6
Chicken neuropeptide Y receptor Y2: structural and pharmacological di¡erences to mammalian Y2 1 Erik Salaneck a , Sara K.S. Holmberg a , Magnus M. Berglund a , Timothy Boswell b , Dan Larhammar a; * a Department of Neuroscience, Unit of Pharmacology, Uppsala University, P.O. Box 593, SE 751 24 Uppsala, Sweden b Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, UK Received 6 October 2000; accepted 18 October 2000 First published online 26 October 2000 Edited by Ned Mantei Abstract Here we report the molecular cloning of the chicken (Gallus gallus) neuropeptide Y (NPY) receptor Y2, the first non- mammalian Y2 receptor. It displays 75^80% identity to mammalian Y2 and has a surprisingly divergent cytoplasmic tail. Expression of the receptor protein in a cell line showed that the receptor did not bind the mammalian Y2 selective antagonist BIIE0246. Furthermore, porcine [Leu 31 , Pro 34 ]NPY, which binds poorly to mammalian Y2, exhibited an unexpectedly high affinity for chicken Y2. In situ hybridisation revealed expression in the hippocampus. Thus, the chicken Y2 receptor exhibits substantial differences with regard to sequence and pharmacological profile in comparison to mammalian Y2 receptors, while the expression pattern in the central nervous system resembles that observed in mammals. ß 2000 Federation of European Biochemical Soci- eties. Published by Elsevier Science B.V. All rights reserved. Key words: G-protein coupled receptor ; Neuropeptide Y ; Cloning ; Expression ; In situ hybridization ; Chicken 1. Introduction The neuropeptide Y (NPY) family of peptides consists in tetrapods of NPY, peptide YY (PYY) and pancreatic poly- peptide (PP). NPY and PYY show a high degree of sequence identity and are found in all vertebrates [1]. The physiological e¡ects of the peptides are mediated through receptors belong- ing to the G-protein coupled receptor superfamily. So far, ¢ve receptor subtypes have been cloned in mammals [2,3]. The Y1, Y2 and Y5 genes are localised on the same chro- mosome in human HSA4 [4,5] and pig SSC8 [6] suggesting, together with the low degree of identity, 30% at the amino acid level, that these subtypes resulted from ancient local du- plications early in vertebrate evolution. Subtypes Y4 and y6 are found on human chromosome HSA10 and 5, respectively, and are approximately 50% identical to Y1 and each other. These genes presumably arose by duplication of the chromo- some harbouring the Y1 gene [6], as well as Y2 and Y5 whose duplicates were probably lost. Indeed, the human genome has several segments that signify early chromosome or genome duplications [7]. Thus, Y1, Y4 and y6 form a subfamily within the NPY receptor family. The ¢ve subtypes di¡er extensively in their replacement rates as shown by comparisons across orders of mammals. Y1 and Y2 are the most highly conserved (each has 95% identity) whereas Y4 and y6 evolve quite rap- idly (75^85% identity) [6]. Y5 is intermediate ; however, its variability is mostly restricted to the large third cytoplasmic loop [6]. The physiological e¡ects of the NPY family of peptides are diverse. The stimulation of appetite, regulation of circadian rhythm and blood pressure and inhibition of anxiety are among the most prominent [8,9]. Certain e¡ects are believed to be mediated primarily by the Y2 receptor subtype, includ- ing regulation of circadian rhythms, inhibition of presynaptic transmitter release in the central nervous system as well as the peripheral nervous system, modi¢cation of the electrophysio- logical properties of hippocampal neurons, nasal congestion and gastro-intestinal and renal epithelial secretion [10^12]. NPY has been shown to bind to all Y receptors, albeit with low a/nity to Y4. PYY binds to, and can mimic the actions of, NPY on all subtypes except Y3 [13], which has not yet been cloned. The mammalian Y2 receptors exhibit the same pharmacological pro¢le binding both NPY and PYY and a number of truncated NPY analogues as well as the Y2 selec- tive antagonist BIIE0246. None of the previously character- ised Y2 receptors bind endogenous PP from rat (r) and hu- man (h), or the modi¢ed NPY analogue porcine (p) [Leu 31 , Pro 34 ]NPY, which have therefore been used to di¡erentiate between the Y2 and other NPY receptor subtypes [14^16]. The complexity and diversity of the biological actions of the NPY family of peptides and their receptors is not fully under- stood. Characterisation of the receptors in more distantly re- lated species may help resolve both the evolution of the re- ceptor^peptide system and the primary physiological functions, as well as assist in generating further sequence in- formation in order to clone and characterise receptor subtypes in other more primitive vertebrate species. The present study describes a chicken (ch) clone encoding a Y2 receptor. We present here the sequence, anatomical mRNA distribution, as well as the pharmacological pro¢le using a number of analogues, fragments and derivatives of NPY. 0014-5793 / 00 / $20.00 ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved. PII:S0014-5793(00)02164-5 *Corresponding author. Fax: (46)-18-511540. E-mail: [email protected] 1 The nucleotide sequence reported in this paper has been submitted to the GenBank1/EMBL with the accession number: AF309091. Abbreviations : NPY, neuropeptide Y; PYY, peptide YY; ch, chick- en; h, human; p, porcine; r, rat FEBS 24268 FEBS Letters 484 (2000) 229^234

Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y2

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Page 1: Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y2

Chicken neuropeptide Y receptor Y2: structural and pharmacologicaldi¡erences to mammalian Y21

Erik Salanecka, Sara K.S. Holmberga, Magnus M. Berglunda, Timothy Boswellb,Dan Larhammara;*

aDepartment of Neuroscience, Unit of Pharmacology, Uppsala University, P.O. Box 593, SE 751 24 Uppsala, SwedenbRoslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, UK

Received 6 October 2000; accepted 18 October 2000

First published online 26 October 2000

Edited by Ned Mantei

Abstract Here we report the molecular cloning of the chicken(Gallus gallus) neuropeptide Y (NPY) receptor Y2, the first non-mammalian Y2 receptor. It displays 75^80% identity tomammalian Y2 and has a surprisingly divergent cytoplasmictail. Expression of the receptor protein in a cell line showed thatthe receptor did not bind the mammalian Y2 selective antagonistBIIE0246. Furthermore, porcine [Leu31, Pro34]NPY, which bindspoorly to mammalian Y2, exhibited an unexpectedly high affinityfor chicken Y2. In situ hybridisation revealed expression in thehippocampus. Thus, the chicken Y2 receptor exhibits substantialdifferences with regard to sequence and pharmacological profilein comparison to mammalian Y2 receptors, while the expressionpattern in the central nervous system resembles that observed inmammals. ß 2000 Federation of European Biochemical Soci-eties. Published by Elsevier Science B.V. All rights reserved.

Key words: G-protein coupled receptor; Neuropeptide Y;Cloning; Expression; In situ hybridization; Chicken

1. Introduction

The neuropeptide Y (NPY) family of peptides consists intetrapods of NPY, peptide YY (PYY) and pancreatic poly-peptide (PP). NPY and PYY show a high degree of sequenceidentity and are found in all vertebrates [1]. The physiologicale¡ects of the peptides are mediated through receptors belong-ing to the G-protein coupled receptor superfamily. So far, ¢vereceptor subtypes have been cloned in mammals [2,3].

The Y1, Y2 and Y5 genes are localised on the same chro-mosome in human HSA4 [4,5] and pig SSC8 [6] suggesting,together with the low degree of identity, 30% at the aminoacid level, that these subtypes resulted from ancient local du-plications early in vertebrate evolution. Subtypes Y4 and y6are found on human chromosome HSA10 and 5, respectively,and are approximately 50% identical to Y1 and each other.

These genes presumably arose by duplication of the chromo-some harbouring the Y1 gene [6], as well as Y2 and Y5 whoseduplicates were probably lost. Indeed, the human genome hasseveral segments that signify early chromosome or genomeduplications [7]. Thus, Y1, Y4 and y6 form a subfamily withinthe NPY receptor family. The ¢ve subtypes di¡er extensivelyin their replacement rates as shown by comparisons acrossorders of mammals. Y1 and Y2 are the most highly conserved(each has 95% identity) whereas Y4 and y6 evolve quite rap-idly (75^85% identity) [6]. Y5 is intermediate; however, itsvariability is mostly restricted to the large third cytoplasmicloop [6].

The physiological e¡ects of the NPY family of peptides arediverse. The stimulation of appetite, regulation of circadianrhythm and blood pressure and inhibition of anxiety areamong the most prominent [8,9]. Certain e¡ects are believedto be mediated primarily by the Y2 receptor subtype, includ-ing regulation of circadian rhythms, inhibition of presynaptictransmitter release in the central nervous system as well as theperipheral nervous system, modi¢cation of the electrophysio-logical properties of hippocampal neurons, nasal congestionand gastro-intestinal and renal epithelial secretion [10^12].

NPY has been shown to bind to all Y receptors, albeit withlow a¤nity to Y4. PYY binds to, and can mimic the actionsof, NPY on all subtypes except Y3 [13], which has not yetbeen cloned. The mammalian Y2 receptors exhibit the samepharmacological pro¢le binding both NPY and PYY and anumber of truncated NPY analogues as well as the Y2 selec-tive antagonist BIIE0246. None of the previously character-ised Y2 receptors bind endogenous PP from rat (r) and hu-man (h), or the modi¢ed NPY analogue porcine (p) [Leu31,Pro34]NPY, which have therefore been used to di¡erentiatebetween the Y2 and other NPY receptor subtypes [14^16].

The complexity and diversity of the biological actions of theNPY family of peptides and their receptors is not fully under-stood. Characterisation of the receptors in more distantly re-lated species may help resolve both the evolution of the re-ceptor^peptide system and the primary physiologicalfunctions, as well as assist in generating further sequence in-formation in order to clone and characterise receptor subtypesin other more primitive vertebrate species.

The present study describes a chicken (ch) clone encoding aY2 receptor. We present here the sequence, anatomicalmRNA distribution, as well as the pharmacological pro¢leusing a number of analogues, fragments and derivatives ofNPY.

0014-5793 / 00 / $20.00 ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 0 1 4 - 5 7 9 3 ( 0 0 ) 0 2 1 6 4 - 5

*Corresponding author. Fax: (46)-18-511540.E-mail: [email protected]

1 The nucleotide sequence reported in this paper has been submittedto the GenBank1/EMBL with the accession number: AF309091.

Abbreviations: NPY, neuropeptide Y; PYY, peptide YY; ch, chick-en; h, human; p, porcine; r, rat

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Page 2: Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y2

2. Materials and methods

2.1. Screening of genomic libraryApproximately 700 000 clones representing roughly four genome

equivalents from a library in the vector EMBL3 were screened usingtwo polymerase chain reaction (PCR) generated 32P-labelled fulllength Y2 fragments from rat and human. Hybridisation was carriedout at 42³C in 25% formamide, 6USSC, 10% dextran sulphate,5UDenhardt's solution and 0.1% SDS over night. The ¢lters werewashed twice in 2USSC/0.1% SDS at room temperature for 5 min,and twice in 0.5USSC/0.1% SDS for 30 min at 42³C. One clonehybridising to two duplicate ¢lters was selected and digested usingEcoRI. Southern blot and subsequent hybridisation with the humanand rat Y2 probes revealed two bands with sequence homology to theprobe fragments. A 2.1 kb fragment containing 800 bp of the chY2gene was cloned in pBluescript KS+vector and sequenced. DNA prep-arations from the selected phage clone were sequenced using speci¢csequencing probes designed from the cloned fragment. Primers werethen designed in order to obtain a full length sequence.

2.2. DNA sequencing and amino acid alignmentSequence determinations were performed using ABI PRISM Dye

Terminator cycle sequencing kit according to the manufacturer's di-rections (Perkin Elmer) and analysed by an automated ABI-310 £uo-rescent-dye sequencer (Applied Biosystems). Sequence alignmentswere performed using Lasergene DNASTAR Megalign software.Transmembrane regions were based upon Schwartz et al. [17]. A se-quence distance tree was made using alignments with Y2, Y1 and Y5sequences and, as an outgroup, bradykinin B1 and B2 receptor aminoacid sequences retrieved from GenBank.

2.3. Cloning in expression vectorA fragment containing the entire coding region of the chY2 gene

was generated with Platinum Pfx DNA polymerase (Gibco BRL),using speci¢c primers containing EcoRI and BamHI sites. Cycle:20 s at 95³C, 30 s at 50³C and 1 min 20 s at 68³C for 35 cycles.The PCR fragment was puri¢ed by Qiagen PCR product puri¢cationkit (Qiagen), digested using EcoRI and BamHI. The 1.2 kb fragmentwas then puri¢ed with QIAquick PCR puri¢cation kit (Qiagen) andthen ligated into a pTEJ8 expression vector to give the clone chY2-pTEJ8. The construct was sequenced and found to be identical to thegenomic clone.

2.4. Transfection protocolFor transient transfections HEK293 (EBNA) cells were transfected

with the construct chY2-pTEJ8 using FuGENE1 Transfection Re-agent (Boehringer Mannheim, Germany), diluted in OptiMEM me-dium (Gibco BRL, Stockholm, Sweden) according to the manufactur-er's recommendations. After transfection, cells were grown inDulbecco's modi¢ed Eagle's medium (DMEM)/Nut Mix F-12 w/oL-glutamine (Gibco BRL) containing 10% foetal calf serum (BiotechLine AS, CA, USA), 24 mM L-glutamine (Gibco BRL) and 250 Wg/mlG-418 (Gibco BRL), penicillin^streptomycin (100 U penicillin, 100 Wgstreptomycin/ml) (Gibco BRL) until harvesting by centrifugation,after 48 h. Cell membrane pellets were frozen in aliquots at 380³C.To obtain semi-stable cells for studies of cyclic AMP (cAMP),HEK293 (EBNA) cells were grown in DMEM containing 10% foetalcalf serum and 250 Wg/ml G-418. Cells were transfected with 6 WlFuGENE1 and 2 Wg of the construct chY2FLAG-pCEP4 (whichcontains the nine amino acid FLAG epitope in the very carboxyterminus). After 24 h, the cells were split and 400 Wg/ml hygromycinB (Gibco BRL) was added to the medium. A control plate with un-transfected cells was treated the same way and after 7 days all cells inthe control plate were dead while the transfected plate was con£uent.The cells from the transfected plate were grown under hygromycinselection and subsequently checked for 125I-pPYY binding. The phar-macology of the stably expressed chY2 receptor was indistinguishablefrom that of the transiently expressed receptor reported in this paper.

2.5. Peptides and peptidic and non-peptidic antagonistsPorcine NPY, p[Leu31, Pro34]NPY, pNPY2^36, pNPY3^36,

pNPY18^36, hPP and rPP were purchased from Bachem, King ofPrussia, PA, USA; p[D-Trp32]NPY from Peninsula Laboratories,CA, USA. BIBP3226 [18] was synthesised at Dr. Karl Thomae's lab-oratories. SR120819A [19] was provided by Sano¢, chicken PP and

PYY were purchased from Schafer-N, Copenhagen, Denmark. Non-peptidic Y2 antagonist BIIE0246 [20] was provided by Boehringer-Ingelheim PharmaKG, Biberach an der Riss, Germany.

2.6. Binding assaysThe thawed aliquots of membranes were resuspended in 25 mM

HEPES-bu¡er (pH 7.4) containing 2.5 mM CaCl2, 1 mM MgCl2and 2 g/l bacitracin and homogenised using an Ultra-Turrax homog-eniser. Saturation experiments were performed in a ¢nal volume of100 Wl with 4^5 Wg protein and 125I-pPYY (Amersham, UK) for 2 h atroom temperature. This radioligand is iodinated at tyrosines 21 and27 and has a speci¢c activity of 4000 Ci/mmol. Saturation experimentswere carried out with serial dilutions of radioligand. Non-speci¢cbinding was de¢ned as the amount of radioactivity remaining boundto the cell homogenate after incubation in the presence of 100 nMunlabelled pNPY. Competition experiments were performed in a ¢nalvolume of 100 Wl. Various concentrations of the peptides pNPY, hPP,rPP, chPP, chPYY, p[Leu31, Pro34]NPY, pNPY2^36, pNPY3^36,pNPY13^36, pNPY18^36, p[D-Trp32]NPY, two non-peptidic Y1 an-tagonists SR120819A and BIBP3226, and one non-peptidic Y2 antag-onist BIIE0246 were included in the incubation mixture along with125I-pPYY. Incubations were terminated by ¢ltration through GF/C¢lters, Filtermat A (Wallac Oy, Turku, Finland), that had been pre-soaked in 0.3% polyethyleneimine, using a TOMTEC (Orange, CT,USA) cell harvester. The ¢lters were washed with 5 ml of 50 mM Tris(pH 7.4) at 4³C and dried at 60³C. The dried ¢lters were treated withMeltiLex A (Wallac) melt-on scintillator sheets and the radioactivityretained on the ¢lters counted using the Wallac 1450 Microbeta coun-ter. The results were analysed using the Prism 2.0 software package(Graphpad, San Diego, CA, USA). Protein concentrations were mea-sured using a Bio-Rad Protein Assay (Bio-Rad, Solna, Sweden) withbovine serum albumin as standard.

2.7. Inhibition of forskolin stimulated cAMP synthesisCyclic AMP was assayed on stably transfected HEK293 (EBNA)

cells. The cells were detached by pipetting with media, diluted to 1000cells/Wl and treated for 30 min at 37³C with 250 WM isobutylmethyl-xanthine (Sigma). About 200 000 cells (200 Wl) were incubated with10 WM forskolin (Sigma) and various concentrations of chPYY,pNPY, p[Leu31, Pro34]NPY, pNPY2^36, or pNPY18^36 for 20 minat 37³C in a total volume of 250 Wl. The ability of BIIE0246 to inhibitthe e¡ect of 100 nM pNPY was also tested. Reactions were termi-nated by adding 25 Wl HClO4 (4.4 M) and the suspension was neu-tralised by adding 40 Wl KOH (5 M). Membranes were pelleted bycentrifugation and 50 Wl of the supernatant was used to quantifycAMP with a radioassay using 3H-cAMP (Amersham Pharmacia Bio-tech) as a competitor. After 2 h, bound 3H-cAMP was separated fromfree by the addition of 150 Wl of a 1% charcoal suspension (50 mMTris, 4 mM EDTA). The tubes were vortexed and spun at maximumspeed for 2 min, 300 Wl of the supernatant was rapidly removed andcounted in a liquid scintillation analyser (Packard). Cyclic AMP bind-ing protein was extracted from the bovine adrenal cortex.

2.8. In situ hybridisationBrains from four Bantam chickens (two male, two female; Roslin

Institute £ock) were processed for in situ hybridisation. The birdswere maintained on a photoperiod of 14L10D and had free accessto food and water. Brains were rapidly dissected from birds killed bycervical dislocation and were immediately frozen in powdered dry ice.The tissue was stored at 370³C before being sectioned on a cryostat(Shandon, Model OT) at 15 Wm thickness. Coronal sections werethaw-mounted onto microscope slides (Superfrost Plus, Cellpath,Hemel Hempstead, Herts, UK) and stored at 370³C. Slides bearingbrain sections were processed by sequential immersion at room tem-perature in: 4% paraformaldehyde (5 min); twice in 0.1 M sodiumphosphate (5 min each); water (6 5 s); 0.1 M triethanolamine, pH 8.0(TEA, 6 5 s); 0.15 M acetic anhydride in 0.1 M TEA (10 min);2USSC; 70, 95 and 100% ethanol (3 min each); and blow-dried.Slides were then stored at room temperature until application of thehybridisation solution. A mixture of two oligonucleotide probes com-plementary to chicken Y2 receptor mRNA was used for in situhybridisation. The oligonucleotide sequences were: 5PCGATCACC-CCCAACAGAATAATGGAACAGTAAGCAAAGATGAGGAT-3Pand 5PCTGATACGCTTAGAGATTTTGCTTTCCAAGTGATAGA-CAATACCACGAT-3P. The probes were labelled at the 3P end with[35S]dATP using terminal deoxynucleotidyl transferase (Amersham,

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Page 3: Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y2

Little Chalfont, Bucks, UK) and were puri¢ed through QIAquick spincolumns (Qiagen, Crawley, West Sussex, UK). Speci¢c activity of theprobes was 108 cpm/Wg oligonucleotide. Hybridisation bu¡er con-sisted of 50% formamide, 4USSC, 10% dextran sulphate, 1% N-laur-ylsarcosine, 1UDenhardt's solution, 200 mM dithiothreitol, and0.1 mg/ml yeast tRNA. A 100 Wl aliquot of hybridisation solutioncontaining 3 ng of each labelled probe was applied to each sectionand covered with a Para¢lm coverslip. Control sections received hy-bridisation solution containing a 100-fold excess of each unlabelledprobe. Hybridisation was performed for at least 16 h in humidi¢edboxes at 42³C. After hybridisation, the sections were washed (4U45min) in 1USSC at 56³C, and brought to room temperature while inthe ¢nal wash. The tissue was then dehydrated through graded alco-hols and apposed to Kodak Biomax MR ¢lm for 2^3 weeks at roomtemperature. Films were developed using an automatic processor (X-ograph Imaging Systems, Tetbury, Gloucestershire, UK).

3. Results

A chicken genomic phage library was screened using twofull length Y2 PCR products, human Y2 and rat Y2, underlow stringency conditions. A strongly hybridising clone wasisolated and an 800 bp fragment isolated from the phage wassubcloned in order to obtain a partial length chicken Y2product. The fragment was sequenced and used to designprimers with which the phage clone was sequenced by se-quence primer walking.

The open reading frame encodes a protein of 385 aminoacid residues which displays the characteristic features of aG-protein coupled receptor, i.e. seven putative transmem-brane regions, a cysteine pair linking extracellular loop 1and loop 2, and a cysteine in the carboxy terminal tail wherepalmitoylation could serve as an anchor to the membrane andform a pseudo fourth loop (Fig. 1). The receptor also containsan asparagine residue close to the amino terminal, which hasbeen identi¢ed as a putative glycosylation site in mammalianY2 receptors. The protein has 78^79% identity to the human,murine, porcine and bovine Y2 receptors and 75% identity tothe guinea pig Y2 subtypes. The mammalian subtypes share90^94% identity between each other. When compared to otherNPY receptor subtypes the chicken Y2 receptor exhibits con-

Fig. 2. Distance tree for the characterised NPY Y2 receptors, threeY5 and three Y1 receptors. Branch lengths correspond to sequencedivergence using the neighbour joining method of the LasergeneDNASTAR Megalign software. The human bradykinin B1 and B2receptors were used as outgroup to root the tree.F

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Page 4: Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y2

siderably less identity: 23^26% to Y1, Y4, Y5 and y6 subtypesin mammals (Fig. 2). The alignment reveals that divergence tomammalian Y2 has primarily occurred in the carboxy andamino terminals while the receptor is more highly conservedin the transmembrane and loop domains. The genomic clonedescribed here was found to be intronless in the coding regionas is the case for the Y2 genes in mammals.

The coding sequence of the genomic clone was transferredto the expression vector pTEJ8 [21] and control sequenced.The chY2-pTEJ8 construct was transfected into HEK293(EBNA) cells and membranes prepared from the transfectedcells were then used for radioligand binding assays. The mem-brane fraction exhibited concentration dependent binding of125I-pPYY with an a¤nity constant (Kd) of 25.6 pM(S.E.M. = 1.7, N = 3) and a Bmax of 303 fmol/mg protein(S.E.M. = 68, N = 3) (Fig. 3). All tested NPY variants,pNPY and chPYY were potent inhibitors of 125I-pPYY. Theinhibition constants (Ki) are presented in Table 1. Weak inhi-bition was seen with p[D-Trp32]NPY (Ki = 400 nM) and noinhibition was detected with the Y1 antagonists BIBP3226and SR120819A, rPP, hPP or the Y2 antagonist BIIE0246(Ki s 1 WM). Functional coupling to G-proteins was shownby inhibition of cAMP synthesis. Chicken PYY(pEC50 = 9.12 þ 0.09, n = 6), pNPY (9.02 þ 0.18, n = 5), and

pNPY2^36 (8.95 þ 0.28, n = 3) were equally potent whileNPY18^36 (8.27 þ 0.40, n = 5) and p[Leu31, Pro34]NPY(8.27 þ 0.21, n = 5) were about four-fold less potent (Fig. 4).The Y2 antagonist BIIE0246 up to a concentration of 1 WMdid not a¡ect the inhibition from 100 nM pNPY (data notshown).

In situ hybridisation with oligonucleotide probes to thechicken Y2 receptor revealed a prominent signal in the hippo-campus (Fig. 5a). No signal was observed in this region aftercompetition with excess unlabelled probe (Fig. 5b).

4. Discussion

NPY receptors previously cloned in species from severalorders of mammals have revealed that the rates of evolutiondi¡er considerably between subtypes [6]. To obtain a greaterunderstanding of the evolution of the NPY receptor genefamily, and the function of the Y2 subtype in particular, wehave undertaken the molecular cloning of a non-mammalianY2 receptor. The chicken Y2 receptor described in this paperexhibits 75^80% identity with mammalian Y2 sequences,which is in agreement with the 90^95% identity between mam-mals and further supports that it is a well conserved receptor(Fig. 1).

A higher degree of identity was seen within the postulatedtransmembrane regions, whereas some divergence can be seenin the loop regions, and particularly in the amino and carboxytermini. In addition to the DRH motif located at the carboxyterminal end of the third transmembrane region typical forNPY Y2 subtypes, the amino acid translation reveals con-served postulated structurally important amino acids for di-sulphide bridges, palmitoylation and glycosylation [1]. A phy-logenetic tree with other NPY receptor sequences places thechicken Y2 sequence closest to the mammalian Y2 sequences,and at a substantial distance from the other receptor subtypes(Fig. 2).

Cloning into a eukaryotic expression vector allowed us toexpress the receptor gene in a mammalian cell line. A widevariety of peptides, truncated peptides and analogues wereused to determine the pharmacological pro¢le in competitionwith iodinated pPYY. The properties proved to be deviant

Fig. 3. Saturation binding isotherm and Scatchard (inset) analysesof 125I-pPYY binding to membranes prepared from HEK293(EBNA) cells transfected with the chY2 expression plasmid chY2-pTEJ8. The ¢gure shows results from a representative experiment.

Table 1Inhibition of 125I-pPYY binding to membranes from HEK293(EBNA) cells transfected with the chY2-pTEJ8 expression constructusing NPY, truncated NPY peptides and non-peptide analogues

Ligand pKI (3log M) S.E.M. n

chPYY 9.20 0.08 3pNPY 9.46 0.06 3pNPY2^36 9.26 0.06 3pNPY3^36 9.13 0.06 3pNPY13^36 8.91 0.08 3pNPY18^36 7.70 0.10 3[Leu31, Pro34]NPY 8.25 0.05 3rPP 6 6.0 3hPP 6 6.0 3chPP 6 6.0 3BIBP3226 6 6.0 3SR120819A 6 6.0 3p[D-Trp32]NPY 6.42 0.11 3BIIE0246 6.20 0.06 3

Fig. 4. Inhibition of forskolin stimulated cAMP synthesis. Baselinerepresents unstimulated cells and 100% represents cells after stimula-tion of 10 WM forskolin. The ¢gure shows one representative experi-ment.

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from previously characterised Y2 receptors [15,16,22^24], withthe following binding in rank order of potency: pNPY = ch-PYY = pNPY2^36 = pNPY3^36s pNPY13^36s pNPY18^3-6 = p[Leu31, Pro34]NPYDp[D-Trp32]NPY (Table 1). No bind-ing (Ki s 1 WM) was observed for BIBP3226, SR120819A,rPP, hPP and BIIE0246. Two of the tested ligands displayedbinding a¤nities that di¡er considerably from known interac-tions with mammalian Y2 receptors: BIIE0246, a potent an-tagonist at the human [20], rat [25] and guinea pig [26] Y2receptors, did not exhibit speci¢c binding at the chY2 recep-tor; and p[Leu31, Pro34]NPY that does not bind to previouslycharacterised Y2 receptors, e.g. Ki 6 1 WM, binds to the chY2receptor with relatively high a¤nity. Functional coupling toG-proteins was also studied. Chicken PYY, pNPY andpNPY2^36 inhibited cAMP synthesis in stably transfectedHEK293 (EBNA) cells with EC50 values of about 1 nM(Fig. 4). The truncated peptide pNPY18^36 displayed anEC50 of 5 nM, i.e. four times higher than the Ki value. Inter-estingly, p[Leu31, Pro34]NPY was also a full agonist only ¢ve-fold less potent than chPYY. BIIE0246 did not inhibit thee¡ect of 100 nM pNPY in agreement with the competitionstudies. The similarity of chY2 to mammalian Y2 in the abil-ity to inhibit cAMP synthesis indicates that the divergent car-

boxy terminal tail does not alter the interaction with Gi pro-teins.

The p[Leu31, Pro34]NPY peptide does not bind to any ofthe known mammalian Y2 receptors. It was designed to bemore similar to the mammalian PP [27] that primarily bindsto the NPY Y4 subtype [28]. In p[Leu31, Pro34]NPY the Glu34

residue of mammalian NPY has been replaced by a prolineresidue found at the corresponding position of all mammalianPP sequences. This gives the peptide a higher a¤nity for themammalian Y4 subtype, i.e. the PP receptor [28]. Chicken PP,as well as other known avian PP sequences, di¡ers from themammalian PP peptides by having a histidine at position 34[1]. This di¡erence may explain why the avian Y2 receptorwould not have the need to discriminate against the Pro34

residue in the p[Leu31, Pro34]NPY analogue. This reveals animportant di¡erence between the avian and mammalian NPYpeptide^receptor interactions, which should prove useful formutagenesis studies.

A molecular explanation for the lack of binding of theBIIE0246 antagonist cannot yet be given, but consideringthe evolutionary distance between the mammalian and avianlineages, and the fact that BIIE0246 was designed to bind tothe human and rat Y2 receptors [20] this is not unexpected.

Fig. 5. In situ hybridisation of 35S end-labelled oligonucleotides to chicken NPY Y2 receptor mRNA in chicken brain sections. A speci¢c hy-bridisation signal in the hippocampus (Hp) (a) disappears in an adjacent section after competition with an unlabelled oligonucleotide probe (b)while non-speci¢c hybridisation to the optic chiasm and neural tracts is retained. Scale bar = 2 mm.

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Page 6: Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y2

Unfortunately, this means that BIIE0246 cannot be used infunctional studies in chicken. The sequence of the chY2 re-ceptor may prove to be of great value when investigatingbinding sites for BIIE0246 at the human Y2 receptor bysite-directed mutagenesis, and such experiments are in prog-ress.

Although the amino acid sequence and pharmacologicalproperties of the chicken Y2 receptor di¡er signi¢cantlyfrom its mammalian counterparts, the localisation of the re-ceptor to the hippocampus in the present study (Fig. 5) in-dicates that the neuroanatomical distribution is conserved be-tween birds and mammals. Expression of the Y2 receptor inthe hippocampus is consistently reported in mammalian spe-cies including rat [29,30], sheep [31] and human [32]. Theexpression of NPY receptors in the chicken hippocampus isnot unexpected because this region displays high speci¢c bind-ing of radiolabelled NPY [33]. Furthermore, NPY mRNAand peptide have been co-localised in the hippocampus ofchickens and Japanese quail [34]. Collectively, these ¢ndingssuggest that the functions of NPY in this brain region arelikely to have been conserved during evolution. The regula-tory e¡ects of NPY in the chicken hippocampus have notbeen elucidated. However, in mammals, interaction of NPYwith the Y2 receptor in the hippocampus has been implicatedin seizure modulation and in the facilitation of learning andmemory [35]. It is noteworthy that the pattern of Y2 receptorexpression within the hippocampus appears to di¡er betweenbirds and mammals. Thus, in the present study, Y2 receptormRNA was evenly distributed over the hippocampal regionwhereas, in the rat, expression is limited to speci¢c sub-re-gions, particularly CA2 and CA3 pyramidal cells and granulecells [29,36]. The signi¢cance of this is unclear. However, adi¡erence in the distribution pattern is predictable given thatthe neuroanatomical organisation of the hippocampus di¡ersmarkedly between birds and mammals to the extent that sub-divisions applied to the mammalian structure are not recog-nisable in birds [37].

In conclusion, we have cloned and characterised a chickenNPY Y2 receptor and found considerable di¡erences in aminoacid sequence as well as pharmacological binding pro¢le. Thereceptor appears to have a similar pattern of expression in thebrain as mammalian Y2. The sequence information obtainedfrom the chicken Y2 receptor should be of great importancefor achieving a greater understanding of the interactions be-tween NPY as well as NPY-like ligands at the Y2 receptors.

Acknowledgements: We thank Ms. Christina Bergqvist for experttechnical assistance, Dr. Klaus Rudolf and Dr. Heike Wieland atBoehringer Ingelheim KG, Biberach, Germany for providingBIBP3226 and BIIE0246 and Dr. Claudine Serradeil-Le Gal at Sano¢,Toulouse Cedex, France for providing SR120819A. This work wassupported by the Swedish Natural Science Research Council.

References

[1] Larhammar, D. (1996) Regul. Pept. 62, 1^11.[2] Larhammar, D. (1996) Regul. Pept. 65, 165^174.[3] Blomqvist, A.G. and Herzog, H. (1997) Trends Neurosci. 20,

294^298.

[4] Herzog, H., Baumgartner, M., Vivero, C., Selbie, L.A., Auer, B.and Shine, J. (1993) J. Biol. Chem. 268, 6703^6707.

[5] Lutz, C.M., Frankel, W.N., Richards, J.E. and Thompson, D.A.(1997) Genomics 41, 498^500.

[6] Wraith, A., Tornsten, A., Chardon, P., Harbitz, I., Chowdhary,B.P., Andersson, L., Lundin, L.G. and Larhammar, D. (2000)Genome Res. 10, 302^310.

[7] Lundin, L.G. (1993) Genomics 16, 1^19.[8] Dumont, Y., Martel, J.C., Fournier, A., St-Pierre, S. and Qui-

rion, R. (1992) Prog. Neurobiol. 38, 125^167.[9] Wahlestedt, C. and Reis, D.J. (1993) Annu. Rev. Pharmacol.

Toxicol. 33, 309^352.[10] Hazelwood, R.L. (1993) Proc. Soc. Exp. Biol. Med. 202, 44^63.[11] Colmers, W.F., Klapstein, G.J., Fournier, A., St-Pierre, S. and

Treherne, K.A. (1991) Br. J. Pharmacol. 102, 41^44.[12] St-Pierre, J.A., Dumont, Y., Nouel, D., Herzog, H., Hamel, E.

and Quirion, R. (1998) Br. J. Pharmacol. 123, 183^194.[13] Gehlert, D.R. (1994) Life Sci. 55, 551^562.[14] Berglund, M.M., Lundell, I., Eriksson, H., So« ll, R., Beck-Sick-

inger, A.G. and Larhammar, D. (2000) Peptides, in press.[15] Rose, P.M., Fernandes, P., Lynch, J.S., Frazier, S.T., Fisher,

S.M., Kodukula, K., Kienzle, B. and Seethala, R. (1995) J. Biol.Chem. 270, 22661^22664.

[16] Nakamura, M., Aoki, Y. and Hirano, D. (1996) Biochim. Bio-phys. Acta 1284, 134^137.

[17] Schwartz, T.W., Gether, U., Schambye, H.T. and Hjort, S.A.(1995) Curr. Pharm. Design 1, 355^372.

[18] Rudolf, K. et al. (1994) Eur. J. Pharmacol. 271, R11^R13.[19] Serradeil-Le Gal, C. et al. (1995) FEBS Lett. 362, 192^196.[20] Doods, H. et al. (1999) Eur. J. Pharmacol. 384, R3^R5.[21] Johansen, T.E., Scholler, M.S., Tolstoy, S. and Schwartz, T.W.

(1990) FEBS Lett. 267, 289^294.[22] Sharma, P., Holmberg, S.K., Eriksson, H., Beck-Sickinger, A.G.,

Grundemar, L. and Larhammar, D. (1998) Regul. Pept. 75^76,23^28.

[23] Malmstrom, R.E., Hokfelt, T., Bjorkman, J.A., Nihlen, C., By-strom, M., Ekstrand, A.J. and Lundberg, J.M. (1998) Regul.Pept. 75^76, 55^70.

[24] Ammar, D.A., Eadie, D.M., Wong, D.J., Ma, Y.Y., KolakowskiJr., L.F., Yang-Feng, T.L. and Thompson, D.A. (1996) Ge-nomics 38, 392^398.

[25] Dumont, Y. et al. (2000) Br. J. Pharmacol. 129, 1075^1088.[26] Berglund, M.M., Lundell, I., Serradeil-Le Gal, C., Rist, B., Beck-

Sickinger, A.G. and Larhammar, D. (2000) Biochem. Pharma-col., in press.

[27] Fuhlendor¡, J. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 182^186.

[28] Lundell, I. et al. (1995) J. Biol. Chem. 270, 29123^29128.[29] Gustafson, E.L., Smith, K.E., Durkin, M.M., Walker, M.W.,

Gerald, C., Weinshank, R. and Branchek, T.A. (1997) BrainRes. Mol. Brain Res. 46, 223^235.

[30] Parker, R.M. and Herzog, H. (1999) Eur. J. Neurosci. 11, 1431^1448.

[31] Dyer, C.J., Simmons, J.M., Matteri, R.L. and Keisler, D.H.(1997) Domest. Anim. Endocrinol. 14, 295^303.

[32] Caberlotto, L., Fuxe, K., Rimland, J.M., Sedvall, G. and Hurd,Y.L. (1998) Neuroscience 86, 167^178.

[33] Merckaert, J. and Vandesande, F. (1996) J. Chem. Neuroanat.12, 123^134.

[34] Boswell, T., Millam, J.R., Li, Q. and Dunn, I.C. (1998) CellTissue Res. 293, 31^38.

[35] Redrobe, J.P., Dumont, Y., St-Pierre, J. and Quirion, R. (1999)Brain Res. 848, 153^166.

[36] Schwarzer, C., Ko£er, N. and Sperk, G. (1998) Mol. Pharmacol.53, 6^13.

[37] Szekely, A.D. (1999) Behav. Brain Res. 98, 219^225.

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