Monitoring of the internalization of neuropeptide Y on neuroblastoma cell line SK-N-MC.

Neuropeptide Y (NPY) is an important neuromodulator in the central and peripheral nervous system. The peptide acts through different NPY receptor subtypes (Y1-Y5, y6) that belong to the family of G protein-coupled receptors. In general, cellular responses to prolonged exposure to agonists of G protein-coupled receptors are attenuated, often through internalization of the receptors and their bound ligands. In this study, a fluorescent labeled NPY derivative was synthesized and characterized to investigate the internalization of NPY in the human neuroblastoma cell line SK-N-MC. Internalization was proven by binding experiments and subsequent acidic washing as well as by direct visualization by means of confocal laser scanning microscopy. Approximately 20-30% of the fluorescent labeled NPY and a tritium-marked NPY were resistant to acid removal of cell surface-bound ligands indicating internalization. Extracellular fluorescent labeled NPY was found to be distributed heterogeneously in a clustered pattern, which suggests that the ligand-receptor complex is collected in pits and caveolae followed by endocytosis.     

Evidence of NPY Y5 receptor involvement in food intake elicited by orexin A in sated rats

Intracerebroventricular (icv) injections of orexin A stimulate feeding in sated rats. Since neuropeptide Y is a potent orexigenic peptide and orexin-containing neurons are morphologically linked with NPY-producing neurons in the hypothalamus, we evaluated the functional relationship between the two orexigenic peptides. The results show that whereas it was ineffective on its own, a selective NPY Y5 receptor antagonist, injected icv 15 min. before orexin A significantly suppressed orexin A-induced feeding. Since previous investigations demonstrated that an NPY Y1 receptor antagonist also inhibits feeding induced by orexin A, the current results further underscore the existence of a functional link between orexin and NPY producing neurons as the orexin network appears to be capable of influencing NPYergic signaling through Y1 and Y5 receptors to stimulate feeding.     

The SK-N-MC cell line expresses an orexin binding site different from recombinant orexin 1-type receptor.

Orexin A and B (also known as hypocretins), two recently discovered neuropeptides, play an important role in food intake, sleep/wake cycle and neuroendocrine functions. Orexins are endogenous ligands of two G-protein-coupled receptors, termed OX1 and OX2. This work presents the first short orexin A and B analogues, orexin A 23-33 and orexin B 18-28, with high affinity (119 +/- 49 and 49 +/- 23 nm) for OX1 receptors expressed on SK-N-MC cells and indicates the importance of the C-terminal part of the orexin peptides for this ligand-receptor interaction. However, these C-terminal fragments of orexin did not displace the 125I-labelled orexin B from the recombinant orexin 1 receptor stably expressed in Chinese hamster ovary cells. To examine the role of the shortened orexin A 23-33 in feeding, its effects in mimicking or antagonizing the effects of orexin A were studied in rats after administration via the lateral hypothalamus. In contrast with orexin A, which potently induced feeding up to 4 h after administration, orexin A 23-33 neither induced feeding nor inhibited orexin A-induced feeding. Modafinil (Vigil), which was shown earlier to activate orexin neurons, displayed binding neither to the orexin receptor expressed on SK-N-MC cells nor to the recombinant orexin 1 receptor, which indicates that modafinil displays its antinarcoleptic action via another yet unknown mechanism. PCR and subsequent sequencing revealed expression of the full-length orexin 1 receptor mRNA in SK-N-MC and NT-2 cells. Interestingly, sequencing of several cDNA clones derived from RNA of both SK-N-MC and NT-2 cells differed from the published nucleotide sequence at position 1375. Amino acid prediction of this A -->G change results in an isoleucine --> valine substitution at the protein level, which may provide evidence for an editing process.     

Identification of cultured cells selectively expressing Y1-, Y2-, or Y3-type receptors for neuropeptide Y/peptide YY.

Neuropeptide Y (NPY) and peptide YY (PYY) are homologous 36 amino acid amidated peptides that often, but not always, exert similar actions and binding profiles. The present study of cultured cells confirms that both peptides as well as radioiodinated analogs, i.e. 125I-Bolton-Hunter-NPY (125I-BH-NPY) and 125I-peptide YY (125I-PYY), show high affinity to binding sites/receptors of the previously proposed Y1- and Y2-subtypes, selectively expressed by the human neuroblastoma cell lines, SK-N-MC and SK-N-BE(2), respectively. In contrast, bovine adrenal chromaffin cells did not bind 125I-PYY, while displaying high affinity 125I-BH-NPY sites, and may therefore represent a cell type expressing a recently proposed Y3-type of (NPY-preferring) receptors. Several non-labeled fragments/analogs have been used in displacement experiments to further characterize the structural requirements for Y1-, Y2-, and Y3-type binding. In every instance, specific binding was reduced by addition of 5'-guanylylimidodiphosphate [Gpp(NH)p], indicating that the three receptor subtypes belong to the G-protein-coupled superfamily of receptors. Moreover, in both neuroblastoma cell lines, the peptides elicited, with appropriate orders of potency, reduction of forskolin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Finally, NPY-evoked 45Ca2+ influx was observed in SK-N-MC and in chromaffin cells. A common dual coupling mechanism of NPY/PYY receptors, i.e. to reduction of cAMP and to Ca2+ elevation, is therefore suggested to exist, although both phenomena could not be demonstrated in every cell type.     

Characterization of Functional Neuropeptide Y Receptors in a Human Neuroblastoma Cell Line

We identified receptors for neuropeptide Y (NPY) on an established human neuroblastoma cell line, SK-N-MC, which are functionally coupled to adenylate cyclase through the inhibitory guanine nucleotide-binding protein of adenylate cyclase, Gi. Intact SK-N-MC cells bound radiolabeled NPY with a KD of 2 nM and contained approximately 83,000 receptors/cell. Unlabeled porcine and human NPY and structurally related porcine peptide YY (PYY) competed with labeled NPY for binding to the receptors. NPY inhibited cyclic AMP accumulation in SK-N-MC cells stimulated by isoproterenol, dopamine, vasoactive intestinal peptide, cholera toxin, and forskolin. NPY inhibited isoproterenol-stimulated cyclic AMP production in a dose-dependent manner, with half-maximal inhibition at 0.5 nM NPY. Porcine and human NPY and porcine PYY gave similar dose-response curves. NPY also inhibited basal and isoproterenol-stimulated adenylate cyclase activity in disrupted cells. Pertussis toxin treatment of the cells completely blocked the ability of NPY to inhibit cyclic AMP production and adenylate cyclase activity. The toxin catalyzed the ADP-ribosylation of a 41-kDa protein in SK-N-MC cells that corresponds to Gi. The receptors on SK-N-MC cells appeared to be specific for NPY, as other neurotransmitter drugs, such as alpha-adrenergic, dopaminergic, muscarinic, and serotonergic antagonists, did not compete for either NPY binding or NPY inhibition of adenylate cyclase. Thus, SK-N-MC cells may be a useful model for investigating NPY receptors and NPY-mediated signal transduction.     

A Y2 receptor mimetic aptamer directed against neuropeptide Y.

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

A neuropeptide Y receptor Y1-subfamily gene from an agnathan, the European river lamprey. A potential ancestral gene.

We report here the isolation and functional expression of a neuropeptide Y (NPY) receptor from the river lamprey, Lampetra fluviatilis. The receptor displays approximately 50% amino-acid sequence identity to all previously cloned Y1-subfamily receptors including Y1, Y4, and y6 and the teleost subtypes Ya, Yb and Yc. Phylogenetic analyses point to a closer relationship with Y4 and Ya/b/c suggesting that the lamprey receptor could possibly represent a pro-orthologue of some or all of those gnathostome receptors. Our results support the notion that the Y1 subfamily increased in number by genome or large-scale chromosome duplications, one of which may have taken place prior to the divergence of lampreys and gnathostomes whereas the second duplication probably occurred in the gnathostome lineage after this split. Functional expression of the lamprey receptor in a cell line facilitated specific binding of the three endogenous lamprey peptides NPY, peptide YY and peptide MY with picomolar affinities. Binding studies with a large panel of NPY analogues revealed indiscriminate binding properties similar to those of another nonselective Y1-subfamily receptor, zebrafish Ya. RT-PCR detected receptor mRNA in the central nervous system as well as in several peripheral organs suggesting diverse functions. This lamprey receptor is evolutionarily the most distant NPY receptor that clearly belongs to the Y1 subfamily as defined in mammals, which shows that subtypes Y2 and Y5 arose even earlier in evolution.     

Distinct roles for Galphai2, Galphai3, and Gbeta gamma in modulation offorskolin- or Gs-mediated cAMP accumulation and calcium mobilization by dopamine D2S receptors

Previous studies have shown that a single G protein-coupled receptor can regulate different effector systems by signaling through multiple subtypes of heterotrimeric G proteins. In LD2S fibroblast cells, the dopamine D2S receptor couples to pertussis toxin (PTX)-sensitive Gi/Go proteins to inhibit forskolin- or prostaglandin E1-stimulated cAMP production and to stimulate calcium mobilization. To analyze the role of distinct Galphai/o protein subtypes, LD2S cells were stably transfected with a series of PTX-insensitive Galphai/o protein Cys --> Ser point mutants and assayed for D2S receptor signaling after PTX treatment. The level of expression of the transfected Galpha mutant subunits was similar to the endogenous level of the most abundant Galphai/o proteins (Galphao, Galphai3). D2S receptor-mediated inhibition of forskolin-stimulated cAMP production was retained only in clones expressing mutant Galphai2. In contrast, the D2S receptor utilized Galphai3 to inhibit PGE1-induced (Gs-coupled) enhancement of cAMP production. Following stable or transient transfection, no single or pair set of mutant Galphai/o subtypes rescued the D2S-mediated calcium response following PTX pretreatment. On the other hand, in LD2S cells stably transfected with GRK-CT, a receptor kinase fragment that specifically antagonizes Gbeta gamma subunit activity, D2S receptor-mediated calcium mobilization was blocked. The observed specificity of Galphai2 and Galphai3 for different states of adenylyl cyclase activation suggests a higher level of specificity for interaction of Galphai subunits with forskolin- versus Gs-activated states of adenylyl cyclase than has been previously appreciated.     

Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase

Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused a rapid and transient increase in the concentration of free calcium in the cytoplasm as measured by the fluorescent probe, Fura-2. The effect of both peptides was independent of extracellular calcium as addition of EGTA or manganese neither changed the size nor the shape of the calcium response. The calcium response to NPY was abolished by pretreatment with thapsigargin, which can selectively deplete a calcium store in the endoplasmic reticulum. Y1 receptor stimulation, by both NPY and [Leu31,Pro34]NPY, also inhibited the forskolin-stimulated cAMP production with an EC50 of 3.5 nM. There was a close relation between the receptor binding and the cellular effects as half-maximal displacement of [125I-Tyr36]monoiodoNPY from the receptor was obtained with 2.1 nM NPY. The Y2-specific ligand NPY(16-36)peptide had no effect on either intracellular calcium or cAMP levels in the SK-N-MC cells. It is concluded that Y1 receptor stimulation is associated with both mobilization of intracellular calcium and inhibition of adenylate cyclase activity.     

Acute exercise alters Galphai2 protein expressions through the ubiquitin-proteasome proteolysis pathway in rat adipocytes

The effects of acute exercise on the protein expressions of heterotrimeric G protein alpha subunits were examined in rat adipocytes. Galphai2 protein expression was significantly reduced 0 and 3h after exercise but increased 24h after exercise, without alterations in Galphai2 mRNA expressions. The protein expressions of other alpha subunits, Galphas, Galphai1, and Galphai3, were not influenced. Both the 26S proteasome activity and polyubiquitination of Galphai2 protein were significantly increased 0 and 3h after exercise. Whereas, proteasome activity was decreased, and the polyubiquitination of Galphai2 protein was returned to the control level 24h after exercise. The reductions in Galphai2 protein expressions 0 and 3h after exercise were completely prevented by the injection either of a proteasome inhibitor or of a beta-adrenergic receptor blocker prior to exercise. Thus, acute exercise altered the expression of Galphai2 protein via mechanisms which involve the coupling of beta-adrenergic receptors to an agonist with subsequent ubiquitin-proteasome-dependent proteolysis.     

Mediation of adenylyl cyclase sensitization by PTX-insensitive GalphaoA, Galphai1, Galphai2 or Galphai3

Chronic activation of mu-opioid receptors, which couple to pertussis toxin-sensitive Galphai/o proteins to inhibit adenylyl cyclase (AC), leads to a compensatory sensitization of AC. Pertussis toxin-insensitive mutations of Galphai/o subtypes, in which the pertussis toxin-sensitive cysteine is mutated to isoleucine (Galpha ), were used to determine whether each of the Galphai/o subtypes is able to mediate sensitization of AC. Galpha , G , G or G were individually transiently transfected into C6 glioma cells stably expressing the mu-opioid receptor, or transiently co-expressed with the mu-opioid receptor into human embryonic kidney (HEK)293T cells. Cells were treated with pertussis toxin to uncouple endogenous Galphai/o proteins, followed by acute or chronic treatment with the mu-opioid agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO). Each Galphai/o subtype mediated acute DAMGO inhibition of AC and DAMGO-induced sensitization of AC. The potency for DAMGO to stimulate sensitization was independent of the Galphai/o subtype, but the level of sensitization was increased in clones expressing higher levels of Galphai/o subunits. Sensitization of AC mediated by a component of fetal bovine serum, which was also dependent on the level of functional Galphai/o subunits in the cell, was observed. This serum-mediated sensitization partially masked mu-opioid-mediated sensitization when expressed as percentage overshoot due to an apparent increase in AC activity.     

Evidence that NPY Y1 receptors are involved in stimulation of feeding by orexins (hypocretins) in sated rats

Neuropeptide Y (NPY) produced in the arcuate nucleus (ARC) of the hypothalamus stimulates feeding both directly by activating NPY receptors and indirectly through release of the orexigenic peptides, galanin and beta-endorphin (beta-END), in the paraventricular nucleus (PVN) and surrounding neural sites. Orexin A and orexin B, produced outside the ARC in the lateral hypothalamic area (LH), have recently been shown to stimulate feeding. In the present studies we tested the hypothesis that NPYergic signaling may mediate feeding stimulated by orexins. In adult male rats injected intracerebroventricularly (i.c.v.) with orexin A (3, 10, 15 nmol) or orexin B (3, 10, 30 nmol) feeding was stimulated in a dose-dependent manner; maximal feeding was seen after 15 nmol orexin A and 30 nmol orexin B. To determine whether NPY may mediate this orexin stimulated feeding, we used 1229U91, a selective NPY Y1 receptor antagonist (NPY-A). Whereas NPY-A on its own was ineffective, it suppressed NPY-induced feeding. Furthermore, NPY-A completely blocked the feeding evoked by either orexin A (15 nmol) or orexin B (30 nmol). These results show that orexin A and B stimulate feeding and further suggest that these excitatory effects may be mediated by NPYergic signaling through Y1 receptors. These findings are in accord with the view that the orexin-NPY pathway may comprise a functional link upstream from NPY within the hypothalamic appetite regulating network.     

NPY receptor subtype specification for behavioral adaptive strategies during limited food access

The neuropeptide Y (NPY) system in the brain regulates a wide variety of behavioral, metabolic and hormonal homeostatic processes required for energy balance control. During times of limited food availability, NPY promotes behavioral hyperactivity necessary to explore and prepare for novel food resources. As NPY can act via 5 different receptor subtypes, we investigated the path through which NPY affects different behavioral components relevant for adaptation to such conditions. We tested NPY Y1 and Y2 receptor knockout mice and their wild-type littermate controls in a daily scheduled limited food access paradigm with unlimited access to running wheel. Here we show that NPY Y1 receptor deficient mice lack the expression of appetitive behavior and that NPY Y2 receptors control the level of hyperactive behavior under these conditions. Thus, receptor specificity determines the differential expression of NPY-mediated behavioral adaptations to overcome a negative energy status.     

Self-regulation of agonist activity at the Y receptors

Neuropeptide Y (NPY) is one of the most abundant neuropeptides, and is likely to be present at nanomolar levels over extended periods in the synaptic space of many forebrain areas. This might be linked to an evolved generalized toning activity through a number of other peptide receptors that use C-terminally amidated agonists (with LHRH and orexin receptors and GIR as examples). However, the Y1 and Y2 receptors (which constitute the bulk of Y receptors active in the neural matrix) possess subnanomolar affinities that, at saturating NPY levels, could produce excessive signaling, as well as receptor losses via repeated endocytosis. The related Y4 receptor shows an even higher agonist affinity, and faces the same problem in visceral and neural locations accessible to pancreatic polypeptide (PP). An examination of agonist peptide interaction with Y receptors shows that Y1 and Y4 receptors in particular (as located on either the intact cells, or on particulates derived from various cell types) develop a blockade dependent on ligand concentration, with the blocking ranks of [NPY]>[peptide YY] (PYY) for the Y1, and [human PP]>[PYY-related Y4 agonist] for the Y4 receptor. This blockade is also echoed in a concentration-related reduction in biological activity of primary agonists (NPY and PP), resembling a partial agonism, and is influenced especially by the allosteric interactivity of agonists. With the Y2 receptor, the blocking by agonists is less pronounced, but the signaling by NPY-related peptides is apparently less than with PYY-related agonists. The extended occupancy and self-attenuation of primary agonist activity at Y receptors could represent an evolutionary solution contributing to a balancing of metabolic signaling, agonist clearance and receptor conservation.     

Cloning and characterization of Rhesus monkey neuropeptide Y receptor subtypes

Neuropeptide Y (NPY) is a 36 amino acid peptide that is abundant in the brain and peripheral nervous system. NPY has a variety of effects when administered into the brain including a pronounced feeding effect, anxiolysis, regulation of neuroendocrine axes and inhibition of neurotransmitter release. These effects are mediated by up to 6 G protein coupled receptors designated Y1, Y2, Y3, Y4, Y5 and y6. To better understand the phylogeny and pharmacology of NPY in non-human primates, we have cloned and expressed the NPY Y1, Y2 and Y5 receptor subtypes from the Rhesus monkey. No cDNA sequence encoding a Y4 receptor was found suggesting substantial sequence differences when compared to the human sequence. Comparison of these sequences with those from human indicated strong sequence conservation of Y1, Y2 and Y5 between the two species. The displacement of (125)I-PYY binding to the Rhesus monkey and human receptors by various peptides was compared to evaluate the pharmacology of the two species. Similar pharmacologies were noted across the species at the various receptor subtypes. These results indicate the Rhesus monkey and human NPY receptor subtypes have a close amino acid sequence conservation and that the peptide recognition domains are conserved as well.