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General and Comparative Endocrinology 177 (2012) 322–331

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General and Comparative Endocrinology

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Proﬁles in Comparative Endocrinology Characterization of the neuropeptide Y system in the frog Silurana tropicalis (Pipidae): Three peptides and six receptor subtypes

G. Sundström a,1, B. Xu a, T.A. Larsson a,2, J. Heldin a,1, C.A. Bergqvist a, R. Fredriksson a, J.M. Conlon b, a c a, I. Lundell , R.J. Denver , D. Larhammar ⇑ a Department of Neuroscience, Uppsala University, Box 593, SE-75124 Uppsala, Sweden b Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates c Department of Molecular, Cellular and Developmental Biology, The University of Michigan, 3065C Kraus Building, Ann Arbor, MI 48109-1048, USA article info abstract

Article history: Neuropeptide Y and its related peptides PYY and PP (pancreatic polypeptide) are involved in feeding Available online 4 May 2012 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 Keywords: describe here the NPY system of the Western clawed frog Silurana (Xenopus) tropicalis. Three peptides, NPY NPY, PYY and PP, were identified together with six receptors, namely subtypes Y1, Y2, Y4, Y5, Y7 and PYY Y8. Thus, this frog has all but one of the ancestral seven gnathostome NPY-family receptors, in contrast Pancreatic polypeptide to mammals which have lost 2–3 of the receptors. Expression levels of mRNA for the peptide and receptor G-protein-coupled receptor Silurana tropicalis genes were analyzed in a panel of 19 frog tissues using reverse transcriptase quantitative PCR. The pep- Xenopus tropicalis tide 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 char- acterized with binding studies using the three frog peptides. PYY had the highest affinity for all three

receptors (Ki 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. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction the elephant shark (Callorhincus millii) [27]. In mammals, ﬁve of these receptor genes are still present, one of which, Y6, is a pseu- Neuropeptide Y receptors are G-protein coupled receptors in- dogene in several mammals including human. The Y6 gene in volved in numerous physiological processes. In mammals these in- chicken is fully functional [5]. A few NPY receptors have been pre- clude appetite regulation, biological rhythms, anxiety, pain, bone viously reported for frogs after cloning, namely Y1 in the African formation, and release of pituitary hormones [40,50]. We have pre- clawed frog Xenopus laevis [4], and Y7 in the frog Pelophylax escu- viously proposed that the ancestral jawed vertebrate had seven lentus (previously called Rana esculenta) and in the western clawed NPY receptors, Y1, Y2, Y4, Y5, Y6, Y7 and Y8 which can be divided frog Silurana (Xenopus) tropicalis [16]. The decision whether to de- into three distinct subfamilies, the Y1 subfamily (Y1, Y4, Y6 and scribe the western clawed frog as S. tropicalis or Xenopus tropicalis Y8), the Y2 subfamily (Y2 and Y7) and the single gene subfamily remains controversial. Cladistic analysis based upon nucleotide se- Y5 [26,27]. All of these receptors are present in a cartilaginous ﬁsh, quences of mitochondrial genes strongly supports the monophyly of Xenopus + Silurana which are united in the Xenopodinae but

Corresponding author. the use of two genera ‘‘underscores trenchant biological and his- ⇑ E-mail address: [email protected] (D. Larhammar). torical differences between the two clades’’ [15]. 1 Present address: Department of Medical Biochemistry and Microbiology, Uppsala The ligands are members of the NPY family of peptides, which University, Box 582, SE-75123 Uppsala, Sweden. also includes PYY (peptide YY) and PP (pancreatic polypeptide) in 2 Present address: Developmental Biology Unit/Structural and Computational tetrapods. The peptide genes code for pre-propeptides that are pro- Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 cessed by speciﬁc peptidases to generate the active peptides. All of Heidelberg, Germany.

G. Sundström et al. / General and Comparative Endocrinology 177 (2012) 322–331 323 the peptides examined in the NPY family consist of 36 amino acid algorithm to estimate the random starting tree. The number of residues, except for chicken PYY and Burmese python PP that con- substitution rate categories was set to 8 and amino acid substitu- sist of 37 and 35 amino acids, respectively [9,49]. NPY in mammals tion model parameters were estimated from the dataset. Both the is mainly expressed in the brain and is one of the most orexigenic NNI and SPR tree improvement methods were used together with peptides known. It has been shown to induce feeding in different tree topology and branch length optimization. species including Siberian hamster, guinea pig, the frog X. laevis, and goldfish [11,28,29,38]. PYY and PP are mainly expressed in 2.2. Cloning of partial NPY receptor gene sequences in P. esculentus endocrine cells in the gastrointestinal tract in mammals. PYY in its truncated form PYY3–36 and PP have important roles as inhibi- Genomic DNA for the frog Pelohpylax esculentus was kindly pro- tors of feeding in mammals [2,3,24,39]. All three peptides also have vided by Dr. Isabelle Lihrmann, Universit of Rouen, France. This several other roles in neuronal, cardiovascular or gastrointestinal species was previously called R. esculenta and is considered to be contexts [40,50]. Endogenous truncated PYY is not known outside a hybrid between the pool frog Pelophylax lessonae (synonyms Rana mammals where the peptide is cleaved by dipeptidyl peptidase IV lessonae, Rana esculenta lessonae) and the marsh frog Pelophylax rid- [36]. This peptidase cannot cleave between two proline residues ibundus (Rana ridibunda). Degenerate PCR with primers based on and all known non-mammalian PYY sequences have the amino gene sequences for NPY-family receptors from several mammals acids Tyr-Pro-Pro, suggesting resistance to this type of cleavage. and chicken was run on P. esculentus genomic DNA using the stoffel A PYY peptide was isolated from the frog Phyllomedusa bicolor in Taq polymerase (Applied Biosystems) and the following PCR the 1990s [37]. It was named skin PYY due to its main location, but conditions: 120 s at 95 °C for one cycle, thereafter 30 s at 95 °C, alignment with PYY from other species identified it as a regular touch-down from 55 to 42 °C for 45 s, and 60 s at 72 °C for 20 PYY sequence. Subsequently, all three members of the peptide cycles, followed by 20 cycles with 30 s at 95 °C, 45 s at 42 °C and family have been isolated or cloned from a variety of frog species 60 s at 72 °C finishing with 5 min at 72 °C. The PCR products were [6,7,8,18,35,34,41,46]. cloned using the TOPO-cloning kit (Invitrogen), sequenced using The NPY system has been suggested to be involved in several the BigDye V3 terminator sequencing kit (Applied Biosystems) aspects of frog physiology and behaviors including background and the extension products were analyzed on an ABI 310 automatic adaptation [17], visual neurotransmission [44], antimicrobial sequencer. The sequence was compared to the GenBank database activity [14,48] and feeding [10]. To facilitate more detailed studies using the On-Line BLASTX program. of these and other effects of the NPY-family peptides, we decided to clone the NPY-family receptors in a frog. We initially attempted 2.3. Sequencing and expression constructs for S. tropicalis receptors this in the frog P. esculentus and identified four distinct receptors using degenerate PCR primers. However, these sequences were Sequences corresponding to the entire coding regions of six S. not full-length and therefore did not allow functional expression. tropicalis NPY receptors were amplified from genomic DNA. The We therefore turned to the genome of S. tropicalis [21] and were PCR products were directionally cloned into the pcDNA3 vector able to identify three receptor genes orthologous to those of P. at the XhoI and HindIII sites and transformed into chemically com- esculentus, i.e., Y1, Y5, and Y7, plus three additional receptor genes, petent DH5a E. coli cells (Invitrogen). The insert was sequenced Y2, Y4 and Y8. Furthermore, we have identified and synthesized using receptor-specific and T7 vector-specific primers and BigDye the three endogenous NPY-family peptides NPY, PYY and PP. We V3 terminator sequencing kit (Applied Biosystems). The product report here an initial characterization of the NPY system of S. was analyzed on an ABI PRISM 310 Genetic Analyzer (PE Applied tropicalis. Biosystems) automatic sequencer to confirm sequence. Sequences were analyzed using the lasergene software (DNASTAR).

2. Materials and methods 2.4. Transfection

2.1. Identification and phylogenetic analysis of S. tropicalis peptides The human embryonic kidney-derived cell line HEK-293 was and receptors used to express the frog receptors for binding assays. Cells were grown to 95% confluency and transfected with 12–24 lg plasmid The NPY-family peptide and receptor gene sequences were diluted in 750 ll OPTI-MEMÒ mixed with 30 ll Lipofectamine identified by BLAST searches [1] in the genome of S. tropicalis in 2000 according to the manufacturer’s recommendations (Invitro- the Ensembl database (www.ensembl.org) using human and chick- gen). Transfected cells were grown in DMEM/F12 growth medium en peptide and receptor sequences as queries. The identified (Gibco BRL/Invitrogen) at 37 °C (5% CO2, 95% air) for 24 h. The cells receptor DNA sequences were retrieved by PCR and sequenced were harvested in 25 mM HEPES buffer containing 2.5 mM CaCl2, (see below). An alignment was constructed using the Windows 1 mM MgCl and stored at 80 °C. 2 À version of Clustal X 1.81 [45] using standard settings (Gonnet X. laevis cell-lines XTC-2, XL-58 and A6 were also used for trans- weight matrix, gap opening penalty 10.0 and gap extension pen- fection. These were grown in amphibian strength L-15 medium at alty 0.20). The alignment included other vertebrate NPY receptor 25 °C (5% CO2, 95% air). The amphibian strength L-15 medium was sequences and human somatostatin receptor 1 (accession numbers made from Leibovitz L-15 medium with L-glutamine (Invitrogen) are available in Supplementary Table 1). The alignment was cut in considering the frog body fluids osmolarity and added with 10% order to remove poorly aligned sequences in the N- and C-terminal (v/v) fetal calf serum (Gibco) and penicillin–streptomycin (Gibco). regions, resulting in a final alignment spanning from the start of transmembrane region 1 (TM1) to the end of TM7. A neighbor join- 2.5. Peptide synthesis and purification ing tree was constructed in Clustal X 1.81 [45] with 1000 bootstrap replicas and the somatostatin receptor 1 was used as outgroup. The S. tropicalis carboxyterminally amidated peptides NPY, PYY, Phylogenetic maximum likelihood (PhyML) trees were constructed and PP were supplied in crude form by GL Biochem. Ltd. (Shanghai, using the online execution of the PhyML 3.0 algorithm available at China) and were purified to near homogeneity (>98% purity) by re- http://www.atgc-montpellier.fr/phyml/ [20]. The analysis was versed-phase HPLC on a (2.2 25 cm) Vydac 218TP1022 (C-18) Â made using the JTT model of amino acid substitution: ProtTest column. The concentration of acetonitrile in the eluting solvent 2.4 was used to select the model. The BIONJ was selected as was raised from 21% to 56% over 60 min and the flow rate was Author's personal copy

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6 ml/min. The structures of the peptides were conﬁrmed by elec- transcriptase (Invitrogen) and random hexamers as primers fol- trospray mass spectrometry. lowing the manufacturer’s recommendations.

2.8. Primer design and quantitative real-time PCR 2.6. Binding assay Primers for the quantitative PCR were designed using the Bea- Thawed aliquots of transfected cells were diluted in the binding con Designer v4.0 (Premier Biosoft). The annealing temperature buffer containing 2 g/l Bacitracin and thereafter homogenized. All was optimized using PCR on genomic DNA. The real-time PCR reac- binding experiments were performed in a total volume of 100 ll, tions were performed in a MyiQ thermal cycler (Bio-Rad Laborato- with 125I-labeled pPYY (porcine PYY) as radioligand and with an ries, Sweden) under the following conditions: 95 °C for 4 min, incubation time of 3 h at room temperature. Saturation experiments followed by 50 cycles at 95 °C for 15 s, 59 °C for 30 s and 72 °C were performed with 12 different concentrations of radioligand for 30 s. This was followed by 84 cycles at 55 °C for 10 s, increased ranging from 8 to 1000 pM. Competition binding experiments were by 0.5 °C per cycle. Each reaction had a total volume of 20 ll and performed with the synthesized amidated native peptides NPY, PYY contained cDNA synthesized from 5 ng of total RNA, 5 pM of each and PP (Fig. 1) as competing ligands which were diluted to 12 differ- 6 11.5 primer, 8 mM MgCl2, 0.2 mM dNTP, SYBR Green (1:50,000), ent concentrations between 10À and 10À M. Protein concentra- 0.02 l/ll Taq DNA polymerase and buffer (without MgCl2) (Invit- tions were measured with the Bio-Rad Protein Assay (Bio-Rad) with rogen). Absence of genomic DNA in the cDNA was controlled for bovine serum albumin as standard. The incubation was terminated by PCR using a primer pair designed to span a small intron. by rapid filtration through GF/C filters presoaked in 0.3% polyethyleneimine using a TOMTEC cell harvester. The filters were 2.9. Data analysis and relative expression calculations washed with ice-cold 50 mM Tris–HCl (pH 7.4) and dried at 50 °C for 30 min and MeltiLex A (Perkin–Elmer) melt-on scintillator Gene expression data were analyzed and threshold cycle (Ct) sheets were melted onto the dried filters. Radioactivity was values derived using MyIQ software v 1.04 (Bio-Rad Laboratories). measured using a Wallac 1450 Betaplate counter. Results were ana- Differences in the primer efficiency was corrected for using Lin- lyzed using Prism 4.0 software package (GraphPad). Each experi- RegPCR [42], and Grubbs test for outliers (GraphPad) was used to ment was performed at least three times, each time with duplicate calculate the mean primer efficiency for each primer pair and samples. The pK values were compared to each other using one- i detection and exclusion of outliers. The Ct values were transformed way ANOVA followed by Tukey–Kramer Multiple Comparison Test into quantities using the delta Ct method [30] and the highest (GraphPad). expression was set to 1. geNorm [47] was used to identify the most stable housekeeping gene which thereafter was used to normalize 2.7. RNA isolation and cDNA synthesis expression due to differences in cDNA concentration between samples. Adult S. tropicalis frogs were purchased from NASCO and main- tained in the laboratory in well water (25–26 °C) under a 12L:12D 3. Results photoperiod. Frogs were fed frog brittle (NASCO). All procedures involving animals were conducted in accordance with the guide- 3.1. Identification and phylogenetic analysis lines of the University Committee on the Care and Use of Animals of the University of Michigan (animal care and use protocol Using BLAST searches with the human and chicken receptor #09860). RNA was extracted from male and female frog tissues amino acid sequences as queries it was possible to identify the using the Trizol Reagent (Invitrogen) following the manufacturer’s genes encoding three peptides and six receptors in the genome instructions. cDNA was synthesized using M-MLV reverse database of S. tropicalis. The three peptide genes were identified

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. Author's personal copy

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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.

as encoding NPY, PYY and PP, respectively, by comparisons with predicted to contain several introns. Our sequence analysis shows NPY-family peptide sequences previously determined in the that the frog Y5 gene lacks introns like the Y5 gene in other species. tetraploid frog X. laevis [18,23,46] as shown in Fig. 1. Two distinct The Y1 gene has an intron of 92 nucleotides, similar in size to the X. laevis sequences have been reported for each of the three pep- corresponding intron in chicken which is 121 nucleotides and in tides. Although it has not been formally demonstrated the pub- human 97 nucleotides. One gene was identified as a Y2 receptor lished X. laevis sequences correspond to two gene copies (they in the S. tropicalis genome database, but our phylogenetic analysis might be allelic), it is reasonable to assume so. The S. tropicalis se- shows that it is a Y7 receptor (see Fig. 2 and Supplementary Fig. 1). quences conform well to the X. laevis sequences. The S. tropicalis The Y7 receptor is not present in mammals and this might explain NPY sequence is identical to one of the X. laevis sequences, the the misidentification in the database. The true frog Y2 receptor other differs at one position. The PP sequence in S. tropicalis like- ortholog is located on a different scaffold. Fig. 3 shows the identi- wise is identical to one of the X. laevis sequences and has one dif- fied S. tropicalis sequences aligned with the NPY receptors from hu- ference to the other one. The X. laevis PYY sequences differ more man and chicken and the cloned sequences from P. esculentus. In from the S. tropicalis sequence, namely at two and four positions, total four partial receptor sequences were cloned in P. esculentus respectively. For pancreatic polypeptide, we could not find the and identified as Y1, Y5, Y6 and Y7 receptors by blastX and phylo- exon that encodes the last two amino acids of the mature peptide, genetic analyses. All sequences have been submitted to GenBank the subsequent glycine forming the carboxyterminal amide, and except Y1 (due to short length) and their accession IDs are shown the two basic amino acids for proteolytic cleavage from the precur- in Supplementary Table 1. sor. The peptide was therefore synthesized based on the last two residues (plus the amide) found in pancreatic polypeptide in other 3.2. Pharmacological characterization amphibians, i.e., RFamide. Two of the receptors were identified as Y1 and Y5 in our phylo- All six S. tropicalis receptor genes were inserted into expression genetic analyses (Fig. 2 and Supplementary Fig. 1) and are located vectors for functional expression. The receptors Y5, Y7 and Y8 gave next to each other on the same scaffold. In human the Y5 gene is good expression that allowed binding studies for measuring affin- located 20 kb from the Y1 gene in a head-to-head orientation, ities. Table 1 shows the affinities for the three endogenous ligands and in chicken the distance is 18 kb. The distance between Y5 in competition with iodinated porcine PYY. Figs. 4 and 5 show rep- and Y1 in the frog is 86 kb. It should be noted that the Y5 gene resentative binding curves for each receptor. For all three recep- in the database, although correctly identified, has been erroneously tors, PYY had significantly (p < 0.05) higher affinity with Ki values Author's personal copy

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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. Author's personal copy

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Table 1 expression are the Y1 and Y2 receptors as well as mRNA for the The Ki-values and standard error of the mean (SEM) for three different S. tropicalis peptide NPY. Unfortunately, the assay could not be performed for (Sitr) receptors and their native ligands. Y4 in the brain regions due to shortage of material. The NPY mRNA Ligand SitrY5 SitrY7 SitrY8 has a similar expression level in the majority of tissues. The scale of

Ki (nM) ± SEM nKi (nM) ± SEM nKi (nM) ± SEM n the Y-axis also gives an indication of the small differences in SitrNPY 13.0 ± 4.2 5 21.7 ± 6.3 3 0.14 ± 0.015 3 expression between the tissues from this gene. For the other pep- SitrPYY 0.34 ± 0.13 4 0.17 ± 0.03 4 0.042 ± 0.005 3 tides and the receptors there is approximately a 25-fold difference SitrPP 96.8 ± 30.2 5 103.0 ± 37.7 3 0.50 ± 0.11 3 between the expression in the tissues with the lowest expression KD (nM) 0.200 ± 0.007 3 0.097 ± 0.004 3 0.080 ± 0.014 3 compared to the tissues with the highest. The mRNA of the PYY n = Number of experiments performed, each experiment was done with duplicate and PP peptides display almost identical expression proﬁles with samples. highest expression in skin and blood (see Fig. 7).

4. Discussion in the sub-nanomolar range, as compared to NPY and PP. NPY and PP also had high affinity for Y8, at 0.14 and 0.50 nM, respectively. The genome of the diploid frog S. tropicalis has been sequenced For the Y5 and Y7 receptors, NPY had approximately 100-fold low- [21] and we describe here three NPY-family peptides and six NPY- er affinity than PYY, and PP more than 500-fold lower affinity. family receptor genes. The sequences for the three peptides NPY, Although all six receptors were transfected, no binding to the Y1, PYY and PP in S. tropicalis agree well with other amphibian and tet- Y2 and Y4 receptors was detected in either human HEK-293 cells rapod sequences. Both NPY and PP sequences are identical to one or the frog cell-lines XTC-2, XL-58 and A6. Constructs with GFP- of the sequences previously reported for X. laevis and differ from tagged receptors were made in order to allow microscopy observa- the other one at only one position (Fig. 1). Surprisingly, the two tion of the receptors in the cells. However, fluorescence from cells published X. laevis PYY sequences differ from each other at as many transfected with the Y1, Y2 and Y4 receptors did not differ from the as six positions. The single S. tropicalis PYY sequence has two and negative control (data not shown). The Y8 receptor was used as a four differences to the X. laevis PYY sequences, and the most parsi- positive control for GFP detection. monius interpretation is that the two X. tropicalis PYY sequences have acquired two and four replacements, respectively, perhaps 3.3. Expression levels for mRNA indicating a reduced conservative selection pressure after the duplication. We synthesized each of the three S. tropicalis peptides The expression pattern of mRNA was investigated with quanti- with a carboxyterminal amide group for binding studies of the tative PCR in a panel of 19 S. tropicalis organs from a male frog, plus functionally expressed receptors. ovaries. In general there was a similar expression pattern for the Using phylogenetic analyses we were able to identify them as receptors with the highest mRNA expression in skin, muscle, blood the six NPY-family receptor genes encoding receptor subtypes and heart (Fig. 6). The only genes with detectable neuronal mRNA Y1, Y2, Y4, Y5, Y7 and Y8 (Fig. 2 and Supplementary Fig. 1). It

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). Author's personal copy

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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. was not possible to identify any Y6 receptor in the genome data- sponding to 46 amino acids was obtained for Y1). This shows that base. However, we have also cloned by PCR partial sequences for the ancestor of the amphibian lineage had the full repertoire of Y1, Y5, Y6 and Y7 from P. esculentus (only a short segment corre- receptors as that inferred for the gnathostome ancestor [26,27], Author's personal copy

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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.

although the Y6 sequence may represent a pseudogene in P. escu- PP (see Table 1). This differs from the situation in both mammals lentus. One interesting observation in this context is that this [32] and chicken [22] where the three peptides have almost equal amphibian has the same three peptides as other tetrapods, but affinities. It has been found that NPY stimulates food intake par- has more receptor subtypes than mammals (4–5 subtypes). In tially via the Y5 receptor in the paraventricular nucleus in mam- the phylogenetic tree, the Y4 receptor sequences appear in two dif- mals [28,29], but the much higher affinity of PYY for Y5 in a frog ferent clusters, namely amniotes and teleost fishes together with may indicate a somewhat different mechanism or physiological sharks. The S tropicalis sequence clusters together with the amni- role. The Y5 receptor arose already in the ancestor of gnathostomes otes, albeit with low bootstrap support. The teleost receptors were and cyclostomes, as it has been identified in a lamprey [27], and identified as Y4 based upon chromosomal location [26] and their therefore must originally have been a receptor for the NPY/PYY sequences cluster with the shark sequences in the tree, thereby ancestral peptide. In this perspective, it is interesting to note that supporting the identity of these as Y4. The Y4 sequences have an in S. tropicalis, NPY has a much reduced affinity for Y5 whereas accelerated substitution rate in all lineages compared to the other PYY has high affinity. It will be interesting to explore if this reflects receptor subtypes, which explains why Y4 in amniotes and tele- a different function for Y5 in the frog lineage as compared to the osts/sharks do not cluster together. Unfortunately, the S tropicalis mammalian lineage. sequence is located on a small scaffold in the database and the The Y7 receptor in S. tropicalis has a ligand profile which is al- few gene neighbors present do not clarify its identity. most identical to that of the Y5 receptor. This differs from chicken The Y4 receptor has an established partnership with PP in mam- receptor Y7 where NPY had a higher affinity than chicken PYY [5]. mals, because PP has the highest affinity for Y4 among the three Compared to Y7 in zebrafish or rainbow trout [16,25] the most endogenous peptides, while all three peptides in chicken bind with striking observation for the chicken Y7 receptor was its overall equal affinity to the chicken Y4 receptor [31,33]. Despite testing low affinity. several different cell lines, including three cell lines from the clo- The S. tropicalis Y8 receptor stands out from Y5 and Y7 in that all sely related frog X. laevis, it was not possible to functionally express three peptides have higher affinity for this receptor than for the the S. tropicalis Y4 receptor. Previous attempts to express the Y1 other two. PYY had the highest affinity at 0.042 nM. Interestingly, receptor from X. leavis in mammalian cells were also unsuccessful PP has an affinity of 0.50 nM making Y8 the only receptor among [4] and the two frog species differ at only 13 amino acid positions these three where PP seems to have physiologically relevant affin- out of 366. ity. It is interesting to note that Y8 is the only receptor among these The Y5 receptor in S. tropicalis was found to have preference for that belongs to the Y1 subfamily, as does the PP-binding receptor PYY whose affinity was much higher than the affinities of NPY or Y4 in amniotes. The Y8 receptor is missing in all amniote genomes Author's personal copy

330 G. Sundström et al. / General and Comparative Endocrinology 177 (2012) 322–331 in the database and seems to have been lost in this lineage. On the References other hand, the Y8 receptor is present in duplicate (Y8a and Y8b) in the teleost fishes Tetraodon nigroviridis and Takifugu rubripes [26]. [1] S.F. Altschul et al., Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403–410. However, PP is a tetrapod-specific duplicate of PYY. [2] R.L. Batterham et al., Gut hormone PYY(3–36) physiologically inhibits food The six S. tropicalis NPY-family receptors have a fairly similar intake, Nature 418 (2002) 650–654. tissue distribution as measured by quantitative PCR of mRNA [3] R.L. Batterham et al., Pancreatic polypeptide reduces appetite and food (Fig. 6). The most prominent expression was observed in skin, mus- intake in humans, J. Clin. Endocrinol. Metab. 88 (2003) 3989–3992. [4] A.G. Blomqvist et al., Cloning and sequence analysis of a neuropeptide Y/ cle, blood and heart. The stomach always has lower expression peptide YY receptor Y1 cDNA from Xenopus laevis, Biochim. Biophys. Acta 1261 than the small and large intestine. There is a higher expression of (1995) 439–441. the Y7 receptor in the small intestine than in the large intestine, [5] T. Bromee et al., Neuropeptide Y-family receptors Y6 and Y7 in chicken. Cloning, pharmacological characterization, tissue distribution and conserved while the other receptors have a similar expression in both parts synteny with human chromosome region, FEBS J. 273 (2006) 2048–2063. of the gastrointestinal tract. The Y5 receptor appears to have a sim- [6] N. Chartrel et al., Characterization of melanotropin-release-inhibiting factor ilar expression pattern as the Y7 receptor in the small and large (melanostatin) from frog brain: homology with human neuropeptide Y, Proc. Natl. Acad. Sci. USA 88 (1991) 3862–3866. intestine, however the error bars are too large to draw any firm [7] J.M. Conlon et al., Primary structure of frog PYY: implications for the molecular conclusions. In the nervous system, the only receptor genes with evolution of the pancreatic polypeptide family, Peptides 13 (1992) 145–149. detectable mRNA expression are Y1 and Y2, in addition to the [8] J.M. Conlon et al., Amino acid sequence diversity of pancreatic polypeptide among the amphibia, Gen. Comp. Endocrinol. 112 (1998) 146–152. mRNA for NPY. The Y1 receptor shows expression in both [9] J.M. Conlon, F. O’Harte, The primary structure of a PYY-related peptide from telencephalon and diencephalon while only minor expression in chicken intestine suggests an anomalous site of cleavage of the signal peptide diencephalon is detectable for the Y2 receptor. The broad expres- in preproPYY, FEBS Lett. 313 (1992) 225–228. [10] E.J. Crespi et al., Roles of corticotropin-releasing factor, neuropeptide Y and sion of the NPY-family receptors in this frog is similar to that pre- corticosterone in the regulation of food intake in Xenopus laevis, J. viously reported for the two cartilaginous fishes spiny dogfish [43] Neuroendocrinol. 16 (2004) 279–288. and elephant shark [27]. [11] D.E. Day et al., Role of NPY and its receptor subtypes in foraging, food hoarding The distribution of the NPY-family peptides in a few organs from and food intake by Siberian hamsters, Am. J. Physiol. Regul. Integr. Comp. Physiol., 2005. various frogs has previously been described using immunohisto- [12] M. De Falco et al., Different patterns of expression of five neuropeptides in the chemistry methods [12,13,19,35]. In mammals NPY is mainly ex- adrenal gland and kidney of two species of frog, Histochem. J. 34 (2002) pressed by neurons while PYY and PP are released from endocrine 21–26. [13] W.G. Ding et al., Neuropeptide Y and peptide YY immunoreactivities in the cells. Our quantitative PCR analyses of the three peptides in a large pancreas of various vertebrates, Peptides 18 (1997) 1523–1529. organ panel show that all three peptides have surprisingly broad dis- [14] I.A. El Karim et al., Antimicrobial activity of neuropeptides against a range of tribution (Fig. 7). The three peptides display a similar expression micro-organisms from skin, oral, respiratory and gastrointestinal tract sites, J. Neuroimmunol. 200 (2008) 11–16. pattern in non-neuronal organs with high expression in skin. Both [15] B.J. Evans et al., A mitochondrial DNA phylogeny of African clawed frogs: NPY and PYY have been isolated from frog skin and it has been dem- phylogeography and implications for polyploid evolution, Mol. Phylogenet. onstrated that they display antimicrobial properties [14,48]. In the Evol. 33 (2004) 197–213. [16] R. Fredriksson et al., Novel neuropeptide Y Y2-like receptor subtype in nervous system the expression level of NPY is highest in diencepha- zebrafish and frogs supports early vertebrate chromosome duplications, J. Mol. lon, followed by telencephalon. Expression in hindbrain and the Evol. 58 (2004) 106–114. spinal cord is also detectable. Neither PYY nor PP has any expression [17] L. Galas et al., Neuropeptide Y inhibits spontaneous alpha-melanocyte- stimulating hormone (alpha-MSH) release via a Y(5) receptor and suppresses in these tissues. High expression for all three peptides was found in thyrotropin-releasing hormone-induced alpha-MSH secretion via a Y(1) small and large intestine. More detailed studies will be necessary to receptor in frog melanotrope cells, Endocrinology 143 (2002) 1686–1694. see whether NPY is expressed in intestinal neurons and PYY and PP [18] D. Griffin et al., Isolation and characterization of the Xenopus laevis cDNA and in endocrine cells. genomic homologs of neuropeptide Y, Mol. Cell. Endocrinol. 101 (1994) 1–10. [19] R.P. Guedes et al., Complete sciatic nerve transection induces increase of 5. Conclusions neuropeptide Y-like immunoreactivity in primary sensory neurons and spinal cord of frogs, Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 139 (2004) 461– 467. We report here that the NPY system in the frog S. tropicalis is [20] S. Guindon, O. Gascuel, A simple, fast, and accurate algorithm to estimate large comprised of three peptides and six receptors and thereby displays phylogenies by maximum likelihood, Syst. Biol. 52 (2003) 696–704. [21] U. Hellsten et al., The genome of the Western clawed frog Xenopus tropicalis, greater complexity than in mammals. Binding properties and ana- Science 328 (2010) 633–636. tomical distribution overall show similarities to mammals but [22] S.K. Holmberg et al., Pharmacological characterization of cloned chicken there are also some interesting differences that warrant further neuropeptide Y receptors Y1 and Y5, J. Neurochem. 81 (2002) 462–471. [23] J.B. Kim et al., Anomalous rates of revolution of pancreatic polypeptide and studies. An intriguing question for the future is why the NPY sys- peptide tyrosine–tyrosine (PYY) in a tetraploid frog, Xenopus laevis (Anura: tem in mammals has lost two of the receptors still present in frogs, Pipidae), Peptides 22 (2001) 317–322. Y7 and Y8, and what functions those receptors perform in frogs. [24] S. Kojima et al., A role for pancreatic polypeptide in feeding and body weight regulation, Peptides 28 (2007) 459–463. [25] T.A. Larsson et al., Characterization of NPY receptor subtypes Y2 and Y7 in Acknowledgments rainbow trout Oncorhynchus mykiss, Peptides 27 (2006) 1320–1327. [26] T.A. Larsson et al., Early vertebrate chromosome duplications and the evolution of the neuropeptide Y receptor gene regions, BMC Evol. Biol. 8 We thank Isabelle Lihrmann, University of Rouen, France, for pro- (2008) 184. viding genomic DNA. This work was supported by a Grant from the [27] T.A. Larsson et al., Neuropeptide Y-family peptides and receptors in the Swedish ResearchCouncil. We are grateful to Yuliang Ma for provid- elephant shark, Callorhinchus milii confirm gene duplications before the ing technical assistance. This research was supported in part by a gnathostome radiation, Genomics 93 (2009) 254–260. [28] A. Lecklin et al., Receptor subtypes Y1 and Y5 mediate neuropeptide Y induced Grant from the National Science Foundation of USA (IOS 0641587) feeding in the guinea-pig, Br. J. Pharmacol. 135 (2002) 2029–2037. to R.J. Denver. [29] A. Lecklin et al., Agonists for neuropeptide Y receptors Y1 and Y5 stimulate different phases of feeding in guinea pigs, Br. J. Pharmacol. 139 (2003) 1433–1440. Appendix A. Supplementary data [30] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real- time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods 25 (2001) 402–408. Supplementary data associated with this article can be found, [31] I. Lundell et al., Cloning of a human receptor of the NPY receptor family with in the online version, at http://dx.doi.org/10.1016/j.ygcen.2012. high affinity for pancreatic polypeptide and peptide YY, J. Biol. Chem. 270 (1995) 29123–29128. 04.027. Author's personal copy

G. Sundström et al. / General and Comparative Endocrinology 177 (2012) 322–331 331

[32] I. Lundell et al., Cloning and characterization of the guinea pig neuropeptide Y [42] C. Ramakers et al., Assumption-free analysis of quantitative real-time receptor Y5, Peptides 22 (2001) 357–363. polymerase chain reaction (PCR) data, Neurosci. Lett. 339 (2003) 62–66. [33] I. Lundell et al., Chicken neuropeptide Y-family receptor Y4: a receptor with [43] E. Salaneck et al., Three neuropeptide Y receptor genes in the spiny dogfish, equal affinity for pancreatic polypeptide, neuropeptide Y and peptide YY, J. Squalus acanthias, support en bloc duplications in early vertebrate evolution, Mol. Endocrinol. 28 (2002) 225–235. Mol. Biol. Evol. 20 (2003) 1271–1280. [34] D.M. McKay et al., The complete primary structure of pancreatic polypeptide [44] W.W. Schwippert et al., Neuropeptide Y (NPY) or fragment NPY 13–36, but not from the European common frog Rana temporaria, Regul. Pept. 31 (1990) 187– NPY 18–36, inhibit retinotectal transfer in cane toads Bufo marinus, Neurosci. 197. Lett. 253 (1998) 33–36. [35] D.M. McKay et al., The primary structure and tissue distribution of an [45] J.D. Thompson et al., CLUSTAL W: improving the sensitivity of progressive amphibian neuropeptide Y, Regul. Pept. 37 (1992) 143–153. multiple sequence alignment through sequence weighting, position-specific [36] R. Mentlein et al., Proteolytic processing of neuropeptide Y and peptide YY by gap penalties and weight matrix choice, Nucleic Acids Res. 22 (1994) 4673– dipeptidyl peptidase IV, Regul. Pept. 49 (1993) 133–144. 4680. [37] A. Mor et al., Skin peptide tyrosine-tyrosine, a member of the pancreatic [46] M.C.H.M. van Riel et al., Cloning and sequence analysis of hypothalamic cDNA polypeptide family: isolation, structure, synthesis, and endocrine activity, encoding Xenopus preproneuropeptide Y, Biochem. Biophys. Res. Commun. Proc. Natl. Acad. Sci. USA 91 (1994) 10295–10299. 190 (1993) 948–951. [38] Y.K. Narnaware, R.E. Peter, Neuropeptide Y stimulates food consumption [47] J. Vandesompele et al., Accurate normalization of real-time quantitative RT- through multiple receptors in goldfish, Physiol. Behav. 74 (2001) 185–190. PCR data by geometric averaging of multiple internal control genes, Genome [39] J.R. Parkinson et al., PYY3-36 injection in mice produces an acute anorexigenic Biol. 3 (2002) RESEARCH0034. effect followed by a delayed orexigenic effect not observed with other [48] I. Vouldoukis et al., Broad spectrum antibiotic activity of the skin-PYY, FEBS anorexigenic gut hormones, Am. J. Physiol. Endocrinol. Metab. 294 (2008) Lett. 380 (1996) 237–240. E698–E708. [49] Y. Wang et al., Molecular evolution of peptide tyrosine–tyrosine: primary [40] T. Pedrazzini et al., Neuropeptide Y: the universal soldier, Cell. Mol. Life Sci. 60 structure of PYY from the lampreys Geotria australis and Lampetra fluviatilis, (2003) 350–377. bichir, python and desert tortoise, Regul. Pept. 79 (1999) 103–108. [41] H.G. Pollock et al., Isolation of peptide hormones from the pancreas of the [50] L. Zhang et al., The neuropeptide Y system: pathophysiological and therapeutic bullfrog (Rana catesbeiana). Amino acid sequences of pancreatic polypeptide, implications in obesity and cancer, Pharmacol. Ther. 131 (2011) 91–113. oxyntomodulin, and two glucagon-like peptides, J. Biol. Chem. 263 (1988) 9746–9751.