Evolution and Pharmacology of Receptors for Bradykinin and Neuropeptide Y in Vertebrates

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(205) LIST OF PUBLICATIONS. I. Dunér† T, Conlon JM, Kukkonen JP, Akerman KE, Yan YL, Postlethwait JH, Larhammar D. Cloning, structural characterization and functional expression of a zebrafish bradykinin B2-related receptor. Biochem J, 2002. 364(Pt 3): p. 817-24. † has been changed to Bromée. II. Bromée T, Kukkonen JP, Andersson P, Conlon JM, Larhammar D. Pharmacological characterization of ligand-receptor interactions at the zebrafish bradykinin receptor. Br J Pharmacol, 2005. 144(1): p. 11-6. III. Bromée T, Venkatesh B, Brenner S, Postlethwait JH, Yan YL, Larhammar, D. Uneven evolutionary rates of bradykinin B1 and B2 receptors in vertebrate lineages. Submitted. IV. Bromée T*, Sjödin P*, Fredriksson R, Boswell T, Larsson TA, Salaneck E, Zoorob R, Mohell N, Larhammar D. Neuropeptide Y-family receptors Y6 and Y7 in chicken: Cloning, pharmacological characterization, tissue distribution and conserved synteny with human chromosome region. Submitted. * contributed equally to this paper..

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(207) CONTENTS. INTRODUCTION ........................................................................................11 G-protein coupled receptors .....................................................................11 G-proteins and their effectors...................................................................12 The adenylyl cyclase (AC) system ......................................................13 The phospholipase C (PLC) system ....................................................13 Structure and formation of kinins.............................................................14 Bradykinin receptors ................................................................................17 The B1 receptor ...................................................................................17 The B2 receptor ...................................................................................18 Evolution of the kinin system...................................................................18 Peptides of the NPY family......................................................................19 NPY .....................................................................................................20 PYY .....................................................................................................20 PP.........................................................................................................20 NPY receptors ..........................................................................................21 The Y1 subfamily ................................................................................21 The Y2 subfamily ................................................................................22 The Y5 subfamily ................................................................................22 Evolution of the NPY system...................................................................23 AIMS OF STUDY ........................................................................................25 MATERIALS AND METHODS..................................................................26 Identification and isolation of new receptor genes...................................26 Expression vector constructs and transfection .........................................27 Measurements of intracellular calcium release ........................................27 Determination of inositol phosphate formation........................................28 Binding assays..........................................................................................28 Genetic mapping ......................................................................................29 RT-PCR....................................................................................................29 RESULTS AND DISCUSSION ...................................................................30 Paper I. Cloning, structural characterization and functional expression of a zebrafish bradykinin B2-related receptor. .........................................30 Paper II. Pharmacological characterization of ligand-receptor interactions at the zebrafish bradykinin receptor. ........................................................32.

(208) Paper III. Uneven evolutionary rates of bradykinin B1 and B2 receptors in vertebrate lineages................................................................................34 Paper IV. Neuropeptide Y-family receptors Y6 and Y7 in chicken: Cloning, pharmacological characterization, tissue distribution and conserved synteny with human chromosome region................................35 SUMMARY AND CONCLUSIONS ...........................................................38 FUTURE PERSPECTIVES..........................................................................39 ACKNOWLEDGEMENTS..........................................................................40 REFERENCES .............................................................................................42.

(209) ABBREVIATIONS. AC Ala Arg B1 B2 BAC BK c cAMP CHO CNS DAG Dre EC ER GDP Gga GI Gly GPCR G-protein GTP HEK HMW Hsa IP IP3 kb Kd Ki Leu LMW Lys MAPK mRNA NF-N%. adenylyl cyclase alanine arginine bradykinin receptor type 1 bradykinin receptor type 2 bacterial artificial chromosome bradykinin chicken cyclic adenosine monophosphate Chinese hamster ovary central nervous system diacylglycerol Danio rerio effective concentration endoplasmic reticulum guanosine diphosphate Gallus gallus gastrointestinal glycine G-protein coupled receptor guanine nucleotide binding protein guanosine triphosphate human embryonic kidney high molecular weight Homo sapiens inositol phosphate inositol-1,4,5-trisphosphate kilo base dissociation constant inhibition constant leucine low molecular weight lysine mitogen activated protein kinase messenger RNA nuclear factor N%.

(210) nM NPY p PCR Phe PIP2 PKC PLC PP Pro PYY RT Ser TES Thr TM Tni Trp Tru Tyr. nanomolar neuropeptide tyrosine porcine polymerase chain reaction phenylalanine phosphatidylinositol 4,5bisphosphate protein kinase C phospholipase C pancreatic polypeptide proline peptide tyrosine tyrosine reverse transcriptase serine 2-([2-hydroxy1,1bis(hydroxymethyl)ethyl]amino)et hane sulfonic acid threonine transmembrane Tetraodon nigroviridis tryptophan Takifugu rubripes tyrosine.

(211) INTRODUCTION. Biological sciences in the modern times are based on the principle that all life processes have a physical and chemical basis and that all organisms and their characteristics are products of evolution (Futuyma, 1998). Therefore, evolution provides us an important tool for understanding how many biological processes and proteins function now and how they came to be the way they are. The evolutionary approach is particularly useful for studies of complex systems such as neuronal and endocrine cell communication, involving multiple neurotransmitters, hormones and receptors. This thesis aims to describe the structure and cellular signaling of two types of proteins, the bradykinin receptors and the neuropeptide Y receptors, belonging to the superfamily of G-protein coupled receptors. This is done with a comparative evolutionary approach using model animals representing different lineages in vertebrate evolution. The results provide information about what positions in the receptors and their ligands that have been conserved during the evolutionary processes and aim to elucidate the evolution of these complex peptide-receptor systems. This work also aims to investigate how these receptors behave regarding ligand-receptor interactions and intracellular signaling. The results will provide information that may be useful in the development of pharmaceuticals acting on these types of receptors.. G-protein coupled receptors Complex organisms have evolved thanks to the ability of different cell types, tissues and organs to communicate with each other, leading to coordination of the activities of different parts of the organism. This is facilitated by specific receptors signaling across biological membranes. G-protein coupled receptors (GPCR) constitute the largest family of cell surface proteins involved in such signaling, hence representing the initial stage in a cascade of events leading to a biological response. The GPCRs constitute one of the largest gene superfamilies of the human genome (Civelli et al., 2001) and they are encoded by approximately 950 genes, or 2% of the total number of genes in the human genome (Bockaert and Pin, 1999; Fredriksson et al., 2003; Lander et al., 2001; Takeda et al., 2002; Venter et al., 2001). The ligand repertoire for GPCRs includes stimuli such as light, odorants, ions, 11.

(212) lipids, hormones and peptides, for a review see (Bockaert and Pin, 1999). Despite a broad variation in biological responses, the GPCRs share common structures and motifs including seven-D-helix transmembrane regions (7 TM) comprised of 20-25 amino acids linked by intracellular and extracellular loops and with an extracellular amino terminus (N-terminus) and an intracellular carboxy terminus (C-terminus). Many of the basic structural features shared among the GPCRs have been studied by electron microscopy and X-ray diffraction and are interpreted or confirmed from the crystal structure of the bovine rhodopsin receptor (Palczewski et al., 2000; Schertler et al., 1993; Unger et al., 1997). Upon ligand binding the receptor undergoes a conformational change, believed to be induced by the rotation of TM6 away from TM3 (Gether et al., 1997), and further activation and signaling via a heterotrimeric guanylyl nucleotide-binding protein (G-protein) on the cytosolic side of the membrane (Okada et al., 2001; Wess, 1998). The C-terminal part of the receptor contains certain motifs involved in receptor signaling and internalization (Hukovic et al., 1998). The second and third intracellular loops are involved in the selection and activation of G-proteins (Parent et al., 1996; Wong, 2003). Recently, other signaling pathways than via G-proteins have been reported for GPCRs (Heuss and Gerber, 2000; Pierce et al., 2002). In 1994, Kolakowski grouped the members of the GPCR superfamily into six separate families where the members within each family showed at least 20% amino acid identity between their TM regions (Kolakowski, 1994). In the later years additional families have been identified (Fredriksson et al., 2003). Several human diseases involve a dysfunction in receptors belonging to the superfamily of GPCRs. The GPCRs form the largest class of therapeutic targets (Ma and Zemmel, 2002; Sadee et al., 2001; Wise et al., 2002) and approximately 45% of the prescribed drugs on the market act on these receptors (Drews, 2000; Hopkins and Groom, 2002; Wise et al., 2002).. G-proteins and their effectors The G-proteins act as a link between the receptor and its effectors and are membrane proteins interacting with the guanine nucleotides GTP and GDP. The G-proteins are composed of the three subunits D, E and J. At least 20 Dsubunits, 6 E-subunits and 12 J-subunits are known today in mammals (Hamm, 1998). The activated receptor undergoes a conformational change and thus causes the G-protein to exchange D-subunit bound GDP with GTP. The D-GTP complex dissociates from the EJ subunit and both complexes are then free to interact with different target proteins. The G-proteins can be divided into four main families according to the similarity of the D subunit and the respective effector pathways they activate, 12.

(213) namely the GDs, GDi, GD12/13 and GDq (Cabrera-Vera et al., 2003; Hamm, 1998). GDs mainly activates adenylyl cyclase (AC) and further induces production of cAMP whereas GDi inhibits AC and hence decreases the cAMP production. GDq mainly stimulates phospholipase C (PLCE) and further the formation of IP3 and diacylglycerol (DAG) with a resultant intracellular rise in Ca2+. The GD12/13 family has only recently been identified with interaction partners such as the small GTP-binding protein Rho (Hermans, 2003; Kristiansen, 2004; Plonk et al., 1998). In recent years it has been clear that also the EJ subunit can interact with a range of effectors such as PLCE (Katz et al., 1992) and AC (Tang and Gilman, 1991). The two effector pathways experimentally investigated in this thesis is PLC (paper I and II) and AC (paper IV) and they are described below.. The adenylyl cyclase (AC) system The membrane bound enzyme AC exists in at least nine different isoforms in mammals (AC1-AC9) (Cali et al., 1994; Paterson et al., 1995; Premont et al., 1996) and contains an ATP-binding cassette. AC converts ATP to cAMP, which acts as a second messenger within the cell. The cAMP is eventually degraded by phosphodiasterases hence terminating the signal. All isoforms of AC can be stimulated by GDs (Simonds, 1999) and hence increase the production of cAMP in the cell. All isoforms except AC9 (Premont et al., 1996) are potentially activated by forskolin, an agent commonly used in experimentally studies to boost the cAMP production in the cell. AC1, AC3, AC5 and AC6 are inhibited by GDi (Chen and Iyengar, 1993; Taussig et al., 1993; Taussig et al., 1994) leading to a decrease in cAMP production. The activity of GDi on AC can be inhibited by pertussis toxin (Gilman, 1995). cAMP regulates many functions within the cell such as ion transport, cell division, enzymes involved in energy metabolism and proteins involved in contraction of smooth muscles. Most of these effects are mediated by binding of cAMP to protein kinases, specifically protein kinase A (Lander et al.). Some regulators such as protein kinase C (PKC) (Ebina et al., 1997; Jacobowitz et al., 1993) and Ca2+ and calmodulin (Choi et al., 1992; Tang et al., 1991) are believed to affect AC and hence contribute to an integrating network of signals from different receptors.. The phospholipase C (PLC) system PLC is an enzyme that can be divided into E, J, G, H and ] depending on its isoform (McCudden et al., 2005). GPCRs mainly act on PLCE, which splits phoshatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (Venter et al., 2001) and 1,4,5-trisphosphate (IP3). After the cleavage, DAG is phosphorylated to form phosphatidic acid while IP3 is dephosphorylated and joined with phosphatidic acid to form PIP2 again. Both DAG and IP3 can act as 13.

(214) second messengers. The main target for IP3 is the IP3 receptor, a ligand-gated calcium channel in the membrane of the endoplasmatic reticulum (ER). Hence, IP3 controls the intracellular levels of Ca2+. Increased Ca2+ levels trigger events like contraction, as well as secretion and enzyme activation. The main function of DAG is to activate protein kinase C (PKC), which in turn activates several intracellular proteins by phosphorylation. One group of PKC-activated proteins are the mitogen-activated protein kinases (MAPK) which in turn control gene expression, cell proliferation and differentiation, for a review see (Lopez-Ilasaca, 1998).. Structure and formation of kinins The kinins are comprised of bradykinin and natural variants of this nonapeptide. Full-length peptides include bradykinin (BK) and Lys-BK (kallidin). Active metabolites of BK include C-terminally truncated BK (des-Arg9-BK) and kallidin (des-Arg10-BK). The kinins are activated at the site of action by the cleavage of the precursor protein kininogen by a group of serine proteases named the kallikreins, for a review see (Bhoola et al., 1992). The kinins are shortlived peptides, which undergo rapid degradation by carboxy-, amino-, and endopeptidases found in biological fluids and tissues (Campbell, 2000; Kuoppala et al., 2000; Murphey et al., 2000). Human kininogen is encoded by a single gene but can be released in two different forms, high molecular weight (HMW) kininogen or low molecular weight (LMW) kininogen, which arise by alternative splicing (Farmer, 1997; Kitamura et al., 1985). The kallikreins in turn are activated from either plasma prekallikrein or tissue prekallikrein (Fig. 1). Plasma prekallikrein is activated by the interaction with the Hageman factor (factor XII) in the blood coagulation cascade, for a review see (Margolius, 1995; Margolius, 1996; Regoli et al., 1997). Bradykinin is generated by the action of plasma kallikrein on HMW kininogen and kallidin is generated by the cleavage of tissue kallikrein on LMW kininogen (Clements, 1994; Raspi, 1996) (Fig. 1). The kinins are peptide hormones and they produce the classical signs of inflammation such as redness, localized heat, swelling and pain at their site of action. The local heat and redness occur because of local endotheliumdependent vasodilation. In response to kinin activation the endothelial cells increase their permeability and induce the exudation of fluid from the blood circulation into the inflamed tissue, which results in swelling. The exposure of bradykinin receptors on pain fiber terminals produces the pain, see (Bhoola et al., 1992; Couture et al., 2001; Dray and Perkins, 1993; Geppetti, 1993; Regoli and Barabe, 1980). Today, much interest is focused on the development of stable kinin analogs for treatment of inflammatory states, 14.

(215) chronic pain, diabetes, cancer and circulatory complications (Marceau and Regoli, 2004). The effects of the kinin-kallikrein system in non-mammals are largely unknown. Bradykinin-related peptides, however, have been generated in plasma, by the addition of exogenous proteases, of species from different vertebrate taxa (Conlon, 1999) (Table 1). In teleost fish, intra-arterial injections of [Arg0, Trp5, Leu8]-BK exhibited effects on the cardiovascular system in cod (Platzack and Conlon, 1997), trout (Olson et al., 1997) and eel (Takei et al., 2001) as well as inhibition of drinking in seawater-adapted eel (Takei et al., 2001). The high conservation of the BK sequence within teleost fish suggests an important function of BK in this group.. Table 1. Structure of BK from various species as presented in Conlon et al., 1999, or as retrieved from the Ensembl database. Name of species/group 0. Residue position 4 5 6 Gly Phe Ser Thr Thr Thr Thr Thr Trp Trp Trp -. 7 Pro -. 8 Phe Leu Leu Leu Leu. 9 Arg -. -. -. Leu. -. Thr. -. Leu. -. T. rubripes a Arg Trp Thr Leu Lungfish Tyr Gly Ala Pro Gar Trp Sturgeon Met Met a as retrieved from Ensembl database. (-) indicates that residue is identical to mammals.. -. Mammals Chicken Alligator Turtle Python Rattlesnake Trout Cod Eel Zebrafish a T. nigroviridis. a. Arg Arg Arg. 1 Arg Ala Val -. 2 Pro -. 3 Pro -. Arg. -. -. -. -. Trp. Arg. -. -. -. -. Trp. 15.

(216) 16 H2 N. Kininogen molecule. Met. Lys. Arg. Pro. Pro. Gly. Phe. Ser. Pro. Phe. Tissue Kallikrein. Kininogen COOH molecule. Arg. Pro. Pro. Gly. Phe. Ser. Pro. Phe. Arg. Bradykinin. Arg. Pro. Pro. Gly. Phe. Ser. Pro. Phe. Arg. Lys-Bradykinin ( Kallidin). Kininase II ( ACE) Kininase I. Lys. Ser. Tissue Kallikrein Plasma Kallikrein. Plasma Kallikrein. Lys. Arg. Highly select ive f or t he B2 recept or. Arg. Pro. Pro. Gly. Phe. Ser. Pro. Phe. Des-Arg 9 -Bradykinin. Arg. Pro. Pro. Gly. Phe. Ser. Pro. Phe. Des-Arg 9 -Kallidin. Highly select ive f or t he B1 recept or. Inact ive product s. Fig. 1. Formation of kinins by enzyme cleavage of the precursor protein kininogen. Angiotensin converting enzyme (ACE)..

(217) Bradykinin receptors Bradykinin receptors belong to the superfamily of G-protein coupled receptors and exist in mammals as two subtypes, B1 and B2, encoded by separate genes (Regoli and Barabe, 1980). Both receptors are involved in the onset and maintenance of nociceptive and inflammatory processes (Calixto et al., 2004; Couture et al., 2001; Proud and Kaplan, 1988). The two receptors display an overall amino acid identity of 35%. B1 and B2 receptors couple via the same G-protein subunit, mainly GDq, which results in IP formation and intracellular Ca2+ mobilization, and to some extent via GDi with a resultant decrease in cAMP formation (Austin et al., 1997; de Weerd and LeebLundberg, 1997). The B1 and B2 genes are located in tandem and the distance between them has been determined in human (12 kb), mouse (7.8 kb) and rat (9.5 kb) (Cayla et al., 2002). Two non-mammalian bradykinin receptors have been cloned and characterized, the first one was a B2-like receptor in chicken (Schroeder et al., 1997) and the second one was the B1 ortholog in zebrafish (paper I and paper II). The chromosomal location of the two subtypes has been determined in zebrafish, two species of pufferfish (Takifugu rubripes and Tetraodon nigroviridis), as well as chicken. This suggests that the two subtypes arose by a local tandem duplication before the divergence of fish from the lineage leading to mammals (paper III). Despite the similarities between the two receptor subtypes described above, they differ in a variety of aspects (see below).. The B1 receptor The B1 receptor is generally expressed at low levels in normal tissues and synthesized de novo under pathological conditions by the activation of kinins deprived of their C-terminus, mainly des-Arg9-BK and des-Arg10-kallidin, which are increased at sites of inflammation (McEachern et al., 1991; Menke et al., 1994; Raymond et al., 1995). The induction of B1 expression is also believed to be controlled by the mitogen-activated protein kinase (MAP kinase) and by the transcriptional factor nuclear factor N% (NF-N%) (Campos et al., 1999; Larrivee et al., 1998; Ni et al., 1998b; Schanstra et al., 1998). A transcriptional regulatory site for NF-N% has been demonstrated in the promoter region of the rat B1 receptor (Bachvarov et al., 1996; Ni et al., 1998a). The B1 receptor has been implicated in the prolonged phase of pain and inflammatory responses (Dray and Perkins, 1993). B1 receptors are believed to be induced on certain cells like macrophages, fibroblasts and endothelial cells and further mediate the release of prostaglandins, cytokines and nitric oxide that in turn activates nociception (Davis et al., 1996; Farmer, 1997). The inflammatory effects of the B1 receptors include leukocyte recruitment, edema and pain (McLean et al., 2000).. 17.

(218) The B1 receptor preferentially binds the C-terminally truncated peptides desArg9-BK or des-Arg10-kallidin (Farmer and Burch, 1992; Marceau et al., 1998; Regoli and Barabe, 1980) in contrast to the B2 receptor, which has high affinity for full-length BK and Lys-BK (kallidin), for a review see (Calixto et al., 2000). B1, in contrast to B2, does not internalize or sequester in response to agonist stimulation (Faussner et al., 1998). In addition, the B1 receptor has a very slow ligand dissociation and long-term stimulation increases the receptor number, hence favoring persistent signaling (Faussner et al., 1999).. The B2 receptor B2 receptors are distributed throughout central and peripheral tissues and are constitutively expressed (Regoli and Barabe, 1980). The B2 receptor is believed to be involved in the acute phase of pain and inflammatory responses (Dray and Perkins, 1993; Farmer, 1997; Vasko et al., 1994). B2 receptor expression has been detected in primary sensory neurons in rat (Laneuville and Couture, 1987; Lopes et al., 1995; Lopes et al., 1993) and the direct action of BK on these receptors is believed to account for the initial nociceptive response (Laneuville and Couture, 1987). Acute signs of inflammation include increased vascular permeability, vasoconstriction, arterial dilation and pain (McLean et al., 2000). As mentioned above, B2 differs from B1 in having high affinity for the fulllength BK peptide or Lys-BK (kallidin) (Regoli and Barabe, 1980), for a review see (Calixto et al., 2000). After the B2 receptor responds to activation it becomes desensitized resulting in a reduced affinity for BK and rapid ligand dissociation. The receptor is sequestered by short-term exposure to an agonist and down-regulated upon long-term exposure of agonist (Faussner et al., 1999; Marceau et al., 2001; Munoz et al., 1993; Phagoo et al., 1999).. Evolution of the kinin system The kinin system has been studied extensively in mammals but has gained little attention in non-mammals. The only cloned and characterized bradykinin receptors in non-mammals are a B2 ortholog in chicken (Schroeder et al., 1997) and a BK receptor in zebrafish (paper I and paper II) (Fig. 2). Single copy genes encode both B1 and B2 receptors as well as kininogen in mammals, except rat that has two kininogen gene copies due to a putative species-specific duplication (Damas, 1996). 18.

(219) Agnat ha. Hagf ishes. Lampreys. Fishes. Cart ilaginous f ishes Bony f ishes. ( puf f erf ish zebraf ish). Amphibians. Gallus gallus. ( chicken). Rept iles. 450. 400 360. 300. Mammals. 425. 600. 500. 400. 300. 200. 100. Tet rapods. Birds. Gnat host omat a. Takif ugu rubripes Tet raodon nigroviridis Danio rerio. ( several species). Mya. Fig. 2. Evolution of vertebrates. Species where the bradykinin receptors have been identified are indicated by an arrowhead. Million years ago (Mya).. In humans, B1 and B2 receptors are located in close proximity to each other in tandem on chromosome 14q32. This organization is well preserved on homologous chromosomes in all species investigated to date (paper III). This suggests that the B1 and B2 receptors arose by a local tandem duplication of an ancestral bradykinin receptor gene before the divergence of bony fishes and tetrapods.. Peptides of the NPY family The Neuropeptide Y (NPY) family of peptides (Cerda-Reverter and Larhammar, 2000; Larhammar, 1996b) in tetrapods includes neuropeptide tyrosine (NPY), peptide tyrosine tyrosine (PYY) and pancreatic polypeptide (PP), which all consist of 36 amino acids with the exception of chicken PYY that has an additional amino acid at the N-terminal (Conlon and O'Harte, 1992). Intermediate degradation products, such as NPY2-36, NPY3-36 (Grandt et al., 1996) and PYY3-36 (Grandt et al., 1994), exist as a result from enzymatic cleavage. All of these peptide fragments still possess affinity for the Y2 and Y5 receptors. The different peptides and their naturally truncated forms bind to the NPY receptor subtypes with differing selectivity and have been implicated in a number of physiological functions including regulation of appetite, circadian rhythms, reproduction, cardiovascular and gastrointestinal functions (Michel, 19.

(220) 2004; Pedrazzini et al., 2003). PYY3-36 has been demonstrated to inhibit feeding by its action on the Y2 receptor in the hypothalamus (Batterham et al., 2002).. NPY NPY is widespread in both the central and peripheral nervous system, but is most abundant in the hypothalamus (Kalra et al., 1999). This peptide mainly works as a neurotransmitter and is released locally from the synapses. Peripheral effects of NPY include induction of vasoconstriction (Shine et al., 1994) hence causing an increase in blood pressure. It also increases gastric motility by its contractile effects on the GI tract (Ferrier et al., 2000; Pheng et al., 1999). Central effects include stimulation of appetite (Clark et al., 1984), regulation of the release of pituitary hormones involved in reproduction (Kalra and Kalra, 1996), regulation of circadian rhythms (Harrington and Schak, 2000), anxiolysis (Bannon et al., 2000; Wahlestedt et al., 1993), influence on alcohol intake, for a review see (Thiele and Badia-Elder, 2003), and regulation of mood and memory (Heilig and Widerlov, 1990; Wahlestedt et al., 1989).. PYY In mammals, PYY is present in the endocrine cells of the epithelium in the GI tract. The release and subsequent concentration of PYY increases after ingestion of a meal. Circulating PYY is mainly hydrolyzed by the enzyme dipeptidylpeptidase IV with a resultant local and circulating production of PYY3-36 (Medeiros and Turner, 1994). PYY3-36 has been demonstrated to be the major satiety factor in man and rodents (Batterham et al., 2002). PYY regulates gall bladder and pancreatic secretions as well as gut motility (Ferrier et al., 2000; Hazelwood, 1993).. PP PP is a peptide hormone and it is released from endocrine cells in the pancreas in response to ingestion of a meal (Schwartz, 1983). The effects of PP have not been demonstrated until recently, where it was found to mediate anti-secretory effects in human and murine colon via binding to the Y4 receptor (Cox and Tough, 2002). PP has also been demonstrated to be involved in appetite inhibition (Batterham et al., 2003).. 20.

(221) NPY receptors The NPY receptors belong to the G-protein coupled receptors and consist of the subtypes Y1 through Y6 in mammals (Michel et al., 1998). The Y3 receptor has been characterized only by pharmacological studies and most likely does not exist as a separate gene (Blomqvist and Herzog, 1997). The distribution varies among the subtypes and includes both central and peripheral parts of the nervous system. The NPY receptors can further be divided into 3 subfamilies, namely Y1, Y2 and Y5 according to sequence identity. The members of the Y1 subfamily display approximately 50% identity to each other and includes the Y1, Y4 and Y6 subtypes in mammals and birds (chicken) as well as the Yb (Lundell et al., 1997) in teleost fish. The Y2 subfamily also display 50% amino acid identity between its members and consists of the Y2 subtype in mammals and birds and the Y7 receptor, which is missing in mammals but can be found in chicken, teleost fish and amphibians (Fredriksson et al., 2004). The Y5 subfamily consists only of the Y5 subtype which can be found in mammals and birds but has been lost in teleost fish (Larsson et al., 2005).. The Y1 subfamily This subfamily includes the Y1, Y4, Y6 and the Yb receptor subtypes. The human Y1 receptor was the first receptor of the NPY family of receptors to be cloned and functionally expressed. It was found to bind NPY and PYY with much higher affinity than PP (Herzog et al., 1992; Larhammar et al., 1992). The Y1 gene is located in close proximity to the Y2 and Y5 genes on Hsa4 (Larhammar et al., 2001). The coding region of Y1 harbors an 100 bp intron after TM5 (Herzog et al., 1993) and this intron has been demonstrated to enhance the expression of Y1 and Y5 receptors in vitro (Marklund et al., 2002) The Y1 receptor is responsible for vascular effects (Capurro and Huidobro-Toro, 1999; Zukowska-Grojec et al., 1998), decreased anxiety (Wahlestedt et al., 1993) and depression (Kask et al., 2001; Redrobe et al., 2002), ethanol intake (Thiele et al., 2002), arousal (Naveilhan et al., 2001) and regulation of feeding (Kanatani et al., 2001; Larsen et al., 1999; Mullins et al., 2001). The Y4 receptor is unique in the sense that it binds PP with high affinity but not NPY and PYY. Chicken Y4 has equal affinity for NPY, PP and PYY (Lundell et al., 2002). This receptor is mainly present in pancreas, intestines and prostate gland (Lundell et al., 1995). The Ya receptor in teleost is the ortholog of the mammalian Y4 (Fig. 3). The Y6 receptor is functional in rabbit and mouse but is a pseudogene in humans and other primates due to a frameshift mutation where a single base 21.

(222) in the third intracellular loop causes an in-frame stop codon after TM6 resulting in a truncated receptor unable to bind any peptide (Gregor et al., 1996). The Y6 gene is absent in rat (Burkhoff et al., 1998). The physiological effects of Y6 are unknown and is sometimes indicated by designating the receptor y6 (Michel et al., 1998). In paper IV we describe the cloning and characterization of a full-length Y6 receptor from chicken, however, no functional response could be demonstrated. The Yb and Yc receptors have been cloned in teleost fish and it has been proposed that their ancestral gene arose from the second tetraploidization (see below, Evolution of the NPY system) and was again duplicated in the teleost lineage giving rise to Yb and Yc (Larhammar and Salaneck, 2004). The physiological relevance of these receptors is still unknown.. The Y2 subfamily The Y2 subfamily includes the Y2 and Y7 receptor subtypes. The Y2 receptor displays higher affinity for NPY and PYY compared to PP. The mammalian and chicken Y2 receptors (Michel et al., 1998) show high affinity for truncated fragments of NPY and PYY whereas the zebrafish Y2 (Fredriksson et al., 2004) does not. The Y2 receptor is mainly expressed in the brain and is believed to function as an autoreceptor, hence inhibiting the release of neurotransmitters (Caberlotto et al., 2000). The Y2 receptor decreases food intake in contrast to Y1 and Y5, which stimulates appetite (Batterham et al., 2002; Challis et al., 2003). Further it increases anxiety and arousal as well as blood pressure (Morton et al., 1999; Nakajima et al., 1998). The Y2 has also been implicated a role in circadian rhythms (Gribkoff et al., 1998), gastric emptying (Ishiguchi et al., 2001) and regulation of bone formation (Baldock et al., 2002). In 2004, the Y7 receptor was cloned in teleost fish and amphibians (Fredriksson et al., 2004). In paper IV we demonstrate the cloning and characterization of the Y7 in chicken. Surprisingly, neither of the cloned Y7 receptors nor the zebrafish Y2 bind truncated fragments of the NPY family of peptides (paper IV) (Fredriksson et al., 2004). The functional role of Y7 is still unknown. However, mRNA expression of the zebrafish Y7 is widely dispersed primarily in GI tract, eye and brain while chicken Y7 mRNA transcripts were detected only in the adrenal gland (paper IV).. The Y5 subfamily The Y5 receptor is the single member of the Y5 subfamily. The Y5 gene locates to Hsa4 where it overlaps with the promoter region for the Y1 gene and the two genes are transcribed in opposite directions. However some 22.

(223) parts of the transcriptional regulation seem to be coordinated (Herzog et al., 1997). The Y5 receptor is mainly expressed in the brain (hypothalamus, hippocampus and amygdala), (Parker and Herzog, 1998; Parker and Herzog, 1999) where it is involved in regulation of feeding. Y5 is also present in the periphery in organs such as testis, spleen and pancreas (Statnick et al., 1998).. Evolution of the NPY system Early in vertebrate evolution the whole genome, or large parts of the genome, is believed to have undergone two rounds of duplications or tetraploidizations (Ohno, 1970). This is sometimes referred to as the 2R theory (Escriva et al., 2002; Furlong and Holland, 2002; Lundin et al., 2003) and explains why some genes, or clusters of genes, are being part of paralogons, or with other words exist in four copies in vertebrates but only in one copy in urochordates and cephalochordates (Leveugle et al., 2004). The two rounds of large scale duplications early in vertebrate history induced the radiation of many new genes, which developed new functions or became very specialized and divided the function of the mother gene between them (subfunctionalization). However, many of the genes translocated to other positions in the genome, accumulated deleterious mutations and developed into pseudogenes or simply disappeared. NPY and PYY are believed to have been generated from an ancestral gene by a large block duplication (tetraploidization) before the radiation of vertebrates whereas in tetrapods PYY underwent a local duplication to generate PP (Larhammar, 1996a; Soderberg et al., 2000). The PYY-PP genes have furthermore undergone an additional and independent duplication in primates, cattle, sheep (Couzens et al., 2000; Herzog et al., 1995). Teleost fish are believed to have undergone an extra tetraploidization (Taylor et al., 2003; Van de Peer et al., 2003) and two copies of PYY are present in both zebrafish and pufferfish as well as two copies of NPY in pufferfish (Sundstrom et al., 2005). The NPY receptors are believed to have arisen from the two rounds of tetraploidizations and subsequently all subtypes belong to a paralogon. In humans the Y1, Y2 and Y5 genes are localized to Hsa4. The Hsa5 harbors the Y6 gene and Hsa10 the Y4 gene. The fourth chromosome related to this paralagon is Hsa8 although this chromosome contains no Y receptors (Wraith et al., 2000). The Y7 receptor has most likely been lost in mammals but can be found in teleost fish and chicken (on chromosome 13) in a paralogous position with the Y2. Teleost fish seem to have lost the Y1 and 23.

(224) ancest ral Y. Local duplicat ion. 1 st t et raploidizat ion. ancest ral Y2. ancest ral Y1 / Y5. ancest ral Y2. ancest ral Y1 / Y5. ancest ral Y5. Local duplicat ion 1 st t et raploidizat ion ancest ral NPY/ PYY ancest ral NPY/ PYY. ancest ral Y2 / Y7. ancest ral Y1 / Y6. ancest ral Y5. ancest ral Y2. ancest ral Y4 / Yb. ancest ral Y5. Early vert ebrat e ancest or. 2 nd t et raploidizat ion 2 nd t et raploidizat ion. NPY Y2. Y1. Y5. Y7. Y6. Y5. Early vert ebrat e ancest or. ancest ral. NPY PYY. Y4. ancest ral. Yb. PYY. Y2 subf amily NPY. PP. Tet rapods. PYY. Gga4 Gga1 3. Gga6. ?. Y2 Y2. Y7. Y2 subf amily. Y5 Y1 subf amily subf amily Y1. Y5. Hsa4. Y6. Hsa5. Y4. Hsa1 0. Yb. Y2. Y1. Y7. Hsa8. Y2 subf amily. Y1 Y5 subf amily subf amily. Y5 Y1 subf amily subf amily. Tni1 8. Y2. Y1. Y6. Tni1. Y7. Y6. Y4. Tni1 7. Y4. Yb. Tni1 2. Yb. ?. Yc. Y5. Y5. addit ional t et raploidizat ion?. Birds. Mammalian. Teleost s. Fig. 3 . Gene duplicat ion scenarios f or t he neuropept ide Y recept ors and pept ides. The genes marked wit h a cross were probably lost af t er t he duplicat ion event . Specif ic chromosomes are indicat ed by numbers and Gga ( Gallus gallus) , Hsa ( Homo sapiens) and Tni ( Tet raodon nigroviridis) .. Y5 receptors but seem to have retained the fourth member of the Y1 family, the Yb. Furthermore, in the teleost specific tetraploidization the Yc receptor was generated from Yb. (Fig. 3).. 24. ancest ral NPY/ PYY.

(225) AIMS OF STUDY. The aims of this thesis were to: x. clone and functionally express bradykinin receptor(s) in zebrafish.. x. investigate the zebrafish bradykinin receptor with regard to ligand-receptor interactions and signal transduction.. x. identify and analyze phylogenetically and cytogenetically bradykinin and its receptors in different vertebrate species in order to elucidate the evolution of the bradykinin system.. x. clone and pharmacologically characterize the NPY-family receptor subtypes Y6 and Y7 in chicken in order to shed further light on their evolutionary origin and to find clues about their function since these subtypes have been lost in human.. 25.

(226) MATERIALS AND METHODS. This section aims to give a brief description of the methods used in the different papers. For detailed information on materials and methods see relevant sections in the respective papers.. Identification and isolation of new receptor genes In paper I, PCR primers were designed based on conserved regions from sequence alignments of all known B2 sequences including the ornithokinin receptor. The primers were used to obtain a PCR product from genomic zebrafish DNA. Touchdown PCR was performed and a PCR product of 640 base pairs was isolated, reamplified and cloned into the pCR®2.1 TOPO vector followed by heat shock transformation into DH5D cells using the TOPO™TA Cloning® kit. Positive clones were isolated and sequenced on an ABI PRISM 310 Genetic Analyzer. The PCR product was identified as a BK receptor-like fragment and was further labeled with 32P and used as a probe to screen a genomic zebrafish bacterial artificial chromosome (BAC) library (Genome Systems Inc.) in order to isolate a full-length clone. Positive BAC clones were sequenced by primer walking to obtain the full-length sequence of the coding region. The chicken Y6 receptor gene in paper IV was isolated by the same procedure as described above for the zebrafish bradykinin receptor gene. However, the receptor gene was isolated from genomic chicken DNA with degenerate PCR primers based on several mammalian and nontetrapod Y1-subfamily members. The full-length gene was obtained by screening of a chicken genomic BAC library (RZPD, Heidelberg). In paper III and IV orthologous genes were localized in the different genomes with the Ensembl database (www.ensembl.org/) and the chicken Y7 receptor gene was further isolated from genomic DNA with specific primers. The respective releases of genomes were v30.4c (zebrafish), v30.2e (fugu), v30.1b (Tetraodon), v30.1f 8 (chicken) and v30.35c (human).. 26.

(227) Expression vector constructs and transfection The coding regions of the zebrafish bradykinin receptor gene (paper I) and chicken Y6 receptor gene (paper IV) were both ligated into a modified (Marklund et al., 2002) version of the expression vector pCEP4 (Invitrogen, Groningen, Netherlands). The chicken Y7 receptor gene (paper IV) was ligated into a pcDNA3 expression vector (Invitrogen, Stockholm, Sweden). The respective receptor genes in the expression vectors were fully sequenced and found to be identical with its genomic sequence. For cell surface expression of the zebrafish bradykinin and chicken Y6 receptors, human embryonic kidney cells with the Ebstein Barr nuclear antigen element (HEK293-EBNA) were transfected with the pCEP4 vector constructs, respectively, and selected for with 150-500 Pg ml-1 hygromycin (Invitrogen AB, Sweden) for semi-stable expression. Chinese hamster ovary (CHO) cells were transfected with the pcDNA3 construct containing the chicken Y7 gene. The transfections were performed with FuGENE™6 Transfection Reagent (Roche, Stockholm, Sweden) diluted in OptiMEM medium (Gibco BRL, Stockholm, Sweden) according to manufacturer’s recommendations. Transfected cells were maintained in Dulbecco´s modified Eagle´s medium (DMEM/Nut Mix F-12) without L-glutamine (Gibco BRL) and supplemented with 10% fetal calf serum (Biotech Line, AS, USA), 250-500 Pg ml-1 active G-418 (Geneticin), 100 U ml-1 of penicillin, 100 µg ml-1 streptomycin, 2 mM Lglutamine (GibcoBRL).. Measurements of intracellular calcium release HEK293-EBNA cells with a semi-stable expression of the cloned zebrafish bradykinin receptor were assayed for intracellular calcium release upon stimulation with fish bradykinin and derivatives (paper I). Both single-cell and population measurements were performed. The method is based on the fluorescent Ca2+ indicator Fura-2 AM, where AM indicates the acetoxymethylester of Fura-2, which makes it permeable to the plasma membrane. Inside the cell Fura-2 AM is cleaved by unspecific esterases to its ionic form, which is able to bind Ca2+. Upon Ca2+ binding Fura-2 will change maximal excitation wavelength from 380 nm to 340 nm and hereby produce a ratiometric fluorescence value related to intracellular Ca2+levels. For single cell measurements the addition of the ligands were made by perfusion. Fluorescence emission from Fura-2 loaded cells was analyzed using the Intracellular Imaging InCyt2 fluorescence imaging system (Cincinnati, OH, USA). The cells were excited by alternating wavelengths of 340 nm and 380 nm.. 27.

(228) Determination of inositol phosphate formation This method is based on radiolabeling cells with 3H-inositol followed by agonist stimulation and subsequent production of tritiated inositol phosphates (Venter et al.). In brief, HEK293-EBNA cells were grown on culture dishes and loaded with 3 µCi ml-1 myo-[2-3H] inositol (1 mCi ml-1) (Amersham Pharmacia Biotech, Buckinghamshire, UK) (paper II). Cells were harvested, suspended in TES buffered medium (TBM). Stimulations were started by dispensing the cell suspension onto previously prepared 96-well plates containing BK peptides. After 20 min of stimulation at 37qC the reactions were stopped and the cells were lysed in perchloric acid, samples were neutralized and the IP fractions were isolated by anion exchange chromatography.. Binding assays Thawed aliquots of membrane were resuspended in 25 mM HEPES buffer (pH 7.4) containing 2.5 mM CaCl2, 1.0 mM MgCl2 and 2 g/L (Y6) or 0.2 g/L (Y7) bacitracin and homogenized using an Ultra-Turrax homogenizer (paper IV). Saturation experiments were performed in a volume of 100 PL. The reactions were incubated for 2 h at room temperature with 125I-pPYY (Amersham Biosciences, Sweden) as radioligand. Saturation experiments were carried out with serial dilutions of the radioligand, with nonspecific binding defined as the amount of radioactivity binding to the cell homogenate with 100 nM non-labeled pPYY included in the reactions. The incubations were terminated by rapid filtration through GF/C filters (Filtermat A from Wallac Oy, Turku, Finland) that had been presoaked in 0.3% polyethyleneimine, using a TOMTEC (Orange, CT) cell harvester. The filters were washed with 5 mL of 50 mM Tris-HCl pH 7.4 at 4°C and dried at 60ºC. The dried filters were treated with MeltiLex A (Perkin Elmer, USA) melt-on scintillator sheets, and the radioactivity retained on the filters counted using the Wallac 1450 Betaplate counter (Wallac, Finland). The results were analyzed with non-linear regression curve fitting using the GraphPad Prism software package (GraphPad, San Diego, CA). For chicken Y7, competition experiments were performed in a final volume of 100 Pl. Various concentrations of the competitor; i.e. cPYY, pPYY, pNPY, pNPY3-36, pNPY13-36, cPP, pNPY[Leu31, Pro34], BIIE0246, BIBP3226 were included in the incubation mixture along with 125I-pPYY. Saturation experiments were also analyzed with linear regression using Scatchard transformation. Hill coefficients were calculated for each individual competition experiment.. 28.

(229) Genetic mapping Meiotic mapping was performed of the genes for zebrafish kininogen and receptors B1 and B2 (paper I and paper III). This method is based on genetic polymorphisms segregating in the heat shock (Hsieh and Stewart) meiotic mapping panel using single strand conformation polymorphism. In brief, a zebrafish mapping panel built on homozygous diploid individuals (or heat shock diploids) is produced by a heat shock treatment of haploid embryos during the one cell stage and hence giving rise to diploid individuals who are homozygous at all loci. This construction simplifies linkage analysis and the use of dominant markers. A panel of polymorphic markers is constructed and genes of interest are mapped in relation to the markers.. RT-PCR Total RNA preparations from various organs in zebrafish (Danio rerio), pufferfish (Takifugu rubripes) and chicken were prepared according to procedures described in paper I, paper III and paper IV. Total RNA was RNasefree and DNase treatment was performed in order to eliminate genomic DNA contaminations as a possible template in RT-PCR reactions. Total RNA was purified and reverse transcribed into cDNA according to manufacturer’s recommendations.. 29.

(230) RESULTS AND DISCUSSION. Paper I. Cloning, structural characterization and functional expression of a zebrafish bradykinin B2-related receptor. This article describes the cloning and characterization of the first piscine bradykinin receptor, isolated from the zebrafish, Danio rerio. The deduced amino acid sequence of the zebrafish receptor is 360 amino acids in length and shows 35% overall identity to human B2 and the ornithokinin receptor, whereas it has 30% identity to human B1. The receptor gene was mapped to linkage group 17 in the zebrafish genome, which is syntenic to the human B2-B1 gene region on Hsa 14q32. The coding region of the zebrafish BK receptor was ligated into a modified (Marklund et al., 2002) version of the expression vector pCEP4 and the receptor was functionally expressed in HEK293-EBNA cells. Radiolabeled BK peptides from non-mammals are commercially not available. However, kallikrein or trypsin treated plasma from several species of teleost fish, namely eel (Takei et al., 2001), cod (Platzack and Conlon, 1997) and trout (Conlon et al., 1996) have generated [Arg0,Trp5,Leu8]-BK and in this study we iodinated trout BK ([Arg0,Trp5,Leu8]-BK) with 125I and exposed the zebrafish BK receptor to nanomolar concentrations to determine the binding affinity of the agonist. However, no specific binding was detected in the nanomolar range. Possible explanations for the lack of specific binding could be that the labeling with iodine was not optimal for this peptide or that the ligand had lower affinity than expected for the zebrafish bradykinin receptor. Trout BK is devoid of tyrosine residues and the Bolton-Hunter method (Bolton and Hunter, 1973) was applied since this methodology does not require free tyrosine residues. However, the use of Bolton-Hunter modifies the N-terminal amino acid by acylation and hereby removes the charge of this residue, which is an Arg in trout BK. The modification may change the structure of the peptide and hence the affinity for the receptor. Instead, we investigated the functional response of the receptor by performing Ca2+ assay experiments. Unlabeled trout BK activated the zebrafish BK receptor with an 30.

(231) EC50 of 6.6 nM (pEC50= 8.18 ± 0.06). The pharmacological profile of the receptor was further investigated using the derivative des-Arg0-troutBK ([des-Arg0,Trp5,Leu8]-BK) and the putative B1-selective analog des-Arg9troutBK ([Arg0,Trp5,Leu8,des-Arg9]-BK). Both of these peptides were significantly less potent than trout BK. Des-Arg0-troutBK activated the receptor with an EC50 of 370 nM (pEC50= 6.43 ± 0.08) and the maximum response to trout BK and des-Arg0-troutBK was not significantly different. The activity of des-Arg9-troutBK was so low that neither the maximum response nor the EC50 value could be reliably estimated. The receptor was identified as a B2 ortholog in accordance with phylogenetic analysis and pharmacological properties. Reverse transcriptase PCR with primers flanking the complete coding region of the receptor was applied on zebrafish cDNA from, eye, muscle, brain, and gastrointestinal tract. A prominent band was readably visible on the gel in the eye cDNA lane as well as a faint band in brain, but the assay was not designed to allow stringent quantification. A Southern blot of the same gel to a nylon filter probed with part of the zebrafish receptor clone confirmed these results and revealed faint bands also in muscle and GI lanes. Thus, the B2 receptor is broadly expressed in zebrafish, suggesting involvement in multiple physiological functions. These functions in teleost fish have yet to be fully elucidated but complex in vivo effects of [Arg0,Trp5,Leu8]-BK on cardiovascular function have been demonstrated in trout (Olson et al., 1997) and cod (Platzack and Conlon, 1997) and the peptide exerts an antidipsogenic effect in the eel (Takei et al., 2001). The present study suggests that a BK-related peptide may also be important in neurotransmission/neuromodulation in the CNS of teleost as well as play a role in ocular function. After the publication of this study the zebrafish genome was released and we identified a second zebrafish BK receptor with an even more B2-like sequence (paper III). We were also able to identify zebrafish bradykinin and could conclude that it was identical to trout, cod and eel bradykinin. We have reassessed the identity of this first BK receptor to be most likely a B1 ortholog according to its localization in the genome and as deduced from the lack of an intron in the amino terminal region as in the mammalian, chicken and pufferfish B1 genes (see paper III below). Its identity was obscured by a higher replacement rate and somewhat deviating pharmacological profile.. 31.

(232) Paper II. Pharmacological characterization of ligandreceptor interactions at the zebrafish bradykinin receptor. This article describes further analyses of the zebrafish BK receptor and presents the first detailed characterization of a piscine bradykinin receptor with regard to ligand interactions as measured by increased inositol phosphate production in transfected HEK293-EBNA cells. We tested an extensive panel of peptides and analogs including zebrafish BK, mammalian BK, ornithokinin, zebrafish des-Arg0-BK, zebrafish des-Arg9-BK and HOE140. Ornithokinin is the endogenous ligand of the chicken B2 receptor (ornithokinin receptor) (Schroeder et al., 1997). Zebrafish BK activated the receptor with a pEC50 of 6.97r0.1. Mammalian BK and ornithokinin both differ from zebrafish BK in two positions, Phe5 and Phe9 (mammals) and Thr6 and Leu9 (chicken). In addition zebrafish BK has an extra Arg0 at the N-terminal. Mammalian BK was almost inactive (pEC505) at the zebrafish BK receptor while ornithokinin acted as a full agonist with reduced potency (pEC50=6.39r0.19). Interestingly, the only position identical in ornithokinin and zebrafish BK but different from mammalian BK is position 9, which is a Phe in mammals but a Leu in zebrafish and chicken. The results indicate an important role of zebrafish BK Leu9 in species-specific selectivity. Zebrafish des-Arg9-BK is the putative B1 selective ligand but it was almost inactive at the zebrafish BK receptor. The modified peptide HOE140 acts as an antagonist on the human B2 while it is a potent agonist of the ornithokinin (chicken) receptor. However it was almost inactive at the zebrafish receptor. In addition to the above mentioned analogs we also included a complete alanine- and D-amino acid scan of zebrafish BK, where each amino acid, one at a time, was replaced with either an alanine or its D-amino acid form. The mutated positions provide insight into which amino acids in the peptide are important in the interaction with the zebrafish BK receptor. Alanine has a small non-polar side chain. Consequently, amino acids substituted with an alanine will lose their original charge and character. The D-amino acid form is the stereoisomer of the naturally existing L-amino acids that all proteins exclusively consist of. The D-isomer may provide the peptide with new characteristics like antagonistic properties. When Pro7 in mammalian BK is replaced with its D-isomer the peptide adapts antagonistic properties (Hsieh and Stewart, 1999). Analogs substituted with Ala1, Ala4, Ala7, Ala8, or Ala9 acted as partial agonists and with a reduced potency. Peptides substituted with D-Ser6, D-Pro7, 8 9 D-Leu , and D-Arg either gave no response or showed very low potency. In contrast, the potency of the analog substituted with Ala at Pro3 was almost 10-fold higher (pEC50=7.53r0.16) compared to wild type BK. 32.

(233) All analogs that were inactive at the receptor were tested for their antagonistic properties and hence dose-response curves for zebrafish BK were constructed in the presence of the inactive analogs at 1PM but the potency of zebrafish BK was unchanged (pEC50=6.97r0.1). Therefore it was concluded that the inactive analogs were devoid of antagonistic properties and thus had lost their affinity for the receptor. The overall results suggest important roles for receptor interaction for residues Gly4, Ser6, Pro7, Leu8 and Arg9 in zebrafish BK whereas residue Pro3 is proposed to have a limiting effect on receptor activation. Interestingly, the putative B1 selective zebrafish des-Arg9-BK peptide was almost inactive at the receptor as has previously been demonstrated in the inability of the peptide to stimulate intracellular accumulation of Ca2+ (paper I). It has been proposed that TM3 and TM6 of human B1 and B2 receptors contain important sites for interaction with BK agonists and HOE140-related molecules (Fathy et al., 2000; Leeb-Lundberg et al., 2001; Nardone and Hogan, 1994). Human B2 positions Phe259 and Thr263 (Leeb et al., 1997) in TM6 are conserved in all B2 receptors, including the zebrafish receptor investigated here (see corresponding zebrafish positions Phe265 and Thr269 in Fig. 1 in paper I). Interestingly, the human B1 receptor differs at the corresponding positions with Tyr266 and Ala270, suggesting that these positions may account for the higher affinity of des-Arg9-BK for human B1 than human B2 and the zebrafish BK receptor. The residues Tyr295 (TM7) and Gln288 (TM7) in human B2 also play a part in the interaction with nonpeptide ligands where Tyr295 also has a subtle function in receptor activation (Marie et al., 2001). Gln288 is conserved in all BK receptor subtypes while Tyr295 is substituted by Phe in human B1, the ornithokinin receptor and the zebrafish BK receptor. Further, the human B2 receptor displays two cysteines in the C-terminal part while human B1 and the zebrafish BK receptors only has one. Cysteines are putative palmitoylation sites and may be important in the anchoring of the cytoplasmic tail to the cell membrane. Thus, the zebrafish BK receptor has features of both mammalian B1 and mammalian B2 receptors. In paper I we designated the zebrafish BK receptor as B2, based upon phylogenetic analysis and the potency of putative B1 and B2 selective agonists. However, this extended pharmacological analysis shows that the pharmacological properties of the zebrafish BK receptor are distinct from both the mammalian B1 and B2 receptors.. 33.

(234) Paper III. Uneven evolutionary rates of bradykinin B1 and B2 receptors in vertebrate lineages. In this paper we aimed to elucidate the relationships between the B1 and B2 receptor subtypes in fish by phylogenetic analyses and comparisons between homologous chromosomal regions. We identified both the B1 and B2 subtypes in the completed pufferfish genomes of Takifugu rubripes and Tetraodon nigroviridis as well as the true B2 ortholog in Danio rerio and the B1 receptor in chicken. In addition, we identified the gene for kininogen, the precursor of bradykinin, in zebrafish and in the two species of pufferfish. RT-PCR was performed on isolated tissues from Takifugu rubripes with specific primers for the B1 and B2 receptor genes and kininogen. The coding region of the B1 receptor is contained within a single exon in all of the above species, which is in accordance with B1 receptors in mammals. The B2 receptor, in contrast, harbors an intron in the region encoding the amino-terminal part of the receptor before TM1, which has previously been described in all mammalian B2 receptors investigated. The B1 and B2 receptor genes are localized in a conserved tandem formation on homologous chromosomes in all of the species investigated in this paper. The zebrafish B1 receptor has been cloned and characterized and the results are discussed in paper I and II. The zebrafish B2 ortholog consists of 345 amino acids and the coding region of the gene is interrupted after 4 codons by a 19 kb intron. We suggest that an intron is present also in the chicken B2 (ornithokinin receptor) gene, although it was initially not identified (Schroeder et al., 1997). The additional exon and several additional codons after the intron together extend chicken B2 by 40 amino acids compared to the initial prediction. Functional experiments will be required to see if this extension influences the pharmacological properties of chicken B2. In Takifugu rubripes single gene copies of B1 and B2 were identified 5 kb apart. The B2 receptor consists of 352 amino acids and the gene is interrupted by an 861 bp intron after 7 codons. The coding region of B1 is contained within a single exon and the receptor consists of 366 amino acids. In Tetraodon nigroviridis the B2 and B1 receptor genes are separated by 9.7 kb on chromosome 20 and consist of 352 and 333 amino acids, respectively. The B2 gene is interrupted by an intron of 871 bp after the first 7 codons. The B1 and B2 receptor genes, as well as the gene for the bradykinin precursor, named kininogen, are located in well-conserved regions in the different genomes and thus display extensively conserved synteny. Phylogenetic analyses in combination with chromosome region comparisons strongly suggest that the B1 and B2 receptor genes arose by a local duplication of an ancestral BK receptor gene before the split of ray-finned fish (actinopterygians) and lobe-finned fish (sarcopterygians). 34.

(235) The analysis of mRNA distribution of bradykinin receptors and kininogen in Takifugu rubripes performed by RT-PCR revealed a similar distribution of B1 and B2 receptor mRNA in brain, eye, gill, heart, kidney and spleen. Kininogen mRNA was detected as a strong band in liver, as expected from its production in the liver (Marceau and Regoli, 2004) and release into the bloodstream in mammals. However, it will be interesting to further explore the functions and regulation of the bradykinin system in fish.. Paper IV. Neuropeptide Y-family receptors Y6 and Y7 in chicken: Cloning, pharmacological characterization, tissue distribution and conserved synteny with human chromosome region. This paper describes the initial pharmacological characterization of the chicken Y7 receptor and the tissue distribution and phylogeny as well as chromosomal localization of the chicken Y6 and Y7 receptors. The discoveries of the NPY-family receptors Y6 and Y7 (Fredriksson et al., 2004; Weinberg et al., 1996) came as complete surprises as neither of them had been predicted from physiological or pharmacological studies. However, their existences were predicted from evolutionary studies. Both were found thanks to their sequence similarity to other Y receptors and the sequence comparisons suggested that both Y6 and Y7 arose before the radiation of gnathostomes in evolution (Larhammar and Salaneck, 2004; Larhammar et al., 2001; Salaneck et al., 2003). However, Y6 is a pseudogene in some mammals (Starback et al., 2000) but appears to be functional in the shark Squalus acanthias (Salaneck et al., 2003). Y7 has not been found in any mammal. The chicken Y6 sequence was obtained from chicken genomic DNA by degenerate PCR and was used to screen a chicken BAC library at high stringency. The coding part of the Y6 gene is contained within one exon and encodes a protein of 374 amino acids. The overall identity between chicken and those mammalian Y6 sequences that appear to be functional (mouse, rabbit and peccary) is 61-63%. The coding region of chicken Y6 was transferred to a modified pCEP4 (Marklund et al., 2002) vector and expressed in human HEK-293 EBNA cells. Functional expression of the chicken Y6 gene followed by saturation binding experiments showed that the Kd value of radiolabeled pPYY was around 0.80r0.36 nM. To avoid having to rely on a high-affinity radioligand for determination of the receptor's pharmacological profile, we performed a number of functional assays including cAMP, intracellular calcium release, inositol phosphate production, and extracellular acidification to see if we could detect changes in signal transduction in re35.

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