Ingrid Lundell

Origins of the many NPY-family receptors in mammals

The NPY system has a multitude of effects and is particularly well known for its role in appetite regulation. We have found that the five presently known receptors in mammals arose very early in vertebrate evolution before the appearance of jawed vertebrates 400 million years ago. The genes Y(1), Y(2) and Y(5) arose by local duplications and are still present on the same chromosome in human and pig. Duplications of this chromosome led to the Y(1)-like genes Y(4) and y(6). We find evidence for two occasions where receptor subtypes probably arose before peptide genes were duplicated. These observations pertain to the discussion whether ligands or receptors tend to appear first in evolution. The roles of Y(1) and Y(5) in feeding may differ between species demonstrating the importance of performing functional studies in additional mammals to mouse and rat.

[125I]Leu31, Pro34-PYY is a High Affinity Radioligand for Rat PP1/Y4 and Y1 Receptors: Evidence for Heterogeneity in Pancreatic Polypeptide Receptors

Cloned receptors for the PP-fold peptides are subdivided into Y1, Y2, PP1/Y4, Y5 and Y6. NPY and PYY have similar affinity for Y1, Y2, Y5 and Y6 receptors while PP has highest affinity for PP1. Pro34-substituted analogs of NPY and PYY have selectivity for Y1 and Y1-like receptors over Y2 receptors. In the present study, we found the putative Y1-selective radioligand, [125I]Leu31, Pro34-PYY, also binds with high affinity to the rat PP1 receptor in cell lines expressing the receptor. However, in rat brain sections, [125I]Leu31, Pro34-PYY does not appear to bind to the interpeduncular nucleus, a brain region containing a high density of [125I]-bPP binding sites. Therefore, it appears there is additional heterogeneity in receptors recognizing PP.

Receptor subtypes Y1 and Y5 mediate neuropeptide Y induced feeding in the guinea-pig

Neuropeptide Y (NPY) is one of the most potent stimulants of food intake. It has been debated which receptor subtype mediates this response. Initially Y1 was proposed, but later Y5 was announced as a ‘feeding’ receptor in rats and mice. Very little is known regarding other mammals. The present study attempts to characterize the role of NPY in feeding behaviour in the distantly related guinea‐pig. When infused intracerebroventricularly, NPY dose‐dependently increased food intake. PYY, (Leu31,Pro34)NPY and NPY(2 – 36) stimulated feeding, whereas NPY(13 – 36) had no effect. These data suggest that either Y1 or Y5 receptors or both may mediate NPY induced food intake in guinea‐pigs. The Y1 receptor antagonists, BIBO 3304 and H 409/22 displayed nanomolar affinity for the Y1 receptor (Ki values 1.1±0.2 nM and 5.6±0.9 nM, respectively), but low affinity for the Y2 or Y5 receptors. When guinea‐pigs were pretreated with BIBO 3304 and H 409/22, the response to NPY was inhibited. The Y5 antagonist, CGP 71683A had high affinity for the Y5 receptor (Ki 1.3±0.05 nM) without having any significant activities at the Y1 and Y2 receptors. When CGP 71683A was infused into brain ventricles, the feeding response to NPY was attenuated. The present study shows that NPY stimulates feeding in guinea‐pigs through Y1 and Y5 receptors. As the guinea‐pig is very distantly related to the rat and mouse, this suggests that both Y1 and Y5 receptors may mediate NPY‐induced hyperphagia also in other orders of mammals.

Neuropeptide Y receptor subtype with unique properties cloned in the zebrafish: the zYa receptor

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.

Agonists for neuropeptide Y receptors Y1 and Y5 stimulate different phases of feeding in guinea pigs.

The stimulatory effect of neuropeptide Y (NPY) on food intake is well established but the roles of the receptor subtypes Y1 and Y5 have been difficult to define. We have studied the effects of two novel Y1‐preferring and two Y5‐preferring agonists on feeding in guinea pigs. The Y1‐preferring receptor agonists [Arg6,Pro34]pNPY and [Phe7,Pro34]pNPY had high affinity for the Y1 receptor (Ki values 0.07 and 0.04 nM, respectively) and nanomolar affinity for the Y5 receptor. Administration of either compound into the third brain ventricle increased food intake equally to NPY. The Y5 agonist [Ala31,Aib32]pNPY displayed a moderate affinity for the Y5 receptor (Ki 7.42 nM) and a low affinity for Y1 (Ki 1.7 μM). This compound had only a modest effect on feeding. The other Y5‐preferring peptide [cPP1–7,NPY19–23,Ala31,Aib32,Gln34]hPP had a higher affinity at the Y5 receptor (Ki 1.32 nM) and also at the Y1 receptor (Ki 85 nM). It potently stimulated feeding: the food consumption after administration of this peptide was two‐fold compared to NPY. Our results support the view that both the receptor subtypes Y1 and Y5 are involved in the stimulation of feeding. As the action profiles of the Y1 and Y5 agonists on feeding parameters were different, it seems that they influence different phases of eating.

Agonist internalization by cloned Y1 neuropeptide Y (NPY) receptor in Chinese hamster ovary cells shows strong preference for NPY, endosome-linked entry and fast receptor recycling.

In Chinese hamster ovary (CHO) cells expressing the cloned guinea-pig Y1 receptor, the saturable, receptor-linked internalization of NPY (NPY)-related peptides showed the rank order of human/rat neuropeptide Y (hNPY)>pig/rat peptide YY (pPYY)>=(Pro(34))human PYY>(Leu(31),Pro(34))hNPY>(Leu(31),Pro(34))hPYY>>BVD-11 (a selective Y1 antagonist). All agonists accessed similar numbers of Y1 sites in particulates from disrupted cells, with relatively small affinity variation. The rate of internalization could significantly depend on the overall interactivity of the agonist peptide (reflected in sensitivity to chaotropic agents, as well as in the level of non-saturable binding and internalization). Concentration-dependent inhibition of the agonist-driven CHO-Y1 internalization was found with filipin III (a cholesterol-complexing macrolide), and confirmed with inhibitors of clathrin lattice formation, phenylarsine oxide (PAO) and sucrose. In the concentration range affecting Y1 internalization, none of the above treatments or agents significantly alter agonist affinity for Y1 cell surface or particulate receptors. Largely similar responses to the above inhibitors were observed in CHO-Y1 cells for internalization of human transferrin. Internalization of CHO-Y1 receptor apparently is driven by NPY in strong preference to other naturally encountered agonists. At 37 degrees C, most of the internalized receptor is rapidly recycled through endosome-like membrane elements, detectable in Percoll gradients.

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.

Complex gene organization of synaptic protein SNAP-25 in Drosophila melanogaster

The evolutionarily conserved protein SNAP-25 (synaptosome-associated protein 25 kDa (kilodaltons)) is a component of the protein complex involved in the docking and/or fusion of synaptic vesicles in nerve terminals. We report here that the SNAP-25 gene (Snap) in the fruit fly Drosophila melanogaster has a complex organization with eight exons spanning more than 120 kb (kilobases). The exon boundaries coincide with those of the chicken SNAP-25 gene (Bark, 1993). Only a single exon 5 has been found in Drosophila, whereas human, rat, chicken, zebrafish and goldfish have two alternatively spliced versions of this exon. In situ hybridization and immunocytochemistry to whole mount embryos show that SNAP-25 mRNA and protein are detected in stage 14 and later developmental stages, and are mainly localized to the ventral nerve cord. Thus, Snap has an evolutionarily conserved and complex gene organization, and its onset of expression in Drosophila melanogaster correlates with a time in neuronal development when synapses begin to be formed and when other synapse-specific genes are switched on.

Blockade of pancreatic polypeptide-sensitive neuropeptide Y (NPY) receptors by agonist peptides is prevented by modulators of sodium transport. Implications for receptor signaling and regulation

Ligand binding to rodent pancreatic polypeptide-responding neuropeptide Y (NPY) receptors (here termed PP/NPY receptors), or to cloned Y4 or Y5 receptors, is selectively inhibited by amiloride, peptide or alkylating modulators of sodium transport. The PP/NPY and Y4 receptors are also selectively blocked by human or rat pancreatic polypeptide (PP) and the blocking peptides are not dissociated by high concentrations of alkali chlorides (which restore most of the binding of subtype-selective agonists to Y1 and Y2 sites). The PP/NPY receptors could also be blocked by NPY and related full-length peptides, including Y1-selective agonists (IC50 300-400 pM). The cloned Y(4) receptors from three species are much less sensitive to NPY or PYY. The sensitivity of both the PP/NPY sites and the Y(4) sites to Y2-selective peptides is quite low. The ligand attachment to PP/NPY sites is also very sensitive to peptidic Y1 antagonist ((Cys31,NVal34NPY27-36))2, which however blocks these sites at much higher molarities. Blockade of PP/NPY and Y4 sites by agonist peptides can be largely prevented by N5-substituted amiloride modulators of Na+ transport, and by RFamide NRNFLRF.NH2, but not by Ca2+ channel blockers, or by inhibitors of K+ transport. Protection of both PP/NPY and Y4 sites against blockade by human or rat pancreatic polypeptide is also afforded by short N-terminally truncated NPY-related peptides. The above results are consistent with a stringent and selective activity regulation for rabbit PP/NPY receptor(s) that may serve to differentiate agonists and constrain signaling, and could involve transporter-like interactants.

Studies of the human, rat, and guinea pig Y4 receptors using neuropeptide Y analogues and two distinct radioligands.

The neuropeptide Y-family receptor Y4 differs extensively between human and rat in sequence, receptor binding, and anatomical distribution. We have investigated the differences in binding profile between the cloned human, rat, and guinea pig Y4 receptors using NPY analogues with single amino acid replacements or deletion of the central portion. The most striking result was the increase in affinity for the rat receptor, but not for human or guinea pig, when amino acid 34 was replaced with proline; [Ahx(8-20),Pro(34)]NPY bound to the rat Y4 receptor with 20-fold higher affinity than [Ahx(8-20)]NPY. Also, the rat Y4 tolerates alanine in position 34 since p[Ala(34)]NPY bound with similar affinity as pNPY while the affinity for hY4 and gpY4 decreased about 50-fold. Alanine substitutions in position 33, 35, and 36 as well as the large loop-deletion, [Ahx(5-24)]NPY, reduced the binding affinity to all three receptors more than 100-fold. NPY and PYY competed with (125)I-hPP at Y4 receptors expressed in CHO cells according to a two-site model. This was investigated for gpY4 by saturation with either radiolabeled hPP or pPYY. The number of high-affinity binding-sites for (125)I-pPYY was about 60% of the receptors recognized by (125)I-hPP. Porcine [Ala(34)]NPY and [Ahx(8-20)]NPY bound to rY4 (but not to hY4 or gpY4) according to a two-site model. These results suggest that different full agonists can distinguish between different active conformations of the gpY4 receptor and that Y4 may display functional differences in vivo between human, guinea pig, and rat.