Ligands of the neuropeptide Y Y2 receptor

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

Angiotensin signaling and receptor types in teleost fish

Despite advances characterizing mammalian angiotensin receptors, the phylogeny of fish angiotensin receptors remains unclear. Three aspects of receptor function: (1) the nature of the ligand; (2) the second messenger system activated by it; and (3) the pharmacological profile of specific antagonists, are examined to provide insight into the fish receptor. (1) The octapeptide sequences of fish and mammalian angiotensin II (ANG II) are nearly homologous, differing only at the first and fifth residues. Both peptides are almost equally efficacious and equipotent in heterologous systems and both contain key agonist switches Tyr(4) and Phe(8) necessary to activate mammalian AT(1)-type receptors. (2) ANG II increases inositol trisphosphate production, and elevates intracellular calcium in fish tissues consistent with activation of the AT(1) receptor. (3) However, the specific mammalian sartan-type AT(1) antagonists, e.g. losartan, produce inconsistent results in fish often acting as partial agonists, or inhibiting only at elevated concentrations. Because sartans and ANG II act at distinct sites on the AT(1) receptor, we propose that the teleost receptor is an AT(1)-type receptor that is fairly well conserved with respect to both the ANG binding site and coupling to the second messenger system, whereas the sartan binding site has been poorly conserved. The evidence for non-AT(1) type ANG II receptors in teleosts is limited. Mammalian AT(2) receptor antagonists are generally ineffective but may block at elevated, non-specific doses. Truncated ANG II fragments, ANG III and ANG IV, are often less potent than ANG II, however, their receptors have not been examined. Preliminary studies in trout indicate that angiotensin 1-7 may have a mild vasodilatory effect; additional work is needed to determine if non-AT(1)-type receptors are involved.     

Birth and death of orphan genes in Rickettsia

The origin and evolution of the thousands of species-specific genes with unknown functions, the so-called orphan genes, has been a mystery. Here, we have studied the rates and patterns of orphan sequence evolution, using the Rickettsia as our reference system. Of the Rickettsia conorii orphans examined in this study, 80% were found to be short gene fragments or fusions of short segments from neighboring genes. We reconstructed the putative sequences of the full-length genes from which the short orphan fragments are thought to have originated. One of the genes thus reconstructed displays weak similarity to the ankyrin-repeat protein family, an identification that is strongly supported by comparative molecular modeling. Studies of the patterns of gene fragmentation underscore the importance of short repeated sequences as targets for recombination events that result in sequence loss and the formation of short, transient open reading frames. Our analysis demonstrates that gene sequences present in the common ancestor can be inferred even in cases when no full-length open reading frame is present in any of the contemporary species. Such reconstructions support the identification of lost protein functions and hint at important lifestyle changes.     

Cloning and characterization of the guinea pig neuropeptide Y receptor Y5

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

Surface masking shapes the traffic of the neuropeptide Y Y2 receptor

The neuropeptide Y (NPY) Y2 receptor shows a large masked surface population in adherent CHO cells or in forebrain cell aggregates, but not in dispersed cells or in particulates from these sources. This is related to adhesion via acidic motifs in the extracellular N-terminal domain. Masking of the Y2 receptor is lifted by non-permeabilizing mechanical dispersion of cells, which also increases internalization of Y2 agonists. Mechanical dispersion and detachment by EDTA expose the same number of surface sites. As we have already shown, phenylarsine oxide (PAO), a cysteine-bridging agent, and to a lesser extent also the cysteine alkylator N-ethylmaleimide, unmask the surface Y2 sites without cell detachment or permeabilization. We now demonstrate that unmasking by permeabilizing but non-detaching treatment with cholesterol-binding detergents digitonin and edelfosine compares with and overlaps that of PAO. The caveolar/raft cholesterol-targeting macrolide filipin III however produces only partial unmasking. Depletion of the surface sites by N-terminally clipped Y2 agonists indicates larger accessibility for a short highly helical peptide. These findings indicate presence of a dynamic masked pool including majority of the cell surface Y2 receptors in adherent CHO cells. This compartmentalization is obviously involved in the low internalization of Y2 receptors in these cells.     

Structure-activity relationships of neuropeptide Y Y1 receptor antagonists related to BIBP 3226

Analogues of BIBP 3226, (R)-N(alpha)-diphenylacetyl-N-(4-hydroxybenzyl)argininamide, were synthesized and investigated for Y1 antagonism (Ca2+-assay, HEL cells) and binding on Y1, Y2 and Y5 receptors. Replacing the benzylamino by a tetrahydrobenzazepinyl group preserves most of the Y1 activity. Combination with a N(G)-phenylpropyl arginine and a N(alpha)-p-biphenylylacetyl moiety shifted the NPY receptor selectivity towards Y5.     

Multiple and highly divergent IL-11 genes in teleost fish

Interleukin-11 (IL-11) is a key cytokine in the regulation of proliferation and differentiation of hematopoietic progenitors and is also involved in bone formation, adipogenesis, and protection of mucosal epithelia. Despite this prominent role in diverse physiological processes, IL-11 has been described in only four mammalian species, and recently, in rainbow trout (Oncorhynchus mykiss). Here we report the presence of IL-11 in common carp (Cyprinus carpio), a bony fish species related to zebrafish. IL-11 is expressed in most carp organs and tissues. In vitro expression of IL-11 in cultured macrophages is enhanced by stimulation with lipopolysaccharide and is markedly inhibited by cortisol. A detailed and systematic scan of several fish genome databases confirms that IL-11 is present in all fish, but also reveals the presence of a second, substantially different IL-11 gene in the genomes of phylogenetically distant fish species. We designated both fish paralogues IL-11a and IL-11b. Although sequence identity between fish IL-11a and IL-11b peptides is low, the conservation of their gene structures supplemented by phylogenetic analyses clearly illustrate the orthology of both IL-11a and IL-11b genes of fish with mammalian IL-11. The presence of IL-11 genes in fish demonstrates its importance throughout vertebrate evolution, although the presence of duplicate and divergent IL-11 genes differs from the single IL-11 gene that exists in mammals.     

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

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

Reduced anxiety-like and depression-related behavior in neuropeptide Y Y4 receptor knockout mice

Neuropeptide Y (NPY) acting through Y1 receptors reduces anxiety- and depression-like behavior in rodents, whereas Y2 receptor stimulation has the opposite effect. This study addressed the implication of Y4 receptors in emotional behavior by comparing female germ line Y4 knockout (Y4−/−) mice with control and germ line Y2−/− animals. Anxiety- and depression-like behavior was assessed with the open field (OF), elevated plus maze (EPM), stress-induced hyperthermia (SIH) and tail suspension tests (TST), respectively. Learning and memory were evaluated with the object recognition test (ORT). In the OF and EPM, both Y4−/− and Y2−/− mice exhibited reduced anxiety-related behavior and enhanced locomotor activity relative to control animals. Locomotor activity in a familiar environment was unchanged in Y4−/− but reduced in Y2−/− mice. The basal rectal temperature exhibited diurnal and genotype-related alterations. Control mice had temperature minima at noon and midnight, whereas Y4−/− and Y2−/− mice displayed only one temperature minimum at noon. The magnitude of SIH was related to time of the day and genotype in a complex manner. In the TST, the duration of immobility was significantly shorter in Y4−/− and Y2−/− mice than in controls. Object memory 6 h after initial exposure to the ORT was impaired in Y2−/− but not in Y4−/− mice, relative to control mice. These results show that genetic deletion of Y4 receptors, like that of Y2 receptors, reduces anxiety-like and depression-related behavior. Unlike Y2 receptor knockout, Y4 receptor knockout does not impair object memory. We propose that Y4 receptors play an important role in the regulation of behavioral homeostasis.     

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

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

Birth and death of duplicated genes in completely sequenced eukaryotes

Gene and genome duplications are commonly regarded as being of major evolutionary significance. But how often does gene duplication occur? And, once duplicated, what are the fates of duplicated genes? How do they contribute to evolution? In a recent article, Lynch and Conery analyze divergence between duplicate genes from six eukaryotic genomes. They estimate the rate of gene duplication, the rate of gene loss after duplication and the strength of selection experienced by duplicate genes. They conclude that although the rate of gene duplications is high, so is the rate of gene loss, and they argue that gene duplications could be a major factor in speciation.     

Conformation of neuropeptide Y receptor antagonists: structural implications in receptor selectivity

Two NPY analogue peptides, BVD10 (Ile-Asn-Pro-Ile-Tyr-Arg-Leu-Arg-Tyr-OMe) and BVD15 (Ile-Asn-Pro-Ile-Tyr-Arg-Leu-Arg-Tyr-NH(2)) were characterized conformationally by NMR, CD and molecular dynamics simulations. The two peptides exhibit different secondary structure characteristics in trifluoroethanol. BVD10 exhibits a structure with two consecutive beta-turns at Asn2-Pro3-Ile4-Tyr5 and Ile4-Tyr5-Arg6-Leu7. BVD15 exhibits a helical type of structure along with a beta-turn at Asn2-Pro3-Ile4-Tyr5. Molecular modeling studies suggested that the C-terminus Tyr9 is oriented in different directions in the two peptides. The difference in the structures of peptides observed may contribute to the Y(1) selectivity of BVD10 relative to BVD15.     

Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y2(1)

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.     

Association of neuropeptide Y receptor Y5 polymorphisms with dyslipidemia in Mexican Americans

We examined the genetic association of neuropeptide Y receptor Y5 (NPY5R) single nucleotide polymorphisms (SNPs) with measures of the insulin resistance (metabolic) syndrome. We genotyped 10 NPY5R SNPs in 439 Mexican American individuals (age=43.3+/-17.3 years and BMI=30.0+/-6.7 kg/m2) distributed across 27 pedigrees from the San Antonio Family Diabetes Study and performed association analyses using the measured genotype approach as implemented in Sequential Oligogenic Linkage Analysis Routines (SOLAR). Minor alleles for five (rs11100493, rs12501691, P1, rs11100494, rs12512687) of the NPY5R SNPs were found to be significantly (p<0.05) associated with fasting plasma triglyceride concentrations and decreased high-density lipoprotein concentrations. In addition, the minor allele for SNP P2 was significantly associated (p=0.031) with a decreased homeostasis model assessment of beta-cell function (HOMA-%beta). Linkage disequilibrium between SNP pairs indicated one haplotype block of five SNPs (rs11100493, rs12501691, P1, rs11100494, rs12512687) that were highly correlated (r2>0.98). These preliminary results provide evidence for association of SNPs in the NPY5R gene with dyslipidemia (elevated triglyceride concentrations and reduced high-density lipoprotein levels) in our Mexican American population.     

Role of sulfhydryl groups in Y2 neuropeptide Y receptor binding activity.

Benextramine, a tetramine disulfide, irreversibly inhibits neuropeptide Y (NPY) binding to the 50-kDa Y2 NPY receptor in bovine hippocampus (Li, W., MacDonald, R. G., and Hexum, T. D. (1991) Eur. J. Pharmacol. 207, 89-91). Evidence is presented that this inhibition occurs through a thiol-disulfide exchange. Treatment of bovine hippocampal membranes with benextramine inhibited NPY affinity cross-linking to the 50-kDa receptor. This inhibition of labeling was not affected by washing the membranes, but could be completely reversed by the addition of several thiol reducing reagents, including reduced glutathione, beta-mercaptoethanol, and cysteine. Benextramine inhibited 70% of NPY-specific labeling and was much more effective than other sulfhydryl reactive agents, such as oxidized glutathione, cystamine, and 5,5'-dithio-bis(2-nitrobenzoic acid). Furthermore, the sulfhydryl-modifying agents N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid specifically decreased NPY affinity labeling. Finally, NPY labeling of the 50-kDa receptor was reduced by the heavy metal ions Zn2+, Cu2+, and Hg2+. Preincubation with NPY prevented Y2 receptors from being inactivated by either 400 microM N-ethylmaleimide or 1 mM benextramine. These results suggest that one or more benextramine-sensitive sulfhydryl groups on the Y2 receptor are important for NPY binding activity.     

The cloned guinea pig neuropeptide Y receptor Y1 conforms to other mammalian Y1 receptors.

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.     

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

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

Rapid internalization and recycling of the human neuropeptide Y Y(1) receptor.

Desensitization of G protein-coupled receptors (GPCRs) involves receptor phosphorylation and reduction in the number of receptors at the cell surface. The neuropeptide Y (NPY) Y(1) receptor undergoes fast desensitization. We examined agonist-induced signaling and internalization using NPY Y(1) receptors fused to green fluorescent protein (EGFP). When expressed in HEK293 cells, EGFP-hNPY Y(1) receptors were localized at the plasma membrane, desensitized rapidly as assessed using calcium responses, and had similar properties compared to hNPY Y(1) receptors. Upon agonist challenge, the EGFP signal decreased rapidly (t(1/2) = 107 +/- 3 s) followed by a slow recovery. This decrease was blocked by BIBP3226, a Y(1) receptor antagonist, or by pertussis toxin, in agreement with Y(1) receptor activation. Internalization of EGFP-hNPY Y(1) receptors to acidic endosomal compartments likely accounts for the decrease in the EGFP signal, being absent after pretreatment with monensin. Concanavalin A and hypertonic sucrose, which inhibit clathrin-mediated endocytosis, blocked the decrease in fluorescence. After agonist, intracellular EGFP signals were punctate and co-localized with transferrin-Texas Red, a marker of clathrin-associated internalization and recycling, but not with LysoTracker Red, a lysosomal pathway marker, supporting receptor trafficking to recycling endosomes rather than the late endosomal/lysosomal pathway. Pulse-chase experiments revealed no receptor degradation after internalization. The slow recovery of fluorescence was unaffected by cycloheximide or actinomycin D, indicating that de novo synthesis of receptors was not limiting. Use of a multicompartment model to fit our fluorescence data allows simultaneous determination of internalization and recycling rate constants. We propose that rapid internalization of receptors via the clathrin-coated pits recycling pathway may largely account for the rapid desensitization of NPY Y(1) receptors.