cloning and characterization of neuropeptide y receptors of
TRANSCRIPT
Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 948
_____________________________ _____________________________
Cloning and Characterization of Neuropeptide Y receptors of the Y1 Subfamily in Mammals and Fish
BY
PAULA STARBÄCK
ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2000
Dissertation for Degree of Doctor of Medical Science in Neuroscience presented atUppsala University in 2000
Abstract
Starbäck, P. 2000. Cloning and characterization of NPY receptors of the Y1 subfamilyin mammals and fish. Acta Universitatis Upsaliensis.Comprehensive summaries ofUppsala Dissertations from the Faculty of Medicine 948. 43 pp. Uppsala. ISBN91-554-4786-4
Neuropeptide Y (NPY) is an abundant neurotransmitter in the nervous system andforms a family of evolutionarily related peptides together with peptide YY (PYY),pancreatic polypeptide (PP) and polypeptide Y (PY). These peptides are ligands toa family of receptors that mediate a wide range of physiological effects includingstimulation of appetite. This work describes the molecular cloning of four novel NPYreceptors.
In rat a receptor called PP1, later renamed Y4, was cloned and characterized. Itdisplays the highest amino acid sequence identity to the Y1 receptor. Rat Y4 differsextensively from human Y4, cloned subsequently, in both pharmacological properties,tissue distribution, and amino acid sequence with only 75% identity. Rat and humanY4 are the most diverged orthologues in the NPY receptor family.
In guinea pig, the y6 receptor gene was found to be a pseudogene with severalframeshift mutations. The gene is a pseudogene in human and pig too, but seemsto give rise to a functional receptor in mouse and rabbit. This unusual evolutionarysituation may be due to inactivation of the gene in a mammalian ancestor and thenrestoration of expression in mouse and rabbit, but perhaps more likely due to indepen-dent inactivations in guinea pig, human and pig.
In zebrafish, two new intronless receptor genes were cloned. Sequence compar-isons suggest that both receptors are distinct from the mammalian receptors Y1, Y4
and y6, hence they were named Ya and Yb. Chromosomal localization provides fur-ther support that Ya and Yb may be distinct subtypes.
The discoveries of the rat Y4 and zebrafish Ya and Yb receptors were unexpectedand show that the NPY receptor family is larger than previously thought.
Key words: G-protein, neuropeptide Y, panacreatic polypeptide, neuropeptide Y re-ceptor, ortholog, zebrafish, guinea pig, pseudogene, evolution.
Paula Starbäck, Department of Neuroscience, Unit of Pharmacology, Biomedical Cen-ter, Uppsala University, Box 593, SE-751 24 Uppsala, Sweden
c Paula Starbäck 2000
ISSN 0282-7476ISBN 91-554-4786-4
Printed in Sweden by University Printers, Ekonomicum, Uppsala 2000
Main references
This thesis is based on the following papers, which will be referred to by theirRoman numerals:
I. I. Lundell, M. A. Statnick, D. Johnson, D. A. Schober, P. Starbäck, D. R.Gehlert, and D. Larhammar. The cloned rat pancreatic polypeptide re-ceptor exhibits profound differences to the orthologous receptor. ProcNatl Acad Sci U S A, 93(10):5111–5, 1996.
II. I. Lundell, M. M. Berglund, P. Starbäck, E. Salaneck, D. R. Gehlert, andD. Larhammar. Cloning and characterization of a novel neuropeptide Yreceptor subtype in the zebrafish. DNA Cell Biol, 16(11):1357–63, 1997.
III. P. Starbäck, I. Lundell, R. Fredriksson, M. M. Berglund, Y. L. Yan,A. Wraith, C. Söderberg, J. H. Postlethwait, and D. Larhammar. Neu-ropeptide Y receptor subtype with unique properties cloned in the ze-brafish: the zYa receptor. Brain Res Mol Brain Res, 70(2):242–52, 1999.
IV. P. Starbäck, A. Wraith, H. Eriksson, and D. Larhammar. NeuropeptideY receptor gene y6: multiple deaths or resurrections? Manuscript, 2000.
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Contents
Abstract 2
Main references 3
Abbreviations 6
1 Introduction 71.1 The NPY family . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.1.1 Neuropeptide Y (NPY) . . . . . . . . . . . . . . . . . 81.1.2 Peptide YY (PYY) . . . . . . . . . . . . . . . . . . . 91.1.3 Pancreatic polypeptide (PP) . . . . . . . . . . . . . . 91.1.4 Polypeptide Y (PY) . . . . . . . . . . . . . . . . . . . 91.1.5 NPY-family evolution . . . . . . . . . . . . . . . . . 9
1.2 The NPY family of receptors . . . . . . . . . . . . . . . . . . 91.2.1 G-protein coupled receptors . . . . . . . . . . . . . . 10
1.3 The NPY receptor subtypes in mammals . . . . . . . . . . . . 111.3.1 Y1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111.3.2 Y2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131.3.3 Y4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131.3.4 Y5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131.3.5 y6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141.3.6 What about Y3? . . . . . . . . . . . . . . . . . . . . . 15
1.4 NPY receptors in non-mammalian species . . . . . . . . . . . 151.4.1 Fishes . . . . . . . . . . . . . . . . . . . . . . . . . . 151.4.2 Other vertebrates . . . . . . . . . . . . . . . . . . . . 151.4.3 Invertebrates . . . . . . . . . . . . . . . . . . . . . . 16
1.5 NPY and regulation of feeding . . . . . . . . . . . . . . . . . 161.6 Molecular evolution . . . . . . . . . . . . . . . . . . . . . . . 17
1.6.1 Duplication of the genome . . . . . . . . . . . . . . . 17
2 Aim of the present study 192.1 Pseudogenes in vertebrates . . . . . . . . . . . . . . . . . . . 19
3 Summaries of the papers 223.1 Paper I: The cloned rat pancreatic polypeptide receptor ex-
hibits profound differences to the orthologous receptor . . . . 223.2 Paper II: Cloning and characterization of a novel neuropeptide
Y receptor subtype in the zebrafish . . . . . . . . . . . . . . . 23
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3.3 Paper III: Neuropeptide Y receptor subtype with unique prop-erties cloned in the zebrafish: the zYa receptor . . . . . . . . . 24
3.4 Paper IV: Neuropeptide Y receptor gene y6: multiple deaths orresurrections? . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4 Discussion 264.1 The rat Y4 receptor . . . . . . . . . . . . . . . . . . . . . . . 264.2 The zebrafish receptors . . . . . . . . . . . . . . . . . . . . . 264.3 Molecular evolution: questions answered? . . . . . . . . . . . 28
4.3.1 The zYa and zYb receptors and evolution . . . . . . . 294.3.2 The y6 receptor and evolution . . . . . . . . . . . . . 29
5 Concluding remarks 31
Acknowledgements 32
References 33
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Abbreviations
ARC arcuate nucleusb bovinecAMP cyclic adenosine-mono-phosphatecDNA complementary DNACHO chinese hamster ovaryDMN dorsomedial nucleusDNA deoxyribonucleic acidgp guinea pigGPCR G-protein coupled receptorh humanHOX homeoboxHSA Homo sapiensLG linkage groupmRNA messenger RNAMCH melanin-concentrating hormoneMSH melanocyte-stimulating hormoneNA noradrenalineNPY neuropeptide Yp porcinePCR polymerase chain reactionPVN paraventricular nucleusPY polypeptide YPYY peptide YYPP pancreatic polypeptider ratRNA ribonucleic acidRT-PCR reverse transcriptase-PCRTM transmembranez zebrafish
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1 Introduction
The last ten years of the twentieth century were worldwide declared the Decadeof the Brain. Never before had the brain and its neuronal extensions been somuch in focus. Intelligence, emotional quotients and gender differences of thebrain were discussed in media while scientists continued to publish new andastonishing data. As a reflection of the fin de siècle, selfproclaimed prophetsappeared, each declaring a different aspect of the brain; the mind, as a tool forreaching the innermost parts of consciousness. This outburst of neurologicalmumbo-jumbo demands an even sharper scientific approach so that myths andurban legends about the brain can be eliminated by scientific explanations andtranslated into a language comprehensible to everyone.
To understand the complexity of the central and peripheral nervous system,all their existing parts have to be identified and studied. It has been verified thateven small changes, both internal and environmental, may result in complexalterations of mood, behaviour or interpretations of experiences. One exampleis depression, where insufficient production of the neurotransmitter serotonin,may lead to this very common mental disorder. Another example is ketamine,used as an anaesthetic, that may evoke a special kind of hallucinations duringsurgical operation, resembling so called out of body experiences. The chem-istry of the nervous system is examined piece by piece and continually newpieces in the gigantic puzzle of the brain are discovered and put in a suitableopening.
This thesis focuses on one aspect of the intricacies of the brain’s informa-tion flow: receptors. Receptors bind neurotransmitters, which are one type ofsignaling molecules in the nervous system. Neurotransmitters are importantmediators that convey information between nerve cells as well as from nervecells to non-neuronal cells. Today, more than a hundred neuropeptides areknown, in addition to a dozen classical transmitters. Neuropeptide Y (NPY),one of the most abundant neuropeptides in the brain, was isolated by Tatemotoin 1982 [89]. NPY and its related neuropeptides bind to a specific family ofreceptors, some of which have been cloned and studied in this thesis.
1.1 The NPY family
The NPY family consists of four evolutionarily related peptides: neuropeptideY (NPY), peptide YY (PYY), pancreatic polypeptide (PP) and in certain fishspecies polypeptide Y (PY). Together they display a wide variety of effectssuch as regulation of blood pressure, anxiety, release of pituitary hormones,food intake as well as energy balance and electrolyte homeostasis [30].
The three-dimensional structure of the NPY-family members is known as
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Figure 1: Human neuropeptide Y: The 36 amino acids of NPY adopt a hairpin-like structure called the PP-fold, characterized by a amino terminus, a prolinehelix, a �-turn followed by an �-helix and the amidated carboxy terminal tail.
the PP-fold, consisting of an extended polyproline helix and an �-helix con-nected by a �-turn (Figure 1). The fold is stabilized by hydrophobic interac-tions between the two helices and is important for receptor binding [46].
The different family members show widely different degrees of conserva-tion. NPY is extraordinary well conserved with 22 identical amino acid posi-tions of 36 in all species studied. PYY has 15 positions conserved and PP hasonly 7 constant positions [46].
1.1.1 Neuropeptide Y (NPY)
NPY is widely distributed in the central as well as peripheral nervous system,where it is a potent regulator of cardiovascular function [95]. When injectedintravenously it causes a vasoconstriction enhancing the effect of noradrenaline(NA). NPY and NA are often co-localized in nerve cells and released at thesame time. NPY is also a potent feeding inducer in several vertebrates and
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seems to be a regulator of alcohol consumption, as the NPY knock-out mouseconsumed more alcohol than the wild type. [91]. Regulation of reproduction[45], regulation of circadian rhythms [1], mood and memory [33, 93] are someother important features of NPY.
1.1.2 Peptide YY (PYY)
PYY acts as a hormone and is mainly synthesized and released by intestinalendocrine cells [10]. It has also been detected in a few neuronal populations[68, 83, 84]. PYY inhibits gallbladder secretion, gut motility and pancreaticsecretion [88, 32].
In the bovine genome a duplication of the PYY gene has produced an ad-ditional member of this family, seminalplasmin [34], which is expressed intestis.
1.1.3 Pancreatic polypeptide (PP)
PP is mainly produced in and secreted from the pancreatic islets in tetrapodsas a response to meals. It has almost the same effects as PYY on gut motilityand pancreatic secretion, but acts probably more indirectly through the centralnervous system [98].
1.1.4 Polypeptide Y (PY)
PY is found in the pancreas and in the brain in some teleost fish species. Fishlack PP, but it seems like PY has arisen as an independent gene duplication infish from PYY [12] (Figure 2).
1.1.5 NPY-family evolution
Purification and cloning of NPY-family peptides from a large variety of specieshas allowed a hypothetical evolutionary tree to be deduced [46, 84]. Briefly,NPY and PYY arose from a common ancestor by a chromosomal duplication.PP was generated by a local duplication of the PYY gene at an early stagein tetrapod evolution. PY may be the result of an independent duplication,probably of PYY, in a lineage of teleost fish (Figure 2).
1.2 The NPY family of receptors
Pharmacological studies have shown that several different receptor subtypesmediate the effects of the NPY-family peptides. The receptors to which the
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geneduplication
geneduplication
INVERTEBRATES CHORDATES AGNATHANS TETRAPODS
PY
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PYY
Ancestralpeptide
NPY
Ancestralpeptide
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Figure 2: Schematic diagram of the evolution of the NPY/PYY/PY/PP familygenes.1 Redrawn from Hoyle [39].
NPY-family peptides bind belong to the superfamily of G-protein coupled re-ceptors [60].
1.2.1 G-protein coupled receptors
The G-protein coupled receptors are characterized by seven transmembraneregions [4]. This type of receptor has an extracellular amino terminus, sevenhydrophobic alpha helices traversing the cell membrane linked by loops bothextra- and intracellularly, and a intracellular carboxy terminus. Sometimes theG-protein coupled receptors are referred to as heptahelix receptors or seven-transmembrane (7TM) receptors due to this structure (Figure 3).
The ligand usually binds in a pocket created by the seven transmembraneregions and induces a conformational change that leads to activation of G-proteins, which after a cascade of events either stimulate or inhibit cyclicadenosine-mono-phosphate (cAMP) synthesis or trigger other events in thecytoplasm. The NPY receptors all act by inhibiting cAMP production [60],but have also been shown to increase intracellular Ca2+.
This group of receptors appear to have arisen very early in evolution; othermembers of this superfamily are found in plants, yeasts and slime mold [42,41].
1There is no time scale on the x-axis in this figure!
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MNSQ S F L TN E VHSVNFSEKNAQLLA
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Figure 3: Neuropeptide Y/peptide YY receptor Y1 displaying the typical seventransmembrane regions of a G-protein coupled receptor.
1.3 The NPY receptor subtypes in mammals
Five NPY family receptors have been cloned in mammals so far: Y1, Y2,Y4, Y5 and y6. The whole set of receptors has so far been cloned in fourspecies: human, mouse pig and guinea pig. Sequence analyses have revealeda considerable degree of divergence between the receptor subtypes (Figure4). As an example, receptor subtype Y1 in human shares only 42% of theamino acids with its closest functional relative, the Y4 subtype. The amino acididentity between Y1 and the in human non-functional y6 is 52%. Comparisonswith human Y2 and Y5 give identities of only 31% and 35% respectively [8].Hence, the NPY-receptor family is one of the most divergent in the G-proteincoupled receptor superfamily.
1.3.1 Y1
Receptor subtype Y1 was initially cloned in rat [19] and subsequently in human[48, 35] and some other species. Most recently it was cloned in domesticpig (Sus scrofa) [100] and guinea pig (Cavia porcellus) [7]. The sequenceidentity between different orders of mammals is as high as 95%. Y1 differs
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Figure 4: Distance tree for mammalian NPY receptors using the neighbour-joining method of the DNASTAR Megalign software. The tree was calculatedwithout the large third cytoplasmic loop of the Y5 receptors and without theinsertions in the amino terminal extracellular domain in the mouse Y5 receptorand all of the Y2 receptors. The Lymnaea stagnalis NPY receptor was used asoutgroup to root the tree, accession code X82088.
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from the other NPY receptor genes in the family by having an intron in thecoding region. This intron is located in the region that codes for the thirdintracellular loop.
Y1 seems to be involved in vasoconstriction in the periphery [31] and tomediate at least a part of NPY’s anxiolytic effect [93]. Messenger RNA for Y1has been found in the arcuate nucleus of the hypothalamus in the brain [62],thereby indicating that Y1 might be important in the regulation of feeding.Thisis supported by several in vivo studies. The mRNA levels of hypothalamicY1 are lowered during fasting [13] also indicating involvement in the feedingresponse.
The Y1 receptor binds NPY, PYY and [Leu31, Pro34]NPY with approxi-mately equal preference. When one amino acid is removed from NPY (NPY2–36) affinity decreaces dramatically.
1.3.2 Y2
The Y2 receptor was first cloned in human [25, 75, 72]. Subsequently, Y2 hasbeen found in several other mammals. The Y2 receptor is very well conservedbetween the mammals studied and shows more than 95% identity.
Y2 is localized mainly presynaptically, where it may act as a inhibitor ofneurotransmitter release. Studies show that the Y2 receptor in the brain isinvolved in memory [21] and in circadian rhythms [40].
This receptor binds NPY and PYY, but [Leu31, Pro34]NPY displays a lowaffinity. NPY13–36 shows no changes in affinity to the Y2 receptor as opposedto the Y1 receptor.
1.3.3 Y4
The Y4 receptor was first known as the PP1 receptor, due to the high affinitythe receptor showed for pancreatic polypeptide (PP) [6](paper I). This receptoris the most divergent among the NPY family receptors and shows extensivedifferences between the species studied.
The physiological functions of the Y4 are still unclear, but one study iden-tifies Y4 as the receptor responsible for contractions in the ileum of rabbits[20].
Y4 preferably binds PP. NPY and PYY also bind, but with lower affinity.
1.3.4 Y5
The Y5 receptor has received much attention since its discovery. This be-cause it is assumed to play an important role in NPY-induced feeding. It was
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first cloned from a hypothalamic cDNA library [26]. In the hypothalamus themRNA levels of Y5 are 200–400 times higher than the mRNA levels of Y1 andY2-receptors. [86].
The Y5 receptor is much larger than the other known NPY receptors dueto 100 extra amino acids in the third intracellular loop [100].
The binding profile for Y5 shows equal preferences for NPY, PYY andNPY2–36.
1.3.5 y6
The y6 receptor gene was first cloned in mouse [29, 96] and rabbit [59]. Lateron, the human y6 receptor was cloned [28, 76] along with several other pri-mates (chimpanzee, gorilla and marmoset)[59]. The receptor has also beenfound in pig [100] and peccary [99]. The gene has not been possible to iden-tify in rat despite the availability of a mouse probe, suggesting that y6 haspossibly been lost from the rat genome [11].
The receptor seems to be functional in mouse and rabbit, but there is aframeshift mutation (single base deletion) in human and in the monkey speciesstudied which makes the receptor non-functional. In pig the y6 receptor genehas two 2-bp deletions [100] causing frameshifts and a loss of function. Onthe other hand, the peccary y6 receptor gene does not have this mutation, indi-cating that the receptor most likely is functional.
The y6 receptor shows 78–81% a identity between mammals and displaysa widely different pharmacological profile in mouse and rabbit, where the re-ceptor is functional. In mouse the pharmacological characterization shows amuch higher affinity for PP than PYY and NPY, which is similar to that ofthe mouse Y4 receptor [29]. On the other hand, results for the same receptorfrom another laboratory the same year [96] showed a different pharmacolog-ical profile: NPY and PYY are primarily bound, resembling an atypical Y1
receptor. In rabbit, the y6 receptor prefers PYY and to some extent NPY2–36and NPY13–36. At the same time the affinity for PP is low, giving it a moreY2-like profile. After correction of the frameshift in human, specific bindingof the NPY family peptides were still not enabled.
This receptor has earlier been called PP2, Y2b and even Y5. Now the namey6 is established and due to absence of physiological correlate of this receptor,the International Union of Pharmacology recommends that the y6 receptor iswritten by a lower case y.
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1.3.6 What about Y3?
Compared to the other subtypes of the NPY receptors, little is known about theY3 receptor. The Y3 receptor has never been cloned and has only been demon-strated to exist from pharmacological data from heart muscle cells [3], thebrainstem [27] and bovine chromaffin cells [94]. The Y3 receptor has been re-ported to be present in the nucleus tractus solitarius (NTS), a subset of neurons[51]. It is possible that the Y3 binding properties may result from modificationof one of the already cloned receptors [8].
1.4 NPY receptors in non-mammalian species
NPY receptors have also been detected in other vertebrates as well as inverte-brates.
1.4.1 Fishes
In the zebrafish (Danio rerio) three different NPY receptors have been cloned:zebrafish Ya (zYa) (paper III), zebrafish Yb (zYb) (paper II) and zebrafish Yc(zYc) [73]. Comparisons of the amino acid sequences show that zYb andzYc have a higher degree of identity (75%) than the identity to zYa (50%),indicating that zYb and zYc share a more recent ancestor. In the Atlantic cod(Gadus morhua) a receptor named cod Yb has been cloned [2]. This receptoris equally identical to both the zYb and zYc. Recently, the Y2 receptor has alsobeen cloned in zebrafish [22]. The Y2 has also been found in Siberian sturgeon(Acipenser baeri) [50].
Probably due to low levels of expression it has been difficult to outline thedistribution of the fish receptors. Yet some messenger RNA for zYb has beendetected in the brain, in the eye and in the intestine by reverse transcriptionand PCR (paper II).
1.4.2 Other vertebrates
The hunt for additional NPY receptors continues. The first to be found was theY1 receptor from a frog (Xenopus laevis) [9]. The Y2 receptor has also beencloned in another frog (Rana ridibunda) [22] and in chicken (Gallus gallus)all five receptor subtypes have been cloned: Y1[37], Y2 [80], Y4 [56], Y5 [37]and y6 [23], but still no functional characterization has been performed.
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1.4.3 Invertebrates
An NPY receptor has been cloned in the pond snail (Lymnea stagnalis) [90].Candidate NPY receptors have been reported for other invertebrates such asthe fruitfly (Drosophila melanogaster) [53], a soil nematode (Caenorhabditiselegans) [15]. They all show a low amino acid identity (approximately 30%)to the mammalian NPY receptors, i.e. the same identity as the Y1, Y2 and Y5
receptors in mammals show to each other. However, no NPY-like ligand hasbeen detected in any of these invertebrates.
1.5 NPY and regulation of feeding
NPY is one of the strongest and most important appetite-stimulating agent inthe nervous system. Under normal conditions, release of NPY represents afundamental part of the pathway of the hypothalamic integration of energyhomeostasis [44]. There are NPY-producing neurons both in the brainstem, aswell as in the arcuate nucleus (ARC) and in the dorsomedial nucleus (DMN) ofthe hypothalamus which are handling daily control of food intake [43]. Thus,there is an orexigenic network of several other appetite-stimulating or inhibit-ing signals such as galanin, orexin, �-MSH, MCH, opioid peptides etc.
The NPY-producing neurons in the brain stem their cell bodies have theiraxon in different parts of the hypothalamus, such as arcuate nucleus, ventro-medial nucleus, paraventricular nucleus (PVN) and other neighbouring areas[79, 78]. Rat brain studies have shown that Y1and Y5 receptor subtypes arethe mediators of the appetite stimulation of NPY in the hypothalamus [44].As the Y5 previously is considered being the feeding receptor due to antisenseknock-down studies [81] as well as binding studies with Y5-selective agonists[26, 102] the localization in the hypothalamus, the feeding centre of the brain,makes it even more important for further studies.
Leptin, which is produced and released from adipose tissue to inhibit feed-ing or feeding behaviour, have displayed an interesting connection to NPY.Leptin seems to inhibit NPY gene expression in the ARC as shown by admin-istering leptin to ob/ob mice (mice with a mutated non-functional leptin gene)[87, 82] and suppresses NPY-induced feeding acutely and on a long term basis[103].
Knock-out studies of the NPY gene have produced different and puzzlingresults. In one study [18] the knocked-out mice were still phenotypically nor-mal with no changes in neither metabolism nor feeding, When disrupting theY1, Y2 and Y5 receptor gene, there was a remarkable late on-set increase inbody weight [64]. In another study of NPY knock-out mice the feeding was ac-tually decreased compared to homozygous wild-type mice [5]. Most recently
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a fatty acid synthase inhibitor, C-75, reduces eating with a an involvement ofNPY [54].
1.6 Molecular evolution
Gene families, like the NPY receptor family, have arisen by duplications, bothof individual genes and chromosomal segments as well as the entire genome.
1.6.1 Duplication of the genome
Crucial for the development of the large group of NPY receptors is the dupli-cation of the genome that seems to have occurred at least twice in vertebrates.The first duplication occurred most likely before agnathans (jaw-less fish) di-verged from amphioxus. The second round of duplication probably took placebefore the elasmobranches (cartilage fish) diverged from agnathans, some 420million years ago.
One theory for the duplication of the whole genome, is that the ancestralgenome underwent large-scale duplications early in the vertebrate evolution[57, 36, 85] and by this the evolution of complex anatomical construction ofthe vertebrates was facilitated [36, 85]. Evidence for this is, for example, foundin the three or four copies of certain large gene clusters in the mammaliangenome, for example the homeobox (HOX) clusters which are present in fourcopies on four different human chromosomes [36, 77]. It seems like NPY andPYY have arisen from a common ancestor through a chromosomal duplication,as the NPY gene is located close to the HOXA cluster and PYY is close to theHOXB cluster [38, 46, 84].
One example of local gene duplications is the human hemoglobin genecluster. The �-like and �-like subunits are encoded by a small family of geneseach that are differentially expressed during development. The �-like and �-like globin genes are located on different chromosomes, but each is linked ina cluster and transcribed from the same DNA strand. This and the fact thatadjacent genes in each cluster are structurally related indicates that there havebeen a local gene duplications [70].
Bringing together the sequences of all known full length clones of NPYreceptors from mammals and fish to a distance tree gives an interesting viewof the relationship between the subtypes (Figure 4).
The Y1, Y2 and Y5 show a low degree of identity (27–31% overall iden-tity). Still they are located on the same chromosome, pointing out a probablecommon ancestral NPY/PYY-binding receptor gene, which was duplicated atleast twice The Y1, Y4 and y6 are more similar to each other, and are in figure4
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clustered together, distinct from the Y2 and Y5 receptor subtypes. The clus-ter of Y1, Y4 and y6 and the higher degree of identity within these receptorscompared to Y2 and Y5 the indicate that the three genes have arisen from acommon ancestral receptor (Figure 4).
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2 Aim of the present study
The aim of this study was to clone and characterize NPY-family receptors tooutline their evolution as well as their functional properties in vitro. NPYshows diverse physiological effects, including vasoconstriction, stimulationof feeding behaviour, regulation of metabolic rate and regulation of ethanolconsumption. The approach was molecular cloning of the receptors by PCRfollowed by screening of genomic libraries. The nucleotide sequences andthe amino acid sequences were determined and analyzed. Comparisons ofthe sequences and chromosomal mapping was used to deduce the evolution-ary relationships. The clones were then stably expressed in cell lines and thepharmacological profile determined.
In this thesis, four new NPY family receptor genes are reported. Thecloning took place in three quite diverse species that are commonly used ex-perimental animals. The rat belonging to the order rodents, and the guinea pig,a non-rodent, equally distant related to rodents as to humans and the zebrafishbelonging to teleost fishes.
The rat (Rattus norvegicus) is a well studied and commonly used labora-tory animal, with a well documented gene map. It is very frequently used inresearch concerning behavioural investigations and is the most popular animalused in studies of physiology.
The guinea pig (Cavia porcellus) is also widely used as a laboratory ani-mal. It has recently become even more valuable thanks to studies showing itshould no longer be regarded a rodent as it displays quite divergent sequencesboth for mitochondrial [16] and nuclear genes [52]. The guinea pig divergedfrom humans approximately at the same time that it diverged from rodents, soit should at least be considered a very early branch in the rodent tree.
The zebrafish (Danio rerio) has lately become a frequently utilized modelanimal. One reason is that the embryo is transparent and is therefore easy touse in developmental studies, allowing visualization of organ systems. Anotherreason is that it is easy to keep and breed at a low expense and the geneticmap has been improved in the last years. It has also been discovered thatthe zebrafish and human genome share several loci of chromosomal synteny,which facilitates positional cloning [69].
2.1 Pseudogenes in vertebrates
As the y6 gene was found to be a pseudogene in guinea pig as well as in human,it might be valuable to review the existence of pseudogenes in general terms.
Pseudogenes are common particularly in vertebrates, but have been identi-fied in bacteria, insects and plants [24, 71, 55].
19
�
� �
Figure 5: a) Rat (Rattus norvegicus)2 b) Zebrafish (Danio rerio) c) Guinea pig(Cavia porcellus).
A pseudogene is a “sequence which is present in the genome of a givenpopulation and typically is characterized by close similarities to one or moreparalogous genes, yet is non-functional” [61]. To separate pseudogenes fromtheir functional paralog, the pseudogene is often represented with the Greeksymbol (psi) as a prefix, for example PGK-1, or with a capital P as asuffix, like CYP21P. Pseudogenes are common, but it is hard to calculate theactual number. Sequence data from human chromosome 22 indicates that 19%of the coding sequence were pseudogenes and of these 82% were processedpseudogenes [17]. Vertebrate gene families differ greatly in the number ofpseudogenes.
Pseudogenes arise by gene duplication in two different ways: either byretrotransposition or by duplication of genomic DNA. When retrotranspositionoccurs, the resulting pseudogenes are called either processed pseudogenes orretro-pseudogenes. These retro-pseudogenes lack both introns and 50-promotersequence, but are flanked by direct repeats and contain a 30-polyA stretch [92].They originate from single-stranded RNA [92, 74, 97] and are transcribed intoa double-stranded DNA [58]. The other kind of pseudogenes, generated by du-plication of DNA segments, usually once had the same introns and regulatoryregions as the mother gene [61].
2Picture of rat (Madelein) kindly from Filip Sebek, Mälardalens Högskola.
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The gpy6 receptor gene apparently belongs to the type of pseudogenes thatarose by duplication as shown by its chromosomal location in a large clusterof duplicated genes. Pseudogene transcripts have been detected for instancethe human y6 gene. This might happen if the transcriptional control elementsof the gene still are intact [14].
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3 Summaries of the papers
When this project was initiated, several receptors had been defined by clas-sical pharmacology, but only the Y1 receptor had been cloned. By using thesequence of the Y1 receptor from mammals and frog, then recently cloned inour lab, degenerate primers were made and used for PCR on genomic DNA,primarily from human, rat and zebrafish. This led to the discovery of the recep-tors described in papers I–III. Additional receptor genes were cloned in otherlabs and paper IV describes our cloning of one these, y6, in the guinea pig. Theresults have broadened the perspective on NPY-family receptors and revealedfar greater complexity than expected.
3.1 Paper I: The cloned rat pancreatic polypeptide receptor ex-hibits profound differences to the orthologous receptor3
PCR was run on rat genomic DNA with degenerate primers based on Y1 se-quences and a product of the expected size was obtained. Sequencing revealeda sequence similar to but distinct from Y1. The PCR fragment was used as aprobe to screen a rat genomic library. The full-length sequence obtained wasexpressed in a stable cell line for pharmacological characterization. A North-ern blot was done to determine the distribution of the receptor and a Southernhybridization was performed to investigate if the rat receptor was orthologousto the human PP1 (hPP1) receptor, now renamed hY4. The human Y4 as wellas the rat Y4 (rY4) got their names due to their high affinity for the PP.
The rat Y4 receptor has a greater amino acid sequence identity to the Y1receptors than to any other receptor known at the time of its discovery. Theoverall identity is 46% and in the transmembrane regions the identity is 56%.The amino acid sequence identity between the rY4 receptor and the hY4 re-ceptor is only 75%. In contrast to Y1, there is no intron in the coding re-gion. A Southern blot confirmed an orthologous relationship as both the ratand human receptor probes hybridized strongly to the rat receptor gene. Phar-macologically, the rY4 receptor bound iodinated bovine PP (bPP) with highaffinity (Kd=0.038 nM), did not bind iodinated porcine PYY (pPYY) (unlikethe human Y4). Comparison of the two Y4 receptors revealed that rY4 has aconsiderably lower affinity also for NPY than the human receptor. In stablytransfected chinese hamster ovary (CHO) cell lines, the rY4 receptor inhibitscAMP synthesis. There are also differences between species in the distribu-tion of the receptor. Rat Y4 mRNA surprisingly is most abundant in lung andtestis, with additional weak signals in colon and total brain. For hY4, there is
3The PP1 receptor is now renamed Y4 receptor.
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no transcript in these organs except for the colon.Thus, the Y4 receptor differs between human and rat in many ways. The
sequence, the binding profile and the mRNA distribution differs between thesespecies, but they share the preference for binding PP. Interestingly, the pre-ferred ligand PP also differs greatly in sequence between human and rat.
3.2 Paper II: Cloning and characterization of a novel neuropep-tide Y receptor subtype in the zebrafish
In order to clone additional NPY receptors, degenerate primers from the Y1receptor was used in PCR on genomic zebrafish DNA. Several clones with thesame restriction enzyme pattern were isolated and sequenced (paper III), oneof which showed a different nucleotide sequence. We used the PCR fragmentof this clone, named zebrafish Yb (zYb), as a probe to screen for the full-lengthreceptor in both a genomic zebrafish library and in a cDNA (complementaryDNA) library. Clones encoding a receptor of 375 amino acids were found. Thepharmacological profile of zYb was investigated by expressing the gene in acell line and by using several peptides that are well established ligands. Thedistribution of the receptor was investigated with Northern blots from brainand intestine and reverse transcriptase PCR (RT-PCR) performed on mRNAfrom brain, eye and intestine.
The zYb receptor has an identity of 50% and 52% respectively to the Y1receptor and the Y4 (PP1) receptor. This is much lower than what is normallyobserved for orthologues (species homologues) between mammals and fish,suggesting that zebrafish Yb receptor is a distinct subtype. There is no intronin the coding region of this receptor.
The iodinated porcine PYY bound to the zYb receptor with an affinityconstant (Kd) of 3.7 pM. When competing with both zebrafish and porcineNPY and PYY, the ligands showed strong inhibition of binding of iodinatedporcine PYY (pPYY) in the low picomolar range. The inhibition constant(Ki) of zNPY was 3.4 pM and of the zPYY 42 pM.
By RT-PCR, mRNA was detected in brain, eye and intestine, but no tran-scripts were seen in Northern blots. In Southern blots, a single band was de-tected, indicating that there is only one copy of the zYb receptor gene in thezebrafish genome.
In summary, the zYb receptor seems to be a novel receptor so far uniquefor the zebrafish.
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3.3 Paper III: Neuropeptide Y receptor subtype with unique prop-erties cloned in the zebrafish: the zYa receptor
Several identical clones (mentioned in paper II) were detected by PCR withdegenerate primers on zebrafish genomic DNA. After screening a zebrafishgenomic library, sequencing of the resulting clones revealed that several ofthem had an identical sequence and the receptor was called the zebrafish Ya(zYa) receptor. The receptor contains 377 amino acids.
This new intronless zYa receptor gene was compared with other isolatedNPY receptors from mammals as well as zebrafish. All three known zebrafishreceptors, including zYb and zYc [73], were mapped and compared with thehuman gene map. The pharmacological profile of zYa was investigated byexpressing the gene in a stable cell line and by using several peptides and wellestablished ligands.
The zYa receptor has equally high overall amino acid sequence identity ofapproximately 50% to Y1 and to the zebrafish receptors zYb and zYc. In thetransmembrane regions the identity of zYa to human Y1, human Y4, mouse y6,zYb and zYc is about 60%. The identity to the Y2 and Y5 receptors is only27%.
Pharmacologically, a unique binding profile was displayed where iodinatedporcine PYY bound concentration-dependently to the zYa receptor with anaffinity constant (Kd) of 28 pM and mammalian and zebrafish NPY and PYYbound with equally high affinity. By measuring extracellular acidification witha microphysiometer a functional response was observed. However no cAMPresponse was detected.
The chromosomal localization of the three known zebrafish NPY receptorszYa, zYb and zYc displayed a possible orthologous relationship between zYcand mammalian y6, but no orthologue for zYa. The zYb gene is a recent copyof zYc in the fish lineage.
In summary, zYa seems to be a novel NPY receptor subtype, which has noknown orthologue in mammals, but the existence of one is suggested.
3.4 Paper IV: Neuropeptide Y receptor gene y6: multiple deathsor resurrections?
A genomic guinea pig DNA library was screened with a mouse y6 probe inorder to obtain a clone containing the guinea pig y6 (gpy6) receptor. The guineapig receptor gene was subcloned into a plasmid vector and sequenced.
The guinea pig y6 nucleotide sequence exhibited ten frame shift mutationsleading to three premature termination sites in the coding region, thereforeneither binding studies nor expression studies were performed. There was an
24
amino acid identity of 65-73% to previously cloned full length y6 receptorsfrom other species (human, mouse, pig, peccary and rabbit).
Thus, the y6 receptor gene is a pseudogene in the guinea pig, as well as inhuman and in pig. Interestingly, the inactivating mutations seem to be distinctin guinea pig, human and pig. This difference together with the loss of y6 in ratsuggest that the y6 gene has been inactivated independently in these lineages.
More likely is that an unknown regulatory mutation event, before the ra-diation of the mammalian orders, might be the original inactivating mutation.However, this would imply that the y6 gene has later been resurrected in somespecies, such as mouse and rabbit. The y6 gene constitutes a very interestingevolutionary case which will require information from additional species inorder to be resolved.
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4 Discussion
4.1 The rat Y4 receptor
The rat Y4 receptor, called rat PP1 in paper I, was found to be the orthologueto the human Y4 receptor. Surprisingly, the amino acid sequence identity be-tween the human and rat receptor only 75%, a number that usually is higherbetween orthologues. Both species show a preference for binding the PP, rel-ative to NPY and PYY, but differ in affinity constants. Also the receptor’spreferred PP shows high variability in sequence between human and rat. Be-tween mammals, birds and frogs there is only 50% identity, making PP one ofthe most variable neuroendocrine peptides known.
It seems like the ligand PP and its receptors are co-evolving rapidly [47, 49]This will be a very interesting case to study co-evolution receptor and ligand.
This was the first Y4 receptor to be discovered, but second to be publishedas we used the rat probe screen a human library. The discovery of an Y4 ortho-logue from a rodent enhanced the probability of finding additional receptors,both in rat and in other species.
4.2 The zebrafish receptors
The finding of the zebrafish Ya (zYa) and the zebrafish Yb (zYb) receptor, to-gether with the more recently discovered zebrafish Yc (zYc) [73], exposed anew Y1 receptor-like NPY receptor, this far only present in the fish lineage.The zYa shows a unique pharmacological profile, which distinguishes it fromthe zYb and the zYc, which seems to closely related to each other by a simi-lar pharmacological profile and a higher identity in the amino acid sequences.The zYa receptor has been located, utilizing chromosomal mapping, to linkagegroup 17 (LG17), the zYb to LG8 and the zYc to LG10. Studies of neigh-bouring genes gave a probable orthologue to zYc in the y6 gene. None suchequivalent is found to the zYa or the zYb. On the other hand, the sequencephylogenies suggest that zYb and zYc duplicated after the divergence of theAtlantic cod and the zebrafish lineages, approximately 140 million years ago.One possibility is that zYb arose by reverse transcription of a zYc mRNA andwas then inserted in LG8.
The chromosomal localization of the mammalian NPY receptor genes hasdisclosed a probable scheme of evolution (Figure 6). The Y1, Y2 and Y5 areall located on the human chromosome 4 (HSA4), while the Y4 and the y6is located on the human chromosome 10 (HSA10) and human chromosome5 (HSA5), respectively. Yet there is a higher sequence identity between Y1,Y4 and y6. It seems likely that the Y1, Y2 and the Y5 arose from a common
26
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Figure 6: The proposed scheme for the evolution of the NPY receptor familyand their current arrangement in the human genome. Redrawn from AmandaWraith [100].
ancestor and through local duplications intra-chromosomally early during ver-tebrate evolution and became the three receptors on HSA4. The close sequencerelationships of Y1, Y4 and y6 is presumably due to a more recent common an-cestor, which was duplicated in a more large-scale manner, where segments ofor entire chromosomes were doubled.
The y6 receptor is the black sheep of the NPY receptor family. The lackof function of this receptor in guinea pig, pig and human indicates a complexhistory of activation and/or reactivation of the gene during evolution, as it isfunctional in other mammals.
The most obvious inactivating mutations in the y6 receptor gene, the frameshifts, are distinct in human, pig and guinea pig. The frame shift in the pri-mates studied situated after the sixth transmembrane region [59, 76], in the pigy6 gene is the first frame shift in the region encoding the third extracellularloop [100] and the guinea pig y6 sequence presented has its first frame shiftmutation in the cytoplasmic loop between TM3 and TM4. None of the frameshifts are shared between primates, pig and guinea pig. These differences to-gether with the loss of y6 in rat suggest that the y6 gene has been inactivatedindependently in these four lineages of mammals. If the y6 gene had beeninactivated independently in the lineages studied, this would be a quite rareevent. Two other examples are the deficiency of L-gulono-gamma-lactone ox-
27
idase [67, 66] and probably urate oxidase [101], each having two independentinactivations.
Alternatively, the y6 gene might have been inactivated before the mam-malian orders diverged from each other 80–100 million years ago. This couldhave been caused by a less obvious mutation in the promotor region or in anenhancer region. Subsequently, secondary frame-shift mutations may havefollowed independently in primates, pig and guinea pig. Other lineages couldhave regained expression, i.e., rabbit, mouse, and collared peccary (albeit func-tional studies have not been performed in the peccary to see if its y6 gene isfunctional). This scenario would presumably involve a fairly long period ofnon-expression in these lineages which seems unlikely.
The alternative with four independent inactivation events is favoured, be-cause the accumulation of mutations in the guinea pig gene suggests that theguinea pig gene was much earlier inactivated than human and pig. The piggene was probably inactivated after the divergence from peccary, less than 30million years ago. The rat gene was probably lost after the divergence frommouse, less than 12–15 million years ago. The inactivation time point for hu-man (hence the primates) y6 is hard to estimate.
4.3 Molecular evolution: questions answered?
Receptor cloning and functional characterization of the kind described in thisthesis gives important clues to gene evolution. Two new mammalian geneshave been studied, Y4 and y6. The relationship to the mammalian receptorsremains unclear. The chromosomal localization of the zYa and zYb genessuggest they are distinct subtypes. However high replacement rates and chro-mosomal rearrangements may obscure orthologous relationships. Data fromadditional species will help resolve this matter.
The present study does not address how certain vertebrate species have di-verged from common ancestors nor how different species are related to eachother, but in due time and with data from more species this project will un-doubtedly shed light on these aspects. The kinds of questions that can beanswered are how and when new family members arose and how they haveevolved new functions and from the answers to that question, how other genefamilies have evolved. Other issues to which this information can contributeare the time points for the postulated genome duplications and how this af-fected the function of the genes and the organism carrying the genes.
28
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Figure 7: Proposed scheme for evolution of subtypes of Y1-like receptor sub-types in mammals and fish.
4.3.1 The zYa and zYb receptors and evolution
The common ancestor for zYb, zYc and the cod Yb seems very feasible whenstudying distance trees like figure 4. Still there are just theories how the recep-tor subtypes in fish lineage arose. It is possible to consider the gene duplicationtheory of mammals and speculate (Figure 7).
We determine that the Y1 is considered as the ancestral receptor, displayingtwo genome duplications. The Y1 receptor diverged into Y1/Y6 and Y4/Ybc.The Y4/Ybc diverged into Y4 in mammals and in zebrafish to Ya. The Ybc inthe fish lineage, could in mammals be a still unknown and undetected receptor.
This suggests that the zYa receptor may be the zebrafish ortholog of themammalian Y4 receptor, which does not agree with the chromosomal locationstudies of zebrafish in paper III. In any case, it is likely to find mammalianreceptor genes corresponding to the fish lineage Ybc-receptor genes, althoughthey may not be functional.
4.3.2 The y6 receptor and evolution
The y6 receptor protein has not yet been found to be expressed at any de-tectable amount in any region of the mouse nervous system during the embry-onic development [65], but it has been characterized in vitro in this species.In paper IV, two different hypotheses of death and resurrection of the y6 re-ceptor are presented. In connection to this, some discussion about the possiblephysiological importance of this receptor seem pertinent, to consider why it isfunctional in mouse and rabbit, in contrast to guinea pig, human and pig (andrat).
The y6 receptor gene is very old and arose more than 400 million yearsago. If it remained until the origin of mammals it presumably once had an
29
important functional role. Studies in non-mammals will be required to tracethis function. That question is what happened in the mammalian lineage.
The chromosome segment with the y6 gene copy is the result of a du-plication of the Y5/Y1/Y2 receptor cluster (now present on chromosome 4 inhuman). As far as it is known, y6 is the only gene that survived in this extraset of genes . The Y5-like and Y2-like receptor genes seems to have been losttheir function. It is estimated that about 40% of duplicated genes acquire novelor specialized functions [63], then losing a function of the gene after a dupli-cation is certainly not an uncommon event, which might be the case with they6 receptor gene.
The existence of additional NPY receptor genes, not yet discovered norcloned, is undoubtedly probable. The access to the complete human genomewill change the ability to search for new receptors via databases and the huntfor additional receptor genes will accelerate and many more genes, both func-tional and pseudogenes, will be discovered in a short period of time.
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5 Concluding remarks
Evolution of receptor gene families is just a small part of the gigantic evolu-tionary jig-saw puzzle. In this thesis new discoveries of the vertebrate recep-tors have been presented that provide information of the NPY-family receptorsin an evolutionary perspective. Every scientist’s contribution to our increasedknowledge will be of greatest importance and will in due time be a part ofthe complex full-scale map of the function and importance of each and everypart of the nervous system. The scientist may also make sure that everyone,other scientists and laymen alike, will understand new discoveries irrespectiveof subject studied.
31
Acknowledgements
I would like to give my sincere gratitude to my supervisor Dan Larhammar,who with advice and patience let me—despite my numerous pregnancies andmaternal leaves—finish this thesis.
I would also like to thank:
� Prefect and professor Lars Oreland for running the best unit ever!
� Present and past colleagues in Dan’s group and Lars’ group as well aspresent and past co-workers at the department. It is impossible to nameyou all, but I have had great time!
� My former summer workers Monica and Karin for invaluable help.
� The proof-readers of this thesis: Magnus Berglund, Earl Larson, MariaRingvall, Per Starbäck and Lotta Söderberg.
� The girls at Zoofys, especially Maria Ringvall, who I sometimes believeis my evil twin.
� My mother and my late father for their cool support and their trust in meand my sister.
� My sister Petra, who always says she wants a “doktor” in the family.Well, at least you got a doctor of medical science!
� My parents-in-law, Karin and Orvar, for valuable support and baby-sitting.
� My typographic expert and (of course) gorgeous husband Erik, for beingthe best!
� And—last but not least—my favourite boys Fredrik and Linus. A smilefrom you and nothing is boring anymore!
32
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