Starbäck, P. 2000. Cloning and characterization of NPY receptors of the Y ½ subfamily in mammals and fish. Acta Universitatis Upsaliensis. Comprehensive summaries of Uppsala Dissertations from the Faculty of Medicine 948. 43 pp. Uppsala. ISBN 91-554-4786-4 Neuropeptide Y (NPY) is an abundant neurotransmitter in the nervous system and forms a family of evolutionarily related peptides together with peptide YY (PYY), pancreatic polypeptide (PP) and polypeptide Y (PY). These peptides are ligands to a family of receptors that mediate a wide range of physiological effects including stimulation of appetite. This work describes the molecular cloning of four novel NPY receptors. In rat a receptor called PP1, later renamed Y , was cloned and characterized. It displays the highest amino acid sequence identity to the Y ½ receptor. Rat Y differs extensively from human Y , cloned subsequently, in both pharmacological properties, tissue distribution, and amino acid sequence with only 75% identity. Rat and human Y are the most diverged orthologues in the NPY receptor family. In guinea pig, the y receptor gene was found to be a pseudogene with several frameshift mutations. The gene is a pseudogene in human and pig too, but seems to give rise to a functional receptor in mouse and rabbit. This unusual evolutionary situation may be due to inactivation of the gene in a mammalian ancestor and then restoration of expression in mouse and rabbit, but perhaps more likely due to independent inactivations in guinea pig, human and pig. In zebrafish, two new intronless receptor genes were cloned. Sequence comparisons suggest that both receptors are distinct from the mammalian receptors Y ½ , Y and y , hence they were named Ya and Yb. Chromosomal localization provides further support that Ya and Yb may be distinct subtypes. The discoveries of the rat Y and zebrafish Ya and Yb receptors were unexpected and show that the NPY receptor family is larger than previously thought. The cloned rat pancreatic polypeptide receptor exhibits profound differences to the orthologous receptor. and characterization of a novel neuropeptide Y receptor subtype in the zebrafish.ropeptide Y receptor subtype with unique properties cloned in the ze-brafish: the zYa receptor.

The neuropeptide Y family of G-protein-coupled receptors consists of five cloned members in mammals. Four genes give rise to functional receptors in all mammals investigated. The y6 gene is a pseudogene in human and pig and is absent in rat, but generates a functional receptor in rabbit and mouse and probably in the collared peccary (Pecari tajacu), a distant relative of the pig family. We report here that the guinea pig y6 gene has a highly distorted nucleotide sequence with multiple frame-shift mutations. One evolutionary scenario may suggest that y6 was inactivated before the divergence of the mammalian orders and subsequently resurrected in some lineages. However, the pseudogene mutations seem to be distinct in human, pig, and guinea pig, arguing for separate inactivation events. In either case, the y6 gene has a quite unusual evolutionary history with multiple independent deaths or resurrections.

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 Kd of 28 pM for porcine 125I-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 strived 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.

Annals of the New York Academy of SciencesVolume 839, Issue 1 p. 515-517 Cloning of Neuropeptide Y Receptors in Zebrafisha INGRID LUNDELL, INGRID LUNDELL Department of Medical Pharmacology, Uppsala University, Box 593, S-751 24 Uppsala, SwedenSearch for more papers by this authorMARIA RINGVALL, MARIA RINGVALL Department of Medical Pharmacology, Uppsala University, Box 593, S-751 24 Uppsala, SwedenSearch for more papers by this authorPAULA STARBÄCK, PAULA STARBÄCK Department of Medical Pharmacology, Uppsala University, Box 593, S-751 24 Uppsala, SwedenSearch for more papers by this authorERIK SALANECK, ERIK SALANECK Department of Medical Pharmacology, Uppsala University, Box 593, S-751 24 Uppsala, SwedenSearch for more papers by this authorMAGNUS BERGLUND, MAGNUS BERGLUND Department of Medical Pharmacology, Uppsala University, Box 593, S-751 24 Uppsala, SwedenSearch for more papers by this authorDAN LARHAMMAR, DAN LARHAMMAR Department of Medical Pharmacology, Uppsala University, Box 593, S-751 24 Uppsala, SwedenSearch for more papers by this author INGRID LUNDELL, INGRID LUNDELL Department of Medical Pharmacology, Uppsala University, Box 593, S-751 24 Uppsala, SwedenSearch for more papers by this authorMARIA RINGVALL, MARIA RINGVALL Department of Medical Pharmacology, Uppsala University, Box 593, S-751 24 Uppsala, SwedenSearch for more papers by this authorPAULA STARBÄCK, PAULA STARBÄCK Department of Medical Pharmacology, Uppsala University, Box 593, S-751 24 Uppsala, SwedenSearch for more papers by this authorERIK SALANECK, ERIK SALANECK Department of Medical Pharmacology, Uppsala University, Box 593, S-751 24 Uppsala, SwedenSearch for more papers by this authorMAGNUS BERGLUND, MAGNUS BERGLUND Department of Medical Pharmacology, Uppsala University, Box 593, S-751 24 Uppsala, SwedenSearch for more papers by this authorDAN LARHAMMAR, DAN LARHAMMAR Department of Medical Pharmacology, Uppsala University, Box 593, S-751 24 Uppsala, SwedenSearch for more papers by this author First published: 07 February 2006 https://doi.org/10.1111/j.1749-6632.1998.tb10855.x a This work was supported by a grant from the Swedish Natural Science Research Council. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Volume839, Issue1TRENDS IN COMPARATIVE ENDOCRINOLOGY AND NEUROBIOLOGY: FROM MOLECULAR TO INTEGRATIVE BIOLOGYMay 1998Pages 515-517 RelatedInformation

Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) form a family of structurally related peptides. As we have previously isolated clones for NPY and PYY from the zebrafish (Danio rerio), we wished to clone the receptors for these peptides to allow correlation of ligand and receptor distribution. We describe here the cloning and functional expression of a receptor with equally high identity to the NPY-Y1 receptor as to the recently cloned Y4/PP1 and Y6 receptors with an overall amino acid sequence identity of approximately 50%. Furthermore, the zebrafish receptor gene lacks the intron present in the coding region in vertebrate Y1 genes. These features strongly suggest that the zebrafish receptor represents a separate subtype. Hence, we have named it zYb for zebrafish Y-receptor b. (We have also discovered a unique receptor called zYa.) The zYb receptor has a binding profile that is reminiscent of Y1 with affinities for NPY and PYY in the low picomolar range, whereas affinities for Y2-selective ligands are considerably lower. It couples to adenylyl cyclase by inhibiting cAMP synthesis. Receptor mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) in brain, eye, and intestine. The binding profile and amino acid identity show that the zebrafish zYb receptor is related to Y1 but represents a distinct subtype that is likely to be present also in mammals.

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(i)) of 0.017 nM, while the affinities for peptide YY and neuropeptide Y are substantially lower with K(i) 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.