in chimera 2-36[K4,RYYSA(19-23)]PP where the affinity for the Y5 subtype increased by ninefold over that from NPY. Several of these compounds were also tested for their ability to stimulate food intake in a rat model. Interestingly, again 2-36[K4,RYYSA(19-23)]PP showed the most dramatic effect with a major increase on food intake over a range of doses compared to NPY suggesting a possible synergistic effect of several Y-receptors on feeding behaviour.     

Structure and dynamics of micelle-bound neuropeptide Y: comparison with unligated NPY and implications for receptor selection

The biological importance of the neuropeptide Y (NPY) has steered a number of investigations about its solution structure over the last 20 years. Here, we focus on the comparison of the structure and dynamics of NPY free in solution to when bound to a membrane mimetic, dodecylphosphocholine (DPC) micelles, as studied by 2D (1)H NMR spectroscopy. Both, free in solution and in the micelle-bound form, the N-terminal segment (Tyr1-Glu15) is shown to extend like a flexible tail in solution. This is not compatible with the PP-fold model for NPY that postulates backfolding of the flexible N terminus onto the C-terminal helix. The correlation time (tau(c)) of NPY in aqueous solution, 5.5 (+/-1.0) ns at 32 degrees C, is only consistent with its existence in a dimeric form. Exchange contributions especially enhancing transverse relaxation rates (R(2)) of residues located on one side of the C-terminal helix of the molecule are supposed to originate from dimerization of the NPY molecule. The dimerization interface was directly probed by looking at (15)N-labeled NPY/spin-labeled [TOAC34]-[(14)N]-NPY heterodimers and revealed both parallel and anti-parallel alignment of the helices. The NMR-derived three-dimensional structure of micelle-bound NPY at 37 degrees C and pH 6.0 is similar but not identical to that free in solution. The final set of 17 lowest-energy DYANA structures is particularly well defined in the region of residues 21-31, with a mean pairwise RMSD of 0.23 A for the backbone heavy atoms and 0.85 A for all heavy atoms. The combination of NMR relaxation data and CD measurements clearly demonstrates that the alpha-helical region Ala18-Thr32 is more stable, and the C-terminal tetrapeptide becomes structured only in the presence of the phosphocholine micelles. The position of NPY relative to the DPC micelle surface was probed by adding micelle integrating spin labels. Together with information from (1)H,(2)H exchange rates, we conclude that the interaction of NPY with the micelle is promoted by the amphiphilic alpha-helical segment of residues Tyr21-Thr32. NPY is located at the lipid-water interface with its C-terminal helix parallel to the membrane surface and penetrates the hydrophobic interior only via insertions of a few long aliphatic or aromatic side-chains. From these data we can demonstrate that the dimer interface of neuropeptide Y is similar to the interface of the monomer binding to DPC-micelles. We speculate that binding of the NPY monomer to the membrane is an essential key step preceeding receptor binding, thereby pre-orientating the C-terminal tetrapeptide and possibly inducing the bio-active conformation.     

Recent developments in our understanding of the physiological role of PP-fold peptide receptor subtypes

The three peptides pancreatic polypeptide (PP), peptide YY (PYY), and neuropeptide Y (NPY) share a similar structure known as the PP-fold. There are four known human G-protein coupled receptors for the PP-fold peptides, namely Y1, Y2, Y4, and Y5, each of them being able to bind at least two of the three endogenous ligands. All three peptides are found in the circulation acting as hormones. Although NPY is only released from neurons, PYY and PP are primarily found in endocrine cells in the gut, where they exert such effects as inhibition of gall bladder secretion, gut motility, and pancreatic secretion. However, when PYY is administered in an experimental setting to animals, cloned receptors, or tissue preparations, it can mimic the effects of NPY in essentially all studies, making it difficult to study the effects of PP-fold peptides and to delineate what receptor and peptide accounts for a particular effect. Initial studies with transgenic animals confirmed the well-established action of NPY on metabolism, food-intake, vascular systems, memory, mood, neuronal excitability, and reproduction. More recently, using transgenic techniques and novel antagonists for the Y1, Y2, and Y5 receptors, NPY has been found to be a key player in the regulation of ethanol consumption and neuronal development.     

Spinal and peripheral modulation of gastric acid secretion and arterial pressure by neuropeptide Y, peptide YY, and pancreatic polypeptide in rats

Spinal and peripheral modulation of pentagastrin-stimulated gastric acid secretion by the pancreatic polypeptide-fold (PP-fold) peptides, neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP), in urethane-anesthetized rats was evaluated. Neuropeptide Y, PYY, and PP (400 pmol) were administered via intravenous (IV) and intrathecal (IT) injections. The ₂ antagonist, yohimbine, was used to evaluate the role of the ₂ adrenergic receptors in the modulation of pentagastrin-stimulated gastric acid secretion by NPY, PYY, and PP. Peptide YY and PP (IV) rapidly increased pentagastrin-stimulated gastric acid secretion. Peptide YY and PP (IT) increased pentagastrin-stimulated gastric acid secretion following administration into the thoracic (T8–T10) region of the spinal cord. The ₂ adrenergic receptor antagonist, yohimbine, did not modify the increases in pentagastrin-stimulated gastric acid secretion following PYY and PP (IV or IT) administration. Neuropeptide Y (IT) decreased pentagastrin-stimulated gastric acid secretion. However, in the presence of ₂ adrenergic receptor blockade, pentagastrin-stimulated gastric acid secretion was potentiated by NPY (IT) administration. Therefore, the inhibitory effect of NPY (IT) on pentagastrin-stimulated gastric acid secretion required the activation of ₂ adrenergic receptors in the spinal cord of rats. Mean arterial blood pressure (MAP) was increased immediately following NPY and PYY (IV) administration. During the same time period, PP (IV) decreased MAP in anesthetized rats. Mean arterial blood pressure was rapidly increased by NPY and PYY (IT) in anesthetized rats. The increase in MAP following PYY (IT) was partially attenuated in the presence of yohimbine. The modulation of MAP and gastric acid secretion by the PP-fold peptides occurred by independent mechanisms at spinal and peripheral sites in the rat. The modulation of pentagastrin-stimulated gastric acid secretion by PYY and PP in rats differed from that of the third member of the PP-fold family, NPY, following spinal and peripheral administration.     

Electron paramagnetic resonance backbone dynamics studies on spin-labelled neuropeptide Y analogues.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammalians. NPY acts by binding to at least five G-protein coupled receptors (GPCRs) which have been named Y1, Y2, Y4, Y5 and Y6. Three spin-labelled NPY analogues containing the nitroxide group of the amino acid TOAC (2.2.6.6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe were synthesized by solid-phase peptide synthesis. Synthetic problems owing to the sensitivity of nitroxide towards acidic and reducing conditions have been overcome by using a cleavage cocktail that contains anisole and cresol scavengers. Concerning the receptor binding preferences, the analogues [TOAC34]-pNPY and [Ala31, TOAC32]-pNPY showed a marked selectivity for the Y5 receptor, while [TOAC2]-pNPY maintained a significant binding also to the Y2 receptor subtype. The modifications of the native peptide structure caused by the introduction of TOAC were examined by circular dichroism. In order to determine the rotational correlation time of the spin probes, electron paramagnetic resonance measurements were performed in solution and in the presence of liposomes. This allowed us to evaluate the backbone dynamics of the different parts of the NPY molecule in the free and membrane bound states. The results of these studies showed that NPY Interacts with liposomes by using the C-terminal alpha-helix while the N-terminal tail retains a flexibility that is comparable to that of the peptide in solution as already shown by NMR studies on DPC micelles. Furthermore, we demonstrated that TOAC-labelllng is a valuable tool to investigate changes in the backbone conformation and dynamics. This may be of major importance for peptides and small proteins when they bind to cell membranes.     

Non-specific binding and general cross-reactivity of Y receptor agonists are correlated and should importantly depend on their acidic sectors

Non-specific binding of Y receptor agonists to intact CHO cells, and to CHO cell or rat brain particulates, is much greater for human neuropeptide Y (hNPY) compared to porcine peptide Y (pPYY), and especially relative to human pancreatic polypeptide (hPP). This binding of hNPY is reduced by alkali cations in preference to non-ionic chaotrope urea, while the much lower non-specific binding of pPYY is more sensitive to urea. The difference could mainly be due to the 10-16 stretch in 36-residue Y agonists (residues 8-14 in N-terminally clipped 34-peptides), located in the sector that contains all acidic residues of physiological Y agonists. Anionic pairs containing aspartate in the 10-16 zone could be principally responsible for non-specific attachments, but may also aid the receptor site binding. Two such pairs are found in hNPY, one in pPYY, and none in hPP. The hydroxyl amino acid residue at position 13 in mammalian PYY and PP molecules could lower conformational plasticity and the non-selective binding via intrachain hydrogen bonding. The acidity of this tract could also be important in agonist selectivity of the Y receptor subtypes. The differences point to an evolutionary reduction of promiscuous protein binding from NPY to PP, and should also be important for Y agonist selectivity within NPY receptor group, and correlate with partial agonism and out-of group cross-reactivity with other receptors.     

Analogs of pancreatic polypeptide and peptide YY with a locked PP-fold structure are biologically active

Pancreatic polypeptide (PP), peptide YY (PYY) and neuropeptide Y (NPY), members of the PP-fold family share a high degree of sequence homology. Nuclear magnetic resonance (NMR) and X-ray crystallography studies have shown these peptides can adopt a tightly organized tertiary structure called the PP-fold, which has long been assumed to be the active structure of this family of peptides. To date, however, no studies have been completed with PYY and PP which confirm if the PP-fold structure is important for their physiological actions. The aim of the study was to test if PYY and PP locked into the PP-fold maintained biological activity. Therefore, we designed and produced analogs of PP and PYY in a cyclic conformation with two cysteine amino acid substitutions at the N-terminus and at position 27. These were oxidized to form a cysteine disulfide bond locking the peptides into the PP-fold structure. Studies demonstrate that the cyclic analogs have both similar in vivo activity to their parent molecules, and affinity for the Y2 and Y4 receptors. Results suggest that the proposed PP and PYY-fold is likely to be their biologically active conformation.     

FMRFamides exert a unique modulation of rodent pancreatic polypeptide sensitive neuropeptide Y (NPY) receptors

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.     

Neuropeptide Y, peptide YY, and pancreatic polypeptide modulate duodenal and colonic motility at a thoracic spinal site in rats.

Neuropeptide Y, PYY, and PP (200 pmol) alter intraluminal pressure in the duodenum and colon of rats following their administration into the thoracic (T8-T10) region of the spinal cord. Neuropeptide Y decreases the tone of the duodenum and the colon following intrathecal (T8-T10) administration prior to an increase in tone to baseline or greater. There is no effect on intraluminal pressure of either the duodenum or the colon following intrathecal administration of NPY or PP into the lumbar (L4-L5) region of the spinal cord. Following intrathecal (T8-T10) administration of PYY and PP, increases in intraduodenal pressures are observed (+2.1 and +3.0 mmHg from saline baseline). Phasic contractions of the duodenum are increased following intrathecal administration of PYY into the thoracic spinal cord of rats. Neuropeptide Y, PYY, and PP increase intracolonic pressure +2.2, +3.3, and +3.7 mmHg from saline baseline, respectively. Phasic contractions of the colon are increased following PP intrathecal thoracic administration. Responsiveness of the duodenum or colon to the different ligands of the PP-fold peptide family in the absence of alpha-adrenergic blockade did not vary. The increases in intraluminal pressure of the duodenum and colon following intrathecal administration of the PP-fold peptides are attenuated by both alpha-1 adrenergic (prazosin) and alpha-2 adrenergic (yohimbine) blockade. There is a difference in responsiveness of the colon between the ligands of the PP-fold family in the presence of the alpha-2 adrenergic blockade. The findings of this study indicate that duodenal and colonic motility are modulated by the PP-fold peptides at thoracic spinal sites via alteration of sympathetic outflow.     

Solution structure of monomeric peptide YY supports the functional significance of the PP-fold

Peptide YY (PYY) belongs to a family of peptides including neuropeptide Y (NPY) and pancreatic peptide (PP) that regulate numerous functions through both central and peripheral receptors. The solution structure of these peptides is hypothesized to be critically important in receptor selectivity and activation, based on prior demonstration of a stable tertiary conformation of PP called the "PP-fold". Circular dichroism (CD) spectra show a pH-dependent structural transition in the pH range 3-4. Thus we describe the tertiary structure of porcine PYY in water at pH 5.5, 25 degrees C, and 150 mM NaCl, as determined from 2D (1)H NMR data recorded at 500 MHz. A constraint set consisting of 396 interproton distances from NOE data was used as input for distance geometry, simulated annealing, and restrained energy minimization calculations in X-PLOR. The RMSDs of the 20 X-PLOR-generated structures were 0.71 +/- 0.14 and 1.16 +/- 0.17 A, respectively, for backbone and heavy atom overlays of residues 1-34. The resulting structure consists of two C-terminal helical segments from residues 17 to 22 and 25 to 33 separated by a kink at residues 23, 24, and 25, a turn centered around residues 12-14, and the N-terminus folded near residues 30 and 31. The well-defined portions of the PYY structure reported here bear a marked similarity to the structure of PP. Our findings strongly support the importance of the stable folded structure of this family of peptides for binding and activation of Y receptor subtypes.     

Recognition of neuropeptides FMRFamide and LPLRFamide by chicken cerebellum avian pancreatic polypeptide binding sites

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.     

Multiple Y receptors mediate pancreatic polypeptide responses in mouse colon mucosa

A functional study has been performed to characterise the Y receptors responsible for NPY, PYY and PP-stimulated responses in mouse colonic mucosal preparations. Electrogenic ion secretion was stimulated with VIP following which NPY, PYY and PP analogues were, to varying degrees, inhibitory. PYY(3-36), hPP, Gln(23)hPP and rPP were effective but less potent than full length PYY, NPY or their Pro(34)-substituted analogues, while the Y(5) agonist Ala(31), Aib(32)hNPY was the least active peptide tested. The Y(1) antagonists, BIBP3226 and BIBO3304 virtually abolished Pro(34)PYY and PYY responses while PYY(3-36) responses were selectively inhibited by the Y(2) antagonist, BIIE0246. A combination of BIBO3304 and BIIE0246 also partially attenuated hPP responses, leaving residual effects that were most probably Y(4)-mediated. Thus we conclude that Y(1), Y(2) and Y(4) receptors attenuate ion secretion in mouse colon.     

Pharmacological characterization of cloned chicken neuropeptide Y receptors Y1 and Y5

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

Bovine pancreatic polypeptide (bPP) undergoes significant changes in conformation and dynamics upon binding to DPC micelles

The pancreatic polypeptide (PP), a 36-residue, C-terminally amidated polypeptide hormone is a member of the neuropeptide Y (NPY) family. Here, we have studied the structure and dynamics of bovine pancreatic polypeptide (bPP) when bound to DPC-micelles as a membrane-mimicking model as well as the dynamics of bPP in solution. The comparison of structure and dynamics of bPP in both states reveals remarkable differences. The overall correlation time of 5.08ns derived from the 15N relaxation data proves unambiguously that bPP in solution exists as a dimer. Therein, intermolecular as well as intramolecular hydrophobic interactions from residues of both the amphiphilic helix and of the back-folded N terminus contribute to the stability of the PP fold. The overall rigidity is well-reflected in positive values for the heteronuclear NOE for residues 4-34.The membrane-bound species displays a partitioning into a more flexible N-terminal region and a well-defined alpha-helical region comprising residues 17-31. The average RMSD value for residues 17-31 is 0.22(+/-0.09)A. The flexibility of the N terminus is compatible with negative values of the heteronuclear NOE observed for the N-terminal residues 4-12 and low values of the generalized order parameter S(2). The membrane-peptide interface was investigated by micelle-integrating spin-labels and H,2H exchange measurements. It is formed by those residues which make contacts between the C-terminal alpha-helix and the polyproline helix. In contrast to pNPY, also residues from the N terminus display spatial proximity to the membrane interface. Furthermore, the orientation of the C terminus, that presumably contains residues involved in receptor binding, is different in the two environments. We speculate that this pre-positioning of residues could be an important requirement for receptor activation. Moreover, we doubt that the PP fold is of functional relevance for binding at the Y(4) receptor.     

Determination of affinity and activity of ligands at the human neuropeptide Y Y4 receptor by flow cytometry and aequorin luminescence

Fluorescence-labeled neuropeptide Y (NPY) has been used in flow cytometric binding assays for the determination of affinity constants of NPY Y1, Y2, and Y5 receptor ligands. Because the binding of fluorescent NPY is insufficient for competition studies at the human Y4 receptor (hY4R), we replaced Glu-4 in hPP with Lys for the derivatization with cyanine-5. Because cy5-[K(4)]hPP has high affinity (Kd 5.6 nM) to the hY4R, it was used as a probe in a flow cytometric binding assay. Specific binding of cy5-[K(4)]hPP to hY4R was visualized by confocal microscopy. The hY(4)R, the chimeric G protein G(qi5) and mitochondrially targeted apoaequorin were stably coexpressed in CHO cells. Aequorin luminescence was quantified in a microplate reader and by a CCD camera. By application of these methods 3-cyclohexyl-N-[(3-1H-imidazol-4-ylpropylamino)(imino)methyl]propanamide (UR-AK49) was discovered as the first nonpeptidic Y4R antagonist (pKi 4.17), a lead to be optimized in terms of potency and selectivity.     

Characterization of NPY receptor subtypes Y2 and Y7 in rainbow trout Oncorhynchus mykiss

We report the cloning and pharmacological characterization of two neuropeptide Y (NPY) receptor subtypes, Y2 and Y7, in rainbow trout (Oncorhynchus mykiss). These subtypes are approximately 50% identical to each other and belong to the Y2 subfamily of NPY receptors. The binding properties of the receptors were investigated after expression in human HEK-293 EBNA cells. Both receptors bound the three zebrafish peptides NPY, PYYa, and PYYb, as well as porcine NPY and PYY, with affinities in the nanomolar range that are similar to mammalian Y2. The affinity of the truncated porcine NPY fragments, NPY 13-36 and NPY 18-36 was markedly lower compared to mammalian and chicken Y2. This suggests that mammalian and chicken Y2 are unique among NPY receptors in their ability to bind truncated peptide fragments. The antagonist BIIE0246, developed for mammalian Y2, did not bind either of the two rainbow trout receptors. Our results support the proposed expansion of this gene family by duplications before the gnathostome radiation. They also reveal appreciable differences in the repertoire and characteristics of NPY receptors between fish and tetrapods stressing the importance of lineage-specific gene loss as well as sequence divergence after duplication.