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Characterization of the neuropeptide Y system in the frog Silurana tropicalis (Pipidae): three peptides and six receptor subtypes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. (Larhammar)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology. (Larhammar)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology. (Larhammar)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. (Larhammar)
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2012 (English)In: General and Comparative Endocrinology, ISSN 0016-6480, E-ISSN 1095-6840, Vol. 177, no 3, 322-331 p.Article in journal (Refereed) Published
Abstract [en]

Neuropeptide Y and its related peptides PYY and PP (pancreatic polypeptide) are involved in feeding behavior, regulation of the pituitary and the gastrointestinal tract, and numerous other functions. The peptides act on a family of G-protein coupled receptors with 4-7 members in jawed vertebrates. We describe here the NPY system of the Western clawed frog Silurana (Xenopus) tropicalis. Three peptides, NPY, PYY and PP, were identified together with six receptors, namely subtypes Y1, Y2, Y4, Y5, Y7 and Y8. Thus, this frog has all but one of the ancestral seven gnathostome NPY-family receptors, in contrast to mammals which have lost 2-3 of the receptors. Expression levels of mRNA for the peptide and receptor genes were analyzed in a panel of 19 frog tissues using reverse transcriptase quantitative PCR. The peptide mRNAs had broad distribution with highest expression in skin, blood and small intestine. NPY mRNA was present in the three brain regions investigated, but PYY and PP mRNAs were not detectable in any of these. All receptor mRNAs had similar expression profiles with high expression in skin, blood, muscle and heart. Three of the receptors, Y5, Y7 and Y8, could be functionally expressed in HEK-293 cells and characterized with binding studies using the three frog peptides. PYY had the highest affinity for all three receptors (K(i) 0.042-0.34 nM). Also NPY and PP bound to the Y8 receptor with high affinity (0.14 and 0.50 nM). The low affinity of NPY for the Y5 receptor (100-fold lower than PYY) differs from mammals and chicken. This may suggest a less important role of NPY on Y5 in appetite stimulation in the frog compared with amniotes. In conclusion, our characterization of the NPY system in S. tropicalis with its six receptors demonstrates not only greater complexity than in mammals but also some interesting differences in ligand-receptor preferences.

Place, publisher, year, edition, pages
2012. Vol. 177, no 3, 322-331 p.
Keyword [en]
NPY, PYY, G-protein-coupled receptor, Silurana tropicalis, evolution
National Category
Biological Sciences
URN: urn:nbn:se:uu:diva-129517DOI: 10.1016/j.ygcen.2012.04.027ISI: 000306390100005PubMedID: 22565163OAI: oai:DiVA.org:uu-129517DiVA: diva2:344156

Erratum in General and Comparative Endocrinology 2015:215, doi:10.1016/j.ygcen.2014.11.014.

Available from: 2010-08-18 Created: 2010-08-18 Last updated: 2015-07-03Bibliographically approved
In thesis
1. Evolutionary and Pharmacological Studies of NPY and QRFP Receptors
Open this publication in new window or tab >>Evolutionary and Pharmacological Studies of NPY and QRFP Receptors
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The neuropeptide Y (NPY) system consists of 3-4 peptides and 4-7 receptors in vertebrates. It has powerful effects on appetite regulation and is involved in many other biological processes including blood pressure regulation, bone formation and anxiety. This thesis describes studies of the evolution of the NPY system by comparison of several vertebrate species and structural studies of the human Y2 receptor, which reduces appetite, to identify amino acid residues involved in peptide-receptor interactions.

The NPY system was studied in zebrafish (Danio rerio), western clawed frog (Xenopus tropicalis), and sea lamprey (Petromyzon marinus). The receptors were cloned and functionally expressed and their pharmacological profiles were determined using the native peptides in either binding studies or a signal transduction assay. Some peptide-receptor preferences were observed, indicating functional specialization.

A receptor family closely related to the NPY receptors, called the QRFP receptors, was investigated. A QRFP receptor was cloned from amphioxus, Branchistoma floridae, showing that the receptor arose before the origin of the vertebrates. Evolutionary studies demonstrated that the ancestral vertebrate had as many as four QRFP receptors, only one of which remains in mammals today. This correlates with the NPY receptor family, located in the same chromosomal regions, which had seven members in the ancestral vertebrate but only 4-5 in living mammals. Some vertebrates have considerably more complex NPY and QRFP receptor systems than humans and other mammals.

Two studies investigated interactions of NPY-family peptides with the human Y2 receptor. Candidate residues, selected based on structural modeling and docking, were mutated to disrupt possible interactions with peptide ligands. The modified receptors were expressed in cultured cells and investigated by measuring binding and functional responses. Several receptor residues were found to influence peptide-receptor interactions, some of which are involved in maintaining receptor structure. In a pilot study, the kinetics of peptide-receptor interaction were found to be very slow, of the order several hours.

In conclusion, this thesis clarifies evolutionary relationships for the complex NPY and QRFP peptide-receptor systems and improves the structural models of the human NPY-family receptors, especially Y2. These results will hopefully facilitate drug design for targeting of NPY-family receptors.

Place, publisher, year, edition, pages
Uppsala, Sweden: Acta Universitatis Upsaliensis, 2014. 59 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1040
Neuropeptide Y, genome duplication, Evolution, vertebrate, Pharmacology, Modelling, Kinetics
National Category
Evolutionary Biology Pharmacology and Toxicology Cell and Molecular Biology Neurosciences Biochemistry and Molecular Biology Cell Biology Structural Biology
Research subject
Bioinformatics; Biology with specialization in Evolutionary Genetics; Biology with specialization in Evolutionary Functional Genomics; Pharmaceutical Pharmacology
urn:nbn:se:uu:diva-233461 (URN)978-91-554-9059-1 (ISBN)
Public defence
2014-11-21, C2, 305, Husargatan 3, BMC, Uppsala, 13:15 (English)
Available from: 2014-10-31 Created: 2014-10-06 Last updated: 2015-02-02

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