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*.* !/0-#/.*./ 1*,/-!/ 11*,* 2 Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Neuroscience presented at Uppsala University in 2001

ABSTRACT

Holmberg, S.K.S. 2001. Y receptors in human, guinea pig and chicken. Cloning, in vitro pharmacology and in situ hybridization. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1097. 55 pp. Uppsala. ISBN 91-554- 5160-8.

Neuropeptide Y (NPY) is known to influence a vast number of physiological and behavioral processes such as vasoconstriction, circadian rhythms, feeding, anxiety and memory. of the NPY family bind to five different cloned G-protein coupled subtypes (Y1, 2, 4-6). The studies compiled in this thesis present inter-species comparisons of sequence similarities, binding properties and expression patterns among receptors of the NPY family. Cloning of Y1 and Y2 receptor subtypes from guinea pigs revealed strong binding profile similarity to the corresponding human receptors. Previously demonstrated atypical binding profiles in the caval vein of guinea pigs were concluded to result from other receptors than the cloned Y1 and Y2 receptors, or possibly combinations of distinct receptor subtypes. The guinea pig Y5 receptor was found to be expressed in regions of the brain that have been indicated as important for regulation of food intake. Expression in the , amygdala and brain stem was noticed, similar to studies in rats and humans. In other brain regions, such as the striatum and hippocampus, some species differences were observed. Mutagenesis studies of the human Y1 receptor indicated sites important for binding both of endogenous and synthetic antagonists. Putative new sites of interaction with the Y1 antagonists BIBP3226 and/or SR120819A were recognized. The data were used to construct a three- dimensional structure model, based on a high-resolution bovine model. Cloning of the chicken (Gallus gallus) Y1, Y2 and Y5 receptors revealed high sequence similarities with mammalian receptors. Most endogenous ligands bound with similar affinities as to mammalian receptors. The strongest exception was the discovery of high-affinity binding to chicken Y2 of [Leu31, Pro34]NPY, which was previously considered to bind non-Y2 receptors only. The new human Y1 receptor model provides a basis for further investigations of ligand-receptor interactions, which will be aided by information on NPY receptors from other taxa. Guinea pigs are concluded to be a good complement to rats and mice for studying NPY signaling. These results demonstrate the benefits of species comparisons for pharmacological studies.

Key words: G-protein coupled receptor, , , ortholog, chicken, guinea pig, mutagenesis, receptor computer modeling, mRNA in situ hybridization.

Sara K.S. Holmberg, Department of Neuroscience, Unit of Medical Pharmacology, Biomedical Center, Uppsala University, Box 593, SE-751 24 Uppsala, Sweden

 Sara K.S. Holmberg 2001

ISSN 0282-7476 ISBN 91-554-5160-8

Printed in Sweden by Uppsala University, Tryck och Medier, Uppsala 2001 To my family This doctoral thesis is based on the following communications, which will be referred to by their Roman numerals. Reprints were made with permissions from the following publishers: Regulatory Peptides (I), Elsevier Science (II, V)

I: Cloning and functional expression of the guinea pig neuropeptide Y, Y2 receptor. Sharma, P., Holmberg, S.K.S., Eriksson, H., Beck-Sickinger, A., Grundemar, L., and Larhammar, D. Regulatory Peptides, 75-76, pp. 23-28 (1998). II: The cloned guinea pig neuropeptide Y receptor Y1 conforms to other mammalian Y1 receptors. Berglund, M.M., Holmberg, S.K.S., Eriksson, H., Gedda, K., Maffrand, J., Serradeil-Le Gal, C., Chhajlani, V., Grundemar, L., and Larhammar, D. Peptides, 20(9), pp. 1043-53 (1999). III: Localization of neuropeptide Y receptor Y5 mRNA in the guinea pig brain by in situ hybridization. Holmberg, S.K.S., Johnson, A.E., Bergqvist, C., Källström, L., and Larhammar, D. (Manuscript). IV: Structural model of human neuropeptide Y receptor Y1 based on interactions of mutated receptors with NPY and two antagonists. Holmberg, S.K.S., Sjödin, P., Berglund, M.M., Fredriksson, R., Mohell, N., and Larhammar, D. Structural model of human neuropeptide Y receptor Y1 based on interactions of mutated receptors with NPY and two antagonists. (Manuscript). V: Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y2. Salaneck, E., Holmberg, S.K.S., Berglund, M.M., Boswell, T., Larhammar, D. FEBS Letters, 484, pp. 229-234 (2000). VI: Pharmacological characterization of cloned chicken neuropeptide Y receptors Y1 and Y5. Holmberg, S. K. S., Mikko, S., Boswell, T., Zoorob, R., and Larhammar, D. Pharmacological characterization of cloned chicken neuropeptide Y receptors Y1 and Y5. (Submitted). TABLE OF CONTENTS

ABBREVIATIONS 4

INTRODUCTION 5 Neuropeptide Y and its related peptides belong to the PP-fold family 6 Receptors of the NPY-family peptides are G-protein coupled 9 The Y1 receptor subtype is the best characterized 10 The Y2 receptor can bind truncated fragments but not PP 11 The Y4 receptor is a quickly evolving subtype 12 The Y5 receptor exhibits the binding profile of a "feeding receptor" 13 The y6 receptor is a pseudogene in many species 14 Issues to be addressed considering our current knowledge of the NPY receptor 14 Pharmacological studies of the human Y1 receptor 15 Guinea pig phylogeny and its implications for an additional animal model for feeding studies. 16 Chicken NPY receptor cloning and the value of an evolutionary approach to pharmacological studies 16 RESEARCH AIMS 17

RESULTS 19 Cloning and pharmacological characterization of the guinea pig Y1 and Y2 receptors (Papers I and II) 19 Distribution of Y5 receptor mRNA expression in the guinea pig brain (Paper III) 20 Structural model of the human Y1 receptor (Paper IV) 23 Cloning and pharmacological characterization of the chicken Y1, Y2 and Y5 subtypes (Papers V and VI 34 DISCUSSION 36 Guinea pig Y1 and Y2 receptors (Papers I and II) 36 Distribution of Y5 receptor mRNA in guinea pig brain (Paper III) 37 The human Y1 receptor model (Paper IV) 40 Chicken Y1, Y2 and Y5 receptors (Papers V and VI) 42 Species comparisons of the Y1 receptor may help identify antagonist interaction sites (Paper IV and VI) 45 CONCLUSIONS 46

FUTURE PERSPECTIVES 47

ACKNOWLEDGEMENTS 48

REFERENCES 50 ABBREVIATIONS

10 dorsal motor nucleus of the vagus AD anterodorsal thalamic nucleus AH anterior hypothalamic area AP area postrema Arc arcuate hypothalamic nucleus AStr amygdalostriatal transition area b bovine BL basolateral amygdala bp base pair BST bed nucleus of the stria terminalis CA1 field CA1 of hippocampus CA3 field CA3 of hippocampus cAMP cyclic AMP Ce central nucleus of the amygdala ch chicken CPu caudate putamen DG dentate gyrus DM dorsomedial hypothalamic nucleus EL extracellular loop fi fimbria of the hippocampus GI gastro-intestinal gp guinea pig GPCR G-protein coupled receptor h human Kd dissociation constant Ki inhibition constant mnPO median preoptic nucleus MPA medial preoptic area NPY neuropeptide Y p porcine Pa paraventricular hypothalamic nucleus PP PV paraventricular thalamic nucleus PYY peptide YY r rat Re reuniens thalamic nucleus SCh suprachiasmatic nucleus SFi septofimbrial nucleus SO supraoptic nucleus Sol nucleus of the solitary tract st stria terminalis TM trans-membrane VMH ventromedial hypothalamic nucleus wt wildtype INTRODUCTION

Understanding how cell signalling is propagated by membrane-bound receptors is occupying the time and minds of researcher worldwide. Communication among cells is accomplished by secreted transmitters that will bind receptors in a specific manner. During the last twenty years rapid advances in molecular biology techniques have resulted in a tremendous expansion in neurobiology research. This is a multidisciplinary field that includes expertise from many aspects of human medicine, pharmacology, biology, chemistry and computer science. Despite great differences in complexity of the nervous system among animal groups, where the human brain seems to have little in common with the few neurons governing the sensory and motoric functions of an invertebrate, such as Drosophila melanogaster (a fruit fly) or Caenorhabditis elegans (a worm), the cellular components display, surprising to some, many similarities. All neurons are able to communicate via changes in electron potentials propagated through their axons. In the axon terminus, transmitter molecules are released in the synaptic cleft between neurons and bind to cell surface receptors. Changes in transmitter release are known in several psychiatric and neurological disorders, the pathology of which could possibly be reversed by drugs influencing transmitter signalling. Pharmaceutical companies are naturally exploring this possibility on a large scale. In order to study the binding properties of a certain receptor, it is typically cloned and expressed in vitro in cultured cells. Binding studies of the cloned receptor in combination with computer-aided structure modeling and mutagenesis may lead to insights regarding the three-dimensional structure of the ligand-binding pocket. Selective receptor ligands are important to enable correct interpretations when performing pharmacological studies in vivo. The molecular properties of receptor binding and how a receptor attains an active (signalling) state are interesting not only to drug design, but also in terms of basic research. An understanding of receptor activation is of fundamental importance that would help us understand how biological processes can be governed by cell signalling. For neuropeptide Y (NPY) receptors, the particular role of each subtype (five different known in mammals) in conveying the effects of the peptide ligand has been partially clarified using a combination of physiological studies and mapping of receptor 5 subtype distributions. However, much work remains to define the exact roles and mechanisms of each receptor subtype. More generally, molecular studies can promote the development of highly selective drugs for each receptor for treatment of diseases thought to involve the NPY system, and also confer knowledge of common structure and activation patterns of G-protein coupled receptors (GPCRs). A main goal of studying the NPY system is to understand its functions in humans. However, species differences in receptor function and distribution complicate extrapolations from research animals. A comparative approach is useful, where similar observations in different animal models increase confidence of inferring the condition in humans. Pharmacological comparisons of cloned receptors from different taxa can also provide hypotheses of the structural requirements for ligand binding.

Neuropeptide Y and its related peptides belong to the PP-fold family NPY is a C-terminally amidated 36-amino acid peptide that was first purified from porcine brain (Tatemoto, et al. 1982). It is among the most abundant signalling peptides in the mammalian brain. The precursor protein is encoded by a gene with four exons (Minth, et al. 1986; Larhammar, et al. 1987). Mature NPY is generated from its precursor protein through cleavage by multiple enzymes (Paquet, et al. 1996). NPY is often co-released with catecholamines in the central and peripheral nervous system of vertebrates and is extremely well conserved among all vertebrate species so far studied. NPY-like peptide and receptor genes have even been cloned from the invertebrates Drosophila melanogaster (Brown, et al. 1999) and Lymnaea stagnalis (Cox, et al. 1997). Several studies have revealed diverse physiological roles of NPY in feeding, blood-pressure regulation, anxiety, seizure control, alcohol intake, memory, circadian rhythms, gastro-intestinal (GI) secretion and nociception (Clark, et al. 1984; Flood, et al. 1987; Heilig, et al. 1989; Duggan, et al. 1991; Hua, et al. 1991; Erickson, et al. 1996; Kalra, et al. 1998; Bannon, et al. 2000; Harrington and Schak 2000; Malmstrom 2000; Klemp and Woldbye 2001; Reibel, et al. 2001). Some of the above mentioned effects are also reviewed by (D. R. Gehlert 1998; Kalra, et al. 1999). NPY belongs to a family of structurally related peptides (Larhammar 1996; Cerda- Reverter, et al. 2000). Mammals and birds have NPY, peptide YY (PYY) and pancreatic polypeptide (PP). X-ray crystallography data from avian PP resolved the three-

6 dimensional structure, see Fig. 1, proposed to be common to all so called “PP-fold family peptides” or, as further referenced here, the NPY family. This was later consolidated by nuclear magnetic resonance (NMR) studies of bovine PP (Li, et al. 1992), NPY (Darbon, et al. 1992) and PYY (Keire, et al. 2000). PYY is found in the GI tract of mammals, where it is released in response to meals. It inhibits gut motility, pancreatic and gall bladder secretion. Also, neurons in the lower brain stem and spinal cord of rat and dorsal root ganglia of rat express PYY, indicating functions of PYY in the central nervous system (Pieribone, et al. 1992; Jazin, et al. 1993). PP is expressed and secreted by endocrine pancreatic cells and like PYY is released in response to meals. PP is only found in tetrapods and it is highly variable among species. The amino acid sequence identity is only 50% between mammals, birds and amphibians. Binding sites for PP have been found in the hypothalamus, forebrain, amygdala, thalamus and brain stem of rats indicating a possible central regulatory function (Whitcomb, et al. 1997). See Fig. 2 for amino acid sequences of NPY, PYY and PP.

Figure 1: Schematic structure of PP-fold peptides, illustrated by human NPY. Residues 1-9 form a proline helix followed by a beta-turn and an amphiphilic alpha helix comprised of residues 14-30. The C-terminal residues 31-36 form a flexible tail. This basic structure is implicated for all NPY-family peptides. Adapted from (Allen, et al. 1987).

7 Figure 2: Sequence alignments of NPY, PYY and PP sequences from human, pig, guinea pig, rat, chicken and trout. Adapted from (Larhammar, et al. 1993). Sequences are written beginning at the amino-terminus. Amino acids shown in grey are identical within each peptide group. Dashes represent missing amino acids.

NPY human -YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY-amide NPY pig -YPSKPDNPGEDAPAEDLARYYSALRHYINLITRQRY-amide NPY guinea pig -YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY-amide NPY rat -YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY-amide NPY chicken -YPSKPDSPGEDAPAEDMARYYSALRHYINLITRQRY-amide NPY trout -YPVKPENPGEDAPTEELAKYYTALRHYINLITRQRY-amide

PYY human -YPIKPEAPGEDASPEELNRYYASLAHYLNLVTRQRY-amide PYY pig -YPAKPEAPGEDASPEELSRYYASLAHYLNLVTRQRY-amide PYY guinea pig -YPSKPEAPGSDASPEELARYYASLAHYLNLVTRQRY-amide PYY rat -YPAKPEAPGEDASPEELSRYYASLAHYLNLVTRQRY-amide PYY chicken AYPPKPESPGDAASPEEIAQYFSALRHYINLVTRQRY-amide PYY trout -YPPKPENPGEDAPPEELAKYYTALRHYINLITRQRY-amide

PP human -APLEPVYPGDNATPEQMAQYARDLRRYINMLTRPRY-amide PP pig -APLEPVYPGDDATPEQMAQYARELRRYINMLTRPRY-amide PP guinea pig -APLEPVYPGDDATPQQMAQYAREMRRYINMLTRPRY-amide PP rat -APLEPMYPGDYATHEQRAQYETQLRRYINTLTRPRY-amide PP chicken -GPSQPTYPGDDAPVQDLIRFYNDRQQYLNVVTRHRY-amide

Figure 3: The evolutionary tree shows estimated time of divergence for humans, pigs, cattle,guinea pigs, rats, mice and birds. MYA = Million years ago. UC = Upper Carboniferous, P = Permian, Tr = Triassic, J = Jurassic, K = Cretaceous, T = Tertiary.

8 Receptors of the NPY peptide family are G-protein coupled All peptides of the NPY family signal through GPCRs. These are characterized by seven trans-membrane (TM) hydrophobic helices usually of 20 – 25 amino acid residues, and downstream signalling through various G-proteins. GPCRs can be activated by many different types of ligands such as neurotransmitters, peptides, odor or taste molecules, and light. The NPY receptor family is one of the largest and most diverse among the known peptide GPCR families (Larhammar, et al. 2001). Differing affinities of endogenous peptides, a variety of peptide analogues, and synthetic compounds have been used to distinguish different receptor subtypes, reviewed by (Michel, et al. 1998) (Table 1). Major efforts have been put into cloning and in vitro expression of the different subtypes in order to develop new subtype-selective antagonists and agonists. Five receptor subtypes (Y1, 2, 4-6) (Michel, et al. 1998) have been cloned from mammals and chickens (Papers V and VI; personal communication, T. Boswell, R. Fredriksson, I. Lundell, E. Salaneck, D. Larhammar) and three distinct receptor subtypes have been cloned from teleosts (Ya, b, c) (Lundell, et al. 1997; Ringvall, et al. 1997; Arvidsson, et al. 1998; Sharma, et al. 1999; Starbäck, et al. 1999). The evolutionary events leading to the many and diverse members of this receptor family have to some extent been explored, reviewed by (Larhammar, et al. 2001). There are strong indications that the NPY receptor gene clusters arose through duplication events early in vertebrate evolution. Further explorations of this theory as well as investigations into ligand-receptor interaction and co-evolution will require cloning of peptides and receptor subtypes from multiple taxa. All NPY-family receptor subtypes seem to couple to in an inhibitory fashion, probably through Gi or Go proteins (Michel, et al. 1998). Depending on cell type the coupling can also take alternative pathways, such as phosphatidyl inositol- mediated elevation of intracellular calcium levels (Herzog, et al. 1992), Gβγ-subunit receptor crosstalk (Selbie, et al. 1997) and ion-channel inhibition or activation, for review see (Michel, et al. 1998). Any connections and/or hierarchical relationships among these pathways remain to be shown. Transcription regulatory proteins such as mitogen- activated protein kinase (MAPK) (Benchekroun, et al. 1992; Nie and Selbie 1998; Hansel, et 9 al. 2001) and cyclic (c) AMP response element binding (CREB) protein (Sheriff, et al. 1998) have also been proposed in the NPY signalling cascade. The Y1 and Y4 subtypes also exhibit -driven internalization (in CHO cells) as a means of receptor regulation (S. L. Parker, et al. 2001). Knowledge about intracellular signalling and receptor regulation will increase in the near future, since cloned receptor subtypes can be studied in vitro.

Table 1: Typical binding profiles of mammalian NPY receptors. NPY2-36 through NPY13-36 are depicted "C-terminal NPY-fragment".

Subtype Typical binding profile of mammalian receptors

Y1 NPY, PYY > [Pro34]-substituted analogs >> C-terminal NPY-fragment > PP Y2 NPY, PYY, C-terminal NPY-fragment >> [Pro34]-substituted analog > PP Y3 (not cloned) NPY, [Pro34]-substituted analogs, C-terminal NPY-fragment >> PYY, PP Y4 PP > NPY, PYY, [Pro34]-substituted analogs Y5 NPY, PYY, [Pro34]-substituted analogs, NPY2-36 >> NPY13-36 y6 no consensus

The Y1 receptor subtype is the best characterized The Y1 receptor was the first subtype to be cloned (Eva, et al. 1990; Herzog, et al. 1992; Larhammar, et al. 1992) and it is highly conserved among species (Michel, et al. 1998; Larhammar, et al. 2001). This receptor is characterized pharmacologically by its equal affinity to both NPY and PYY, a lower affinity to PP and a requirement for an intact amino-terminus (amino-terminally truncated peptides exhibit dramatically lowered affinity) (Michel, et al. 1998). Its expression is abundant both in the central nervous system and in the periphery (Larsen, et al. 1993; Wharton, et al. 1993; Nakamura, et al. 1995; Jackerott and Larsson 1997; Kopp, et al. 1997). The Y1 receptor has been implicated in a multitude of NPY-mediated effects such as anxiolysis (Wahlestedt, et al. 1993; Heilig 1995; Kask, et al. 2001), potentiation of anaesthetic sedation (Naveilhan, et al. 2001), antinociception (Zhang, et al. 2000), increase in ethanol self-administration (Kelley, et al. 2001), vasoconstriction and blood-pressure

10 regulation (Michel and Rascher 1995; Nilsson, et al. 1996; Capurro and Huidobro-Toro 1999; Modin, et al. 1999; Abrahamsson 2000; Malmstrom 2000), hypothermia (Lopez- Valpuesta, et al. 1996), neuroproliferation (Hansel, et al. 2001), DNA synthesis (Nilsson and Edvinsson 2000), inhibition of release in the medial preoptic area (Silveira and Franci 1999), luteinizing hormone release (Leupen, et al. 1997; Jain, et al. 1999), inhibition of vagally stimulated acid secretion (Lloyd, et al. 1996) and inhibition of secretion seems to be mediated by peripheral Y1 receptors (Morgan, et al. 1998). Y1 antisense oligodeoxynucleotide treatment enhances food intake in energy-deprived rats (Schaffhauser, et al. 1998). Also, Y1 receptor-deficient mice exhibit obesity without hyperphagia, possibly as a result of dysfunctions in glucose metabolism and signalling (Kushi, et al. 1998; Kanatani, et al. 2000; Burcelin, et al. 2001).

The Y2 receptor can bind truncated peptide fragments but not PP Earlier pharmacological studies with truncated and modified peptide fragments had clearly indicated the existence of a receptor with lower sensitivity to truncated fragments than Y1. NPY and PYY were first shown to bind the Y2 receptor with equally high affinities. Indicative of this subtype is that fragments as small as NPY13-36 can bind the receptor with high affinity, while PP does not bind Y2. The isolation of a Y2 receptor gene by expression cloning was reported in 1995 and 1996 by two different groups (Gerald, et al. 1995; Rose, et al. 1995). The cloned receptor exhibited only 35% sequence identity to the human Y1 receptor, thereby explaining the failed attempts to isolate DNA clones by homology screening. Gene expression is mainly localized to the brain, but low expression levels have also been observed in the GI tract. Indeed, the Y2 receptor, as described below, seems to exhibit a different binding profile in the periphery. In the intestine, a PYY- preferring receptor subtype has been implicated. Recently, this receptor was shown to be identical with the formerly cloned Y2 receptors in both rats and humans. Apparently, binding profiles of the cloned receptor differ depending on the cell type in which it is expressed (Goumain, et al. 2001). No PYY preference was detected for the human nor the guinea pig Y2 receptor expressed in COS cells (Rose, et al. 1995; Gerald, et al. 1996; Sharma, et al. 1998). The chicken Y2 receptor expressed in HEK293 cells also exhibited equal preference for NPY as for PYY. This implies a flexibility in the binding profile that

11 could account for some reports of not-yet cloned receptor subtypes. Considering recent theories on a cell's available signalling repertoire in terms of G-protein composition and other putative pathways such a scenario is not unlikely (Kenakin 1995b;a; Leff, et al. 1997). Another explanation to differing ligand preference could be receptor-associated proteins analogous to receptor activity modifying proteins (RAMPs) (Sexton, et al. 2001). The proposed central effects of Y2 receptor activity are quite diverse. They include influence on GI motility regulated from the hypothalamus or dorsal vagal complex, phase shifting of circadian rhythm mediated through the suprachiasmatic nucleus (SCh), inhibition of glutaminergic excitatory synaptic transmission in areas CA1 and CA3 of the hippocampus, thus influencing memory and seizure control, nociception, vasopressor activity mediated through the nucleus of the solitary tract stimulation of vasopressin and oxytocin release, regulation of prolactine release and other pituitary hormones. These effects are excellently reviewed by (Kaga, et al. 2001). Y2 deficient mice (Naveilhan, et al. 1999) showed an attenuated response to leptin administration, surprisingly gained more weight on high fat diet and became mildly obese on a normal fat diet, exhibited increased food intake and reduced activity, compared to wildtype littermates, thus indicating a role of the Y2 receptor in regulation of food intake and energy expenditure.

The Y4 receptor is a quickly evolving subtype PCR using degenerated primers directed to conserved regions of the Y1 receptor and subsequent screening of a human genomic library resulted in cloning of the Y4 receptor (Lundell, et al. 1995). Y4 is the fastest evolving functional subtype and PP, its preferred ligand, is the most rapidly evolving of the NPY-family peptides (Larhammar 1996). Localization of Y4 is mainly peripheral in humans and rats (Lundell, et al. 1995; Lundell, et al. 1996) but a restricted expression in rat brainstem, hypothalamus and hippocampus has been reported (R. M. Parker and Herzog 1999) (Larsen and Kristensen 1997). It has also been implicated in regulation of GI motility (Pheng, et al. 1999). The Y4 receptor has not been studied extensively in terms of pharmacology. The peptide dimer 1229U91 or GR231118 (Daniels, et al. 1995) has been shown to function as an agonist on the Y4 receptor (E. M. Parker, et al. 1998; Schober, et al. 1998). However, the usefulness of this ligand is limited since it also binds Y1 and y6 receptors. Structure-activity studies,

12 reviewed by (Cabrele and Beck-Sickinger 2000)) revealed that a substitution of glycine in position 34 by leucine yielded an analog that was 18-fold more potent than NPY. The development of a Y4-selective antagonist would be very useful to further determine the role of Y4.

The Y5 receptor exhibits the binding profile of a "feeding receptor" The Y5 receptor is the most recently cloned receptor subtype, discovered by expression cloning from rat hypothalamus (Gerald, et al. 1996; Hu, et al. 1996). Some of the peptides known to induce feeding (NPY, PYY and moderately aminoterminally truncated fragments of NPY) exhibit high affinity for the Y5 receptor. The Y5 receptor structure differs from other subtypes by a large and highly variable third intracellular loop. Except for the variable loop, the TM regions are highly conserved among mammals with an amino acid sequence identity of 98-100% (Larhammar, et al. 2001). Alternative splicing has been observed in the 5' - untranslated region of humans, rats and pigs (E. M. Parker and Xia 1999; Wraith 1999). Y5 also seems to play a role in natural food intake, though any dramatic effects on feeding over the Y1 receptor remain to be shown. Both the Y5 and Y1 knock-out mice exhibited reduced food intake in response to NPY (Marsh, et al. 1998; Kanatani, et al. 2000). Other studies have also indicated a varying participation of Y5 receptor in feeding regulation (Schaffhauser, et al. 1997; Criscione, et al. 1998; Tang-Christensen, et al. 1998; Wyss, et al. 1998; Flynn, et al. 1999; Hwa, et al. 1999; Yokosuka, et al. 2001). More likely, to some extent Y1, Y2 and Y5 are all involved in the regulation of feeding. Phase shifting of suprachiasmatic nucleus circadian rhythms (Yannielli and Harrington 2000), seizure control (Marsh, et al. 1999; Vezzani, et al. 2000; Reibel, et al. 2001) (Woldbye, et al. 1997) and inhibition of luteinizing hormone secretion (Raposinho, et al. 1999) have also been attributed to Y5. The localization of Y5 mRNA and protein have been studied in human, rat, mouse, guinea pig, vervet monkey and marmoset (Widdowson, et al. 1997; Dumont, et al. 1998b; Naveilhan, et al. 1998; Statnick, et al. 1998; Nichol, et al. 1999; R. M. Parker and Herzog 1999; Durkin, et al. 2000). Expression was generally observed in hypothalamic, amygdaloid and hippocampal areas of the brain. The studies also report various species

13 differences, as well as discrepancies between the detection of mRNA expression and Y5 receptor binding within the same species. [D-Trp32]NPY is being used as a selective Y5 agonist, but its affinity is relatively low. New selective Y5 agonists have been reported, but their usefulness for studies in vivo remain to be seen (Cabrele and Beck-Sickinger 2000). CGP71863A, one of the few reported high-affinity Y5 non-peptide antagonists has been studied by several research groups. Unfortunately, it seems to exhibit some affinity to non-NPY receptors and perhaps even toxicity (Criscione, et al. 1998; Zuana, et al. 2001).

The y6 receptor is a pseudogene in many species A functional y6 receptor was cloned from a rabbit hypothalamic cDNA library in 1996 (Matsumoto, et al. 1996). At the same time, a mouse y6 receptor was cloned independently (Gregor, et al. 1996; Weinberg, et al. 1996). Later studies revealed that the y6 receptor gene is a pseudogene in all mammalian species thus far studied except mouse and rabbit (Matsumoto, et al. 1996) and the collared peccary, a distant relative of the pig (Wraith 1999). In rats, the gene seems to be absent (Burkhoff, et al. 1998). Interestingly, in the species that have a non-functional y6 gene, it seems to have been inactivated through independent events. The frameshift mutations are positioned differently in primates, pigs (Wraith 1999) and in guinea pigs (in which there are multiple frameshift mutations) (Starback, et al. 2000). The cloned chicken y6 receptor is functional ( R. Fredriksson, D. Larhammar, personal communication). Because of its non-functionality and therefore lack of selective constraints, the y6 gene is very divergent among species. Conflicting pharmacological profiles have been published, even when the receptor was expressed in the same cell type (COS-7) (Gregor, et al. 1996; Weinberg, et al. 1996). A recent study (expression in HEK 293) rejects the y6 as a feeding receptor in mice (Mullins, et al. 2000). Neither Weinberg et al. nor Mullins et al. observed any PP binding, while Gregor et al. reported such binding in their study.

Issues to be addressed considering our current knowledge of the NPY receptors For some time, only the Y1 and Y2 subtypes were known. The later cloning of subtypes Y4, Y5 and y6 revealed binding profiles partially similar to Y1 and Y2. Older studies of

14 receptor distribution and studies of physiology using non-specific ligands would therefore need to be re-evaluated. Species differences and also intra-species discrepancies between mRNA and protein levels and localization have been reported. The reasons and implications of these observations remain yet to be seen. Reports continue to emerge on physiological responses to NPY, PYY or PP not matching the binding repertoires of the cloned subtypes. The implicated NPY-preferring receptor, since long designated as the Y3 subtype, has still eluded cloning (Blomqvist and Herzog 1997; Michel, et al. 1998). Pharmacological studies have brought speculations about yet additional subtypes (O'Shea, et al. 1997). The Y2 receptor expressed in rat and human intestine exhibits a tissue specific binding profile, and this could very well be the case for other PYY preferring or atypical receptors, as well. Yet, the possibility of other subtypes, known or unknown, still remains.

Pharmacological studies of the human Y1 receptor The Y1 receptor is by all accounts the most well studied receptor in the NPY family. It has been cloned for the longest time and has been ascribed many diverse physiological effects. The human receptor is naturally of particular interest in terms of drug design. Mutagenesis studies combined with computer-aided receptor-ligand modeling have been performed (Walker, et al. 1994; Munch, et al. 1995; Munoz, et al. 1995; Sautel, et al. 1995; Sautel, et al. 1996; Du, et al. 1997). One model, utilizing vaccinia virus expression in HeLa cells suggested that the basic residues of endogenous peptide ligands interact with four aspartic residues in the three extracellular loop (EL) regions (D104, D194, D200, D287) (Walker, et al. 1994). Later studies, expressing the mutant receptors in E. coli, showed that the D194A and D200A mutants exhibited unaltered binding of NPY, suggesting that these residues were not involved in ligand interaction (Munch, et al. 1995). Difficulty of expressing the mutant receptors may have been the reason for initially conflicting data. Further studies proposed a hydrophobic binding pocket constituted of residues in TM2, TM6 and EL3 (Y100, F286 and H298) of the receptor to interact with the carboxy-terminus of NPY (Sautel, et al. 1995). The antagonist BIBP3226 (Rudolf, et al. 1994) was developed relatively early and is therefore one of the most well characterized Y1-selective non-peptide antagonists to date.

15 It was used in earlier studies to determine the three-dimensional structure of the receptor’s antagonist-binding site (Sautel, et al. 1996; Du, et al. 1997).

Guinea pig phylogeny and its implications for an additional animal model for feeding studies Guinea pigs (Cavia porcellus) have long been used as research animals. However, rats (Rattus rattus) and mice (Mus musculus) have been used to a much larger extent than guinea pigs in the NPY field, as in many other areas. If one wishes to extrapolate the observations from a laboratory animal to humans, one has to consider the extent to which each NPY receptor's physiological role is conserved among species. When studying complex behaviors such as feeding there will be qualitative and quantitative differences species to species. Also, the differential rate of evolution of the various NPY receptor subtypes implies an advantage of studying additional animal species apart from rats and mice. Unequal evolutionary branch lengths are consistent with the observation in many comparative studies that overall similarity does not always equal homology. Recent phylogenetic studies using both genetic and morphological data place guinea pigs (Caviomorpha) as very distant relatives to rats and mice (Myomorpha) among the rodents (Adkins, et al. 2001) (Huchon and Douzery 2001) (Fig. 3). Of interest to this study, the guinea pig Y4 receptor subtypes has been shown to be more similar to the human receptor than to rat and mouse. Further, guinea pig PP is more similar to human PP than to rat and mouse PP (Eriksson, et al. 1998). Another reason for studying the guinea pig NPY receptor subtypes was the puzzling observation that guinea pig caval vein contractile response to amino-terminally truncated NPY could be blocked by BIBP3226 (Grundemar and Ekelund 1996), suggesting the possibility of a deviating Y1 receptor or a novel receptor subtype. The need to clone and characterize all NPY receptor subtypes in guinea pigs was therefore identified as an important research project.

Chicken NPY receptor cloning and the value of an evolutionary approach to pharmacological studies The cloning of Y1, Y2, Y4, Y5 and y6 subtypes from chicken (Gallus gallus) was initiated by an interest both in the pharmacology and the evolution of the NPY receptor family in a

16 non-mammalian species. Chickens are suitable for pharmacological studies since they are domesticated and initial studies of feeding and reproduction have been undertaken. Also, the genetics of this species has been studied extensively. NPY has been shown to initiate feeding in chickens (Kuenzel, et al. 1987) and it has been shown to be upregulated in feed- restricted broilers (Boswell, et al. 1999). NPY-like immunoreactive neurons in the infundibular nucleus have been implicated in the control of sexual maturation (Walsh and Kuenzel 1997). Furthermore, different breeds are available with variance in feeding and reproductive behaviors. Receptor autoradiography studies have shown differences in NPY receptor expression levels in broilers (fast-growing breed) compared to leghorn chicken (egg-laying breed) (Merckaert and Vandesande 1996). Evolutionarily conserved amino acids are candidate sites for ligand interaction. The sequence conservation between Y1, Y2 and Y5 is very low, but all three receptors can still bind NPY and PYY. Conserved sites in all three receptor subtypes could therefore indicate sites important for ligand binding. In the case of the Y1, Y2 and Y5 subtypes there is a high degree of sequence conservation within mammals. Therefore, the cloning of subtypes from more distantly related taxa would further our knowledge about sequence conservation within each subtype.

RESEARCH AIMS

When starting up the projects described here the focus was on cloning new receptor subtypes of the NPY family, and on elucidating in greater detail the molecular properties of ligand binding. The Y1, Y2 and Y4 subtypes had been cloned, but not yet the Y5 and y6 receptors. During the past several years, pharmacological companies have put much effort into high-throughput screening in order to find new subtype-selective receptor ligands. Yet, to this date, only a few specific antagonists have been developed. The diverse NPY system, influencing so many different aspects of physiology, has been investigated in this thesis by studies on molecular evolution and pharmacology. The extent of sequence conservation was assessed by cloning receptors from different animal species. An evolutionary approach has been used for generating ideas regarding receptor- 17 ligand interactions. Similarities and differences in ligand preference would help identify sites important for subtype-specific ligand binding. Most previous computer-generated structure models of GPCRs were based on the low-resolution bovine rhodopsin or the bacterial rhodopsin model, now known to differ significantly from the recently published high-resolution structure of bovine rhodopsin. More recently, the field has included receptor second messenger activation and receptor internalization studies. Receptor cloning and mutagenesis, in vitro receptor binding, in situ mRNA hybridization and computer aided receptor structure modeling have been used in further described projects in order to obtain knowledge about NPY signalling.

Specific aims k To investigate NPY receptors in guinea pigs regarding primary structure, functional expression, in vitro pharmacology and anatomical distribution. This species could provide a complementary animal model to rats and mice for studying food intake and other behaviors. Therefore it was important to characterize each receptor subtype with regard to binding properties. k To perform ligand-receptor binding studies of the human Y1 receptor in order to generate new ideas about the ligand-binding pocket, to be compared and contrasted with previously presented models. k To investigate NPY receptors in chicken. The results would aid in generating working hypotheses for receptor binding and provide insight into the divergence of receptor subtypes through evolutionary time.

18 RESULTS

Cloning and pharmacological characterization of the guinea pig Y1 and Y2 receptors (Papers I and II) The guinea pig (gp) Y1 receptor gene was cloned from a guinea pig genomic library. The open reading frame, contained in two exons, encoded a protein sequence of 384 amino acids. The sequence identity was 92-93% to other mammalian Y1 receptors. The gene was subcloned into the pTEJ-8 expression vector, and the receptor protein was stably expressed in CHO cells. Saturation and competition studies were performed with iodinated pPYY as radioligand. Radioligand binding recognized a single binding site with a Kd of 37 ± 3 pM (S.E.M., n=3) and a Bmax of 177 ± 11 fmoles/mg protein. The receptor exhibited a typical Y1 receptor binding profile, shown in Table 2, with porcine (p) NPY, pPYY, p[Leu31, Pro34]NPY binding with high affinity, while amino-terminally truncated NPY fragments bound with much lower affinities. The two Y1 antagonists SR120819A and BIBP3226 bound with affinities comparable to the rat (r) Y1 receptor. Guinea pig PP bound with 75-fold lower affinity than NPY and p[D-Trp32]NPY did not bind the receptor. Inhibition of cyclic AMP (cAMP) production was not observed, but CHO cells exhibited a concentration-dependent increase in extracellular acidification rate upon agonist stimulation. The gpY2 receptor gene was cloned from a guinea pig genomic library. The open reading frame was contained in an uninterrupted sequence encoding a protein of 376 amino acids. The sequence identity to other mammalian Y2 receptors was 90-95%. The isolated receptor gene was subcloned in an expression vector and transiently expressed in COS-7 cells. The binding profile was assessed through binding assays in vitro using iodinated pPYY as a radioligand. Saturation binding studies revealed that PYY binds to a single, high-affinity site with a Kd of 6.4 ± 2.1 pM (S.E.M., n=6) and a Bmax of 168.1 ± 11.9 fmoles/mg protein (S.E.M., n=6). Porcine NPY, pPYY, bovine PP, modified NPY analogues (p[Leu31, Pro34]NPY, p[Pro30, Tyr32, Leu34]NPY and pNPY[D-Trp32]), amino- terminally truncated and also centrally deleted NPY fragments (pNPY3-36, pNPY13-36, pNPY18-36, pNPY22-36, pNPY26-36, Ahx(8-20)NPY, Ahx(5-24), Ahx(8-20)[Pro34]) were

19 each tested in competition assays with iodinated pPYY. See Table 2 for inhibition constants (Ki). Porcine NPY, pPYY, the N-terminally truncated fragments of pNPY (down to pNPY22-36) and the centrally truncated analogs Ahx(8-20)NPY and Ahx(5-24)NPY functioned as high-affinity ligands. Bovine PP, p[Leu31, Pro34]NPY, pNPY[D-Trp32], Ahx(8- 20)[Pro34]NPY and p[Pro30, Tyr32, Leu34]NPY28-36 bound poorly. The Y1 antagonist BIBP3226 did not bind to the gpY2 receptor. Alignments of Y1 and Y2 receptor sequences, respectively, are shown in Figs. 4 and 5.

Distribution of Y5 receptor mRNA expression in the guinea pig brain (Paper III) Y5 receptor expression was observed in regions of the hypothalamus previously shown to be of importance for feeding regulation, such as the arcuate hypothalamic nucleus (Arc), the paraventricular hypothalamic nucleus (Pa), the ventromedial (VMH) and dorsomedial hypothalamus (DM). Moderate expression levels were observed in median preoptic areas thought to be involved in reproduction. High expression levels were also observed in the dentate gyrus (DG) and CA3 regions of the hippocampus, while CA1 exhibited lower expression levels. In the brain stem high to moderate expression was noticed in the area postrema (AP), the dorsal vagal complex (10) and the nucleus of the solitary tract (Sol). Moderate expression levels were also observed in the central nucleus (Ce) and basolateral region (BL) of the amygdala and the caudate putamen (CPu). The thalamic area exhibited low expression levels, with some elevated signals in the paraventricular thalamic nucleus (PV) and the reuniens thalamic nucleus (Re) (Table 3, Figs. 6-10).

20 Figure 6: In situ hybridization with Y5 probe in guinea pig brain. The bright-field picture shows silver grain staining over hippocampal cells. Emulsion-dipped section, counterstained with hematoxylin.

Table 3: The expression of guinea pig Y5 mRNA in areas if the brain and brain stem.+ corresponds to low levels of hybridization signal, ++ to moderate levels, +++/ ++++ to high -very high levels.

Brain region Intensity Brain region Intensity CPu ++ AStr + SFi ++ DG +++ MPA ++ CA3 ++++ SCh + CA1 + SO ++ AD ++ Pa ++ AH + BL ++ Re ++ Ce ++ AP ++ st +++ Sol +++ fi ++ 10 +++ DM + BST + VMH ++ mnPO ++ Arc +++ PV +

21 Figure 7 (top left): Localization of Y5 mRNA by in situ hybridization. Scanned autoradiogram (1,200 ppi) of guinea pig brain, coronal section ca -2.80 mm from Bregma (Plate 29 in Paxinos and Watson). The most intense signals were observed over the stria terminalis (st) and the arcuate hypothalamic nucleus (Arc). Moderate signals were also localized to the caudate putamen (CPu), the central nucleus of the amygdala (Ce), the basolateral amygdala (BL) and the ventromedial hypothalamic nucleus (VMH).

Figure 8 (top right): Localization of Y5 mRNA by in situ hybridization. Scanned autoradiogram (1,200 ppi) of guinea pig brain, coronal section ca -2.12 mm from Bregma (Plate 26 in Paxinos and Watson). Very intense signals were observed in field CA3 of the hypothalamus (CA3) and the stria terminalis (st). Moderate signals were also detected in the anterodorsal hypothalamic nucleus (AD), the paraventricular hypothalamic nucleus (Pa), the supraoptic nucleus (SO) and the caudate putamen (CPu). Low signals were observed in field CA1 of the hippocampus (CA1) and the anterior hypothalamic area (AH).

Figure 9 (bottom left): Localization of Y5 mRNA by in situ hybridization. Scanned autoradiogram (1,200 ppi) of guinea pig brain, coronal section ca -1.80 mm from Bregma (Plate 25 in Paxinos and Watson). Very intense signals were observed in field CA3 of the hippocampus (CA3), moderate levels were observed in anterodorsal hypothalamic nucleus (AD), the fimbria of the hippocampus (fi), the caudate putamen (CPu), the reuniens thalamic nucleus (Re) and the supraoptic nucleus (SO). Low signals were seen in the suprachiasmatic nucleus (SCh) and the paraventricular thalamic nucleus (PV).

Figure 10 (bottom right): Localization of Y5 mRNA by in situ hybridization. Scanned autoradiogram (1,200 ppi) of guinea pig brain, coronal section ca -14.60 mm from Bregma (Plate 76 in Paxinos and Watson). Very intense signals were observed in the nucleus of the solitary tract (Sol) and the dorsal motor nucleus of the vagus (10). Moderate hybridzation was also seen in the area postrema (AP).

22 Structural model of the human Y1 receptor (Paper IV) The human (h) Y1 receptor was investigated by site-directed mutagenesis. Binding studies on mutant and wildtype (wt) receptors expressed in vitro were performed with the endogenous agonist NPY and two non-peptide antagonists, BIBP3226 or SR120819A. Positions for mutagenesis were selected after inter- and intra-species sequence comparisons with other NPY receptors (Larhammar, et al. 2001). A three-dimensional model of the hY1 receptor was calculated, based on the bovine rhodopsin structure at 2.8 Å resolution (Palczewski, et al. 2000), shown in Fig. 11. The wildtype receptor displayed a Kd of 0.1 ± 0.01 nM (S.E.M., n=3) and a Bmax value of 68.0 ± 9.8 fmoles/mg protein, deduced from saturation assays with 125I-pPYY. Competition with pNPY gave a Ki of 0.2 ± 0.03 nM (S.E.M., n=3). The results are summarized in Table 4 and Figs 12 - 14. Four extracellular aspartic acid residues were initially suggested to be involved in agonist binding (Walker, et al. 1994). Three of these, D194, D200 and D287 were mutated in Paper IV, (shown in Figs. 4, 15, 16) and loss of binding was observed for D287A1 (Table 4). However, the peptide ligands bind D194A and D200A with high affinity in agreement with two other studies (Munch, et al. 1995; Sautel, et al. 1996). The antagonist BIBP3226 has more than four-fold lower affinity to the D200A mutant as compared to the wild-type receptor in contrast to a previous report that observed higher affinity for this mutant (Sautel, et al. 1996). The other Y1 antagonist, SR120819A, is unaffected by this mutation. Sequence comparisions gave reasons to scrutinize some earlier mutagenesis results. Three positions have been proposed to form a hydrophobic pocket that interact with the amidated carboxy-terminus of the peptide ligands, namely Y100 in TM2, F286 in TM6 and H298 in EL3 (Sautel, et al. 1995). Amino acids in the corresponding positions are not conserved across receptor subtypes despite the fact that they all interact with the carboxy- terminal part of the peptides. Y4 receptors exhibits lower affinity to NPY than the Y1 subtype and mammalian Y4 receptors all have glutamic acid at the position corresponding to F286 in the outer part of TM6 (Figs. 15 -16). The F286E mutant showed 15-fold reduced affinity for NPY in competition with PYY, while a previous publication reported that binding of NPY to a F286A mutant was unaltered (Du, et al. 1997). BIBP3226 bound the

1 The mutated receptors are shown as "wt amino acid;position;mutated amino acid", like in this case D287A 23 F286A mutant with a five-fold reduction in affinity (Sautel, et al. 1996). BIBP3226's affinity to the F286E mutant, as expected, showed a more dramatic drop by approximately 25-fold and the other antagonist, SR120819A, lost binding completely. The F173A mutant studied in Paper IV is not significantly different from the wt receptor with regard to peptide binding. It was initially reported that NPY and BIBP3226 could not bind to F173A (Sautel, et al. 1996) but another report found only a 2.5-fold loss in affinity of PYY (Kanno, et al. 2001).

Figure 12: Inhibition binding of 125I-pPYY binding to membranes from EBNA1-HEK293 cells transfected with wildtype human Y1 receptor. Competition binding assays were performed with NPY, BIBP3226 or SR120819A.

24 Figure 13: Saturation and Scatchard (inset) analyses of 125I-pPYY binding to membranes from EBNA1-HEK293 cells transfected with wt hY1 receptor. Results from one typical experiment are shown.

Figure 14: Saturation and Scatchard (inset) analyses of 125I-pPYY binding to membranes from EBNA1-HEK293 cells transfected with N116A hY1 receptor. Results from one typical experiment are shown.

25 Sequence comparisons show that both Y2 and Y5 have a leucine at the corresponding position and therefore it would seem unlikely that the phenylalanine in Y1 is crucial for peptide binding. Binding of both antagonists was abolished in competition with radio-labeled peptide in the concentration range tested. Peptide binding to N116A appears intact, while BIBP3226 and SR120819A showed lowered affinity, indicating a role of this amino acid for Y1 antagonist selectivity. This was in agreement with sequence comparisons, since this position is conserved in all known Y1 sequences (Fig. 4) but differs in all other subtypes. The positions A90, V167 and L214 were chosen since all of these amino acid replacements change a hydrophobic residue to a polar (threonine in chicken) and are conserved in all known mammalian Y1 receptors, but not in chY1 (Fig. 4). SR120819A does not bind the chY1 receptor and therefore it might be possible to identify amino acids responsible of the loss of binding. Nevertheless, all tested ligands bind to all three mutants with unaltered affinity. Thus, none of these amino acids is likely to contribute to SR120819A's lack of affinity for the chicken receptor. T212 is conserved in all Y1 receptors and is a threonine or serine in other subtypes. Peptide binding to the T212A mutant receptor is virtually unaffected, in agreement with previously published results (Kanno, et al. 2001). Interestingly, the affinity of SR120819A is drastically reduced. The amino acid residue T213 is conserved in the Y1 subfamily (which includes also Y4 and y6) but differs in Y2 and Y5. Paper IV shows that T213A displays unaltered affinity for SR10819A. Q219 is conserved in the Y1 subfamily and Y5 but differs in Y2. A slight loss of affinity for the peptide ligands was observed whereas a previous publication reported loss of binding to Q219A (Sautel, et al. 1996). A recent study reported that two antagonists other than the ones used in Paper IV had unaltered binding, but PYY binding was not tested (Kanno, et al. 2001). Two highly conserved residues in rhodopsin-like receptors have been shown to be primarily involved in the conformational changes leading to the active conformation of the receptor and the subsequent activation of G-proteins. The corresponding positions in the hY1 receptor are R138 on the inner side of TM3 and W276 in TM6 (Fig. 4). The R138K mutant differs little if at all to the wildtype receptor in peptide binding as well as binding of the two antagonists. W276 is constant in all NPY-family receptors including the PP-

26 preferring Y4 receptor as well as the more divergent Y2 and Y5 receptors. The affinity of NPY for the W276A mutant is somewhat higher than for the wildtype receptor. A previous publication reported unaltered affinity (Du, et al. 1997). The antagonist SR120819A has 10- fold lower and BIBP3226 has 20-fold lower affinity. The affinity of 125I-pPYY to the wt hY1 receptor in this study is much higher than that reported by other groups. Also, the difference in affinity between BIBP3226 and NPY is greater than previously observed (Sautel, et al. 1996) using 125I-NPY and 3H-BIBP3226. This may be because we used BIBP3226 in competition experiments with radio-labeled PYY.

Figure 11: a) side view of receptor, b) top view of receptor Three-dimensional model of the human Y1 receptor based on the rhodopsin high-resolution structure. Four important residues included in this study are highlighted: N116 in TM3, F173 in TM4, and W276 and F286 in TM6. The extracellular loops have been removed for clarity since their three-dimensional structures are unknown.

27 Figure 15: Schematic serpent model of human Y1 highlighting mutated residues

28 Figure 4: Multiple amino acid sequence alignment. The human Y1 receptor serves as master sequence in alignment with the human, pig, dog, guinea pig, mouse, rat, chicken and Xenopus laevis Y1 receptors. GenBank accession numbers are: human: M84755, pig: AF005779, dog: AF005778, guinea pig: AF135061, rat: Z11504, mouse: Z18280, chicken:(submitted, Paper VI.) and Xenopus: L25416. In latter sequences only positions that differ from the human Y1 receptor sequence are shown while dots represent identities. Dashes indicate gaps introduced to optimize alignment. The hydrophobic segments assumed to be embedded in the cell membrane are within boxes.

29 Figure 5: Multiple amino acid sequence alignment. The human Y2 receptor serves as master sequence in alignment with the pig, cattle, guinea pig, rat, mouse, and chicken Y2 receptors. GenBank accession numbers are: human:U32500, cattle: U50144, guinea pig: AF072821, rat: AY004257, mouse: D86238 and chicken: AF309091. In latter sequences only positions that differ from the human Y2 receptor sequence are shown while dots represent identities. Dashes indicate gaps introduced to optimize alignment. The hydrophobic segments assumed to be embedded in the cell membrane are within boxes.

30 Figure 17: Multiple amino acid sequence alignment of Y5 receptors. The human Y5 receptor serves as master sequence in alignment with the pig, dog, guinea pig, rat, mouse and chicken Y5 receptors. GenBank accession numbers are pig: AF106083, human: U56079, pig: AF049328, dog: , guinea pig: AF363240, rat: U56078, mouse: AF022948 and chicken: submitted (Paper VI). In latter sequences only positions that differ from the human Y5 receptor sequence are shown while dots represent identities. Dashes indicate gaps introduced to optimize alignment. The hydrophobic segments assumed to be embedded in the cell membrane are within boxes.

31 Table 4. 32

PYY 125I PYY 125I n pNPY n pNPY BIBP3226 n BIBP3226 SR120819A n SR120819A Mutant Kd (nM) Bmax Ki (nM) Ki/KiWT Ki (nM) Ki/KiWT Ki (nM) Ki/KiWT (fmoles/mg protein) Wildtype 0.1±0.01 68±9.8 3 0.2±0.03 3 1.0 7.5±0.9 3 1.0 4.2±0.2 3 1.0 A90T 0.1±0.2 2 0,6 7.0±0.9 2 0.9 2.3±0.3 3 0,5 N116A 0,18 800 2 0.04±0.01 3 0,2 34±7.4 3 4,5 55±7 3 13 R138K 0.03±0.01 3 0,1 11±4.0 3 1,5 2.9±0.2 3 0,7 V167T 0.09±0.01 3 0,4 10±1.7 3 1,3 5.8±1 3 1,4 F173A 0,15 34 2 0,07 2 0,3 nbd nbd D194A 0.4±0.2 2 2 11±0.7 3 1,5 4.3±0.3 3 1.0 D200A 0,18 86±19 3 1.3±0.5 2 6 27±0.7 3 3,6 3.9±0.3 3 0,9 T212A 0,27 31 1 0.4± 2 2,2 86±0.5 2 20 T213A 4.4±0 2 1 L214T 0.1±0.01 2 0,5 6.6±0.6 3 0,9 3.5±0.4 3 0,8 Q219A >0.4 nbd nbd nbd W276A 0.5±0.07 3 2,2 280±14 3 37 42±2.3 3 10 F286E >0.4 2,8 2 13 150±60 3 20 nbd D287A >0.4 nbd nbd nbd Figure 16: Helical wheel model showing the TM regions. Note that TM helices 1, 3, 5, and 7 go into the plane of the paper in a clockwise fashion whereas the remaining TM regions come out of the plane of the paper in the counterclockwise direction. Mutated positions in the TM regions are highlighted. The loops have been removed for clarity since their structures are highly flexible.

33 Cloning and pharmacological characterization of the chicken Y1, Y2 and Y5 subtypes (Papers V and VI) Genes for the Y1, Y2 and Y5 subtypes were cloned from chicken by various strategies such as screening of a chicken genomic BAC library and degenerate PCR. The phylogenetic tree shown in Fig. 18 was calculated with PAUP*4.04 using maximum parsimony and confirms that these chicken receptors are orthologues of the mammalian Y1, Y2 and Y5 receptor subtypes, respectively. Sequence alignments for Y1, Y2 and Y5 receptors are shown in Figs. 4, 5, 17. The cloned chY1, including the endogenous intron, the chY2 and chY5 receptor were expressed separately in the mammalian cell line HEK 293 EBNA-1. Binding studies in vitro were performed using 125I-pPYY as radioligand. Untransfected cells gave no specific binding. Second messenger signalling was shown to involve the inhibition of cAMP production for chY2. The rank order of potencies for the tested agonists chicken (ch) PYY, pNPY, pNPY2-36, NPY18-36 and p[Leu31, Pro34]NPY) was the same as that of affinities. No inhibition of cAMP production was detected for the chY1 and Y5 receptors. The reasons for this could be the non-homologous expression system. Similarly, no inhibition of cAMP production was detected for the gpY1 receptor as described above. In situ hybridization with antisense oligodeoxynucleotide probes of chicken Y1, Y2 and Y5 receptors revealed expression in the infundibular nucleus of the hypothalamus. The infundibular nucleus is the avian correlate to mammalian arcuate hypothalamic nucleus. Y2 expression was detected in the hippocampus, a site where Y2 is abundant also in mammals. Competition at both sites with an excess of unlabeled probe abolished the signal. The open reading frame of chY1 was 385 amino acids and the four extracellular cysteines in the Y1 receptor probably form two disulfide bonds. There is also one cysteine in the carboxy terminal tail that presumably serves as an anchor to the membrane through palmitoylation. Close to the amino terminus of each receptor there are putative glycosylation sites. In accordance with other receptor genes, the chY1 nucleotide sequence has an intron between the codons of amino acids K233 and I234. The chY1 full-length receptor displays 80-83% amino acid identity to mammalian receptors. Comparing only

34 the putative TM regions of the Y1 receptors gives 90-92% amino acid identity to mammalian receptors as calculated by the DNASTAR Software. Saturation binding assays of the Y1 receptor gave a Kd of 32 ± 7.5 pM (S.E.M., n=3), and a Bmax of 608 ± 201 fmoles/mg protein (S.E.M., n=3). The rank order of affinities of the ligands for the chY1 receptor was; pNPY ≥ p[Leu31,Pro34]NPY > chPYY > BIBP3226 ≥ pNPY(2-36) ≥ pNPY(3-36) ≥ pNPY(13-36) >> SR120819A, BIIE0246, chPP, hPP, rPP, p[D- Trp32]NPY, CGP71863A. Inhibition constants are shown in Table 2. The open reading frame of the chicken Y2 receptor encodes a protein of 385 amino acids. A cysteine pair can be formed between EL1 and EL2 and a cysteine in the carboxy- terminal could anchor the receptor to the cell membrane. The full-length receptor has 75- 79 % identity to mammalian orthologues and only 23-26 % identity to mammalian Y1, Y4, Y5 and y6 subtypes. Saturation binding assays of the Y2 receptor gave a Kd of 25.6 ± 1.7 pM (S.E.M., n=3) and a Bmax of 303 ± 68 fmoles/mg protein (S.E.M., n=3). The rank order of affinities was; chPYY, pNPY, pNPY2-36, pNPY3-36 > pNPY13-36 > p[Leu31, Pro34]NPY > pNPY18-36 > p[D-Trp32]NPY, BIIE0246 > rPP, hPP, chPP, BIBP3226, SR120819A. Inhibition constants are shown in Table 2. The open reading frame of the chicken Y5 receptor gene coded for 443 amino acids. One cysteine pair presumably links EL1 and EL2 of the Y5 receptor and two cysteines in the carboxy-terminus could presumably serve as anchors to the membrane through palmitoylation. The full-length chY5 displays 64-72% amino acid identity to mammalian receptors, but the TM regions display 85-86% amino acid identity to mammalian receptors. Saturation assays of the Y5 receptor gave a Kd of 113 ± 10.1 pM (S.E.M., n=3), and a Bmax of 18 1 ± 5.5 fmoles/mg protein (S.E.M., n=3). The rank order of affinities were; p[Leu31,Pro34]NPY, pNPY, CGP71863A ≥ pNPY(3-36), pNPY(2-36) ≥ hPP ≥ chPYY ≥ p[D- Trp32]NPY ≥ rPP > pNPY(18-36) ≥ chPP >> BIBP3226, SR120819A. Inhibition constants are shown in Table 2.

35 DISCUSSION

Guinea pig Y1 and Y2 receptors (Papers I and II) The guinea pig Y1 and Y2 receptor genes were cloned as part of the goal to provide full information on amino acid conservation as well as pharmacological features of all NPY receptor subtypes in this species. Both of the receptors were highly conserved as compared to their mammalian orthologues. The guinea pig Y1 receptor had been proposed to differ from the human and rat orthologues with respect to pharmacological profile (Grundemar and Ekelund 1996; D. R. C. Gehlert and Gackenheimer 1997) and anatomical distribution (D. R. C. Gehlert and Gackenheimer 1997). Cloning of the guinea pig Y1 receptor revealed a very high degree of sequence similarity to other mammalian subtypes. Also, the receptor pharmacology was that of a typical Y1 receptor, as was shown by binding studies performed in parallel on the rat receptor. No inhibition of cAMP production could be detected when the receptor was expressed in vitro. Nevertheless, changes in cell metabolism were observed when measuring extracellular acidification rates upon agonist stimulation. The two cell clones that were chosen for further studies both seem to signal through pathways other than Gi activation. Y1 receptors have been shown to couple via different intracellular signalling systems depending on cell type (Herzog, et al. 1992). Cloning and pharmacological characterization of the guinea pig Y2 receptor revealed a highly conserved Y2 receptor with a typical binding profile, similar to that of human and rat Y2. The guinea pig caval vein contractile response to amino-terminally truncated NPY, which can be blocked by BIBP3226 (Grundemar and Ekelund 1996), seems therefore to be mediated by other receptors than both the cloned Y1 and Y2. Alternatively, a tissue specific receptor binding profile is caused by associated receptor modifying proteins or the specific G-protein repertoire of NPY receptor expressing caval vein cells. The guinea pig Y4, Y5 and y6 receptors have also been cloned and pharmacologically characterized (Eriksson, et al. 1998; Starback, et al. 2000; Lundell, et al. 2001). They were all shown to be very similar in sequence and binding profile to their human orthologues. The guinea pig Y4 receptor is indeed more similar to the human Y4 than the rat Y4. This could be explained by co-evolution of the Y4 receptor with PP which 36 is more divergent between rats and humans than between guinea pigs and humans (Eriksson, et al. 1998). Taken together, the strong similarities in sequence and pharmacology of the Y1, Y2, Y4 and Y5 receptors indicate that guinea pigs can be used for studies of the NPY system with regard to physiology and pharmacology. Food intake studies in guinea pigs confirm roles for Y1 and Y5 in stimulating food consumption (A. Lecklin and D. Larhammar, personal communication).

Distribution of Y5 receptor mRNA in guinea pig brain (Paper III) Guinea pig brain sections were hybridized with Y5 specific 35S-labeled oligonucleotide probes. The distribution of Y5 mRNA was mapped to regions described in a rat brain atlas (Paxinos and Watson 1997). The results reported in Paper III differed substantially from those of the only published in situ hybridization study mapping Y5 distribution in mouse (Naveilhan, et al. 1998), where very little or no expression was reported throughout the brain. Results in Paper III were more consistent with mapping of brain Y5 mRNA by in situ hybridization in human and rat, where moderate to high Y5 mRNA expression levels were noted in hypothalamic and hippocampal areas (Nichol, et al. 1999; R. M. Parker and Herzog 1999; Durkin, et al. 2000). In the hypothalamus, results in Paper III generally agreed with previous studies in rats, which reported strong to moderate labeling of the paraventricular hypothalamic nucleus (Pa), the SCh and the supraoptic nucleus (SO). Strong Y5 mRNA signals was observed in the guinea pig Arc, in agreement with two studies (Nichol, et al. 1999; R. M. Parker and Herzog 1999) but in contrast to a third study in rats (Durkin, et al. 2000). Moderate labeling of Arc in humans (Jacques, et al. 1997; Nichol, et al. 1999) and a weak labeling in mice have also been reported (Naveilhan, et al. 1998; Broberger, et al. 1999; Nichol, et al. 1999). According to (Dumont, et al. 1998b) and (Statnick, et al. 1998), hypothalamic areas mostly lack Y5 protein while high Y5 mRNA levels have been reported in the Pa and Arc of rat and human (Gerald, et al. 1996; Dumont, et al. 1998a; Dumont, et al. 1998b; Statnick, et al. 1998; Nichol, et al. 1999; R. M. Parker and Herzog 1999). A similar disagreement has been noted in guinea pigs, where Dumont et al. reported low Y5-like binding in the guinea pig hypothalamus, while Paper III describes high mRNA levels in specific hypothalamic

37 nuclei such as the Pa, Arc, DM and VMH. Dumont et al. and Statnick et al. have listed a few reasons, such as that all available Y5 binding sites may not be detected by the receptor autoradiography, the Y5 receptor may be under strict post-transcriptional control, or that Y5 receptor protein is transported to sites distal from those of transcription (Dumont, et al. 1998b; Statnick, et al. 1998). Nevertheless, a study by (Widdowson, et al. 1997) does identify Y5-like binding in the hippocampus, amygdala, hypothalamus and some thalamic structures in rats. Widdowson et al. identified sites as Y5-like if 125I-PYY3-36 binding was detected in competition with BIBP3226, and was not detected in competition with either [Leu31, Pro34]NPY or human PP. Statnick et al. used competition with various ligands, and determined that the binding profile of human hypothalamus homogenates is not Y5-like. Dumont et al. identified [Leu31, Pro34]PYY sites that were insensitive to BIBP3226 as Y5- like. A recent study reports high levels of Y5 receptor immunoreactivity in somal membranes, cellular compartments and in short fiber processes throughout the hypothalamus. The authors discuss the possibility that Y5 protein is stored intracellularly, and can translocate to the cell surface under certain physiological conditions (Campbell, et al. 2001). One could also speculate that the Y5 receptor has a high turn-over rate and that the immunoreactivity represents internalized receptors. Another reason for the discrepancy could be that the Y5 receptor exhibits different binding properties in the hypothalamus than in cultured cells. In the hippocampus, the distribution of Y5 mRNA observed in Paper III differed from previous reports in rats and human. Paper III describes weaker labeling of CA1 than of CA3 and the dentate gyrus (DG), both of which were very strongly labeled, as was also observed in rats (Nichol, et al. 1999). Moderate labeling in both CA1 and CA3 and a somewhat stronger labeling in the DG was observed by (Durkin, et al. 2000), while (R. M. Parker and Herzog 1999) reported strong labeling in each of these areas in rats. Although the estimates of relative intensities differ between rat studies, the overall pattern of hippocampal expression is essentially the same as that observed in guinea pigs. In the human hippocampus, however, the CA1-4 regions exhibited low to moderate expression levels (Nichol, et al. 1999).

38 In the amygdala, Y5 receptor mRNA has been observed in rats and humans (Nichol, et al. 1999; R. M. Parker and Herzog 1999). Similarly in guinea pigs, labeling of the Ce and BL as well as the amygdalostriatal transition area (AStr) was detected. Interestingly, the guinea pig striatum exhibited prominent Y5 mRNA expression in agreement with observations of Y5-like binding by (Dumont, et al. 1998b). These authors also reported Y5 specific binding in marmoset striatum, but not in rats, mice, vervet monkeys or humans. However, Borowsky et al. observed mRNA expression by Northern blots in the caudate putamen and nucleus of humans (Borowsky, et al. 1998). In the brain stem, the observations of Y5 expression in guinea pigs in PaperIII are in general agreement with those reported for other species. Dumont et al. noticed strong Y5 specific labeling of Sol in guinea pigs. (By competition at 125I-[Leu31, Pro34]PYY-binding sites with BIBP3226). Furthermore, Dai et al. reported the presence of NPY immunoreactivity in the dorsal motor nucleus of the vagus and, to a lesser extent, in the AP, both regions where Y5 expression was observed in the present study (Dai, et al. 1986). Parker et al. suggested that the gene organization of the Y1 and Y5 genes with overlapping promoter regions contributes to their similar expression patterns in rats (Herzog, et al. 1997; R. M. Parker and Herzog 1999). Future research will investigate whether such an overlap is also to be found in guinea pigs. Mice seem to differ from other mammals studied so far with respect to Y5 expression. Mice also differ from rats, humans and guinea pigs in having a functional y6 receptor expressed in the hypothalamus (Burkhoff, et al. 1998; Mullins, et al. 2000). The mouse Y5 receptor might have acquired a different physiological role because of greater redundancy in the hypothalamic NPY receptor repertoire. The functional mouse y6 receptor could also be part of the explanation for the observation that Y5-deficient mice developed late-onset obesity. Although it was concluded that the mouse y6 receptor pharmacological profile is not that of a feeding receptor (Mullins, et al. 2000), these aspects suggest that results from studies of the NPY system in mice may not be representative for mammals in general. In all mammalian species examined thus far, the post-transcriptional processing of Y5 mRNA in the hypothalamus is undocumented which leaves open the possibility that observed effects on feeding by the Y5 receptor are governed by other brain regions. Yet,

39 the guinea pig Y5 receptor mRNA is expressed in many brain areas known to be important in the regulation of autonomic functions and is consistent with a role of Y5 in food intake regulation.

The human Y1 receptor model (Paper IV) The new model of hY1 proposed here is based on the recently published high-resolution bovine rhodopsin structure (Palczewski, et al. 2000). Rhodopsin has been used as a model for structure predictions of other rhodopsin-like GPCRs. A notable difference to NPY receptors is that bovine rhodopsin is activated by light which induces a conformational change in the retinal molecule which is covalently bound to the receptor. The conformational change causes the retinal molecule to dissociate from the receptor. Since NPY receptors are activated by large protein ligands, observations on ligand-binding, and probably also on the modes of receptor activation will not be directly transferable. Nevertheless, lacking high-resolution structural information for the Y1 receptor itself, the rhodopsin-model in combination with mutagenesis and computer modeling is helpful in providing theories about ligand-receptor interactions. Previous theories on the hY1 receptor structure, concluded from site-directed mutagenesis studies and other structure models of rhodopsin (based on computer-improved low-resolution structures) are not in full agreement. Binding studies were performed on some previously studied single amino acid substituted hY1 receptors, in order to test and compare the previously proposed theories . By making sequence comparisons between different Y1 receptor subtypes as well as to other NPY receptor subtypes some new and potentially interesting sites, in terms of ligand binding, were mutated. One initial hypothesis (Walker, et al. 1994) was that the basic NPY molecule made electrostatic interactions with aspartic residues in the Y1 receptor, D104 in TM2, D194 and D200 in EL2 and D287 in TM6. Later studies showed that D194 and D200 are probably not involved in ligand binding (Munch, et al. 1995; Sautel, et al. 1995; Kanno, et al. 2001), and the first results were probably due to major difficulties in expressing the hY1 receptor in high amounts. Paper IV confirms loss of binding for mutation D287A, but unaffected binding for D194A and D200A.

40 BIBP3226 binds with four-fold lower affinity to the D200A mutant than to the wildtype receptor. This is in contrast with a previous report (Sautel, et al. 1996) that observed higher affinity for this mutant. SR120819A on the other hand exhibits unaltered affinity compared to D200A. The amidated carboxy-terminus of NPY or PYY has previously been proposed to bind the receptor in a hydrophobic pocket. The ligand-interacting amino acids of the Y1 hydrophobic pocket would be Y100 in TM2, F286 in TM5 and H298 in EL3. Since NPY can bind to all functional receptors of the NPY family, though with lower affinity to Y4, those amino acids would be expected to be similar in all receptors. A sequence comparison makes it clear that no such similarity can be observed. F286 in the outer part of TM6 (Fig. 4) is one of the most variable of those amino acids. In all known Y4 receptors a glutamic acid residue is present in this position, so therefore the mutant F286E was studied in Paper IV. The result of this mutation was a ten-fold reduction in affinity for NPY. BIBP3226 exhibited a 25-fold drop in affinity. A previous study by (Sautel, et al. 1996) demonstrated a five-fold drop in affinity to BIBP3226 of the mutant F286A. Also, SR120819A lost its affinity for F286E. Conflicting results have been reported for the mutation F173A. Sautel et al. demonstrate total loss of affinity by NPY and BIBP3226, while Kanno et al. showed only a 2.5-fold loss of affinity for PYY. The corresponding residue in Y2 and Y5 is leucine, which also indicates a less important role of F173. Results from Paper IV show no significant change compared to wt in peptide binding, while a complete loss of affinity for both antagonists was noticed. Mutations of two polar residues on the outer part of TM5 disrupted binding of peptide ligand and antagonists. SR120819A displayed a lowered affinity to T212A, peptide ligand and SR120819A bound with lowered affinity to Q219A as reported earlier (Sautel, et al. 1996). A previous study showed intact binding of two different antagonists to Q219A (Kanno, et al. 2001). Corresponding residues to R138 and W276 are highly conserved among rhodopsin- like receptors and both of those seem involved in the transition between active and inactive receptor conformations (Marie, et al. 2001). In line with those observations, the residues do not seem to be involved in any direct interaction with endogenous peptides of

41 the hY1 receptor. Antagonists, on the other hand, exhibited lowered affinity to the mutated receptors. An effect of W276 on BIBP3226 binding has previously been reported (Du, et al. 1997). Interactions with antagonists at residues involved in receptor activation, thereby stabilizing the inactive receptor conformation seems likely. Signal transduction experiments will be necessary to determine whether those residues are involved in receptor activation. The two antagonists also had lowered affinity to the mutant N116A. The model of hY1 presented in Paper IV with TM3 located diagonally across the inner side of TM4 would be compatible with a direct interaction of BIBP3226 and TM3. Docking experiments with BIBP3226 and Y1 are currently being performed.

Chicken Y1, Y2 and Y5 receptors (Papers V and VI) The chicken Y1, Y2 and Y5 receptor genes were cloned and pharmacologically characterized. Interestingly, the Y2 receptor exhibited high affinity for [Leu31, Pro34]NPY, used in many previous studies to detect Y1-like binding. Chicken PYY that has an extra alanine at the amino-terminus binds the Y1 receptor with lower affinity than pNPY and pPYY. The extra amino acid does not affect chPYY binding to the Y2 receptor. Thus, the chY1 receptor probably interacts with the aminoterminus of peptide ligands similarly to Y1 in mammals. This might be of importance if PYY competes with NPY in vivo. However, NPY is mainly expressed in the brain while PYY is presumably more abundant than NPY in the GI tract, at least in mammals. In analogy with rat Y5, endogenous PP does not exhibit a very high affinity to the chicken Y5 receptor. Nevertheless, the high degree of conservation of all these three receptor subtypes is more striking than the differences, both in amino acid sequence (Figs. 4, 5, 17) and binding profile, again indicating an important role of NPY signalling. The Y1 antagonist BIBP3226 and also the Y5 antagonist CGP71863A recognize the respective chicken receptors, but the Y1 antagonist SR120819A does not. The expression of Y1 and Y5 in the avian correlate of mammalian Arc, a site strongly implicated in feeding regulation, is indicative of a conserved physiological role of those receptors. The finding of high levels of Y2 expression in chicken hippocampus correlated well with earlier observations in mammals. A study of food intake in broiler

42 chicks (Ando, et al. 2001) reports that injections into the third brain ventricle of mammalian [Leu31, Pro34]NPY, NPY, PYY, and PP elicited feeding. [D-Trp32]NPY induced a delayed feeding response. NPY13-36 injections resulted in a short-lasting and weak feeding response, implying that Y2 is not important for feeding in chickens. Both human and rat PP bind to the chY5 receptor with high affinities, as does [Leu31, Pro34]NPY. It therefore appears likely that Y5 is involved in food intake regulation in chickens but further studies are needed to clarify which subtype is involved in feeding. Possibly, the NPY receptor representation in broilers is different from leghorn chickens (Merckaert and Vandesande 1996). The situation is further complicated by the fact that chicken Y4 can serve as a receptor for both NPY, PYY and PP and is expressed in the brain stem (I. Lundell, T. Boswell, D. Larhammar, personal communication). Also, y6 seems to be functional in chickens, the expression pattern and binding profile of which are currently being investigated (R. Fredriksson, E. Salaneck, T. Boswell, and D. Larhammar, personal communication). The phylogenetic tree (Fig. 18) clusters chicken receptors with their mammalian orthologues. Also, the chicken receptors have been localized to chicken chromosome 4q by fluorescent in situ hybridization to metaphase chromosomes (B. Chowdhary, S. K. S. Holmberg, S. Mikko, and D. Larhammar, unpublished observations). Part of chicken chromosome 4 has been shown to be homologous to human chromosome 4. The existence of all three NPY receptor genes on 4q both in humans and chickens is further evidence that they are homologous. This hypothesis is corroborated by the localization of 40% (12/30) of the currently mapped genes to 4q both in humans and chickens (including the three NPY receptor genes = 50%): ALB (albumin); ANXA5 (AnnexinV or endonexin II);CENPC (centromere protein c); CLOCK; FGB ( B beta polypeptide); GC (Group-specific component Vit D binding protein); IL2 (Interleukin 2); IL8 (Interleukin 8); IRF2 (Interferon regulatory factor-2); SPP1 ( or secreted phosphoprotein 1); and TLR2. (Information about genes localized to chromosome 4 of chickens was taken from ChickGBASE from Roslin Institute, Edinburgh, UK. Available from Internet: URL http:// www.ri.bbsrc.ac.uk/ chickmap/ gbase/ chickgbase.html. Comparisons with human genes were made using GDB™ Human Genome Database (database online). Toronto (Ontario,

43 Canada): The Hospital for Sick Children, Baltimore (Maryland, USA): Johns Hopkins University, 1990-. Updated daily. Available from Internet: URL http://www.gdb.org/).

44 Figure 18: Results of a maximum parsimony analysis of the TM region amino acid sequences for Y1, Y2, Y5, Y4, and y6 receptors. Branch-lengths are proportional to number of evolutionary changes. The Lymnaea stagnalis NPY receptor was used as an outgroup. GenBank accession numbers: For Y1, Y2 and Y5 NPY-receptors see figure legends Fig. 4, 5 and 17, human Y4: Z66526, mouse y6: NM_010935, Lymnaea stagnalis: CAA57620.

Species comparisons of the Y1 receptor may help identify antagonist interaction sites (Papers IV and VI) Since all members of the NPY receptor family bind NPY and PYY, sequence comparisons have proven to be valuable tools for better predicting which amino acids are interacting with the endogenous ligands. Studying sequence differences within one receptor subtype can also prove valuable. As observed both in Paper IV and Paper VI, BIBP3226 and SR120819A seem to interact with the Y1 receptor in different ways. The pharmacological profile of the cloned chY1 receptor suggested some amino acids that could be involved in the differential affinities of BIBP3226 and SR120819A to chY1. Three sites of the human Y1 receptor were mutated, A90T in TM2, V167T in TM4 and L214T in TM5, but none of these confers any changes on their own in antagonist affinity (Paper IV). Further studies along these lines are currently being performed.

45 CONCLUSIONS k Guinea pigs can be used as an informative complementary research animal to better extrapolate animal studies of the NPY system to humans. (Papers I-III) k Guinea pig Y5 receptor expression was observed in brain areas implicated in feeding and reproduction, such as the hypothalamus, amygdala, preoptic tract and brain stem. Y5 expression is conserved between humans, rats and guinea pigs in the hypothalamus and amygdala while other regions of the brain exhibit more species differences. (Paper III) k New possible sites of interaction with the antagonist BIPB3226 in TM3 and TM5 were identified in the human Y1 receptor. This may help explain the compound's antagonistic effect and may provide useful insights for future development of antagonists with improved pharmacological properties. Only two, at the most, of the four aspartic acid residues previously proposed may be involved in peptide binding. Furthermore, the hydrophobic pocket proposed to interact with the amidated carboxy-terminus of NPY must be questioned, but more studies are required to confirm how these residues may interact with the peptide ligand. (Paper IV) k The peptide analog [Leu31, Pro34]NPY, traditionally used to distinguish Y1-like receptor binding from Y2 binds to the chicken Y2 receptor with high affinity. The chicken Y1 and Y5 receptors displayed a similar binding profile to endogenous ligands and their mammalian orthologues. Chicken PYY, with an extra alanine in the amino-terminus, binds to Y1 with lower affinity than to Y2, indicating that the structural requirements of the Y1 receptor are similar to mammals. The antagonists BIBP3226 and CGP71863A developed in mammals bind to chicken Y1 and Y5, respectively. (Paper V and VI) k Sequence comparisons among NPY receptors proved useful for identifying putative sites of ligand interaction. (Paper IV) 46 FUTURE PERSPECTIVES

Feeding studies in guinea pigs are being conducted, aided by the information on receptor binding properties reported in this thesis. As all NPY-family receptors have now been cloned in this species, it would also be interesting to explore other aspects of physiology. Studies of the effects of Y4 receptor activation would be of special interest, since guinea pig Y4 is more similar to the human than to rat and mouse Y4 receptors. More detailed studies of NPY receptor distributions will also help attributing specific functions to each subtype. Inter-species comparisons of pharmacological studies in vivo will be useful to better extrapolate ideas about how the NPY system might function in humans. Feeding studies in chickens have already been performed by other groups, and their work will hopefully be continued, now with higher precision facilitated by the cloning of all NPY receptor subtypes. Distribution studies of NPY receptors in chickens have been initiated (T. Boswell, personal communication). Some receptor antagonists were found to bind the chicken receptors and can be used in functional studies. Investigations of the conservation of receptor function between birds and mammals can give insights about the evolution of NPY family receptors. Mutagenesis studies of cloned receptor subtypes will continue with the improved expression system described in this thesis. Of great interest will be to uncover what structural features distinguish the binding profiles of different NPY receptor subtypes. Studies on intra-cellular signalling could help us understand what determines the coupling to different pathways. It is also desirable to study whether a hierarchical relationship exists among different pathways, if receptors dimerize upon activation, how receptors cooperate with and influence the signalling of other GPCRs, receptor kinases and ion-channels. Some of the outlined ideas are natural immediate proceedings that are already in the making. Other studies suggested here will require more time and development of new techniques, some in the form of collaborations. Also, the field will uncover additional hypotheses , moving the focus of research in new directions. For better or worse, a lot remains to be seen in the field of receptor pharmacology, a challenging task that will continue to surprise us as we go along. 47 ACKNOWLEDGEMENTS

The work underlying this thesis was carried out at the Department of Neuroscience, Uppsala University, Uppsala, Sweden. The support from my colleagues, friends and family was critical for finalizing my projects. My warmest gratitudes to everyone.2

I would especially like to acknowledge:

Dan Larhammar for kind and knowledgeable mentorship. Many thanks for allowing me the flexibility and mobility I have enjoyed during my student years. Many thanks to Helen Larhammar for the wonderful dinners you and Dan treated the lab-group to. I especially remember the parfait with marzipan. (Yum! Even beat Dan's "lab-cleaning-cake" but I will miss both.) Lars Oreland for being a perfect Prefect. My co-authors for all essential and valuable help with the projects.

All past and present members at the Unit of Pharmacology with whom I had the pleasure to share lab-facilities, seminars, coffee breaks and great lab-parties. Many thanks to all of you who willingly shared my many troubles and rare successes. You provided inspiring discussions and great company over the years. Special thanks to Christina B. not only for patiently providing her skilled technical assistance, but also for all the fun times we had working out, going to movies and pubs. Paula Sj. many thanks for working so tremendously and outstandingly at the end of the mutagenesis project! Robert many thanks for helping me trouble-shooting in the lab and servicing my computer in times of despair. Thanks for helping me finalize the mutagenesis project and for help with spikning of my thesis. It was great fun to discuss science with you, preferably over beers. Magnus many thanks for teaching me in vitro pharmacology and helping out with critical reading and comments on manuscripts. Also thanks for helping me keep track of your brother. Spain again? Aha. Sofia for great help with setting up Real-time PCR experiments and fun parties. Also, thanks for letting James borrow your tuxedo - I'll return the shirt at some point soon! Parul for your energetic and inspiring nature. I remember when you stayed in the lab all night willingly trouble- shooting techniques for me. Henrik thank you for mentoring me during my masters project. Mårten for being an excellent project student working on the mutagenesis project and for helping us purify primers. Martin for helping out with the cloning of chicken receptors as a project student. Erik for helping me as a newcomer in the lab. Nina Mohell many thanks for your outstanding expertise on receptor binding. Christina A. for good advise and chats in the cell lab.Lotta G. for inspiring me and advising me, especially as a newcomer in the lab. Also, thanks for the great time I had visiting you in Copenhagen. Ulla for assisting with administration and many helpful advise. Ingrid many thanks for teaching me in vitro pharmacology. Also, thanks for all the time and heart you put in to arranging outings for the lab, dinners in your home etc. Helgi for helpful advice on receptor pharmacology. Paula St. - I enjoyed the theater performances! Lotta S. for help and assistance when I was new in the lab. Thanks also for great company at the Neuroscience meetings. Amanda many thanks for your everlasting patience in providing great scientific and technical advise, even more thanks for the great beer-parties and dinners you arranged on your scarce spare time. Thanks to Aneta, Torun, Malin, Maria R. and Karin for patiently sharing my office and for your cheerful company and helpfulness in the lab Girl-Power! I'll miss you! Thanks to Earl for the

2 I apologize for any mis-spellings of names. Also, it is very possible that I failed to mention everyone by name who deserved it. My apologies for that as well. 48 CDs and for keeping the lab international and to Anne for being interested in guinea pigs - great that we'll stay colleagues in Gainesville.

Allan Johnson and Lillemor Källström for invaluable guidance and technical assistance with in situ hybridizations at a time when I needed it the most. Tomas Hökfelt, Kristina Holmberg and Jutta Kopp many thanks for your help and advise on in situ hybridizations. Timothy Boswell for hosting me during my stay in Edinburgh. Thanks also to your colleagues for the good time I enjoyed during my stay. I look forward to revisiting your beautiful corner of the world. Pushpa Kalra, Satya Kalra and all past and present members of their labgroups who have given me such a warm welcome to my new work-environment. Karl Åkerman and his labgroup for great help with second messenger assays. Anita Morénand Anders Kallin at the Ludwig institute for help with FLAG-epitope and Quickchange protocols. Anna Törnsten for teaching me chicken fiber FISH. Anja Castensson for helping me with Primer Express. Finn Hallböök and the past and present members of your labgroup for good advise and for letting me borrow reagents and materials. Peter Ekblom and his lab group for letting me use your microscope. Tina Kunz for trouble-shooting in situ hybridizations with me and for helping me with CoolPix. Rolf Larsson, Sara Ekelund and Lena Lenhammar for helping me use their Cytosensor, and Yamashita-sensei and Junko Imaki for hosting me during my stay in Tokyo and for introducing me to in situ hybridization techniques. Thanks also to your colleagues and students for their kind help and assistance during my stay.

Everyone I got to know in orchestras and spelmanslag - thanks for all the good times shared with you!

My dear friends who provided relaxation and good times outside the lab. Yes - it was possible. How??? What did we talk about? What did we do? I know for a fact that it wasn't always science! Why did we go for vacation when I could have been home working? Why did we rent a movie when I could have been working? Why did you invite me over for dinner when I could have picked up a pizza at BBB? I really don't know, but I suspect the reason was that you are the best friends one could ever ask for. Special thanks to you who opened your homes for me after I had moved to Gainesville: Malin, Tobbe, Anders, Janne and Gosia. To all of you - ingen nämnd och ingen glömd - många kramar, många minnen.

My beloved family: James, Sven-Åke, Ing-Marie, Beata, Stefan I keep you all very close to my heart. No need to say how much you mean to me - you know it. Kära familjen - jag behöver inte säga vad ni betyder för mig, ni vet.

hhh

49 REFERENCES

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