A number of receptors for the pancreatic polypeptide-fold peptides are proposed based on findings... more A number of receptors for the pancreatic polypeptide-fold peptides are proposed based on findings from pharmacology and molecular biology studies. Neuropeptide Y and peptide YY have similar affinity for neuropeptide Y Y~ and neuropeptide Y Y, while pancreatic polypeptide has highest affinity for pancreatic polypeptide 1. Pro34-substituted analogs of neuropeptide Y and peptide YY have selectivity for neuropeptide Y Yj over neuropeptide Y Y= receptors. In the present study, we found that one such 'neuropeptide Y Yt-selective' radioligand, [~=5I][Leu3~,Pro34]peptide YY, also binds with high affinity to the pancreatic polypeptide 1 receptor. Therefore, caution needs to be exercised when using Pro34-analogs to define the neuropeptide Y Y~ receptor in vivo and using tissue preparations.
1 Neuropeptide Y (NPY) is one of the most potent stimulants of food intake. It has been debated w... more 1 Neuropeptide Y (NPY) is one of the most potent stimulants of food intake. It has been debated which receptor subtype mediates this response. Initially Y 1 was proposed, but later Y 5 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. 2 PYY, (Leu 31 ,Pro 34 )NPY and NPY(2 ± 36) stimulated feeding, whereas NPY(13 ± 36) had no eect. These data suggest that either Y 1 or Y 5 receptors or both may mediate NPY induced food intake in guinea-pigs.
A peptide ending with RFamide (Arg-Phe-amide) was discovered independently by three different lab... more A peptide ending with RFamide (Arg-Phe-amide) was discovered independently by three different laboratories in 2003 and named 26RFa or QRFP. In mammals, a longer version of the peptide, 43 amino acids, was identified and found to bind to the orphan G protein-coupled receptor GPR103. We searched the genome database of Branchiostoma floridae (Bfl) for receptor sequences related to those that bind peptides ending with RFa or RYa (including receptors for NPFF, PRLH, GnIH, and NPY). One receptor clustered in phylogenetic analyses with mammalian QRFP receptors. The gene has 3 introns in Bfl and 5 in human, but all intron positions differ, implying that the introns were inserted independently. A QRFP-like peptide consisting of 25 amino acids and ending with RFa was identified in the amphioxus genome. Eight of the ten last amino acids are identical between Bfl and human. The prepro-QRFP gene in Bfl has one intron in the propeptide whereas the human gene lacks introns. The Bfl QRFP peptide was synthesized and the receptor was functionally expressed in human cells. The response was measured as inositol phosphate (IP) turnover. The Bfl QRFP peptide was found to potently stimulate the receptor's ability to induce IP turnover with an EC50 of 0.28nM. Also the human QRFP peptides with 26 and 43 amino acids were found to stimulate the receptor (1.9 and 5.1nM, respectively). Human QRFP with 26 amino acids without the carboxyterminal amide had dramatically lower potency at 1.3μM. Thus, we have identified an amphioxus QRFP-related peptide and a corresponding receptor and shown that they interact to give a functional response.
Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and acts in... more Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and acts in humans via at least three receptor subtypes: Y1, Y2, and Y5. Whereas selective agonists and antagonists are known for the Y2- and Y5-receptors, the Y1-receptor still lacks a highly selective agonist. This work presents the first NPY-based analogues with Y1-receptor preference and agonistic properties. Furthermore, the importance of specific amino acids of NPY for binding to the Y-receptor subtypes is presented. Amongst the analogues tested, [Phe7,Pro34]pNPY (where pNPY is porcine neuropeptide Y) showed the most significant Y1-receptor preference (> 1 : 3000-fold), with subnanomolar affinity to the Y1-receptor, and Ki values of approximately 30 nM for the Y2- and Y5-subtype, respectively. Variations of position 6, especially [Arg6,Pro34]pNPY and variations within positions 20-23 of NPY were found to result in further analogues with significant Y1-receptor preference (1 : 400-1 : 2000). In contrast, cyclo S-S [Cys20,Cys24]pNPY was found to be a highly selective ligand at the Y2-receptor, binding only threefold less efficiently than NPY. Analogues containing variations of positions 31 and 32 showed highly reduced affinity to the Y1-receptor, while binding to the Y5-receptor was affected less. Inhibition of cAMP-accumulation of selected peptides with replacements within position 20-23 of NPY showed preserved agonistic properties. The NPY analogues tested give insights into ligand-receptor interaction of NPY at the Y1-, Y2- and Y5-receptor and contribute to our understanding of subtype selectivity. Furthermore, the Y1-receptor-preferring peptides are novel tools that will provide insight into the physiological role of the Y1-receptor.
Guinea-pig neuropeptide Y 1 and rat pancreatic polypeptide Y 4 receptors expressed in Chinese ham... more Guinea-pig neuropeptide Y 1 and rat pancreatic polypeptide Y 4 receptors expressed in Chinese hamster ovary cells were internalized rapidly upon attachment of selective peptide agonists. The Y 1 and Y 2 , but not the Y 4 , receptor also internalized the nonselective neuropeptide Y receptor agonist, human/rat neuropeptide Y. The internalization of guinea-pig neuropeptide Y 2 receptor expressed in Chinese hamster ovary cells was small at 37 8C, and essentially absent at or below 15 8C, possibly in connection to the large molecular size of the receptor2ligand complexes (up to 400 kDa for the internalized fraction). The rate of intake was strongly temperature dependent, with essentially no internalization at 6 8C for any receptor. Internalized receptors were largely associated with light, endosome-like particulates. Sucrose dose-dependently decreased the internalization rate for all receptors, while affecting ligand attachment to cell membrane sites much less. Internalization of the Y 1 and the Y 4 receptors could be blocked, and that of the Y 2 receptor significantly inhibited, by phenylarsine oxide, which also unmasked spare cell-surface receptors especially abundant for the Y 2 subtype. The restoration of Y 1 and Y 4 receptors after agonist peptide pretreatment was decreased significantly by cycloheximide and monensin. Thus, in Chinese hamster ovary cells the Y 1 and Y 4 receptors have much larger subcellular dynamics than the Y 2 receptor. This differential could also hold in organismic systems, and is comparable with the known differences in internalization of angiotensin, bradykinin, somatostatin and opioid receptor subtypes. Abbreviations: CHO, Chinese hamster ovary cells; GPCR, G-protein coupled receptor; hNPY, human/rat neuropeptide Y; hPP, human pancreatic polypeptide; hPYY(3±36), human peptide YY(3±36); LP-PYY, (Leu 31 ,Pro 34 ) human peptide YY; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide; MTSET, 2-[(trimethylammonium)ethyl] methanethiosulfonate bromide; PAO, phenylarsine oxide; pPYY, porcine/rat peptide YY; rPP, rat pancreatic polypeptide.
Agonist stimulation readily internalizes neuropeptide Y receptor Y1 while there are contradictory... more Agonist stimulation readily internalizes neuropeptide Y receptor Y1 while there are contradictory results for the Y2 receptor. In order to explore putative functional differences between the Y1 and Y2 receptors we generated reciprocal chimeras by swapping the third intracellular loop, the carboxy terminus or both between human Y1 and Y2. Internalization was studied in a quantitative radioligand binding assay with removal of surface-bound ligand in an acidic-wash procedure. The internalization assay revealed a lower degree of internalization as well as slower kinetics for the Y2 receptor. Generally, reciprocal exchange of receptor segments did not convey properties of the donor receptor but tended to enhance internalization. Surprisingly, insertion of the Y2 carboxy terminus into Y1 gave almost complete internalization (92%), rather than reduced internalization, while the insertion of both segments resulted in internalization equal to the native Y1 receptor. These findings were confirmed by fluorescence microscopy of immuno-stained receptors tagged with a C-terminal FLAG epitope. However, after exposure to high agonist concentrations (100 nM) Y2 was internalized. Studies of Y2 and the closely related Y7 receptor confirmed low internalization for Y2 from chicken and teleost fishes as well as Y7 from two teleosts. The conservation across species of low internalization at physiological concentrations suggests that this is an ancient feature and of vital importance for Y2 function. We propose that amino acid motifs in the third intracellular loop as well as the C terminus of both Y1 and Y2 are able to drive agonist-promoted internalization and that there may be constraining motifs in the Y2 receptor.
In Chinese hamster ovary (CHO) cells expressing the cloned guinea-pig Y1 receptor, the saturable,... more 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 )hPYYHBVD-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 cholesterolcomplexing 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 jC, most of the internalized receptor is rapidly recycled through endosome-like membrane elements, detectable in Percoll gradients. D 0167-0115/02/$ -see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 -0 11 5 ( 0 2 ) 0 0 0 9 4 -0 Abbreviations: NPY, neuropeptide Y; PYY, peptide YY; hNPY, human/ rat neuropeptide Y; pPYY, porcine/rat peptide YY; hPYY, human peptide YY; hPP, human pancreatic polypeptide;
Neuropeptide Y (NPY) and peptide YY (PYY) share ∼ 70% of their 36 amino acids and bind to the sam... more Neuropeptide Y (NPY) and peptide YY (PYY) share ∼ 70% of their 36 amino acids and bind to the same three human receptor subtypes, Y1, Y2 and Y5, even though these receptors only share ∼ 30% sequence identity. Based on our previous investigation of human Y1 we describe here a mutagenesis study of three corresponding positions in human Y2, i.e. Tyr 2.64 , Val 6.58 and Tyr 7.31 . Pharmacological characterization was performed with the four peptide agonists PYY, NPY, PYY(3-36) and NPY(13-36) as well as the non-peptide antagonist BIIE0246. Results from mutants where Tyr 2.64 has been substituted by Ala suggest that Tyr 2.64 is involved in the interaction with all investigated ligands whereas position Tyr 7.31 seems to be more important for interaction with the truncated peptide PYY(3-36) than with intact NPY. Surprisingly, substitution of Tyr 7.31 with His, the corresponding residue in Y1, resulted in total loss of binding of iodinated porcine PYY. The third position, Val 6.58 , did not influence binding of any of the ligands. These findings differ from those obtained for Y1 where Ala substitution resulted in lost or changed binding for each of the three positions. Although Tyr 2.64 and Tyr 7.31 in Y2 are involved in ligand binding, their interactions with the peptide ligands seem to be different from the corresponding positions in Y1. This suggests that the receptor-ligand interactions have changed during evolution after Y1 and Y2 arose from a common ancestral receptor.
Proceedings of the National Academy of Sciences, 1992
Neuropeptide Y (NPY) is an abundant and widespread neuropeptide in the nervous system of mammals.... more Neuropeptide Y (NPY) is an abundant and widespread neuropeptide in the nervous system of mammals. NPY belongs to a family of 36-amino acid peptides that also includes pancreatic polypeptide and the endocrine gut peptide YY as well as the fish pancreatic peptide Y. To study the evolution of this peptide family, we have isolated clones encoding NPY from central nervous system cDNA libraries of chicken, goldfish, and the ray Torpedo marmorata, as well as from a chicken genomic library. The predicted chicken NPY amino acid sequence differs from that of rat at only one position. The goldfish sequence differs at five positions and shows that bony fishes have a true NPY peptide in addition to their pancreatic peptide Y. The Torpedo sequence differs from that of rat at three positions. As Torpedo NPY has no unique positions when compared with the other sequences, it seems to be identical to the NPY of the common ancestor ofcartilaginous fishes, bony fishes, and tetrapods after 420 million years of evolution. The 30-amino acid carboxyl-terminal extension of the NPY precursor also displays considerable sequence conservation. These results show that NPY is one ofthe most highly conserved neuroendocrine peptides.
Proceedings of the National Academy of Sciences, 1996
Pancreatic polypeptide (PP) is produced in the islets of Langerhans and released in response to m... more Pancreatic polypeptide (PP) is produced in the islets of Langerhans and released in response to meals. It belongs to a family of peptides that also includes neuropeptide Y and peptide YY. In the present communication, we describe a rat receptor with high affinity for PP, therefore named PP1. Clones for the PP1 receptor were obtained by PCR using sequence information for the neuropeptide Y receptor Y1 from several species. The PP1 receptor has 46% overall amino acid sequence identity to the rat Y1 receptor and 56% identity in the transmembrane regions. The PP1 receptor displays a pharmacological profile that is distinct from previously described neuropeptide Y-family receptors. In competition with iodinated bovine PP, it binds rat PP with an affinity (K*) of 0.017 nM, while the affinities for peptide YY and neuropeptide Y are substantially lower with K; values of 162 and 192 nM, respectively. In stably transfected CHO cells, the PP1 receptor inhibits forskolin-stimulated cAMP synthesis. Northern blot hybridizations to a panel of mRNAs detected transcripts in testis and lung. A faint band was seen in colon and total brain. In contrast, the human receptor is expressed primarily in colon and small intestine. Whereas rat and human PP1 bind PP with the same affinity, the rat receptor has much lower affinity than its human ortholog for peptide YY and neuropeptide Y. Interestingly, the amino acid sequence identity between rat and human PP1 is only 75%. Thus, the sequence, the tissue distribution, and the binding profile of the PP1 receptor differ considerably between rat and human.
The Y5 receptor has been postulated to be the main receptor mediating NPY-induced food intake in ... more 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 ϭ NPY ϭ NPY2-36 Ͼ gpPP Ͼ rPP Ͼ Ͼ 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.
The NPY system has a multitude of effects and is particularly well known for its role in appetite... more 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.
Ligand binding to rodent pancreatic polypeptide-responding neuropeptide Y (NPY) receptors (here t... more Ligand binding to rodent pancreatic polypeptide-responding neuropeptide Y (NPY) receptors (here termed PP/NPY receptors), or to cloned Y 4 or Y 5 receptors, is selectively inhibited by amiloride, peptide or alkylating modulators of sodium transport. The PP/NPY and Y 4 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 Y 1 and Y 2 sites). The PP/NPY receptors could also be blocked by NPY and related full-length peptides, including Y 1 -selective agonists (IC 50 300 -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 Y 2 -selective peptides is quite low. The ligand attachment to PP/NPY sites is also very sensitive to peptidic Y 1 antagonist ((Cys 31 ,NVal 34 NPY(27-36)) 2 , which however blocks these sites at much higher molarities. Blockade of PP/NPY and Y 4 sites by agonist peptides can be largely prevented by N5-substituted amiloride modulators of Na ϩ transport, and by RFamide NRNFLRF.NH 2 , but not by Ca 2ϩ channel blockers, or by inhibitors of K ϩ transport. Protection of both PP/NPY and Y 4 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.
The neuropeptide Y-family receptor Y4 differs extensively between human and rat in sequence, rece... more 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.
Ligand binding to neuropeptide Y (NPY) receptors Y1, Y2, Y4, and Y5 from guinea-pig was investiga... more Ligand binding to neuropeptide Y (NPY) receptors Y1, Y2, Y4, and Y5 from guinea-pig was investigated using the two NPY-galanin hybrids M32 (galanin1-13-NPY25-36-amide) and M242 ([D-Trp(32)]M32). The affinity of M32 for Y1, Y2, and Y4 receptors was 13, 4, and 30nM, respectively, similar to that of NPY18-36 and NPY22-36 but 40-fold to 300-fold lower than the affinity of intact porcine NPY. M242 bound to the Y1, Y2, and Y4 receptors with 9-fold to 20-fold lower affinity than did M32. The affinities of M32 and M242 for Y5 were 400 and 800 nM, respectively. Thus, M32 seems to gain affinity relative to both of its constituent peptide portions although the NPY25-36 part may be sufficient for NPY-receptor recognition, especially at the Y2 receptor. This suggests that the galanin portion of M32 influences and/or stabilizes the conformation of the NPY portion, similar to the effect seen for the NPY portion of M32 in binding to galanin receptors.
The members of the neuropeptide Y (NPY) family are key players in food-intake regulation. In huma... more The members of the neuropeptide Y (NPY) family are key players in food-intake regulation. In humans this family consists of NPY, peptide YY (PYY) and pancreatic polypeptide (PP) which interact with distinct preference for the four receptors showing very low sequence identity, i.e. Y1, Y2, Y4 and Y5. The binding of similar peptides to these divergent receptors makes them highly interesting for mutagenesis studies. We present here a site-directed mutagenesis study of four amino acid positions in the human Y2 receptor. T 3.40 was selected based on sequence alignments both between subtypes and between species and G 2.68 , L 4.60 and Q 6.55 also on previous binding studies of the corresponding positions in the Y1 receptor. The mutated receptors were characterized pharmacologically with the peptide agonists NPY, PYY, PYY(3-36), NPY(13-36) and the non-peptide antagonist BIIE0246. Interestingly, the affinity of NPY and PYY(3-36) increased for the mutants T 3.40 I and Q 6.55 A. Increased affinity was also observed for PYY to Q 6.55 A. PYY(3-36) displayed decreased affinity for G 2.68 N and L 4.60 A whereas binding of NPY(13-36) was unaffected by all mutations. The antagonist BIIE0246 showed decreased affinity for T 3.40 I, L 4.60 A and Q 6.55 A. Although all positions investigated were found important for interaction with at least one of the tested ligands the corresponding positions in hY1 seem to be of greater importance for ligand binding. Furthermore these data indicate that binding of the agonists and the antagonist differs in their points of interaction. The increase in the binding affinity observed may reflect an indirect effect caused by a conformational change of the receptor. These findings will help to improve the structural models of the human NPY receptors.
Within the neuropeptide Y (NPY) family of peptides, pancreatic polypeptide is the most divergent ... more Within the neuropeptide Y (NPY) family of peptides, pancreatic polypeptide is the most divergent across species. It differs in 20 of 36 positions between human and chicken. In mammals, it binds primarily to the Y4 receptor, to which NPY and peptide YY (PYY) bind with lower affinities. Because of these large sequence differences in pancreatic polypeptide, we decided to characterise the chicken Y4 receptor. We report here that Y4 displays the least sequence conservation among the Y-family receptors, with only 57-60% overall amino acid identity between chicken and mammals, compared with 64-83% for the Y1, Y2 and Y5 receptors. After expression of the chicken Y4 receptor in COS-7 cells, 125 I-labelled porcine (p) PYY bound with a K d of 20 pM. In competition with 125 I-pPYY, chicken pancreatic polypeptide bound with high affinity at 140 pM. Interestingly, chicken PYY bound with even greater affinity at 68 pM. The affinity of NPY, 160 pM, was similar to that of pancreatic polypeptide. Chicken Y4 is less sensitive than is mammalian Y4 to truncation of the amino terminus of the NPY molecule. RT-PCR revealed expression in several peripheral organs, including adipose tissue and oviduct. In brain, Y4 mRNA was detected in the brainstem, cerebellum and hippocampus. In situ hybridisation to brain sections showed expression in the dorsal motor nucleus of the vagus in the brainstem. Thus the chicken Y4 receptor is less selective and anatomically more widespread than that in mammals, probably reflecting the original properties of the Y4 receptor.
The evolutionarily conserved protein SNAP-25 (synaptosome-associated protein 25 kDa (kilodaltons)... more 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.
A number of receptors for the pancreatic polypeptide-fold peptides are proposed based on findings... more A number of receptors for the pancreatic polypeptide-fold peptides are proposed based on findings from pharmacology and molecular biology studies. Neuropeptide Y and peptide YY have similar affinity for neuropeptide Y Y~ and neuropeptide Y Y, while pancreatic polypeptide has highest affinity for pancreatic polypeptide 1. Pro34-substituted analogs of neuropeptide Y and peptide YY have selectivity for neuropeptide Y Yj over neuropeptide Y Y= receptors. In the present study, we found that one such 'neuropeptide Y Yt-selective' radioligand, [~=5I][Leu3~,Pro34]peptide YY, also binds with high affinity to the pancreatic polypeptide 1 receptor. Therefore, caution needs to be exercised when using Pro34-analogs to define the neuropeptide Y Y~ receptor in vivo and using tissue preparations.
A number of receptors for the pancreatic polypeptide-fold peptides are proposed based on findings... more A number of receptors for the pancreatic polypeptide-fold peptides are proposed based on findings from pharmacology and molecular biology studies. Neuropeptide Y and peptide YY have similar affinity for neuropeptide Y Y~ and neuropeptide Y Y, while pancreatic polypeptide has highest affinity for pancreatic polypeptide 1. Pro34-substituted analogs of neuropeptide Y and peptide YY have selectivity for neuropeptide Y Yj over neuropeptide Y Y= receptors. In the present study, we found that one such 'neuropeptide Y Yt-selective' radioligand, [~=5I][Leu3~,Pro34]peptide YY, also binds with high affinity to the pancreatic polypeptide 1 receptor. Therefore, caution needs to be exercised when using Pro34-analogs to define the neuropeptide Y Y~ receptor in vivo and using tissue preparations.
1 Neuropeptide Y (NPY) is one of the most potent stimulants of food intake. It has been debated w... more 1 Neuropeptide Y (NPY) is one of the most potent stimulants of food intake. It has been debated which receptor subtype mediates this response. Initially Y 1 was proposed, but later Y 5 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. 2 PYY, (Leu 31 ,Pro 34 )NPY and NPY(2 ± 36) stimulated feeding, whereas NPY(13 ± 36) had no eect. These data suggest that either Y 1 or Y 5 receptors or both may mediate NPY induced food intake in guinea-pigs.
A peptide ending with RFamide (Arg-Phe-amide) was discovered independently by three different lab... more A peptide ending with RFamide (Arg-Phe-amide) was discovered independently by three different laboratories in 2003 and named 26RFa or QRFP. In mammals, a longer version of the peptide, 43 amino acids, was identified and found to bind to the orphan G protein-coupled receptor GPR103. We searched the genome database of Branchiostoma floridae (Bfl) for receptor sequences related to those that bind peptides ending with RFa or RYa (including receptors for NPFF, PRLH, GnIH, and NPY). One receptor clustered in phylogenetic analyses with mammalian QRFP receptors. The gene has 3 introns in Bfl and 5 in human, but all intron positions differ, implying that the introns were inserted independently. A QRFP-like peptide consisting of 25 amino acids and ending with RFa was identified in the amphioxus genome. Eight of the ten last amino acids are identical between Bfl and human. The prepro-QRFP gene in Bfl has one intron in the propeptide whereas the human gene lacks introns. The Bfl QRFP peptide was synthesized and the receptor was functionally expressed in human cells. The response was measured as inositol phosphate (IP) turnover. The Bfl QRFP peptide was found to potently stimulate the receptor's ability to induce IP turnover with an EC50 of 0.28nM. Also the human QRFP peptides with 26 and 43 amino acids were found to stimulate the receptor (1.9 and 5.1nM, respectively). Human QRFP with 26 amino acids without the carboxyterminal amide had dramatically lower potency at 1.3μM. Thus, we have identified an amphioxus QRFP-related peptide and a corresponding receptor and shown that they interact to give a functional response.
Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and acts in... more Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and acts in humans via at least three receptor subtypes: Y1, Y2, and Y5. Whereas selective agonists and antagonists are known for the Y2- and Y5-receptors, the Y1-receptor still lacks a highly selective agonist. This work presents the first NPY-based analogues with Y1-receptor preference and agonistic properties. Furthermore, the importance of specific amino acids of NPY for binding to the Y-receptor subtypes is presented. Amongst the analogues tested, [Phe7,Pro34]pNPY (where pNPY is porcine neuropeptide Y) showed the most significant Y1-receptor preference (> 1 : 3000-fold), with subnanomolar affinity to the Y1-receptor, and Ki values of approximately 30 nM for the Y2- and Y5-subtype, respectively. Variations of position 6, especially [Arg6,Pro34]pNPY and variations within positions 20-23 of NPY were found to result in further analogues with significant Y1-receptor preference (1 : 400-1 : 2000). In contrast, cyclo S-S [Cys20,Cys24]pNPY was found to be a highly selective ligand at the Y2-receptor, binding only threefold less efficiently than NPY. Analogues containing variations of positions 31 and 32 showed highly reduced affinity to the Y1-receptor, while binding to the Y5-receptor was affected less. Inhibition of cAMP-accumulation of selected peptides with replacements within position 20-23 of NPY showed preserved agonistic properties. The NPY analogues tested give insights into ligand-receptor interaction of NPY at the Y1-, Y2- and Y5-receptor and contribute to our understanding of subtype selectivity. Furthermore, the Y1-receptor-preferring peptides are novel tools that will provide insight into the physiological role of the Y1-receptor.
Guinea-pig neuropeptide Y 1 and rat pancreatic polypeptide Y 4 receptors expressed in Chinese ham... more Guinea-pig neuropeptide Y 1 and rat pancreatic polypeptide Y 4 receptors expressed in Chinese hamster ovary cells were internalized rapidly upon attachment of selective peptide agonists. The Y 1 and Y 2 , but not the Y 4 , receptor also internalized the nonselective neuropeptide Y receptor agonist, human/rat neuropeptide Y. The internalization of guinea-pig neuropeptide Y 2 receptor expressed in Chinese hamster ovary cells was small at 37 8C, and essentially absent at or below 15 8C, possibly in connection to the large molecular size of the receptor2ligand complexes (up to 400 kDa for the internalized fraction). The rate of intake was strongly temperature dependent, with essentially no internalization at 6 8C for any receptor. Internalized receptors were largely associated with light, endosome-like particulates. Sucrose dose-dependently decreased the internalization rate for all receptors, while affecting ligand attachment to cell membrane sites much less. Internalization of the Y 1 and the Y 4 receptors could be blocked, and that of the Y 2 receptor significantly inhibited, by phenylarsine oxide, which also unmasked spare cell-surface receptors especially abundant for the Y 2 subtype. The restoration of Y 1 and Y 4 receptors after agonist peptide pretreatment was decreased significantly by cycloheximide and monensin. Thus, in Chinese hamster ovary cells the Y 1 and Y 4 receptors have much larger subcellular dynamics than the Y 2 receptor. This differential could also hold in organismic systems, and is comparable with the known differences in internalization of angiotensin, bradykinin, somatostatin and opioid receptor subtypes. Abbreviations: CHO, Chinese hamster ovary cells; GPCR, G-protein coupled receptor; hNPY, human/rat neuropeptide Y; hPP, human pancreatic polypeptide; hPYY(3±36), human peptide YY(3±36); LP-PYY, (Leu 31 ,Pro 34 ) human peptide YY; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide; MTSET, 2-[(trimethylammonium)ethyl] methanethiosulfonate bromide; PAO, phenylarsine oxide; pPYY, porcine/rat peptide YY; rPP, rat pancreatic polypeptide.
Agonist stimulation readily internalizes neuropeptide Y receptor Y1 while there are contradictory... more Agonist stimulation readily internalizes neuropeptide Y receptor Y1 while there are contradictory results for the Y2 receptor. In order to explore putative functional differences between the Y1 and Y2 receptors we generated reciprocal chimeras by swapping the third intracellular loop, the carboxy terminus or both between human Y1 and Y2. Internalization was studied in a quantitative radioligand binding assay with removal of surface-bound ligand in an acidic-wash procedure. The internalization assay revealed a lower degree of internalization as well as slower kinetics for the Y2 receptor. Generally, reciprocal exchange of receptor segments did not convey properties of the donor receptor but tended to enhance internalization. Surprisingly, insertion of the Y2 carboxy terminus into Y1 gave almost complete internalization (92%), rather than reduced internalization, while the insertion of both segments resulted in internalization equal to the native Y1 receptor. These findings were confirmed by fluorescence microscopy of immuno-stained receptors tagged with a C-terminal FLAG epitope. However, after exposure to high agonist concentrations (100 nM) Y2 was internalized. Studies of Y2 and the closely related Y7 receptor confirmed low internalization for Y2 from chicken and teleost fishes as well as Y7 from two teleosts. The conservation across species of low internalization at physiological concentrations suggests that this is an ancient feature and of vital importance for Y2 function. We propose that amino acid motifs in the third intracellular loop as well as the C terminus of both Y1 and Y2 are able to drive agonist-promoted internalization and that there may be constraining motifs in the Y2 receptor.
In Chinese hamster ovary (CHO) cells expressing the cloned guinea-pig Y1 receptor, the saturable,... more 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 )hPYYHBVD-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 cholesterolcomplexing 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 jC, most of the internalized receptor is rapidly recycled through endosome-like membrane elements, detectable in Percoll gradients. D 0167-0115/02/$ -see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 -0 11 5 ( 0 2 ) 0 0 0 9 4 -0 Abbreviations: NPY, neuropeptide Y; PYY, peptide YY; hNPY, human/ rat neuropeptide Y; pPYY, porcine/rat peptide YY; hPYY, human peptide YY; hPP, human pancreatic polypeptide;
Neuropeptide Y (NPY) and peptide YY (PYY) share ∼ 70% of their 36 amino acids and bind to the sam... more Neuropeptide Y (NPY) and peptide YY (PYY) share ∼ 70% of their 36 amino acids and bind to the same three human receptor subtypes, Y1, Y2 and Y5, even though these receptors only share ∼ 30% sequence identity. Based on our previous investigation of human Y1 we describe here a mutagenesis study of three corresponding positions in human Y2, i.e. Tyr 2.64 , Val 6.58 and Tyr 7.31 . Pharmacological characterization was performed with the four peptide agonists PYY, NPY, PYY(3-36) and NPY(13-36) as well as the non-peptide antagonist BIIE0246. Results from mutants where Tyr 2.64 has been substituted by Ala suggest that Tyr 2.64 is involved in the interaction with all investigated ligands whereas position Tyr 7.31 seems to be more important for interaction with the truncated peptide PYY(3-36) than with intact NPY. Surprisingly, substitution of Tyr 7.31 with His, the corresponding residue in Y1, resulted in total loss of binding of iodinated porcine PYY. The third position, Val 6.58 , did not influence binding of any of the ligands. These findings differ from those obtained for Y1 where Ala substitution resulted in lost or changed binding for each of the three positions. Although Tyr 2.64 and Tyr 7.31 in Y2 are involved in ligand binding, their interactions with the peptide ligands seem to be different from the corresponding positions in Y1. This suggests that the receptor-ligand interactions have changed during evolution after Y1 and Y2 arose from a common ancestral receptor.
Proceedings of the National Academy of Sciences, 1992
Neuropeptide Y (NPY) is an abundant and widespread neuropeptide in the nervous system of mammals.... more Neuropeptide Y (NPY) is an abundant and widespread neuropeptide in the nervous system of mammals. NPY belongs to a family of 36-amino acid peptides that also includes pancreatic polypeptide and the endocrine gut peptide YY as well as the fish pancreatic peptide Y. To study the evolution of this peptide family, we have isolated clones encoding NPY from central nervous system cDNA libraries of chicken, goldfish, and the ray Torpedo marmorata, as well as from a chicken genomic library. The predicted chicken NPY amino acid sequence differs from that of rat at only one position. The goldfish sequence differs at five positions and shows that bony fishes have a true NPY peptide in addition to their pancreatic peptide Y. The Torpedo sequence differs from that of rat at three positions. As Torpedo NPY has no unique positions when compared with the other sequences, it seems to be identical to the NPY of the common ancestor ofcartilaginous fishes, bony fishes, and tetrapods after 420 million years of evolution. The 30-amino acid carboxyl-terminal extension of the NPY precursor also displays considerable sequence conservation. These results show that NPY is one ofthe most highly conserved neuroendocrine peptides.
Proceedings of the National Academy of Sciences, 1996
Pancreatic polypeptide (PP) is produced in the islets of Langerhans and released in response to m... more Pancreatic polypeptide (PP) is produced in the islets of Langerhans and released in response to meals. It belongs to a family of peptides that also includes neuropeptide Y and peptide YY. In the present communication, we describe a rat receptor with high affinity for PP, therefore named PP1. Clones for the PP1 receptor were obtained by PCR using sequence information for the neuropeptide Y receptor Y1 from several species. The PP1 receptor has 46% overall amino acid sequence identity to the rat Y1 receptor and 56% identity in the transmembrane regions. The PP1 receptor displays a pharmacological profile that is distinct from previously described neuropeptide Y-family receptors. In competition with iodinated bovine PP, it binds rat PP with an affinity (K*) of 0.017 nM, while the affinities for peptide YY and neuropeptide Y are substantially lower with K; values of 162 and 192 nM, respectively. In stably transfected CHO cells, the PP1 receptor inhibits forskolin-stimulated cAMP synthesis. Northern blot hybridizations to a panel of mRNAs detected transcripts in testis and lung. A faint band was seen in colon and total brain. In contrast, the human receptor is expressed primarily in colon and small intestine. Whereas rat and human PP1 bind PP with the same affinity, the rat receptor has much lower affinity than its human ortholog for peptide YY and neuropeptide Y. Interestingly, the amino acid sequence identity between rat and human PP1 is only 75%. Thus, the sequence, the tissue distribution, and the binding profile of the PP1 receptor differ considerably between rat and human.
The Y5 receptor has been postulated to be the main receptor mediating NPY-induced food intake in ... more 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 ϭ NPY ϭ NPY2-36 Ͼ gpPP Ͼ rPP Ͼ Ͼ 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.
The NPY system has a multitude of effects and is particularly well known for its role in appetite... more 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.
Ligand binding to rodent pancreatic polypeptide-responding neuropeptide Y (NPY) receptors (here t... more Ligand binding to rodent pancreatic polypeptide-responding neuropeptide Y (NPY) receptors (here termed PP/NPY receptors), or to cloned Y 4 or Y 5 receptors, is selectively inhibited by amiloride, peptide or alkylating modulators of sodium transport. The PP/NPY and Y 4 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 Y 1 and Y 2 sites). The PP/NPY receptors could also be blocked by NPY and related full-length peptides, including Y 1 -selective agonists (IC 50 300 -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 Y 2 -selective peptides is quite low. The ligand attachment to PP/NPY sites is also very sensitive to peptidic Y 1 antagonist ((Cys 31 ,NVal 34 NPY(27-36)) 2 , which however blocks these sites at much higher molarities. Blockade of PP/NPY and Y 4 sites by agonist peptides can be largely prevented by N5-substituted amiloride modulators of Na ϩ transport, and by RFamide NRNFLRF.NH 2 , but not by Ca 2ϩ channel blockers, or by inhibitors of K ϩ transport. Protection of both PP/NPY and Y 4 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.
The neuropeptide Y-family receptor Y4 differs extensively between human and rat in sequence, rece... more 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.
Ligand binding to neuropeptide Y (NPY) receptors Y1, Y2, Y4, and Y5 from guinea-pig was investiga... more Ligand binding to neuropeptide Y (NPY) receptors Y1, Y2, Y4, and Y5 from guinea-pig was investigated using the two NPY-galanin hybrids M32 (galanin1-13-NPY25-36-amide) and M242 ([D-Trp(32)]M32). The affinity of M32 for Y1, Y2, and Y4 receptors was 13, 4, and 30nM, respectively, similar to that of NPY18-36 and NPY22-36 but 40-fold to 300-fold lower than the affinity of intact porcine NPY. M242 bound to the Y1, Y2, and Y4 receptors with 9-fold to 20-fold lower affinity than did M32. The affinities of M32 and M242 for Y5 were 400 and 800 nM, respectively. Thus, M32 seems to gain affinity relative to both of its constituent peptide portions although the NPY25-36 part may be sufficient for NPY-receptor recognition, especially at the Y2 receptor. This suggests that the galanin portion of M32 influences and/or stabilizes the conformation of the NPY portion, similar to the effect seen for the NPY portion of M32 in binding to galanin receptors.
The members of the neuropeptide Y (NPY) family are key players in food-intake regulation. In huma... more The members of the neuropeptide Y (NPY) family are key players in food-intake regulation. In humans this family consists of NPY, peptide YY (PYY) and pancreatic polypeptide (PP) which interact with distinct preference for the four receptors showing very low sequence identity, i.e. Y1, Y2, Y4 and Y5. The binding of similar peptides to these divergent receptors makes them highly interesting for mutagenesis studies. We present here a site-directed mutagenesis study of four amino acid positions in the human Y2 receptor. T 3.40 was selected based on sequence alignments both between subtypes and between species and G 2.68 , L 4.60 and Q 6.55 also on previous binding studies of the corresponding positions in the Y1 receptor. The mutated receptors were characterized pharmacologically with the peptide agonists NPY, PYY, PYY(3-36), NPY(13-36) and the non-peptide antagonist BIIE0246. Interestingly, the affinity of NPY and PYY(3-36) increased for the mutants T 3.40 I and Q 6.55 A. Increased affinity was also observed for PYY to Q 6.55 A. PYY(3-36) displayed decreased affinity for G 2.68 N and L 4.60 A whereas binding of NPY(13-36) was unaffected by all mutations. The antagonist BIIE0246 showed decreased affinity for T 3.40 I, L 4.60 A and Q 6.55 A. Although all positions investigated were found important for interaction with at least one of the tested ligands the corresponding positions in hY1 seem to be of greater importance for ligand binding. Furthermore these data indicate that binding of the agonists and the antagonist differs in their points of interaction. The increase in the binding affinity observed may reflect an indirect effect caused by a conformational change of the receptor. These findings will help to improve the structural models of the human NPY receptors.
Within the neuropeptide Y (NPY) family of peptides, pancreatic polypeptide is the most divergent ... more Within the neuropeptide Y (NPY) family of peptides, pancreatic polypeptide is the most divergent across species. It differs in 20 of 36 positions between human and chicken. In mammals, it binds primarily to the Y4 receptor, to which NPY and peptide YY (PYY) bind with lower affinities. Because of these large sequence differences in pancreatic polypeptide, we decided to characterise the chicken Y4 receptor. We report here that Y4 displays the least sequence conservation among the Y-family receptors, with only 57-60% overall amino acid identity between chicken and mammals, compared with 64-83% for the Y1, Y2 and Y5 receptors. After expression of the chicken Y4 receptor in COS-7 cells, 125 I-labelled porcine (p) PYY bound with a K d of 20 pM. In competition with 125 I-pPYY, chicken pancreatic polypeptide bound with high affinity at 140 pM. Interestingly, chicken PYY bound with even greater affinity at 68 pM. The affinity of NPY, 160 pM, was similar to that of pancreatic polypeptide. Chicken Y4 is less sensitive than is mammalian Y4 to truncation of the amino terminus of the NPY molecule. RT-PCR revealed expression in several peripheral organs, including adipose tissue and oviduct. In brain, Y4 mRNA was detected in the brainstem, cerebellum and hippocampus. In situ hybridisation to brain sections showed expression in the dorsal motor nucleus of the vagus in the brainstem. Thus the chicken Y4 receptor is less selective and anatomically more widespread than that in mammals, probably reflecting the original properties of the Y4 receptor.
The evolutionarily conserved protein SNAP-25 (synaptosome-associated protein 25 kDa (kilodaltons)... more 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.
A number of receptors for the pancreatic polypeptide-fold peptides are proposed based on findings... more A number of receptors for the pancreatic polypeptide-fold peptides are proposed based on findings from pharmacology and molecular biology studies. Neuropeptide Y and peptide YY have similar affinity for neuropeptide Y Y~ and neuropeptide Y Y, while pancreatic polypeptide has highest affinity for pancreatic polypeptide 1. Pro34-substituted analogs of neuropeptide Y and peptide YY have selectivity for neuropeptide Y Yj over neuropeptide Y Y= receptors. In the present study, we found that one such 'neuropeptide Y Yt-selective' radioligand, [~=5I][Leu3~,Pro34]peptide YY, also binds with high affinity to the pancreatic polypeptide 1 receptor. Therefore, caution needs to be exercised when using Pro34-analogs to define the neuropeptide Y Y~ receptor in vivo and using tissue preparations.
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Papers by Ingrid Lundell