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Gen Comp Endocrinol
2012 Jul 01;1773:322-31. doi: 10.1016/j.ygcen.2012.04.027.
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Characterization of the neuropeptide Y system in the frog Silurana tropicalis (Pipidae): three peptides and six receptor subtypes.
Sundström G
,
Xu B
,
Larsson TA
,
Heldin J
,
Bergqvist CA
,
Fredriksson R
,
Conlon JM
,
Lundell I
,
Denver RJ
,
Larhammar D
.
???displayArticle.abstract??? 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.
Fig. 1.
Alignment of the three NPY-family peptide sequences in S. tropicalis with sequences from the African clawed frog Xenopus laevis [18]; [23] ; [46]. Dots mark residues that differ from the top sequence in each of the three panels. The last two amino acids of pancreatic polypeptide and the glycine forming the carboxyterminal amide could not be predicted because they are encoded on a separate exon that could not be found in the S. tropicalis genome database. We therefore synthesized this peptide to end with RFamide as in PP reported for Xenopus laevis and other frogs.
Fig. 2.
Maximum Likelihood (ML) phylogenetic tree with 100 bootstrap replicates showing the NPY-receptor family. The genes described in this study are marked with a dot. The tree is rooted with human somatostatin receptor 1. Species abbreviations are: Sitr, S. tropicalis; Hosa, Homo sapiens; Cafa, Canis familiaris; Susc, Sus scrofa; Capo, Cavia porcellus; Rano, Rattus norvegicus; Mumu, Mus musculus; Gaga, Gallus gallus; Cami, Callorhinchus milii; Sac, Squalus acanthias; Dare, Danio rerio; Orcu, Oryctolagus cuniculus; and Taru, Takifugu rubripes. A neighbor-joining tree is available in Supplementary Fig. 1.
Fig. 3.
Amino acid alignment of NPY-receptor sequences from Western clawed frog, S. tropicalis (Sitr); the frog Pelophylax esculentus (Pees); chicken, Gallus gallus (Gaga); and human, Homo sapiens (Hosa). Boxes mark the predicted transmembrane (TM) regions.
Fig. 4.
Saturation and Scatchard (inset) analyses of 125I-pPYY binding to the Y5, Y7 and Y8 receptors. Results shown are from one representative experiment (for a summary of all binding experiments see Table 1).
Fig. 5.
Competition assays performed for Y5, Y7 and Y8 receptors using native NPY, PYY and PP and 125I-pPYY as radioligand. Results shown are from one typical experiment for each receptor (for a summary of all binding experiments see Table 1).
Fig. 6.
Expression data for NPY-family receptors in a panel of 19 S. tropicalis organs. Error bars show standard error of the mean. Normalized Ct values were used to calculate the relative expression values. For each transcript the tissue with the lowest expression was used to calculate relative expression. For the different genes, the following tissues had lowest expression: Y1, ovary; Y2, spinal cord; Y4, ovary; Y5, spinal cord; Y7, egg; and Y8, hindbrain. The analysis was performed twice, each time with duplicate samples.
Fig. 7.
Expression data for NPY-family peptides NPY, PYY and PP in a panel of 19 S. tropicalis organs. Error bars show standard error of the mean. Normalized Ct values were used to calculate the relative expression values. For each transcript the tissue with the lowest expression was used to calculate relative expression. For the different genes, the following tissues had lowest expression: NPY, ovary; PYY, telencephalon; and PP, ovary. The analysis was performed twice, each time with duplicate samples.
