Cloning and pharmacological characterization of the neuropeptide Y receptor Y5 in the sea lamprey, Petromyzon marinus

Peptides 39 (2013) 64–70

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Cloning and pharmacological characterization of the neuropeptide Y receptor Y5 in the sea lamprey, Petromyzon marinus Bo Xu a , Görel Sundström a,1 , Shigehiro Kuraku b,2 , Ingrid Lundell a , Dan Larhammar a,∗ a b

Department of Neuroscience, Uppsala University, Box 593, 75124 Uppsala, Sweden Department of Biology, University of Konstanz, Universitätsstrasse 10, 78464 Konstanz, Germany

a r t i c l e

i n f o

Article history: Received 3 October 2012 Received in revised form 13 November 2012 Accepted 14 November 2012 Available online 23 November 2012 Keywords: Lamprey Neuropeptide Y Peptide YY Y5 receptor

a b s t r a c t The neuropeptide Y system is known to have expanded in early vertebrate evolution. Three neuropeptide Y receptors have been proposed to have existed before the two basal vertebrate tetraploidizations, namely a Y1-like, a Y2-like, and a Y5-like receptor, with their genes in the same chromosomal region. Previously we have described a Y1-subfamily and a Y2-subfamily receptor in the river lamprey, Lampetra fluviatilis. Here we report the identification of a Y5 receptor in the genome of the sea lamprey, Petromyzon marinus. In phylogenetic analyses, the Y5 receptor clusters together with gnathostome Y5 receptors with high bootstrap value and shares the long intracellular loop 3. This lamprey receptor has an even longer loop 3 than the gnathostome Y5 receptors described so far, with the expansion of amino acid repeats. Functional expression in a human cell line, co-transfected with a modified human G-protein, resulted in inositol phosphate turnover in response to the three lamprey NPY-family peptides NPY, PYY and PMY at nanomolar concentrations. Our results confirm that the Y1–Y2–Y5 receptor gene triplet arose before the cyclostome-gnathostome divergence. However, it is not clear from the NPY receptors whether cyclostomes diverged from the gnathostome lineage after the first or the second tetraploidization. Duplicates resulting from the tetraploidizations exist for both Y1 and Y2 in gnathostomes, but only a single copy of Y5 has survived in all vertebrates characterized to date, making the physiological roles of Y5 interesting to explore. © 2012 Elsevier Inc. All rights reserved.

1. Introduction The NPY (neuropeptide Y) system has attracted considerable attention due to its role in the regulation of appetite and energy balance, but also in several other biological contexts or diseases, including but not limited to blood pressure, depression, pain, cancer and bone formation [25,29,49]. The physiological functions of the NPY system are exerted by the binding of NPY-family peptides to several receptor subtypes that have different expression patterns [18,34,47]. The roles of the NPY system in appetite regulation are well characterized in mammals: NPY binds to receptors Y1 and Y5 in the hypothalamus to stimulate appetite, whereas the hormones PYY and PP have the opposite effect by

Abbreviations: NPY, neuropeptide Y; PYY, peptide YY; PP, pancreatic polypeptide; PMY, peptide MY; 2R, two rounds of genome doubling; 3R, third rounds of genome doubling; TM, transmembrane region; IP assay, inositol phosphate assay. ∗ Corresponding author. Tel.: +46 184714173; fax: +46 18511540. E-mail address: [email protected] (D. Larhammar). 1 Present address: Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, 75123 Uppsala, Sweden. 2 Present address: Genome Resource and Analysis Unit, RIKEN Center for Developmental Biology (CDB) Kobe, Japan. 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.11.007

binding to Y2 and Y4, respectively, in the basal hypothalamus, the vagus nerve and the brainstem [44,49]. Some functional studies of the NPY system have also been performed in other vertebrate classes like birds, reptiles, bony fish and lampreys, see for instance [3,6,30,33,35], but the information about these lineages is still limited. For detailed characterization of the biological functions in different species, evolutionary studies and identification of the individual NPY-family peptides and their receptors is required. We have therefore identified the genes, synthesized the peptides and cloned and characterized the receptors from a broad range of vertebrates [4,8–11,20–22,26,39,42,43,45,47]. The number of peptides and receptors of the NPY family has expanded through both local gene duplications and genome duplications which are two important mechanisms for the emergence of new genes and gene functions [18,24,46]. NPY and PYY have been identified in all major vertebrate lineages investigated. The tetrapod-specific pancreatic polypeptide (PP) has been confirmed to be a local duplicate of the PYY gene [12,17]. In teleost fishes, an extra gene copy for both NPY and PYY was generated [46] during the teleost-specific tetraploidization [5,13]: two copies of NPY, named NPYa and NPYb, have been identified in several species of teleost fishes, although zebrafish seems to have lost NPYb, and the socalled fish-specific peptide PY has been confirmed to be a duplicate

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Fig. 1. Peptides used for functional assay. Four peptides were used for IP functional assay: L. fluviatilis NPY (LflNPY), P. marinus PYY (PmaPYY) and PMY (PmaPMY) and pig PYY (pPYY). LflNPY was used instead of PmaNPY as they differ by a only single conservative replacement: D16 is an E in PmaNPY.

of PYY, hence renamed to PYYb [46]. In the river lamprey Lampetra fluviatilis, three NPY-family peptides, NPY, PYY and PMY have been identified [40,48]. The sea lamprey Petromyzon marinus peptides have been isolated and PYY has the same sequence as in the river lamprey whereas NPY and PMY differ at a single position between the two species [7,28]. The evolutionary relationship between PYY and PMY in the lamprey lineage has not yet been clarified [46]. It has been proposed that three ancestral neuropeptide Y receptor genes [18] existed before the two basal vertebrate tetraploidizations, called 2R for two rounds of genome doubling [32,38]. This ancestral triplet was generated by local duplications of a single ancestral NPY receptor gene, and resulted in a Y1-like, a Y2-like and a Y5-like gene. A repertoire of seven receptor genes was present in the gnathostome ancestor (after 2R), belonging to three subfamilies based on phylogenetic analyses, Y1-like (Y1, Y4, Y6, Y8), Y2-like (Y2, Y7) and Y5-like (only Y5) [18,23,24]. Through lineagespecific deletions, local duplication and the teleost fish-specific third round genome doubling (3R), different numbers of receptors, from 4 to 7, are now maintained in mammalian, bird, amphibian and teleost fish lineages [4,8–11,20–22,26,39,42,43,45,47]. Except for the euteleost fish lineage, the Y5 gene has been cloned or identified in all these lineages, including basal ray-finned fishes as well as in the lobe-finned fish Latimeria chalumnae and the cartilaginous fish Callorhinchus milii [22,24,43]. Interestingly, the number of receptors in the Y1 and Y2 subfamilies increased during vertebrate evolution, but Y5 is the only member belonging to the Y5 subfamily. A Y1-subfamily [41] and a Y2/Y7-subfamily [24] receptor have been identified in L. fluviatilis, and partial sequences for a Y5-like [24] and another Y1-subfamily gene (unpublished) have also been found in this species. Lampreys constitute a highly interesting and important vertebrate lineage because they diverged from the lineage leading to gnathostomes around the time for the second vertebrate tetraploidization. Whether lampreys diverged after the first or the second tetraploidization is still not clear [16]. Here we report the complete Y5 sequence of P. marinus and functional studies in vitro with the three lamprey peptides, thereby confirming the previously proposed evolutionary scenario for the vertebrate NPY-family receptors.

0.20). The alignment included sequences from following species; Pma, Petromyzon marinus, Hsa Homo sapiens, Ssc Sus scrofa, Rno Rattus norvegicus, Gga Gallus gallus, Cmi Callorhinchus milii, Sac Squalus acanthias, Dre Danio rerio, Ocu Oryctolagus cuniculus, Lch Latimeria chalumnae, Tru Takifugu rubripes and Lfl Lampetra fluviatilis. Accession numbers; HsaY1 – NM 000909, SscY1 – AF106081, RnoY1 – NM 001013032, GgaY1 – NM 001031535, CmiY1 – EU637847, SacY1 – AH012614, DreY1 – EU046342, OcuY6 – D86521, GgaY6 – NM 001044687, LchY6 – ABI94073, SacY6 – AY177271, CmiY6 – EU637851, TruY8a – EU104004, DreY8a – NM 131437, DreY8b – AF030245, TruY8b – EU104005, CmiY8 – EU637853, HsaY4 – NM 005972, SscY4 – AB021678, RnoY4 – U84245, GgaY4 – AF410853, SacY4 – AY177270, CmiY4 – EU637849, DreY4 – AF037400, TruY4 – EU104002, LflY1-like – AAL66410, PmaY1like – ENSPMAG00000009963, HsaY5 – NM 006174, RnoY5 – NM 012869, SscY5 – AF106083, GgaY5 – NM 001031130, LchY5 – ABI94072, CmiY5 – EU637850, DreY2 – XP 001343301, TruY2 – EU104001, SscY2 – AF106082, HsaY2 – NM 000910, RnoY2 – NM 023968, GgaY2 – NM 001031128, CmiY2 – EU637848, LflY2Y7-like – EU743622, GgaY7 – NP 001032913, CmiY7 – EU637852, TruY7 – EU104003, DreY7 – AY585098, HsaSSTR1 – NP 001040. The alignment was cut to remove N-terminal and C-terminal parts, resulting in a final alignment spanning from the start of TM1 (Transmembrane region 1) to the end of TM7. A phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1000 bootstrap replicates in ClustalX 2.012 [19].

2.1.2. Alignment of Y5 receptors The identified sea lamprey amino acid sequence was aligned with other Y5 sequences using ClustalW 2.012 with standard settings (Gonnet weight matrix, gap opening penalty 10.0 and gap extension penalty 0.20). Amino acid sequences with these accession numbers were retrieved using the NCBI database. HsaY5 – NM 006174, RnoY5 – NM 012869, SscY5 – AF106083, GgaY5 – NM 001031130, StrY5 – NP 001072244, LchY5 – ABI94072, CmiY5 – EU637850, ObiY5 – EU46356, AbaY5 – EU046345, PseY5 – EU046360, SacY5 – EU046362. Abbreviations as above but the alignment also includes also these species; Str, Silurana tropicalis, Obi, Osteoglossum bichirossum, Aba, Acipenser baerii, Pse, Polypterus senegalus.

2. Materials and methods 2.1. P. marinus Y5 sequence identification and analysis

2.2. Primer design and coding region amplification

The nucleotide and amino acid sequences of the putative P. marinus Y5 (PmaY5) was identified on the scaffold25143 in the genome assembly PMAR3 (http://genome.wustl.edu/pub/organism/Other Vertebrates/Petromyzon marinus/assembly/Petromyzon marinus3.0/output/) by TBLASTN search using human NPY Y5 as a template.

Primers for PCR amplification were designed based on PmaY5 sequence, a Kozak consensus sequence for initiation of translation was added to the 5’ end of forward primer: 5’-CCT ACC ATG GCC CTC TCC ACG-3’. A reverse primer was designed where the Stop codon was replaced to give a continuous open reading frame with GFP: 5’-CCG CCC GTG ACC CAG GCA G-3’. The GC-rich PCR system, DNTPPack reagent (Roche) was used to amplify the genomic DNA using the program: 95 ◦ C for 5 min and following 30 cycles of 30 s at 95 ◦ C, 30 s at 62.5 ◦ C, and 90 s at 72 ◦ C, followed by 7 min at 72 ◦ C. The PCR product was purified using the MinElute Gel Extraction Kit (Qiagen) according to product instructions.

2.1.1. Phylogenetic analyses The identified PmaY5 amino acid sequence was aligned with other vertebrate NPY receptor sequences and human somatostatin receptor 1 using ClustalW 2.012 with standard settings (Gonnet weight matrix, gap opening penalty 10.0 and gap extension penalty

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Fig. 2. Amino acid sequence alignment of Y5 receptors. Predicted transmembrane (TM) regions are marked with frames. The frames in the aminoterminal region mark the consensus sequences Asn-X-Ser/Thr for N-linked glycosylation. The frames in extracellular loops 2 and 3 mark the cysteines predicted to form a disulfide bridge. The cysteine in the box in the carboxyterminal region is expected to be the target for palmitoylation. Species abbreviations are: Pma, Petromyzon marinus, Hsa Homo sapiens, Ssc Sus scrofa, Rno Rattus norvegicus, Gga Gallus gallus, Cmi Callorhinchus milii, Sac Squalus acanthias, Str, Silurana tropicalis, Obi, Osteoglossum bicirrhossum, Aba, Acipenser baerii, Pse, Polypterus senegalus. For accession numbers see Section 2.

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Fig. 3. Neighbor-joining (NJ) phylogenetic tree for the NPY-receptor family, with bootstrap values (out of 1000) showing on each node. The tree is rooted with human somatostatin receptor 1. Three-letter abbreviations denote species (see below) followed by receptor subtype. Species abbreviations are: Pma, Petromyzon marinus, Hsa Homo sapiens, Ssc Sus scrofa, Rno Rattus norvegicus, Gga Gallus gallus, Cmi Callorhinchus milii, Sac Squalus acanthias, Dre Danio rerio, Ocu Oryctolagus cuniculus, Lch Latimeria chalumnae, Tru Takifugu rubripes and Lfl Lampetra fluviatilis. For accession numbers see Section 2.

2.3. Expression vector cloning

2.4. Cellular expression detection

The purified Y5 coding region DNA was cloned into pcDNA3.1/CT-GFP-TOPO® vector (Invitrogen) using the C-terminal GFP fusion TOPO TA expression kits (Invitrogen) according to the manufacturer’s instructions. Orientation and sequence of the PmaY5 coding region were confirmed by sequencing using T7 forward (5’-TAA TAC GAC TCA CTA TAG GG-3’) and GFP reverse (5’-GGG TAA GCT TTC CGT ATG TAG C-3’) primers. The resulting PmaY5-GFP plasmid was purified using PureLinkTM HiPure Plasmid FilterMaxiprepKit (Invitrogen) for transient transfections.

HEK 293 cells growing on coverslips were transfected with 4 ␮g of plasmids using 5 ␮l of lipofectamine 2000 (Invitrogen) and 500 ␮l of OPTI-MEM (Invitrogen) according to the instructions from the lipofectamine 2000 manual. Cells were grown for 24 h at 37 ◦ C, 5% CO2 . Then the coverslips with cells were washed twice with PBS, 200 ␮l of DAPI was added to the coverslips and incubated at room temperature in the dark for 20 min. After washing twice with PBS, the coverslips were dried and put upside down on a slide with a drop of mounting medium and sealed with nail polish. Cells transfected with no plasmids but using same procedure were used as

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negative control. Pictures were taken using Zeiss LSM 510 Meta confocal microscope, with a 63× oil immersion objective (NA = 1.4), and the LSM software. 2.5. NPY-family peptides used for inositol phosphate (IP) assay The river lamprey L. fluviatilis NPY-family peptides NPY (accession code: P48097) and PYY (AAA21352) as well as the sea lamprey P. marinus NPY-family peptide PMY (AAW47391) were used for the functional assay along with porcine Sus scrofa PYY (P68005). The L. fluviatilis PYY has an identical sequence to P. marinus PYY (AAW47392). For peptide sequence alignment see Fig. 1. The detailed information about synthesis and purification of L. fluviatilis NPY and PYY see [41]. The crude P. marinus PMY was supplied by GL Biochem. Ltd. (Shanghai, China) and was purified to near homogeneity (>98% purity) by reversed-phase HPLC on a 2.2 cm × 25 cm Vydac 218TP1022 (C-18) column. The concentration of acetonitrile in the eluting solvent was raised from 28% to 56% over 60 min and the flow rate was 6 ml/min. The structure of the peptide was confirmed by electrospray mass spectrometry (observed molecular mass 4205.4, calculated molecular mass 4205.9). 2.6. Radioactive inositol phosphate (IP) assay An inositol phosphate (IP) assay was used to study the PmaY5 receptor’s response. A modified G protein plasmid was contransfected with the PmaY5-GFP plasmid: G␣qi4, kindly provided by E. Kostenis [15], encodes the four last amino acids of G␣i at its C-terminus replacing the corresponding G␣q residues, thereby coupling to the inositol phosphate pathway. HEK293 cells about 90–95% dense were co-transfected with the PmaY5-GFP plasmid construct and the G␣qi4 plasmid construct using Lipofectamine 2000 (Invitrogen) according to product instructions. The next day, myo-[2-3 H]inositol at 3 ␮Ci/ml was added. The following day, the cells were detached with PBS-EDTA (0.2 g/L) and resuspended in assay buffer containing 10 mM LiCl (20 mM Hepes, 137 mM NaCl, 5 mM KCl, 0.44 mM KH2 PO4 , 4.2 mM NaHCO3 , 1.2 mM MgCl2 , 1 mM CaCl2 and 10 mM glucose). The cells were preincubated for 10 min and then stimulated with a serial dilution of ligands for 20 min at 37 ◦ C. An equal volume of ice cold 0.8 M PCA was added and incubated on ice for 60 min to lyse the cells. The reaction was terminated by neutralization with KOH–KHCO3 . The generated [3 H] inositol phosphate was isolated by ion exchange chromatography on AG 1-X8 resin (Bio-Rad). The resin was washed with 5 mM Na2 B4 , 60 mM NH4 -formate and eluted with 1 M NH4 -formate, 0.1 M formic acid (method adapted from [14]). After mixing with OptiPhase HiSafe (Perkin-Elmer), the 3 H radioactivity was measured with a liquid scintillation counter. The assay was performed in triplicate for each concentration.

Table 1 EC50 values of different peptides for stimulation of inositol phosphate production in P. marinus Y5-expressing HEK cells. The results are shown as means ±SEM, n represents the number of assays performed. Each assay was performed in triplicate for each ligand concentration. Peptides

EC50 (nM)

SEM (nM)

n

LflNPY PmaPYY PmaPMY pPYY

14.3 18.6 48.1 44.4

4.5 5.2 26.4 19.8

5 6 4 3

other species. Furthermore it contains the characteristic large third cytoplasmic loop which is unique to Y5 in the NPY receptor family. The phylogenetic tree in Fig. 3 shows that PmaY5 clusters with the other vertebrate Y5 sequences with high bootstrap value. 3.2. Pharmacological studies Initially, a radioligand binding assay was performed for the Y5 receptor using the commercially available radioligand 125 I-porcine PYY (125I-pPYY). However, the Kd value could not be determined due to low affinity that would require very high concentration of the radioligand. Instead, a functional assay measuring inositol phosphate (IP) production was used to investigate if Y5 is a functional receptor for the lamprey peptides. It has previously been shown that NPY receptors in mammals preferentially signal through the Gi/o pathway, leading to inhibition of adenylyl cyclase and cAMP production. Depending on the cell type, other pathways are also involved, like the synthesis of inositol phosphates [27,31]. We cotransfected the cells with a modified G-protein that allows G␣i-coupling receptors to give an IP response. We stimulated the cells with either P. marinus PYY or PMY or L. fluviatilis NPY which differs at only a single position from the P. marinus sequence (Fig. 1). The EC50 values for the peptides are 14.3 nM, 18.6 nM and 48.1 nM, respectively (Table 1; a representative response curve is shown in Fig. 4). The pPYY peptide was also used for the functional assay and the EC50 value was 44.3 nM. There were no statistically significant differences between the ligands (ANOVA with Tukey’s Multiple Comparison Test was performed using the pEC50 values, data not shown). 3.3. Cellular expression The PmaY5 construct was tagged with GFP to facilitate microscopic monitoring of receptor expression. After transfection into HEK293 cells, a GFP-positive signal was detected in the cells transfected with Y5 plasmid while the negative control showed no GFP signal. The GFP signal was detected on the cell surface, as expected, but also in cytoplasm to a surprisingly large degree (Fig. 5).

2.7. Data analysis The data were analyzed with Prism 5 software (GraphPad). Sigmoidal dose-response curves were fitted to calculate the EC50 value. 3. Results 3.1. Sequence and phylogenetic analyses The length of the cloned P. marinus Y5 (PmaY5) receptor is 528 amino acids. Compared to the sequence retrieved from the genome database, the cloned Y5 coding region showed a few nucleotide differences without affecting the protein sequence, and a deletion of 6 nucleotides encoding two amino acids in intracellular loop 3, a serine and an alanine. The sequence was aligned with other NPYfamily receptors (Fig. 2) and displays the highest identity to Y5 from

Fig. 4. Representative curve for inosital phosphate (IP) assay Ten different concentrations of PmaPMY peptide were used for each curve. For each concentration, triplicate samples were measured. The functional assay was repeated at least three times for each peptide. Error bars show the standard error of the mean.

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Fig. 5. P. marinus Y5 expression visualized by GFP tag. HEK 293 cells were transfected with the PmaY5-GFP plasmid. Y5 expression is visible on the cell surface (as also demonstrated by the functional assay using intact cells) and in the cytoplasm. Cells transfected with no plasmids were used as negative control (NTC, non-transfected cells).

4. Discussion It has previously been shown that an NPY receptor Y5 arose before the gnathostome radiation by a local duplication of an ancestral Y1/Y5 receptor [18]. However, no Y5 gene has previously been identified in any species that diverged before the origin of the gnathostome lineage. Here we report the identification of a Y5 receptor from the sea lamprey P. marinus which represents the closest extant lineage to the gnathostomes. From the phylogenetic analysis, it is clear that the cloned receptor clusters together with Y5 from the gnathostome lineages, including Mammalia, Aves, Amphibia, and Chondrichthyes, with high bootstrap values (Fig. 3). This shows that the Y5 gene arose before the cyclostome–gnathostome divergence, as predicted in previous analyses of synteny and paralogons in gnathostomes [18,24]. The cloned P. marinus Y5 receptor has the common features of Y5 from other species, including cysteines for disulfide formation and palmitoylation and consensus sites for N-linked carbohydrate AsnX-Ser/Thr (Fig. 2). Compared with other NPY-family receptors, it has a considerably longer cytoplasmic loop 3 and a shorter carboxy terminus. The cytoplasmic loop 3 of this receptor is even longer than in other Y5 sequences, due to a few expanded amino acid regions, including an Asp-rich region, a His- and Gln-rich region, and an Ala-rich region (Fig. 2). The two-amino-acid deletion in intracellular loop 3 as compared to the sequence of P. marinus Y5 in the genome database presumably is an allelic polymorphism. The results of functional expression showed that PmaY5 in addition to cell surface expression also has prominent localization in the cytoplasm. It has previously been shown that N-terminal glycosylations of the rat Y1 receptor are crucial for cell surface expression of Y receptors (Robin-Jagerschmidt et al., 1998), and palmitoylation of cysteine(s) in the cytoplasmic tail can anchor GPCR receptors in the cell membrane and affect the expression [1,36]. In most species, Y5 has two glycosylation sites, except chicken and Latimeria chalumnae that have three [11,22]. As sequences for N-linked glycosylation in the aminoterminal region and a cysteine for palmitoylation in the carboxyterminal tail are present in PmaY5, boxed in Fig. 2, we assume the prominent cytoplasmic expression in the transfected cells may have other reasons. One speculation is that it may be caused by the long and repetitious intracellular loop 3. All known Y5 sequences, including PmaY5 described here, have a shorter cytoplasmic tail than the other NPY receptor subtypes, just a few residues beyond the cysteine assumed to be palmitoylated. The human Y5 receptor has been reported to have very low internalization upon agonist stimulation [2], hence this may apply to the lamprey Y5 receptor as well.

In tetrapods, the Y5 gene is located adjacent to the Y1 gene in a head-to-head orientation. As neither the P. marinus nor the elephant shark genomes have been assembled into larger contigs, it is still not clear if the same gene configuration prevails also in these species. Indeed, a true Y1 ortholog has not been identified in lamprey. The two Y1-like sequences from this lineage shown in Fig. 3 lack the intron found in Y1 in all gnathostomes and they diverge basally in the Y1 subfamily. In order to characterize the binding profile of lamprey NPYfamily peptides to this receptor, a binding assay was performed using 125 I-pPYY to see if this could work as radioligand for competition studies. However, the low affinity of 125 I-pPYY to PmaY5 precluded its use as a tracer ligand to study the affinities of the peptides. Because NPY receptors mainly function through the G␣i/o pathway in mammals [27], an IP assay was performed instead after co-transfection with a hybrid G protein construct. The results showed that all three lamprey peptides could activate the receptor with similar potencies in the low nM range with no statistically significant difference (Table 1). In conclusion, these results show that lampreys have NPY-family receptors from all three subfamilies, namely Y1-like, Y2-like and a Y5 receptor. We have demonstrated that the P. marinus Y5 receptor is functional and couples via the G␣i protein like Y5 in mammals. Y5 in other vertebrates is expressed in the brain [11,22,34], and it has been shown that Y5 is involved in appetite stimulation in response to NPY in the hypothalamus in mammals [49]. A detailed study of the expression pattern of Y5 mRNA in the brain of P. marinus has recently been completed (performed by Juan Pérez-Fernández, Manuel Megías and Manuel A. Pombal, personal communication), as well as their recently reported description of the mRNA distribution for the Y1-like P. marinus receptor [37]. Acknowledgments This work was supported by a grant from the Swedish Research Council. We thank J. Michel Conlon for purification of the synthesized lamprey NPY family peptides. References [1] Adams MN, Christensen ME, He Y, Waterhouse NJ, Hooper JD. The role of palmitoylation in signalling, cellular trafficking and plasma membrane localization of protease-activated receptor-2. PloS ONE 2011;6:e28018. [2] Bohme I, Stichel J, Walther C, Morl K, Beck-Sickinger AG. Agonist induced receptor internalization of neuropeptide Y receptor subtypes depends on third intracellular loop and C-terminus. Cell Signal 2008;20:1740–9. [3] Boswell T, Dunn IC, Corr SA. Neuropeptide Y gene expression in the brain is stimulated by fasting and food restriction in chickens. Brit Poultry Sci 1999;40. Suppl:S42–3.

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