3-Iodothyronamine and its role in aminergic G-protein coupled receptor signaling and neuromodulation

vorgelegt von Dipl.-Ing. Julia Bräunig

von der Fakultät III - Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades

Doktor der Ingenieurwissenschaften - Dr.-Ing. -

genehmigte Dissertation

Promotionsausschuss Vorsitzender: Prof. Dr. Jens Kurreck Gutachter: Prof. Dr. Roland Lauster Gutachterin: Prof. Dr. Heike Biebermann Gutachter: Prof. Dr. Juri Rappsilbe

Tag der wissenschaftlichen Aussprache: 29. März 2019

BERLIN 2019

Abstract

3-Iodothyronamine (T1AM) is an endogenous thyroid hormone metabolite. Its profound phar- macological effects on energy metabolism and thermal homeostasis in rodents have raised interest to elucidate its signaling properties in tissues that pertain to metabolic regulation and thermoge- nesis. Especially its anapyrexic effect could be utilized in emergency medicine to treat disease conditions like stroke. Although the exact molecular mechanism of T1AM-induced anapyrexia has not been fully enlightened, it is assumed that in rodents its regulation is mediated centrally in the brain. Previous studies identified G protein-coupled receptors (GPCRs) and transient receptor potential channels (TRPs) as targets of T1AM in various cell types. However, known T1AM targets cannot explain the anapyrexic effect. In my dissertation, I investigated whether aminergic receptors that are involved in thermoreg- ulation such as receptor 1b (5-HT1b) and receptor 1 (HRH1) are new recep- tor targets for T1AM. 5-HT1b primarily activates the Gαi/o-mediated pathway and (PLC) signaling through the Gβγ-subunit of Gi/o, whereas HRH1 is Gq/11 coupled. Since the expres- sion profiles of TAAR1, the first described GPCR target of T1AM, and 5-HT1b coincide, I evaluated heteromerization of these two GPCRs and their signaling properties under co-expression. Next, I aimed at identifying activated brain areas after the intraperitoneal (i.p.) injection of T1AM in mice. I used the proto-oncogene cFOS (FOS) as a marker for neuronal activation. Further, I used murine neuronal cell lines to study their activation through T1AM regarding Gs and Gi/o signaling. In the first part of my dissertation, I showed that T1AM blocked HRH1 activation through its endogenous histamine. Thus T1AM is an antagonist for HRH1. T1AM induced Gαi/o signal- ing through 5-HT1b at a concentration of 10 µM. Strikingly, T1AM only activated the Gαi/o medi- ated reduction of cAMP accumulation at 5-HT1b, but not PLC signaling through the βγ-subunit.

Therefore, T1AM is a biased ligand for 5-HT1b. Additionally, I confirmed the heterodimerization between TAAR1 and 5-HT1b using fluorescence resonance energy transfer. When co-expressed, only TAAR1 mediated T1AM-induced signaling, whereas 5-HT1b activation was abrogated. In con- clusion, 5-HT1b and HRH1 are new receptor targets for T1AM. Altogether, this indicates a complex interrelation of distinct signaling effects between the investigated GPCRs and respective ligands. In the second part, I observed a significant increase in the amount of FOS expressing neurons in the paraventricular nucleus (PVN) in C57BL/6 mice upon T1AM stimulation. To elucidate the underlying mechanism behind this T1AM signalosome, I used three different murine hypothalamic cell lines (GT1-7, mHypoE-N39 (N39) and mHypoE-N41 (N41)), which are known to express PVN markers. The cell lines expressed various aminergic GPCRs, as well as numerous members of TRP channel superfamily. Effects of T1AM on these cell lines were analyzed for the two GPCR signaling pathways, Gs and Gi/o. Here, I demonstrated that, despite the expression of known GPCR targets of T1AM, this thyroid hormone metabolite had no significant effect on Gi/o signaling and only a minor impact on the Gs pathway in the hypothalamic cell lines N39 and N41.

A faint Gs signaling upon T1AM stimulation might have been the reason for FOS-expressing cells in the PVN. Although, the herein identified T1AM targets HRH1 and 5-HT1b are not Gs-coupled GPCRs my findings confirm that the multi-target ligand T1AM can modulate both histamine and serotonin GPCRs.

Zusammenfassung

3-Iodothyronamin (T1AM) ist ein endogener Schilddrüsenhormonmetabolit. Seine pharmako- logischen Auswirkungen auf den Energiestoffwechsel und die Temperaturregulation in Nagetieren haben Interesse an seiner Signaltransduktion in verschiedensten Organen, welche in den Meta- bolismus und die Thermogenese involviert sind, geweckt. Vor allem seine anapyrexische Wirkung hat das Potential in der Notfallmedizin zur Behandlung von akuten Krankheitsbildern, wie bei ei- nem Schlaganfall, zur Anwendung zu kommen. Der genaue molekulare Mechanismus von T1AM- induzierter Anapyrexie ist nicht vollständig aufgeklärt, wird aber vermutlich bei Nagetieren zen- tral im Gehirn reguliert. Frühere Studien identifizierten G-Protein-gekoppelte Rezeptoren (GPCRs) und transiente Rezeptorpotentialkanäle (TRPs) als Interaktionspartner von T1AM. Bereits bekannte T1AM-Interaktionspartner können die ausgelöste Anapyrexia jedoch nicht erklären. In meiner Dis- sertation untersuchte ich, ob aminerge Rezeptoren, die an der Temperaturregulation beteiligt sind, wie der Serotoninrezeptor 1b (5-HT1b) und der Histaminrezeptor 1 (HRH1), auch Rezeptoren für

T1AM sind. 5-HT1b aktiviert in erster Linie den Gαi/o Signalweg und Phospholipase C (PLC) durch Gβγ von Gi/o, während HRH1 ein Gq/11-gekoppelter Rezeptor ist. Da TAAR1, der erster beschriebene GPCR von T1AM, und 5-HT1b sich in ihrem Expressionsmuster teilweise überschneiden, untersuchte ich eine mögliche Heterodimerisierung zwischen diesen beiden GPCRs und die Signalwege der Rezep- toren bei Ko-Expression. Darüber hinaus identifizierte ich Gehirnareale, welche durch intraperitoneal (i.p.) injiziertes

T1AM aktiviert werden. Dazu verwendete ich das Protooncogen cFOS (FOS) als Marker für stimu- lierte Neuronen. Um die endogene Gs und Gi/o Signaltransduktion durch T1AM-Stimulation zu un- tersuchen verwendete ich murine neuronale Zelllinien.

Im ersten Teil meiner Dissertation konnte ich zeigen, dass T1AM die Aktivierung von HRH1 durch seinen endogenen Liganden Histamin blockierte, wodurch T1AM als ein Antagonist am HRH1 iden- tifiziert werden konnte.

T1AM induzierte Gαi/o-Signalisierung am 5-HT1b in einer Konzentration von 10 µM. Allerdings, aktivierte T1AM am 5-HT1b nur die α- und nicht die βγ-Untereinheit. Dies macht T1AM zu einem funktionell selektiven Liganden. Zudem konnte ich zeigen, dass TAAR1 und 5-HT1b dimerisieren können. Bei der Ko-Expression von TAAR1 und HTR1b wurde die T1AM-Wirkung nur über TAAR1 vermittelt und die Aktivierung von 5-HT1b durch T1AM wurde blockiert. In meinem Exprimenten stellten sich 5-HT1b und HRH1 als neue Rezeptoren für T1AM heraus. Insgesamt deutet dies auf einen komplexen Zusammenhang der Signalwirkungen zwischen den untersuchten GPCRs und den jeweiligen Liganden hin. Im zweiten Teil dieser Arbeit konnte ich beobachten, dass die i.p. Injektion von 50 mg/kg Körper- gewicht an T1AM die Anzahl an FOS exprimierenden Neuronen im Nucleus paraventricularis (PVN) von C57BL/6-Mäusen signifikant erhöhte. Um den zugrunde liegenden Mechanismus hinter die- sem T1AM-induzierten Signalosom aufzuklären, habe ich drei verschiedene murine Hypothalamus- Zelllinien verwendet, da sie verschiedene PVN-Marker exprimieren: GT1-7, mHypoE-N39 (N39) und mHypoE-N41 (N41). In diesen Zelllinien wurden verschiedene aminerge GPCRs sowie TRP-Kanäle exprimiert. Auswirkungen von T1AM auf die hypothalamischen Zelllinien wurden für die zwei GP- CR Signalwege Gs und Gi/o untersucht. Ich konnte zeigen, dass der Schilddrüsenhormon-Metabolit keinen signifikanten Effekt auf die Signalisierung von Gi/o und nur gering Gs Signalisierung in den hypothalamischen Zelllinien N39 und N41 aktiviert.

Im PVN könnte ein Gs -Signal durch die T1AM Stimulation zu FOS-positiven Neuronen führen. Auch wenn die neu identifizierten T1AM Interaktionspartner HRH1 und 5-HT1b nicht Gs gekoppelte GPCRs sind, zeigen meine Forschungsergebnisse dennoch, dass T1AM ein Multi-Target-Ligand ist und sowohl Histamin-, als auch Serotonin-GPCRs modulieren kann. Contents

1 Introduction 6 1.1 Brief overview of ...... 6 1.1.1 The hypothalamus-pituitary-thyroid gland (HPT) axis...... 7

1.1.2 Biosynthesis of T3 and T4 ...... 7 1.1.3 Further regulation of thyroid hormones...... 8 1.2 Historical view on ...... 8 1.3 G-protein coupled receptors (GPCRs)...... 10 1.3.1 G-proteins and signaling pathways...... 10 1.3.2 The structure of GPCRs...... 12

1.4 3-Iodothyronamine (T1AM)...... 12 1.4.1 Effects on metabolism and food consumption...... 14

1.4.2 Cardiac effects of T1AM...... 15 1.4.3 Effects on the thyroid gland...... 16

1.4.4 Thermoregulation of T1AM...... 16 1.5 T1AM and aminergic GPCRs...... 18 1.6 Aim...... 20

2 Materials 22 2.1 General chemicals and buffers...... 22 2.2 Cloning and plasmid purification...... 23 2.3 Cell culture, transfection reagents and functional assays...... 24 2.4 Reagents for immunohistochemistry...... 26

3 Methods 27 3.1 Cell culture...... 27 3.2 Immunohistochemistry...... 27 3.3 Cloning...... 28 3.3.1 PCR amplification...... 30 3.3.2 Restriction and ligation...... 30 3.3.3 Transformation of E. coli ...... 31 3.3.4 Plasmid preparation and sequencing...... 31 3.4 Transfections protocols...... 31 3.4.1 Metafectene...... 32 3.4.2 GeneJuice...... 32 3.5 Evaluation of GPCR and TRP expression via quantitative PCR...... 32 3.5.1 Total RNA isolation via TRIzol™ ...... 32

3 CONTENTS

3.5.2 DNase digestion...... 34 3.5.3 cDNA synthesis...... 34 3.5.4 Quantitative SYBR Green PCR...... 34

3.6 Monitoring of Gs and Gi/o signaling transduction via cellular cAMP...... 35 3.7 Luciferase assays to determine PLC and MAPK activation...... 37 3.8 Protein-Protein-Interaction measured by Fluorescence Resonance Energy Transfer (FRET)...... 37 3.9 Statistical Evaluation...... 39

4 Results 40

4.1 Part I: T1AM and the aminergic GPCRs HRH1 and 5-HT1b...... 40 4.1.1 Establishment of βTAAR1...... 40

4.1.2 T1AM as an antagonist at the histamine 1 receptor (HRH1)...... 43 4.1.3 T1AM is a biased ligand of the serotonin 1b receptor (5-HT1b)...... 44 4.1.4 Summary...... 48

4.2 Part II: T1AM activates the PVN and induces Gαs signaling in hypothalamic cell lines. 48 4.2.1 T1AM induces neuronal activity in the PVN...... 48 4.2.2 Expression profile of GPCRs and TRP channels in murine hypothalamic cell lines 51

4.2.3 T1AM induces FSK-amplified Gαs signaling in mHypoE-N39 and mHypoE-N41 cell lines...... 55 4.2.4 Summary...... 56

5 Discussion 58

5.1 Part I: T1AM and the aminergic histamine 1 receptor (HRH1) and serotonin 1b recep- tor (5-HT1b)...... 58 5.1.1 β-TAAR1 signaling induced by different ligands...... 58

5.1.2 T1AM as an antagonist of the HRH1...... 59 5.1.3 T1AM induced biased signaling at the 5-HT1b...... 60 5.1.4 Co-expression of TAAR1 and 5-HT1b in interplay with T1AM and 5-HT modu- lated the Gαi/o signaling profile...... 61 5.2 Part II: The influence of T1AM on the murine brain and hypothalamic cell lines.... 63 5.2.1 T1AM-induced signalosome activated PVN neurons of C57BL/6 mice...... 64 5.2.2 T1AM slightly stimulated Gαs signaling in murine hypothalamic cell lines.... 65 5.2.3 T1AM activates TRPM8 in hypothalamic cell lines N39 and N41...... 66

6 Conclusion and final remarks 69

Abbreviations 72

List of figures 75

List of tables 77

Literature 78

Appendix 98

A Plasmid maps and gene sequences 98

4 CONTENTS

B Response of hypothalamic cell lines to aminergic ligands 110

C Control assays for cAMP, PLC and MAPK signaling 112

D FRET - controls 116

E cFOS staining of murine brain loci and hypothalamic cell lines 118

Acknowledgment 120

5 Chapter 1

Introduction

Thyroid hormones (TH) accompany one‘s entire life. They regulate a wide range of biological pro- cesses associated with development, metamorphosis, and metabolism. THs are evolutionary con- served and vertebrate, as well as a large number of invertebrates, respond to them. Virtually all species, from plants, fungi, bacteria, and mammals can convert naturally occurring iodate to . From there, the essential molecules of thyroid hormones, mono- and (MIT, DIT), can spontaneously form THs (Nishinaga et al., 1968; Cahnmann and Funakoshi, 1970). In vertebrates,

3,5,3’- (T3) and 3,5,3’,5’-tetrathyrosine (T4) are synthesized in the thyroid gland. They mainly act through genomic signaling involving the binding of their nuclear receptor to control gene expression directly. T3 and T4 can be further metabolized to active and inactive molecules, like thyronamines (TAM, Piehl et al. 2011). These TH metabolites induce amongst other pathways non-genomic signaling through G-protein coupled receptors (GPCRs). The TAM 3-iodothyronamine

(T1AM) has been the focus of research as it rapidly reduces the body temperature without side effects (Scanlan et al., 2004). Therefore T1AM evokes interest to transfer this hypothermic effect from rodents to human emergency medicine. However, the underlying molecular mechanism is not completely understood. Furthermore, T1AM acts centrally in rodents, but the exact brain loci remain subject to speculation (Gachkar et al., 2017). As T1AM is a promiscuous ligand for aminergic GPCRs, I would like to enlighten in this thesis if serotonin and histamine GPCRs may play a role in

T1AM-induced hypothermia.

1.1 Brief overview of thyroid hormones

3,5,3’-Triiodothyronine (T3) and 3,5,3’,5’-tetrathyrosine (tetraiodothyronine, T4) are presumably pre- cursors of TAMs. THs are responsible for a broad spectrum of biological processes. During the first weeks of pregnancy, the fetus receives THs through a transplacental passage from the mother, to ensure a sufficient amount of TH during development. At around 18-20 weeks after postmen- strual age, the fetus can secrete its own THs (de Escobar et al., 2004). THs promote many vital developmental steps such as myelination during neurogenesis (Bhat et al., 1979). After birth, THs regulate several tasks: they maintain bone formation and resorption (Gouveia et al., 2018), and on the metabolic side increase the basal metabolic rate, oxygen consumption, gluconeogenesis, glucose uptake, and lipolysis (Cicatiello et al., 2018). THs also influence cardiomyocytes and vascu- lature controlling heart rate and cardiac output (Razvi et al., 2018). The synthesis and tissue actions of TH are highly regulated to manage their diverse biological functions.

6 1.1 Brief overview of thyroid hormones

Figure 1.1: Biosynthesis of thyroid hormones In the thyroid, I- is transported via the sodium-iodine sym- porter (NIS) (basolateral membrane) and pendrin (apical membrane) into the colloid of a thyroid follicle. Thy- - rocytes secrete thyroglobulin into the colloid, where it is iodinated by oxidized I .T4 is synthesized through the conjugation of two diiodotyrosine (DIT) residues. One DIT and one monoiodotyrosine (MIT) residue result in the formation of T3.T3 and T4, still bound by thyroglobulin, are taken up by endocytosis. Proteases release these THs, and the specific monocarboxylate transporter 8 secretes them into the bloodstream. Figure by Thomsen et al. 2016.

1.1.1 The hypothalamus-pituitary-thyroid gland (HPT) axis

The hypothalamus-pituitary-thyroid gland (HPT) axis highly regulates TH secretion. If thyrotropin- releasing hormone (TRH) is released from neurons of the paraventricular nucleus (PVN, Ishikawa et al. 1988; Merchenthaler and Liposits 1994; Fekete et al. 2000), it activates its receptor in the pi- tuitary gland, which in turn leads to the expression of the thyroid-stimulating hormone (TSH). In the thyroid gland, TSH binds to its receptor, increases the sodium-iodine symporter (NIS) expres- sion and therefore leads to Iodine - transport and consequently to an increased thyroid hormone production (Paire et al., 1997; Kogai et al., 1997; Uyttersprot et al., 1997). Serum T3 and T4 regulate through negative feedback loops hypothalamic TRH and pituitary TSH.

1.1.2 Biosynthesis of T3 and T4 The thyroid gland is located ventral to the trachea and is divided into two lobes connected by the isthmus. The thyroid gland is organized into follicles. Every follicle consists of a colloid surrounded with one layer of thyrocytes. Calcitonin-secreting, parafollicular cells, so-called C cells, are dis- tributed among the thyrocytes. The thyroid gland needs iodide (I-) to generate THs. NIS transports I- from the arteries into the cytosol of the thyrocytes (Fig. 1.1). Presumably, the transporters pen- drin and anoctamin pump I- into the follicle colloid. The thyroid peroxidase then oxidizes it in the presence of H2O2 to iodine monoxide (I0).

7 CHAPTER 1. INTRODUCTION

Meanwhile, the cells synthesize the protein thyroglobulin and secrete it into the colloid. The reactive I0 binds to the tyrosyl residues of thyroglobulin to form monoiodotyrosine (MIT). Free rad- icals of iodine and MIT lead to the formation of diiodotyrosine (DIT). In the final step, two neighbor- ing tyrosyl groups fuse: two DITs result in T4, and DIT and MIT residues result in T3. This mechanism relies on the thyroid peroxidase, on H2O2 consumption and the structural properties of thyroglob- ulin (Deme et al., 1978; Cahnmann et al., 1977; Lamas and Taurog, 1977; Virion et al., 1981). The whole complex re-enters the thyrocytes by endocytosis (Fig. 1.1). The cleavage by various pro- teases releases T3 and T4 from thyroglobulin via the SCL16A2 monocarboxylate transporter 8 into the bloodstream. It is still under investigation if thyrocytes express further TH transporters. The thyroid gland mainly produces T4, which is deiodinated into T3 in the target tissue by deiodinases (DIOs) 1, 2 or 3.

1.1.3 Further regulation of thyroid hormones

Once secreted by the thyroid gland, the bloodstream transports T3 and T4 to their target tissue. About 99.95% of T4 and 99.5% of T3 are bound to thyroxine-binding-globulin, prealbumin/transthyretin and albumin in the serum as backup storage (Robbins, 2000). Therefore, less than 1% of the THs are available for tissue-uptake. Moreover, tissue concentrations of THs do not depend on serum concentrations. The uptake and the actions of THs are regulated by the target cells via a specific gene expression pattern of TH transporters, deiodinases (DIOs), and thyroid hormone receptors

(TRs). They all result in the individual response of every cell type upon T4/T3 stimulation. Once arrived in the target tissue, THs exhibits genomic and nongenomic actions. The classical

TH pathway is genomic by the direct binding of T3 to the TRs. The receptor complex itself binds to specific DNA motifs (TR response element, TRE) and thereby influences gene expression. There are positively and negatively regulated TREs. In mammals, TRH and TSH possess TRE-like hexamers, which are negatively regulated, mainly via the TRβ (Abel et al., 2001; Dupre et al., 2004). Regarding non-genomic actions of TH, T4 and T3 can interact with integrin and activate several pathways in the cytosol, like MAPK or PI3K/AKT pathway (Davis et al., 2016).

For T4 to become T3, the outer phenolic ring has to be 5’-deiodinated by DIO1 or DIO2. T4 and T3 can further undergo sulfation by sulfotransferases and glucuronidation by glucuronyltransferases (Visser et al., 1998; Wu et al., 2005). These modifications enhance the solubility of THs and increase their excretion through bile and urine. In conclusion, the metabolism of T4, as the prohormone, effectively mediates T3 availability.

However, T3 and T4 metabolism do not always lead to inactive biological compounds. Oxida- tive leads to thyronamines (TAMs), while further oxidative deamination results in thyronacetic acids (Hoefig et al., 2015b; Saba et al., 2010). Even though their physiological concen- trations and role are still under debate (Richards et al., 2017; Köhrle and Biebermann, 2019), their pharmacological effects partly oppose T3 and T4 effects, like hypothermia and increased lipolysis (Scanlan et al., 2004; Braulke et al., 2008).

1.2 Historical view on thyronamines

The chemical structure of T3 and T4 consists of a phenolic (outer ring) and a tyrosyl (inner ring) group (Fig. 1.2). Based on this structure, several thyronamines (TAMs) were synthesized, before they were discovered in vivo (Tomita and Lardy, 1956). TAMs share two benzyl rings and the side

8 1.2 Historical view on thyronamines

Figure 1.2: Chemical structures Structures of (A), thyronamines (B), tryronacetic acids (C), (E) and serotonin (F), table of relevant THs, TAMs and TAs (D). chain with thyroid hormones (Fig. 1.2 A+B). They further have a benzyl ring and an alanine side chain in common with aminergic ligands like dopamine and epinephrine (Fig. 1.2 B+E, Meyer and Hesch 1983; Chiellini et al. 2017).

In the 50s, Gross and Pitt-Rivers(1952) discovered T 3 as the bioactive form of T4, but it was still unknown if T3 was the only compound responsible for the TH actions. Therefore, a variety of structural analogs of T4 were under investigation at that time. In 1955, K. Tomita and H. Lardy published bioactivity data on structural T3 and T4 analogs including the TAMs thyroxamine, 3,5,3‘-triiodothyronamine (T3AM) and diiodothyronamine (T2AM, Tomita and Lardy, 1956). Using a standard tadpole assay, thyroxamine and T3AM accelerate metamorphosis, but all three amines fail to prevent goiter formation by thiouracil administration in rats. Meyer and Hesch(1983) and

Cody et al.(1984) revisited the topic of T 3AM. Meyer and Hesch(1983) hypothesized that due to its similar structure with dopamine and epinephrine, T3AM could act on adrenergic receptors. Indeed, T3AM inhibits the binding of β-adrenergic antagonist 3H-dihydroalprenolol with a similar affinity as isoproterenol. Further, T3AM blocks isoproterenol-stimulated cAMP accumulation in turkey ery- throcytes (Cody et al., 1984). These early publications about TAMs demonstrate the initial limitations of this research topic since there was no physiological or pharmacological relevance for TAMs. Not until Scanlan et al.

(2004) published the first data on the effects of 3-iodothyronamine (T1AM) and (T0AM) in rodents together with the first in vivo detection of these TAMs. A high pharmacological dose of

50 mg/kg body weight of T1AM induces a drop in body temperature in C57BL/6J mice of up to 8°C without causing shivering. This temperature effect lasts for several hours. The TAM significantly reduces heart rate and cardiac function for up to 8 h. Afterward, mice recover quickly without any impairments. In T0AM (50 mg/kg body weight) treated mice, the effects are less pronounced and

9 CHAPTER 1. INTRODUCTION have a shorter duration time. The associated receptor 1 (TAAR1) is a receptor for both TAMs and presented as possible effect mediator (Scanlan et al., 2004). As the effect in rodents was more distinct with T1AM-stimulation than with T0AM, it became the mainly studied TAM. Emergency medicine is in search of hypothermia-inducing substances to treat significant trau- mas, with a particular focus on drugs without side effects like severe shivering. Indeed, the ad- ministration of T1AM and T0AM after a stroke reduce the lesion size in mice (Doyle et al., 2007). Also, Frascarelli et al.(2011) showed that T 1AM administration decreases the tissue damage in a cardiac infarct model in rats. In both studies, only the hypothermia without further effects of the

TAMs improves the outcome. To unravel the underlying molecular mechanism, T1AM was tested in TAAR1 knockout (KO) mice, but the hypothermic effect persisted (Panas et al., 2010). Future studies have to solve this mechanism in detail to further translate the beneficial effects of T1AM on trauma patients.

T1AM is a multi-target ligand, especially for aminergic G-protein coupled receptors (GPCRs, Scan- lan et al. 2004; Dinter et al. 2015a,b,c; Laurino et al. 2016). Furthermore, it is assumed that T1AM could be a ligand for additional GPCRs (Kleinau et al., 2011). Several aminergic GPCRs are involved in thermoregulation: histamine, serotonin and dopamine receptors (Brezenoff and Lomax, 1970; Lundius et al., 2010; Heisler et al., 1998; Gudelsky et al., 1986; Oerther and Ahlenius, 2001; Hedlund et al., 2003; Chaperon et al., 2003). One of them might mediate the T1AM-induced hypothermia. However, none of these GPCRs have been tested for T1AM-stimulation so far.

1.3 G-protein coupled receptors (GPCRs)

Since it is assumed that T1AM exerts its action via the activation of GPCRs, it is essential to under- stand the function of these receptors. GPCRs are the largest membrane protein family with more than 800 identified genes in humans. They transmit external stimuli into the cell, amplifying the signal to mediate a suitable cellular response, which enables communication between distant tis- sues but also neighboring cells. The ligands for these receptors range from ions, amines, lipids, , and proteins to light for optical GPCRs. GPCRs are generally divided into five families based on their structure and sequence. Ligands ranging from light, small molecules up to peptides and proteins bind to class A (-like) GPCRs. hormones modulate class B receptors ( family). Ions and small molecules are ligands for class C receptors (Metabotropic glutamate). The ligands of Adhesion GPCRs are mostly unknown. GPCRs are receptors for wingless int-1 (WNT) signaling pep- tides. Even though 30-40% of all currently available drugs aim at GPCRs, available drugs only target less than 10% of the known GPCRs (Wise et al., 2002). Drugs interfere with their endogenous sig- naling by enhancing, blocking or modulating it. The GPCR structure dynamically changes from an inactive to active state, thereby determining its function.

1.3.1 G-proteins and signaling pathways

Upon activation, GPCRs interact with heterotrimeric G-proteins (Fig. 1.4). G-proteins belong to the family of guanosine triphosphate (GTP) binding proteins. G-proteins consist of an α, β and γ subunit. In mammalians, at least 18 α, 5 β, and 12 γ subunits are expressed (see Syrovatkina et al. 2016 for an extensive review on G-proteins). The α subunit harbors a guanosine diphosphate (GDP). Due to the

10 1.3 G-protein coupled receptors (GPCRs)

Figure 1.3: Overview of GPCR Signaling The different Gα and βγ-subunits induce various signaling cascades.

Gαs activates the adenylate cyclase (AC) increasing the cellular cAMP content, while Gα i/o reduces it. This leads to the regulation of protein kinase A (PKA) and the transcription factor CREB and further MAPK signaling. The

βγ-subunit of Gαi/o can further activate ERK signaling via phosphoinositide-3-kinase (PI3K). Gα12/13 inhibits

PI3K and activates RhoA signaling, which leads subsequently to Tau phosphorylation. Gαq/11 activates phos- pholipase C-β (PLC-β), which leads to the increase of inositol-triphosphate as a second messenger and further downstream of either MAPK signaling or the activation of the transcription factor NFκB.

11 CHAPTER 1. INTRODUCTION binding of an active receptor, the conformation of the α subunit changes and GDP is exchanged to guanosine triphosphate (GTP). The heterotrimeric complex dissociates into α and βγ subunits and can induce several intracellular signaling cascades. Every tissue possesses Gαs,Gαi/o,Gαq,Gα12/13 signaling, while Gαolf is limited to olfactory GPCRs and Gαt (transducing) is only expressed in the eye for rhodopsin.

The different Gα-subunits induce various signaling cascades (Fig. 1.3). Gαs activates the adeny- late cyclase (AC), increasing the cellular cAMP content, while Gαi/o reduces AC activity. cAMP con- centrations regulate the protein kinase A (PKA) and the transcription factor CREB. The βγ-subunit of Gαi/o can further activate ERK signaling via phosphoinositide-3-kinase (PI3K). Whereas, Gα12/13 inhibits PI3K and activates RhoA signaling. Gαq/11 activates phospholipase C-β (PLC-β), which leads to the increase of inositol-triphosphate as a second messenger and further downstream of either MAPK signaling or the activation of the transcription factor NFκB. The signaling capacity of vari- ous tissues depends on the expression pattern of G-proteins and GPCRs. Furthermore, homo- and heterodimerization can have an impact on signaling (Lohse, 2010).

1.3.2 The structure of GPCRs

GPCRs consist of a single polypeptide chain sectioned into seven membrane-spanning α-helices (Fig. 1.4). The protein synthesis starts with the extracellular N-terminus, followed by the seven transmembrane domains (TM1 – TM7) connected by three intracellular loops (ICL1, ICL2, ICL3) and three extracellular loops (ECL1, ECL2, ECL3, Latorraca et al. 2017). At the end is an intracellular hydrophilic C-terminus forming an eighth helix. The sequence of the TMs is highly conserved among receptor classes and even species. The TMs form a barrel structure with a ligand binding site opening to the extracellular side. The N-terminus is involved in ligand binding in class C GPCRs. The C-terminus and ICLs contribute to G-protein and arrestin binding (Thomsen et al., 2016). However, there is currently no particular binding sequence or structure known to explain the specific binding to one G-protein. The tertiary structure of a GPCR is not rigid, but dynamic. The activation of a GPCRs induces a change in its conformation, and there is not one active conformational state, but infinite. The binding of a ligand only favors one conformational state, which then leads to the binding of either a specific G-protein or an arrestin. GPCR ligands can be and antagonist acting fully, partial or inversely. An activates signal transduction and antagonist blocks it. Both, a full or a partial agonist, activate the same signaling cascades. However, a full agonist evokes the maximal pathway activation possible, while a partial agonist can only induce a fraction of the full signaling capacity (Vi- lardaga et al., 2005). An reduces the basal activity of a GPCR (Vilardaga et al., 2005). Further, biased ligands are an emerging concept of GPCR signaling. In contrast to endogenous lig- ands, which generally activate the whole signaling spectrum of a GPCR, these functionally selective ligands discriminate either between different heterotrimeric G-proteins or between G-proteins or arrestin leading to enhancement of the selected signaling pathway (Smith et al., 2018). The ligand binding specifies a distinct conformation (Vilardaga et al., 2005; Smith et al., 2018).

1.4 3-Iodothyronamine (T1AM)

Since T1AM could be the lead compound for a hypothermia-inducing drug, every aspect of the pharmacological effects of this TAM should be investigated to design a potent drug to induce hy-

12 1.4 3-Iodothyronamine (T1AM)

Figure 1.4: Structure of GPCRs based on the ADRB2 (A) GPCRs possess seven transmembrane domains connected by intra- and extracellular loops, an extracellular N-terminus and an intracellular C-terminus. (B) Upon ligand binding, a heterotrimeric G-protein attaches to the receptor. The spatial arrangement of the GPCR is based on the crystallography data of the ADRB2 (Rasmussen et al., 2011). Figure by Latorraca et al. 2017.

13 CHAPTER 1. INTRODUCTION

pothermia in humans. A starting point might be to investigate if a GPCR mediates T1AM-induced hypothermia. The first described interaction partner for T1AM was the aminergic GPCR TAAR1 (Scanlan et al., 2004). Since then further GPCRs, but also other proteins, like the apolipoprotein

B100 and the mitochondrial F0F1-ATPase have been described to interact with T1AM (Roy et al., 2012; Cumero et al., 2012). Since T1AM is a multi-target ligand, further effects, besides hypother- mia and a reduced cardiac function, were discovered for the pharmacological application of T1AM: hyperglycemia, increased lipolysis, alterations in food intake, gene expression in the thyroid gland, memory and pain sensitivity (Braulke et al., 2008; Mariotti et al., 2014; Dhillo et al., 2009; Schanze et al., 2017; Manni et al., 2013).

1.4.1 Effects on metabolism and food consumption

After the first publication on T1AM, it quickly became apparent in further experiments that T1AM has metabolic effects. Djungarian hamster and BL6 mice treated with 50 mg/kg T1AM show a de- creased metabolic rate and respiratory quotient followed by ketonuria 8 h indicating a blockage of glucose metabolism (Braulke et al., 2008). T1AM treated hamster show an overall lose weight, while mice show a higher change in body temperature, metabolic rate, and respiratory quotient. Hence the hypothesis that T1AM promotes a shift to lipid metabolism.

Lehmphul et al.(2018) stimulated murine pancreatic cells MIN6 with T 1AM (100 nM), which de- creases the glucose-stimulated secretion. A monoamine oxidase (MAO) inhibitor dampens this effect without completely blocking it (Lehmphul et al., 2018). Several cell types take up T1AM, metabolize it to TA1 via MAOs and then secrete it (Saba et al., 2010; Agretti et al., 2011; Schanze et al., 2017; Lorenzini et al., 2017; Lehmphul et al., 2018). It was already shown for mitochondria of the bovine heart that T1AM inhibits the F0F1-ATPase (Cumero et al., 2012). Furthermore, T1AM decreased mitochondrial ATP-production in MIN6 as pancreas model (Lehmphul et al., 2018). In- deed, T1AM and to a lesser extent its metabolite thyronacetic acid (TA1), reduce the respiratory output of the β-cells. In conclusion, T1AM reduces insulin secretion by being taken up by the cells, getting metabolized partially to TA1 and together with TA1 inhibiting the mitochondrial F0F1-ATPase (Lehmphul et al., 2018).

As the liver is responsible for gluconeogenesis and T1AM induces hyperglycemia, Ghelardoni et al. 2014 investigated the response of HepG2 (human hepatocellular carcinoma cells) as well as isolated rat livers towards T1AM stimulation. T1AM increases glucose production in the cell line and in in situ perfused rat liver in a dose-dependent manner. It reduces ketone body formation, while free fatty acid amounts remain unaltered. Again, the MAO inhibitor iproniazid diminishes the T1AM effects from formerly 25 % to 9 %. As the MAO inhibition is effective in pancreas and liver, TA1 is supposedly the main active compound. However, the external administration of TA1 alone showed only minor effects due to a slower uptake compared to T1AM in all experiments (Lehmphul et al., 2018; Ghelardoni et al., 2014).

T1AM also has effects on adipose tissue. Wistar rats were i.p. injected with 10 mg/kg bodyweight T1AM two times a day for five consecutive days (Mariotti et al., 2014). T1AM upregulates several genes for the lipoprotein metabolism in the adipose tissue (Mariotti et al., 2014).

T1AM was also tested for possible effects on food consumption considering its influence on glucose metabolism. One acute dose (4 nmol/kg body weight) of i.p. administered T1AM dose- dependently increases food intake in Wistar rats (Dhillo et al., 2009). By injecting intracerebroven- tricularly (i.c.v, 1.2 nm/kg body weight) T1AM, neurons in the nucleus arcuatus are c-FOS (FOS) acti-

14 1.4 3-Iodothyronamine (T1AM)

vated. On the contrary, a study showed that an i.c.v injection of 1.3 µg/kg body weight of T1AM lowers food consumption, while 20 µg/kg increases food consumption, and a concentration of 26 µg/kg shows no effect (Manni et al., 2012). Once more, the MAO inhibitor clorgyline dimin- ishes the 20 µg/kg effect of T1AM. In the case of the lowest dose of T1AM, clorgyline blocks the rise in glucose levels and decreases insulin sensitivity in the periphery. Again, the effects appear to be mainly induced by the T1AM metabolite TA1.

A comparison of i.p. vs i.c.v. administration of T1AM and T0AM revealed that during the first 45 min the effects are mainly centrally mediated, whereas later effects descend from the periphery (Klieverik et al., 2009). TAMs affect the hypothalamus–pituitary–adrenal axis as well as the pancreas itself.

TA1 is presumably the effector for the metabolic effects of T1AM: hypoinsulinemia, hyperglycemia, increased lipolysis and diverse effects on food consumption. However, i.p injections of T1AM seem to possess faster pharmacokinetic then TA1. The apolipoprotein B10 binds T1AM in the blood, which favors the cellular uptake of the TAM (Roy et al., 2012; Köhrle and Biebermann, 2019).

1.4.2 Cardiac effects of T1AM

T1AM perfused Wistar rat hearts react quickly by decreased cardiac output, heart rate, systolic pressure, and coronary flow (Chiellini et al., 2007). T1AMs metabolite T0AM only has a minor impact on the cardiac output and the heart rate. Recordings of the heart rate and aortic pressure show that the heart tissue already responds 30 sec after T1AM stimulation (Chiellini et al., 2007).

To unravel the underlying signaling of the cardiac T1AM effect, rat hearts were tested for Gαs signaling by measuring the cAMP increase or using PTX to block Gαi/o signaling. Further, the hearts were stimulated with various blockers, to inhibit protein kinases A and C, the calcium-CaM- dependent kinase II, the phosphatidylinositol-3-kinase, the MAP kinase-2, and the MAP kinase ki- nase (Chiellini et al., 2007). Chiellini et al.(2007) further investigated the stimulation with T 1AM together with insulin or atropine. None of the listed approaches leads to any conclusion of the involved pathways or influences the T1AM induced effects. However, the kinase inhibitor genistein in co-administration with T1AM can enhance T1AM effects significantly, even though genis- tein has no effects when administered alone. On the other hand, the tyrosine phosphatase inhibitor vanadate blocks the decrease in cardiac output induced by T1AM. T1AM reduces the tyrosine phos- phorylation of cytosolic and mitochondrial proteins in rat hearts (Chiellini et al., 2007). Patch clamp experiments with adult rat cardiomyocytes show a reduced amplitude and dura- tion of calcium influx due to depolarization when stimulated with T1AM (Ghelardoni et al., 2009). However, depolarization of potassium channels is also affected by T1AM challenge.

Besides T1AM, also T0AM and the trace amines , β-phenylethylamine (PEA) and oc- topamine (OCT) have negative inotropic effects on rat hearts (Frascarelli et al., 2008). Again, genis- tein potentiates these effects. Since rats express five TAARs in the heart (Chiellini et al., 2007), it is likely that TAARs mainly mediate cardiac T1AM responses as its endogenous ligands, trace amines, show similar effects. However, T1AM or PEA do not activate the highly expressed TAAR8 (Muhlhaus et al., 2014). Moreover, T1AM modulates calcium and potassium homeostasis through an intracel- lular calcium channel, known as the ryanodine receptor in adult rat cardiomyocytes (Ghelardoni et al., 2009).

15 CHAPTER 1. INTRODUCTION

1.4.3 Effects on the thyroid gland

A variety of mechanisms precisely regulate thyroid hormones, including negative feedback loops. In mice and rats, follicular cells express TAAR1 at the apical membrane and within the cytosol

(Szumska et al., 2015). Therefore, pharmacological doses of T1AM might also affect the thyroid gland. However, T1AM (0.1 - 100 µM) stimulation of FRTL5 cells (rat thyroid cell line) does not affect cAMP concentrations, mRNA expression, iodine or glucose uptake (Agretti et al., 2011). Additionally,

Schanze et al.(2017) tested whether repeated injection of 5 mg/kg body weight of T 1AM into male mice affected thyroid function and the HPT-axis. Their study indicates that T1AM decreases NIS, thyroglobulin and pendrin expression without altering thyroid hormone concentrations or the HPT- axis. Experiments with a PCCL3 cell line (rat thyroid epithelial cell line) show the same results 2+ (Schanze et al., 2017). In this cell line, T1AM evokes a Ca influx through the transient potential cation channel member of family M 8 (TRPM8, Schanze et al. 2017). In conclusion, T1AM could have an impact on TH synthesis without altering the HPT axis. The repeated T1AM injections have no effect in THs concentration in the in vivo experiments (Schanze et al., 2017).

1.4.4 Thermoregulation of T1AM

The condition of the mice treated with T1AM was very reminiscent of hibernation or torpor. Mice or rats naturally do not go into hibernation, but other rodents like hamsters do. The physiological mechanism for hibernation is still unknown. Hence, T1AM could play a crucial role. What quickly spoke against this hypothesis was the high concentration of T1AM to induce said effects and its short duration time (Glossmann and Lutz, 2017).

The study of Gachkar et al.(2017) partially explained the mechanism of T 1AM action in ther- moregulation. In this study, T1AM triggers the of the tail vein centrally and not in the periphery. That would require for T1AM or its metabolite to cross the blood-brain barrier. Ian- culescu et al.(2009) suggested several transporters for T 1AM, whereby the blood-brain barrier in rats express the solute carrier transporter 7A1 (SLC7A1) (Enerson and Drewes, 2006) and mice ex- press the SLC7A1 and SLC31A1 (Daneman et al., 2010). Further, i.c.v and i.p. injection of T1AM increases phosphorylation of ERK1/2 and AKT in the hippocampus and hypothalamus (Manni et al.,

2013). Therefore, it is possible for T1AM to cross the blood-brain barrier. Both, the heat dissipation over the tail vein and the temperature drop without shivering re- sponse, suggest that T1AM induces rather anapyrexia than hypothermia. During hypothermia, the body is not able to counteract the heat loss by brown adipose tissue thermogenesis in rodents or muscle shivering in humans. Anapyrexia is the lowering of the set point of the basal body temper- ature. Rodents show hypomobility and no activation in brown adipose tissue thermogenesis after

T1AM injection, which further supports the hypothesis of anapyrexia (Gachkar et al., 2017). The body temperature underlies complex sensory and regulatory mechanisms. The tempera- ture can fluctuate at the body surface. In the viscera and central nervous system, the temperature has to be stable. The brain measures the ambient temperature, the inner body temperature, and its difference to the set point to maintain stable core temperature. It also takes into account the amount of heat generation and the loss over the body surface (Tan and Knight, 2018). Warmth- and cold-sensing neurons are distributed throughout the body to determine the body temperature (Fig. 1.5, Vriens et al. 2014). Brown adipose tissue thermogenesis and muscle shivering generate heat (Morrison et al., 2012; Nakamura and Morrison, 2011). Vasodilation or vasoconstriction control heat dissipation (Johnson et al., 2014). Water evaporation by sweating or panting can also increase

16 1.4 3-Iodothyronamine (T1AM)

Figure 1.5: Basics of thermoregulation Cold-sensing neurons express TRPM8, while warmth-sensing neurons express TRPV1. The preoptic area (POA) processes the main peripheral information, from there further brain loci various responses are evoked. Shivering of the and brown adipose tissue thermogenesis generate heat. Vasodilation leads to heat dissipation over the skin and vasoconstriction reduces this heat loss. Sweating increases heat loss through evaporation chill. Whereas, anpyrexia lowers the setpoint. Warmth- sensing neurons report that the body temperature is too high. Heat-producing mechanisms like brown adipose tissue thermogenesis are reduced, while vasodilation is induced to cool the body. heat dissipation (Jessen, 1985).

T1AM activates the transient receptor potential ion channel melastatin 8 (TRPM8) in human con- junctival epithelial cells (Khajavi et al., 2015). TRPM8 activation leads to an increase in its expression in human corneal epithelial cells (Lucius et al., 2016). The TRPM8 channel senses temperatures be- low 30°C and is expressed on cold-sensing neurons (Peier et al., 2002). Icilin and menthol are further agonists of this TRP channel (Peier et al., 2002; McKemy et al., 2002). These cooling agents have been used to suppress pain sensitivity (Proudfoot et al., 2006). T1AM is therefore also handled as a cooling agent for dry eye syndrome (Khajavi et al., 2015). Further, TRPM8 is overexpressed in several cancer types (Beck et al., 2007). TRPM8 activation in human uveal melanoma by T1AM blocks its counterpart TRPV1. This inhibition reduces neoan- giogenesis in tumors (Walcher et al., 2018). Besides for eye cancer, T1AM induces apoptosis in vitro in the human ovarian cancer cell line OVCAR3 and the human breast adenocarcinoma cell line

MCF7 (Shinderman-Maman et al., 2017; Rogowski et al., 2017). Therefore, T1AM is currently in the discussion for cancer treatment.

17 CHAPTER 1. INTRODUCTION

Figure 1.6: Overview of aminergic GPCRs as T1AM interaction partners

1.5T 1AM and aminergic GPCRs

T1AM interacts with TAAR1. However, there might be further aminergic targets to explain the un- changed T1AM actions in TAAR1 KO mice (Panas et al., 2010). The structure of T1AM has similarities with dopamine and epinephrine (Fig. 1.2 B+E). Therefore, it was assumed that T1AM is possibly ca- pable of interacting with further aminergic receptors. Computational modeling of aminergic GPCRs supports this hypothesis (Kleinau et al., 2011). These GPCRs are a part of class A and include adren- ergic, dopamine, serotonin, histamine, muscarinergic and trace associated amine receptors. Amin- ergic receptors contribute to a variety of signaling cascades. T1AM can be either an agonist or antagonist for these receptors or even a biased ligand (Fig. 1.6). An agonist activates signaling of a GPCR, and an antagonist inhibits its activation. A biased ligand promotes only the activation of a specific G-protein or arrestin and not the complete signaling spectrum of a GPCR induced by its endogenous ligand.

T1AM interacts with trace amine associated receptors. This receptor family consists of 28 sub- families. TAAR1-9 have been found mainly in vertebrates, while the remaining have been identified in teleosts (Gloriam et al., 2005; Hashiguchi and Nishida, 2007; Hussain et al., 2009). Humans ex- press TAAR1, 2, 5, 6, 8 and 9 subfamilies, while mice express the subfamilies TAAR1-9 with a total of 15 receptors (Hashiguchi and Nishida, 2007).

T1AM is, like trace amines, an agonist of the TAAR1 inducing Gαs signaling (Fig. 1.6, Scanlan et al. 2004). TAAR1 is known as a neuromodulator. It regulates the surface expression of the dopamine 2 receptor (DRD2) by heterodimerization (Espinoza et al., 2011). TAAR1 KO mice over- express postsynaptic DRD2 in the striatum, which leads to an increased sensitivity towards DRD2 ligands similar to the phenotype of schizophrenic patients (Espinoza et al., 2015). The activation of TAAR1 leads to an increased expression of itself, which decreases DRD2 surface expression (Stavrou et al., 2018; Gratz et al., 2018). Therefore, T1AM could contribute to this neuromodulation. Using murine dorsal striatal slices, only T1AM induces phosphorylation of the tyrosine hydroxylase and

18 1.5 T1AM and aminergic GPCRs leads to the release of L-dopamine through TAAR1, while the endogenous TAAR1 ligands and phenylethylamine (PEA) have no effects (Zhang et al., 2018). T1AM induces a signaling cascade involving calmodulin-dependent protein kinase II and protein kinase A, which is abolished in TAAR1 knockout mice or by administering TAAR1 specific inhibitor EPPTB (Zhang et al., 2018). Neverthe- less, there are significant differences in the signaling and the surface expression of TAAR1 between the species, even though it is highly conserved (Coster et al., 2015). For instance, the rat TAAR1 has a higher basal activity and a higher surface expression than the human one (Coster et al., 2015). Therefore, it is difficult to relate rodent data to human physiology.

T1AM can interact with further TAARs. Human T and B cells express TAAR1 and TAAR2 (Babusyte et al., 2013). Both receptors contribute to Gαs signaling upon T1AM stimulation (0.1 - 1 nM), which leads to increased IgE production (Babusyte et al., 2013). T1AM is an inverse agonist at the human TAAR5 reducing Gαq and MAPK signaling, while the murine TAAR5 only shows basal activity for Gαs,

Gαq and MAPK signaling (Dinter et al., 2015c). It was speculated that T1AM activates TAAR8a, as it is highly expressed in the rat heart and was assumed to contribute to the cardiac effects of TAMs

(Chiellini et al., 2007). However, neither T1AM nor trace amines, like PEA seem to be a ligand for the human or murine TAAR8 (Muhlhaus et al., 2014).

Besides the trace amine associated receptors, T1AM is an antagonist of the rat muscarinic re- ceptor 3 (CHRM3) by reducing carbachol-induced MAPK signaling (Laurino et al., 2016).

Regarding adrenergic GPCRs, is has already been shown for T3AM to modulate adrenergic sig- naling (Meyer and Hesch, 1983; Cody et al., 1984). The α2- (ADRA2A) signaling is linked with a lower insulin secretion upon T1AM stimulation in mice. I.p. injections of 50 mg/kg body weight of T1AM enforce lower insulin and higher glucagon concentrations resulting in a 250% rise of blood glucose levels in mice (Regard et al., 2007). A RosaPTX mouse model specifically sup- presses Gi/o signaling in the pancreas, which abolishes the increase of blood glucose concentrations (Regard et al., 2007). Experiments with either an α2-adrenergic receptor (ADRA2A) specific blocker or ADRA2A null mice achieve the same results (Regard et al., 2007). In conclusion, T1AM induces a Gαi/o signaling cascade through ADRA2A in the murine pancreas to block insulin secretion. In in vitro experiments, T1AM is a biased ligand at the ADRA2A, inducing only Gαi/o and not MAPK signaling (Dinter et al., 2015b). The hyperglycemic effect is assigned to TAAR1 (Regard et al., 2007).

T1AM enhances (ISOP) stimulation of the β2-adrenergic receptor (ADRB2) by in- 2+ creasing Gαs signaling, but through ADRB1 (Dinter et al., 2015a). Moreover, T1AM induces Ca influx in IOBA-NHC cells, a human conjunctiva cell line, which can be abolished by the non-specific adrenergic blocker timolol (Dinter et al., 2015a).

There are further hints that T1AM and TA1 are involved in the system (Laurino et al., 2015). I.v.c. injections of T11AM induce hyperalgesia with or without MAO inhibitor. The T1AM-induced hyperalgesia was only abolished in a histamine decarboxylase double knockout mice. These mice cannot produce histamine.

However, not only aminergic GPCRs but also aminergic transporters are targets of T1AM. The TAM blocks the dopamine transporter (DAT), transporter (NET) and the vesicular monoamine transporter 2 (VMAT2, Snead et al. 2007). Serval aminergic regions are involved in temperature control, e.g., the serotonergic dorsal raphe nucleus (Ginefri-Gayet and Gayet, 1993). However, so far none of the already described GPCR tar- gets of T1AM are involved in T1AM induced anapyrexia. In TAAR1 knockout mice, the T1AM effect persisted (Panas et al., 2010), which is surprising as TAAR1 mediates hypothermia upon metham- phetamine administration (Miner et al., 2017). The activation of the TRPM8 leads to hyperthermia

19 CHAPTER 1. INTRODUCTION

and not hypothermia (Ding et al., 2008). TA1 seems to be the primary ligand for the metabolic ef- fects of T1AM. Nevertheless, the metabolites TA1 and T0AM do not influence the body temperature (Hoefig et al., 2015a; Harder et al., 2018). Ultimately, no brain locus, GPCR, or ion channels has been proven to mediate the T1AM induced anapyrexia.

1.6 Aim

Presumably, the hypothalamus mediates different effects of T1AM, such as anapyrexia and alter- ations in food consumption (Dhillo et al., 2009; Manni et al., 2012, 2013). As the pharma industry is interested in T1AM-induced anapyrexia and its precise mechanism, it is necessary to find the brain loci mediating this effect and its specific signaling pathway. Even though T1AM has been studied extensively since 2004, so far, it is challenging to match identified targets of T1AM with the rodent phenotype. The TAM activates several aminergic GPCRs, but none of them can be linked to ther- moregulation. Therefore, it is still open whether another member of the aminergic receptor family is responsible for T1AM effects. It should be investigated whether T1AM also interacts with rep- resentatives of the serotonin and histamine receptors (5-HT, HRH), as several of these receptors are involved in thermoregulation: HRH1, HRH3, 5-HT1a, 5-HT1b, 5-HT2c, and 5-HT7 (Brezenoff and Lomax, 1970; Lundius et al., 2010; Heisler et al., 1998; Oerther and Ahlenius, 2001; Gudelsky et al., 1986; Oerther and Ahlenius, 2001; Hedlund et al., 2003). Nevertheless, also further TRP channels, like TRPM2, could be involved (Gachkar et al., 2017). The aminergic receptors, 5-HT1b and HRH1, were chosen as potential candidates.

Therefore, this study aimed at answering the following questions:

Is T1AM a ligand for HRH1 and 5-HT1b? If so, what signaling is induced by T1AM? Can this be transferred to the in vivo effects of T1AM? Where in the brain stimulates T1AM neuronal activity? And can respective neuronal cell lines give conclusions about the molecular mechanism?

Fig. 1.7 shows an overview of the experimental organization. Part I: The aminergic receptors, 5-HT1b and HRH1, will be tested in HEK293 cells for their signal- ing properties concerning T1AM as a ligand. 5-HT1b is Gαi/o coupled and further induces MAPK and PLC activity. HRH1 is Gαq coupled. Cyclic AMP will be monitored using the AlphaScreen™ assay. NFAT, for phospholipase C signaling, and luciferase assays will monitor MAPK activity. Finally, since TAAR1 modulates DRD2 signaling by dimerization, the dimerization between TAAR1 and 5-HT1b will also be tested, together with the impact on their signaling capacities. Dimerization will be tested via fluorescence resonance energy transfer (FRET). PartII: Besides, brain areas are to be identified, which are activated by T1AM stimulation to enlighten the anapyrexic mechanism. The identification of a specific neuronal subtype would limit the number of potential GPCRs and TRP channels as possible interaction partners for T1AM. The protooncogene c-FOS (FOS) is a marker for neuronal activity. Brain slices of T1AM (50 mg/kg body weight) injected C57BL/6J mice will be stained for FOS to identify activated neurons. If activated brain areas are found, matching murine cell lines will be tested for GPCR involvement by measuring

Gαs and Gαi/o signaling.

20 1.6 Aim

Figure 1.7: Experimental Overview The first part should elucidate if T1AM is an interaction partner for HRH1 and 5-HT1b including its signaling profile. The dimerization of TAAR1 and 5-HT1b is under investigation and if this influences their signaling properties concerning T1AM stimulation. Part II deals with the identification of activated brain areas due to i.p. injection of T1AM in mice. Neuronal cell lines might enlighten the underlying mechanism.

21 CHAPTER 2. MATERIALS

Chapter 2

Materials

2.1 General chemicals and buffers

Table 2.1: General chemicals and buffers compound supplier acetone Roth agarose Roth calcium chloride dihydrate (CaCl2∗2H2O) Calbiochem chloroform Merck ethanol 96% Merck ethanol 70% Roth ethylenediaminetetraacetic acid (EDTA) Sigma-Aldrich glucose Sigma-Aldrich isopropanol Roth Hepes buffer Gibco magnesium chloride (MgCl2) Roth magnesium chloride hexahydrate (MgCl2∗6H2O) Roth PBS Dulbecco w/o Ca, Mg (Instamed 9,55 g/l) Biochrom potassium chloride (KCl) Roth sodium bicarbonate (NaHCO3) Merck sodium chloride (NaCl) Sigma-Aldrich sucrose Merck tryptone/peptone from casein Roth yeast extract Roth water HPLC (2,5 L) Fisher Scientific

22 2.2 Cloning and plasmid purification

2.2 Cloning and plasmid purification

Table 2.2: Protocols for culture media, agar plates and buffers medium composition antibiotics

LB medium 0.5 % yeast extract, 1 % tryptone/peptone, 100 µg/mL ampicillin 1 % NaCl, pH 7.4 or kanamycin LB agar plate 1.5 % agarose with LB medium 100 µg/mL ampicillin or kanamycin SOB medium 0.5 % yeast exctract, 2 % tryptone/peptone, 0.05 % NaCl, 25 mM KCl, pH 7.4

SOC medium 20 mM MgCl2, 20 mM glucose in SOB medium stimulation buffer 138 mM NaCl, 6 mM KCl, 1 mM [MgCl2*6H2O], 5.5 mM glucose, 20 mM HEPES,

1 mM [CaCl2*2H2O], 1 mM IBMX

Table 2.3: Consumables for cloning compound supplier

BIOTAQ DNA Polymerase Bioline CutSmart buffer NEB dNTPs Invitrogen NH4 buffer Bioline MaCl Bioline Calf Intestine Phosphatase (CiP) NEB T4 Ligase NEB T4 buffer NEB Ampicillin Sigma-Aldrich Kanamycin Sigma-Aldrich

Table 2.4: Enzymes for cloning compound supplier

EcoR I NEB SpeI NEB Aat II NEB AgeI NEB XhoI NEB BamH I NEB

23 CHAPTER 2. MATERIALS

Table 2.5: Kits for cloning and plasmid purification kit supplier

NucleoBond Xtra Midi (100) Macherey-Nagel PureYield Plasmid Miniprep System Promega PureYield Plasmid Midiprep System Promega TA Cloning Kit Life Technologies Wizard® SV Gel and PCR Clean-Up System Promega

2.3 Cell culture, transfection reagents and functional assays

Table 2.6: Consumables for cell culture compound supplier

6-well plate BD Falcon Corning 96-well plate BD Falcon Corning 96-well plate, white Costar Corning AdvancedMEM Life Technologies Dulbecco’s modified Eagle’s medium Biochrom (4,5 g/L D-glucose, with stable L-glutamin) fetal bovine serum (FBS) Biochrom FluoroDish (∅35 mm) World Precision Instruments MEM with sable L-glutamin Biochrom non-essential amino acids Biochrom Penicillin/Streptomycin 10.000 E/10.000 µg/ml Biochrom Poly-L-lysin Biochrom T25 flask Sarstedt T75 flask Sarstedt Trypsin/EDTA Solution (10X) Biochrom

24 2.3 Cell culture, transfection reagents and functional assays

Table 2.7: Consumables for transfection and functional assays compound supplier

1x passive lysis buffer Promega 3-Isobutyl-1-methylaxanthine (IBMX, 500mM) Sigma-Aldrich ABsolute QPCR Mix, SYBR Geen Thermo Scientific AlphaScreen™ cAMP Assay Kit PerkinElmer Life Science DNase I BioLabs DNase I buffer BioLabs Forskolin (FSK) AppliChem GmbH Gene Juice Transfection Reagent EMP Millipore Corp 3-isobutyl-1-methylxanthine (IBMX) Sigma Luciferase Assay System Promega Metafectene Biontex Oligo dTs Promega Omniscript RT Quiagen Pierce BCA Protein Assay Kit Thermo Scientific Random Hexamers Applied Biosystems RNase free water Quiagen RNAsin®Plus RNase Inhibitor Promega TRIzol™ ligands

3-iodothyronamine (T1AM) Santa Cruz Biotechnology bovine TSH Sigma-Aldrich dopamine (DA) Sigma-Aldrich isoproterenol (ISOP) Sigma-Aldrich histamine (HIS) Sigma-Aldrich L-norepinephrine hydrochloride (NorEpi) Sigma-Aldrich (PEA) Sigma-Aldrich serotonin (5-HT) Sigma-Aldrich

25 CHAPTER 2. MATERIALS

2.4 Reagents for immunohistochemistry

Table 2.8: Reagents for immunohistochemistry compound supplier adhesive microscope slides HistoBond Marienfeld cover slides, thickness No. 1 VWR DAPI Roche porcine skin gelatin Fluka Triton X-100 BIO-RAD Tris Roth Vectashild Hardset Vektor Labortries

Table 2.9: target protein host dilution company anti-FOS rabbit 1:200 Santa Cruz Biotechnology Alexa Fluophor 594 anti rabbit IgG goat 1:200 Jackson ImmunoResearch

26 Chapter 3

Methods

3.1 Cell culture

Cells were kept at 37°C in humidified air containing 5 % CO2. Table 3.1 lists all used cell lines. HEK293 cells were cultured in MEM with 5 % fetal bovine serum (FBS) and non-essential amino acids. GT1-7 (mouse hypothalamic gonadotropin-releasing-hormone neuronal cell line) were pur- chased from MERK. mHypoE-N39 (N39) and mHypoE-N41 (N41), both embryonic mouse hypotha- lamic cell lines, were acquired from Cedarlane, established by Belsham et al.(2004). Screening profiles of neuronal markers of all three cell lines can be found online (https://www.cedarlanelabs. com/Products/Listing/Hypothalamic). All hypothalamic cell lines were cultured in Dulbecco‘s modi- fied Eagle‘s medium (DMEM), supplemented with 10 % FBS and 1 % penicillium/streptavidin.

3.2 Immunohistochemistry

Principle A marker of neuronal activation is the proto-oncogene FOS (FOS). Immunohistochemis- try staining of this marker was used to evaluate the effects of T1AM on the murine brain.

Performance Mice brains were obtained from the Karolinska Institute, Sweden, Department of Cell & Molecular Biology with generous support from Carolin Höfig and Jens Mittag. Stockholm‘s Norra Djurförsöksetiska Nämnd gave ethnic approval under the European Community Council Di- rectives (86/609/EEC). C57BL/6J mice (n = 3) were intraperitoneal (i.p.) injected with 50 mg/kg body weight T1AM solved in 60 % DMSO and 40 % PBS. The volume of injection was 5 µl/g body weight. Control mice were only injected with solvent. After 60 min, mice were transcardially perfused with PBS and 10 % formalin. Mice were dissected, and brains were stored at 4°C. In Berlin, brains were placed successively in 10 %, 20 %, and 30 % sucrose until they reached a higher density and sank to the bottom of the container. They were flash frozen in liquid nitrogen and stored at -20°C until cryosectioning. The brains were cut into 20-25 µm slices with a cryotome (Leica CM 1950). The slices were dried at room temperature for 30 min up to 1 h. Samples were fixed in -20°C cold acetone, washed three times with PBS for 1 min and incubated for 2 h with SUMI buffer (TBS, 0.25 % porcine skin gelatin, 0.5 % Triton X-100) to reduce background fluorescence. The primary was diluted with SUMI buffer and incubated overnight at 4°C. After one further washing step, the secondary antibody was incubated for 2 h at RT. In the end, the brain slides were stained with DAPI for 5 min, washed thoroughly 3 times with TBS and mounted with Vectashild.

27 CHAPTER 3. METHODS

Table 3.1: Cell lines cell lines characteristics

HEK293 human embryonic kidney 293 cells were generated via the transfection of human embryonic kidney cells with sheared DNA of the adenovirus 5, integration of 4.5 kilobase pairs of the virus DNA into the HEK293 genome, adherend growing cells with fibroblast-like morphology mHypoE-N39 murine embryonic hypothalamic cell line N39 was generated by retroviral transfer of the simian virus (SV40) large T-antigene into isolated embryonic neurons by the pZIPNeo SV(X) 1 vector. N39 are adherent growing cells and express the single- minded homologe 1 (SIM1) besides many other hypothalamic markers. mHypoE-N41 murine embryonic hypothalamic cell line N41 was generated by the retroviral transfer of the simian virus (SV40) large T-antigene into isolated embryonic neu- rons by the pZIPNeo SV(X) 1 vector. N39 are adherent growing cells and express the pro- Y (NPY) besides many other hypothalamic markers.

GT1-7 murine mature hypothalamic gonadotropin-releasing hormone (GnRH) neuronal cell line Transgenic mice were generated by coupling the promoter region of GnRH with the coding region of SV40 T-antigene oncogene. Resulting tumors were re- moved and screened for GnRH. GT1-7 are adherent and secrete GnRH in response to depolarization.

After a drying period, slides were photographed with the Keyence BZ-9000 microscope. In Tab. 2.4 the primary and secondary antibody are listed with host species and dilution factors. FOS positive cells were counted with the ImageJ software using two brain slides of each animal and for each brain loci. For the FOS stainings of hypothalamic cell lines, cells were seeded onto poly-L-lysine coated glass slides, and after 48h of cultivation, cells were stimulated either with 0.1 % DMSO or 10-5 M

T1AM in 0.1 % DMSO for 60 min. Cells were fixated in acetone at -20°C for 10 min. Afterward, the immunostaining protocol as stated above was carried out.

3.3 Cloning

A general overview of the cloning workflow can be seen in Fig. 3.1

Principle For this work, the HRH1, and the β-TAAR1 were cloned into the eukaryotic expression vector pcDps (provided by Torsten Schöneberg, University of Leipzig, Germany). 5-HT1b and β- TAAR1 were cloned into the FRET vectors pEYFP-N1 and pECFP-N1 (Clontech Laboratories, USA). All other plasmid constructs were already available in the laboratory. A detailed list of all used or cloned plasmids with genes and antibiotic resistance is presented in Tab. A.1. Primers are listed under Tab. A.2. Gene sequences were taken from NCBI (https://www.ncbi.nlm.nih.gov/) and UCSC Genome Browser (https://genome.ucsc.edu/). Restriction analysis was performed with NEBcutter2 (https://tools.neb.com/NEBcutter2/). The primers were designed with Primer3 (https://primer3.ut. ee/).

28 3.3 Cloning

Figure 3.1: Cloning workflow An amplification PCR was followed by specific digestion. The target vector was digested the same way with additional CIP treatment. After ligation, competent E. coli were transformed with the new vector constructed. Antibiotic resistance is used to identify potential positive clones, which is verified by analytical restriction and Sanger sequencing.

29 CHAPTER 3. METHODS

Different tags were attached to TAAR1 to improve cell surface expression. As β-tag, the first nine amino acids of the β-adrenergic receptor 2 (ADRB2, MGQPGNGSA ) were added N-terminally (Barak et al., 2008; Liberles and Buck, 2006) and as Rho-tag the last nine amino acids of the bovine rhodopsin (TETSQVAPA ) were added C-terminally. For cAMP and luciferase assays, the receptors were cloned into the eukaryotic expression vector pcDps. For FRET, the genes were cloned into pECFP-N1 and pEYFP-N1.

3.3.1 PCR amplification

A PCR was used to amplify genes and thereby adding restriction sides and tags.

PCR mix: 1 µL (< 1000 ng) template 1 x NH4 buffer 2.5 mM MgCl 0.1 mM dNTPs 0.5 µM forward primer 0.5 µM reverse primer 2.5 U BIOTAQ polymerase x µL aqua bidest.

Σ = 50 µL PCR mix

temperature program: ⟳ | 1. 95°C → 1 min | 2. 58°C → 1 min 32x | 3. 72°C → 1 min 4. 72°C → 10 min hold 4°C

The fragments were purified via gel electrophoresis and the Wizard® SV Gel and PCR Clean-Up System by Promega, a silica membrane based affinity chromatography.

3.3.2 Restriction and ligation

Restriction Restriction protocols were generated with the Double Digest Finder tool by New Eng- land Biolabs (https://nebcloner.neb.com/#!/redigest). The vectors were additionally digested with a calf intestine phosphatase (CIP) for 30 min as a dephosphorylation step to prevent religation.

general restriction protocol:

30 3.4 Transfections protocols

1 µg template cDNA 5-10 U restriction enzyme 1 x suitable buffer x µL aqua bidest.

Σ = 50 µL restriction mix incubation for 1 h + 1 µL CIP to the vector for 30 min

Ligation In total 50 ng of DNA were used for the ligation in a ratio of 5 : 1 or 3 : 1 (fragment : vector). The ligation mix was incubated at room temperature overnight.

x µL fragment x µL vector 1 x T4 buffer 400 U T4 ligase

Σ = 20 µL ligation mix

3.3.3 Transformation of E. coli

Performance Competent E. coli were transformed with the ligation mix via heat shock. These cells were frozen with a rubidium chloride solution to permeabilize them for foreign DNA (Hanahan et al., 1991). The cells were thawed on ice for 10 min. Afterward, the ligation mix was gently added to the cells. The solution was incubated for 30 min on ice. Then a heat shock was performed at 42°C for 90 s. 250 µL SOC media was added, and the cells were incubated for 1 h at 37°C. In this resting period, the cells start to produce the antibiotic resistance enzyme. The cells were plated onto an agar plate with ampicillin or kanamycin to select positively transfected cells. The plates were incubated overnight at 37°C. Supposing positive clones were picked and cultured for 8 - 12 h in LB medium. After plasmid isolation with the PureYield Plasmid Miniprep System, an analytical restriction and subsequently sequencing was done to verify the clone. Plasmid stocks were kept at -20°C and dilutions of 100 ng/mL were stored at 4°C.

3.3.4 Plasmid preparation and sequencing

Performance To generate a sufficient amount of plasmid DNA, 250 - 400 mL LB medium was inoc- ulated with transformed E. coli and incubated overnight at 37°C on a shaking platform. Afterward, the plasmid was purified using the PureYield Plasmid Midiprep System or the NucleoBond Xtra Midi kit. Plasmids were sequenced and verified by BigDye-terminator sequencing (PerkinElmer, Inc., Waltham, MA, USA), using an automatic sequencer (ABI 3710xl; Applied Biosystems).

3.4 Transfections protocols

An overview of the transfection and plasmid used for the specific experiments is presented under Tab. 3.2.

31 CHAPTER 3. METHODS

3.4.1 Metafectene

Principle Metafectene is a liposomal transfection reagent, leading to the complexation of plasmid DNA and liposomes. Liposomes are internalized through endocytosis. After endosome degrada- tion, the plasmid DNA is freed and enters the nucleus during the next cell division. This transfection method was used for cAMP and luciferase assays.

Performance HEK293 cells were seeded on poly-L-lysine coated 96 well plates (1.5 × 104 cells/well). For every transfections sample 50 µL Advanced MEM were pre-incubated with 0.45 µL Metafectene for 5 min. Meanwhile, 0.45 ng DNA per plasmid were added to 50 µL advanced MEM and finally pooled with the Metafectene pre-mix. After another 15 min, the transfection mix was added to the cells. After 48 h the cells were used for functional assays.

3.4.2 GeneJuice

Principle GeneJuice is a polyamine reagent that forms complexes with DNA due to its polar prop- erties. As for Metafectene, these complexes are absorbed through endocytosis.

Performance For FRET experiments HEK293 were seeded onto poly-L-lysine coated FluoroDishes (∅35 mm) in concentrations of 1 x 105 cells per dish. Twenty-four hours later, 1 µg DNA and 4.5 µL GeneJuice per dish were incubated in 100 µL additive-free MEM for 10 min and then added to the cells. On the next day, the medium was changed to complete medium. 72 h after transfection cells were used in functional assays.

Table 3.2: Transfection

Metafectene

HEK293 Alpha Screen™ pcDps HEK293 reporter gene assay pGL4.33 [luc2/SRE/Hygro] + pcDps or pGL4.3 [luc2/NFAT-RE/Hygro]

GeneJuice

HEK293 FRET peYFP-N1, peCFP-N1

3.5 Evaluation of GPCR and TRP expression via quantitative PCR

A workflow of all required steps in a qPCR is shown in Fig. 3.2.

3.5.1 Total RNA isolation via TRIzol™

Performance Cell lines GT1-7, N39, and N41 were cultured over several passages. Between pas- sage 3-7 three samples were taken from every cell line. Cells were trypsinated in a T75 flask at an 80 % confluence, washed and centrifuged. The supernatant discarded. 0.5 mL of TRIzol™ was added, and the solution was vortexed. 0.1 mL chloroform was added, the solution was vortexed and incubated for 3 min at room temperature. Afterward, the samples were centrifuged at 12000 x g for 15 min at 4°C. RNA remained in the upper aqueous phase and was gently pipetted into a new 1.5 mL

32 3.5 Evaluation of GPCR and TRP expression via quantitative PCR

Figure 3.2: qPCR workflow Cells were cultivated in T75 flasks up to 80 % confluency. Total RNA was isolated by TRIzol. Genomic DNA was digested, followed by reverse transcription of mRNA to cDNA. Samples with (+RT) and without (-RT) reverse transcription were added together with sequence-specific primers and SYBR Green mix onto a 96-well plate. After the run melting and amplification curves are validated. In the end, data were evaluated with the Δct method.

33 CHAPTER 3. METHODS tube. 0.25 mL isopropanol was added and incubated for 10 min at room temperature. Finally, the solution was centrifuged at 12000 x g for 10 min at 4°C. As a washing step, the supernatant was dis- carded, 0.5 mL 75 % ethanol was added, and samples were again centrifuged (7500 x g, 5 min, 4°C). This washing step was repeated once. Ethanol was removed entirely, and the pellet was air-dried for 10 min. As for the last step the pellet was dissolved in 30 µL HPLC H2O. RNA samples were either stored at -80°C or used for DNase digestion.

3.5.2 DNase digestion

A DNase digestion was performed to remove remaining genomic DNA from the cell line total RNA.

10 µL RNA 1 µL DNase 89 µL DNase buffer

Σ = 100 µL DNase digestion mix

The DNase digestion mix was incubated for 30 min at 37°C. 2.5 µL of 0.5 M EDTA was added, and the solution was heat inactivated for 10 min at 75°C. RNA samples were stored at -80°C.

3.5.3 cDNA synthesis

The Omniscript RT kit by Qiagen was used according to manufactures protocol to generate cDNA from the RNA samples. For each batch of RNA with reverse transcriptase, one was performed without enzyme added to test if the samples still contained genomic DNA, despite DNase digestion.

cDNA mix: 12 µL RNA 2 µL RT buffer 2 µL dNTPs 2 µL oligo-dT 1 µL RNase inhibitor 1 µL reverse transcriptase

Σ = 20 µL cDNA mix

The cDNA mix was incubated for 60 min at 37°C and stored at -20°C.

3.5.4 Quantitative SYBR Green PCR

Principle A quantitative PCR (qPCR) was performed to determine the expression level of several GPCRs and TRPs. With a reference gene, in this case, Pgk1, the gene expression levels can be quan- tified relatively. During the qPCR, SYBR Green dye is used, which is only able to emit light when it intercalates in double strand DNA. In theory, the SYBR Green signal intensity doubles after every amplification cycle. However, the amplification efficiency is often lower due to primer specificity and secondary structures. Therefore, I used the slope of a standard curve to determine the am- plification efficiency for each primer pair (E = 10-1 / slope). QPCR primers including their efficiency are listed in supplementary Tab. A.4. Pgk1 was chosen as the reference gene, as previously recom- mended (Boda et al., 2009).

34 3.6 Monitoring of Gs and Gi/o signaling transduction via cellular cAMP

Performance mRNA was transcribed into cDNA by the Omniscript RT kit using random hexam- ers and OligodTs. Absolute QPCR Mix, SYBR Green, no Rox was used for qPCR on a Stratagene Mx3000P System using 100 nM per primer. The temperature program started with 15 min at 95°C for enzyme activation, followed by 42 cycles of a denaturing step for 15 s at 95°C, followed by an annealing phase for 30 s at 60°C and an elongation step at 72°C for 30 sec. Melting curve analysis was performed to confirm the specificity of the PCR reaction. After the run, the fluorescence in- tensity was set to a threshold (intensity = 5000), which determines the cycle threshold. The relative ct expression of a gene is then calculated using the Δct method (E = Ect (reference) /E (target) ). Each of the three passages of every cell line was measured in duplicates (+RT) together with a sample without reverse transcription (–RT) to exclude genomic DNA contamination.

3.6 Monitoring of Gs and Gi/o signaling transduction via cellu- lar cAMP

A workflow of the cAMP assay is presented in Fig. 3.3.

Principle Activation of Gs signaling leads to the increase in cellular cAMP content, while activation of Gi/o signaling leads to a decrease. Gs and Gi/o signaling were determined by measuring cAMP accumulation using the Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen™ tech- nology). The AlpaScreen™ technology kit is a bead-based competitive chemiluminescent assay to quantify cellular cAMP concentrations. The acceptor bead is coated with antibodies against cAMP, while the donor bead recognizes biotin. If a biotinylated cAMP molecule binds both, acceptor and donor, the beads are spatially close enough so that a singlet oxygen is transferred from the donor to the acceptor upon excitation at 680 nm. The singlet oxygen reacts with thioxene compounds and emits a chemiluminescent signal at 370 nm within the acceptor bead. This signal excites a fluorophore in the same acceptor bead, which then shifts the emission and therefore the detection wavelength to 520-620 nm. The addition of lysed cells leads to a competition of endogenous and biotinylated cAMP at the acceptor beads. Therefore, high cellular level of cAMP leads to a low signal, while low concentrations lead to a high signal at 520-620 nm.

Performance 48 hours after Metafectene transfection, cells were stimulated using a stimulation -5 -12 buffer and the considered ligands in the desired concentration (between 10 - 10 M). For Gi/o pathway examination, cells were additionally co-stimulated with 50 µM forskolin to activate the adenylyl cyclase. Substance incubation was performed at 37°C with 5 % CO2 and stopped after 20 or 45 min by removing the medium. Cells were lysed at 4°C on a shaking platform. Intracellular cAMP accumulation was determined by the AlphaScreen™ assay according to manufactures pro- cedures and measured using a Berthold Microplate Reader. If cAMP values exceeded the possible measurement range, samples were diluted. The results of the hypothalamic cell lines were additionally normalized to the protein content to compare the different cell types with each other. Protein concentrations were measured with the Pierce BCA Protein Assay Kit.

35 CHAPTER 3. METHODS

Figure 3.3: Workflow for an AlphaScreen™ Technology based cAMP assay HEK293 were seeded onto a 96- well plate (1.5 x 10-4 cells per well). On the next day, cells were transfected with Metafectene. 48 h later, cells were stimulated with ligands in stimulation buffer for 45 min (Gαs) or 25 min (Gαs). After lysis cellular cAMP amounts were quantified using a competitive luminescence assay (AlphaScreen™ Technology). Exogenous biotinylated cAMP and endogenous cAMP compete for the binding to the donor bead. If a donor bead is binding to a biotinylated cAMP molecule, an acceptor bead binds to the biotin, which brings it in close proximity to transfer one singlet oxygen. It reacts with thioxene compounds of the acceptor bead, which results in a luminescent signal at 370 nm wavelength. Therefore, a high endogenous cAMP amount leads to a low signal at 370 nm, a low amount leads to a high signal. CAMP amounts can be quantified by using a cAMP standard curve. 36 3.7 Luciferase assays to determine PLC and MAPK activation

3.7 Luciferase assays to determine PLC and MAPK activation

Principle Luciferase assays are based on a reporter plasmid containing a luciferase and a reporter element as a promoter. Reporter elements are specific, short and repetitive sequences known to be activated via distinct transcription factors at the end of a signaling cascade. Upon receptor activation, the luciferase is produced and converts its substrate luciferin to oxyluciferin, under the consumption of O2 and ATP. Thereby light is emitted, which can be quantified by a microplate photometer. The serum response element (SRE) was used for the MAPK signaling pathway and the nuclear factor of activated T-cells (NFAT) response element for PLC activation. For an excessive review describing signal cascades and their response elements see Cheng et al.(2010).

Performance HEK293 were co-transfected with a reporter plasmid firefly luc reporter gene (pGL 4.33 [luc2/ SRE/Hygro], pGL4.3 [luc2/NFAT-RE/Hygro]) and either a receptor or an empty vector (mock transfection) in an equimolar concentration (0.45 µg per plasmid per well). The thyrotropin (TSH) receptor served as a positive control and was stimulated with 100 mU/ml bovine TSH. 48 hours post-transfection, cells were incubated for 6 hours with respective ligands (at 10 µM) in supplement- free MEM at 37°C with 5 % CO2. The media was removed, and cells were lysed for 15 minutes on a shaking platform at room temperature using 50 µl of a 1 x passive lysis buffer. 10 µL sample was transferred to a black 96-well plate to determine the luciferase activity. 40 µL luciferase substrate was automatically injected using the Berthold Microplate Reader and immediately measured.

3.8 Protein-Protein-Interaction measured by Fluorescence Res- onance Energy Transfer (FRET)

Principle The Fluorescence Resonance Energy Transfer (FRET) describes the energy transfer of two close spatial fluorophores (see Fig. 3.4). The emitted light indicates the proximity between two fluorophores. If the fluorophores are linked to proteins, one can detect proximities between proteins to investigate protein-protein-interactions. The method takes advantage of overlapping emission and absorption spectra of two fluorophores, in this case, CFP and YFP. When CFP is excited, it emits light at 475 nm, which in turn can be absorbed by a YFP near the CFP molecule. The now activated YFP emits light in the wavelength of 530 nm. This is called Fluorescence Resonance Energy Transfer (FRET). To measure FRET, I employed the acceptor bleaching method as previously described (Tarnow et al., 2008): YFP is photobleached leading to the disruption of the energy transfer from CFP to YFP. The photobleaching would result in an increasing CFP signal if a FRET signal was present.

Performance A CFP-tagged receptor and a YFP-tagged receptor were co-transfected. Cells with the expression of CFP and YFP-tagged receptors were picked 72 h after transfection. Subsequently, YFP was photo-bleached at 512 nm for 0.5 s for 10 cycles, followed by 20 cycles of 6 s bleaching while measuring CFP and YFP emissions. The increased CFP-emission was measured at excitation at 410 nm for 0.5 s. For evaluation, the first data point of the YFP intensity was set to one, and on this base, all other values were converted. In the case of CFP, the last measurement was set to one, and all other measurements related to this point. FRET efficiency was calculated as follows:

E = (FCFPmax – CFPmin)/FCFPmax. A stable protein-protein interaction is defined by a FRET efficiency

37 CHAPTER 3. METHODS

Figure 3.4: Workflow for FRET experiments Cells are seeded and transfected with either CFP, and YFP tagged receptors. Energy is transferred from CFP to YFP if these molecules are close, which reduces CFP emission and increases YFP fluorescence. After destroying YFP through photobleaching, the energy transfer is disrupted leading to an increase in the measured CFP intensity if there was a FRET interaction between two tagged proteins.

38 3.9 Statistical Evaluation between 8 and 25 % (Rediger et al., 2009). To ensure protein-protein interaction also vice versa experiments were performed. MC3R and GHSR were used as positive control (Rediger et al., 2011), MC3R and CB1R as negative and rCHRM3 as non-interacting protein (NIP).

3.9 Statistical Evaluation

GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA) was used to visualize and analyze the data. Experimental data is presented as the mean ± standard error of the mean (SEM). For the comparison of brain loci, a multiple t-test (one per row) was used. For column graphs, statistical analysis was performed by a one-way ANOVA, followed by Tukey’s honest significant difference post hoc test. To analyze receptor co-expression (grouped), a two-way ANOVA was performed, followed by Sidak’s multiple comparisons test. The statistical significance was set to *p ≤ 0.05, **p ≤0.01, ***p ≤ 0.001 and ****p ≤ 0.0001.

39 Chapter 4

Results

The thesis intended to identify new possible thermoregulatory targets for T1AM and to determine further on which structure in the brain T1AM acts. Therefore, this study aimed at answering the following questions: Is T1AM a ligand for HRH1 and 5-HT1b? If so, what signaling is induced by T1AM? Does this mechanism of action reflect the in vivo effects of T1AM? Where in the brain does T1AM stimulate neuronal activity? Moreover, can matching cell lines give conclusions about the molecular mechanism?

4.1 Part I: T1AM and the aminergic GPCRs HRH1 and 5-HT1b

In part I, the hypothesis was tested whether T1AM as a multi-target ligand also interacts with sero- tonin and histamine GPCRs. From a structural point of view, endogenous aminergic ligands possess an amino side chain, just like T1AM. Therefore, I investigated if GPCRs involved in thermoregula- tion, such as HRH1 and 5-HT1b are targets of T1AM. An overview of G-protein signaling induced by TAAR1, HRH1, and 5-HT1b is displayed in Fig. 4.1. Furthermore, this figure shows which assays were applied to the respective receptor.

4.1.1 Establishment of βTAAR1

4.1.1.1 Concentration-response curves of βTAAR1 and several ligands

It is known that TAAR1 is expressed at very low levels in the cells (Barak et al., 2008). Therefore it is an established procedure for transient transfection experiments, that an N-terminal extension is introduced into TAAR1 to enhance cell surface expression and thereby signaling capacity. For FRET measurements, cAMP and luciferase assays the human TAAR1 was tagged at the N-terminus with the first nine amino acids of the β-2 adrenoreceptor (ADRB2), resulting in β-TAAR1, which according to Barak et al.(2008) enhances protein expression. They could show that this modi fication only leads to a higher membrane expression of the receptor and does not alter the signaling properties. I compared the β-tag to a C-terminal rhodopsin-tag (Rho-tag) concerning their signaling upon

PEA or T1AM stimulation to confirm this statement. Further, I tested if serotonin (5-HT) is capable of activating human TAAR1. Therefore, HEK293 were stimulated with different concentrations (10-5 - -11 10 M) of PEA, T1AM, and 5-HT. In the lower concentration range of PEA from 10-9 M to 10-7 M, the cells over-expressing Rho- TAAR1 or β-TAAR1 showed no difference to the mock control (Fig. 4.2 A). At a PEA concentra-

40 4.1 Part I: T1AM and the aminergic GPCRs HRH1 and 5-HT1b

Figure 4.1: Hypothesis and experimental steps in the elucidation of the role of T1AM at HRH1 and 5-HT1b Overview shows the signaling properties of the receptors TAAR1, HRH1 and 5-HT1b, and their endogenous ligands. Furthermore, it shows which experiments have been carried out for T1AM and the corresponding receptor.

41 CHAPTER 4. RESULTS

Figure 4.2: β-TAAR1 results in a higher cAMP signal without a shift in the IC50 value Cells were transfected via Metafectene either with pcDps as mock control, Rho-TAAR1 or β-TAAR1. The AlphaScreen™ kit was used to evaluate cAMP levels. (A) Cells were stimulated with PEA in a concentration range from 10-9 M until 10-5 M for 45 min (n = 5 measured in triplicates). Cells transfected with β-TAAR1 showed a higher cAMP accumula- tion compared mock transfection then Rho-TAAR1 expressing cells. (B) Cells were stimulated with T1AM in a concentration range from 10-8 M until 10-5 M for 45 min (n = 5 measured in triplicates. β-TAAR1 transfection resulted in a higher cAMP accumulation compared to Rho-TAAR1 transfection. (B) Mock and β-TAAR1 trans- fected cells were stimulated with 5-HT1b (10-11 M until 10-5 M, n = 4 measured in triplicates). Data are shown as ± SEM. As statistical analysis, a Two-way ANOVA was performed, comparing each cell mean with the mean of pcDps on that row. Statistical significance was defined as to *p 0.05, **p 0.01, ***p 0.001 and ****p ≤ ≤ ≤ 0.0001. ≤

42 4.1 Part I: T1AM and the aminergic GPCRs HRH1 and 5-HT1b tion of 10-6 M Rho-TAAR1 reached a cAMP accumulation of 7.9 ± 1.0 nM compared to β-TAAR1 with 22.4 ± 2.0. At the highest PEA stimulation (10-5 M), Rho-TAAR1 over-expression leads to a cAMP ac- cumulation of 10.3 ± 1.2 nM, while β-TAAR1 expressing cells had a cAMP content of 27.5 ± 2.9 nM. In comparison to Rho-TAAR1, β-TAAR1 showed a higher cAMP response to a PEA concentration- response curve without shifting the EC50 value (0.54 µM for Rho-TAAR1 vs. 0.40 µM for β-TAAR1).

Determination of EC50 values was not possible for T1AM stimulation (Fig. 4.2 B), as the highest -7 concentration did not reach saturation of the receptors. T1AM-stimulation with 10 M or lower did not lead to Gαs activation of either mock control, Rho-TAAR1 or β-TAAR1. At a stimulation concentra- -6 tion of 10 M of T1AM, Rho-TAAR1 transfected cells reached 7.7 ± 0.6 nM cAMP and β-TAAR1 trans- -5 fected cells 30.4 ± 5.1 nM cAMP. At 10 MT1AM, Rho-TAAR1 showed a cAMP content of 20.7 ±2.7 nM and β-TAAR1 of 94.0 ± 14.9 nM. So far, 5-HT activation has only been shown for the rat TAAR1. Therefore I tested 5-HT stim- ulation at the human TAAR1. In comparison to a mock control, cAMP concentrations for β-TAAR1 were slight increased between stimulation concentrations of 10-10 M and 10-8 M (2.49 ± 0.14 nM for mock control and 3.92 ± 0.26 nM for β-TAAR1). CAMP levels started to rise for β-TAAR1 transfected cells between 5-HT concentrations of 10-7 M and 10-5 M without reaching saturation. At the highest stimulation concentration, β-TAAR transfected cells showed a 2-fold increase in cAMP compared to the mock control (3.16 ± 0.17 nM cAMP for mock and 6.35 ± 0.54 nM cAMP for β-TAAR1).

Conclusion: The β–tag increased TAAR1-mediated cAMP accumulation and therefore improves

its cell surface expression without changing the EC50 value of the original receptor. Subsequently, β-TAAR1 was used for all further assays and will be referred to as TAAR1. Human TAAR1 was only

slightly activated by 5-HT compared to its other ligands PEA and T1AM.

4.1.1.2 Co-stimulation of serotonin and T1AM at the TAAR1

Here, TAAR1-transfected cells were stimulated with 10 µM T1AM resulting in cAMP accumulation that is significantly higher than basal signaling (Fig. 4.3 A). Mock transfection did not lead to a change in cAMP amounts due to T1AM-stimulation (Suppl. Fig. C.1 A). Even though 5-HT in the concentration-response curves slightly activated TAAR1, here the co-stimulation did not enhance

Gαs signaling. Besides, an antagonistic activity of 5-HT at TAAR1 unlikely as co-stimulation of T1AM and 5-HT in equimolar concentrations resulted in signaling identical to T1AM stimulation alone (Figure 4.3 A).

To measure Gαi/o, HEK293 were pretreated with forskolin to elevate basal cAMP concentrations, as this G-protein inhibits the adenylate cyclase, which decreases cellular cAMP. In parallel, the 5- HT1b receptor was stimulated with 5-HT (10 µM) as a positive control (Fig 4.5 B). This reduced the forskolin-activated adenylate cycles to 50 % activity. TAAR1 stimulated with 10 µM 5-HT, or T1AM revealed no Gαi/o signaling (Fig. 4.3 B), an effect that is comparable to mock-transfected cells (Suppl. Fig. C.1 B).

4.1.2T 1AM as an antagonist at the histamine 1 receptor (HRH1)

T1AM has been reported to modulate the histaminergic system (Laurino et al., 2015). Therefore, I first investigated if T1AM is a ligand for the histamine 1 receptor (HRH1). HRH1 is a Gαq coupled GPCR. Its signaling can be measured via an NFAT luciferase assay. Upon Gαq activation, PLC is activated and induces the conversion of phosphatidylinositol-triphosphate to inositol triphosphate 2+ (IP3). IP3 leads to a Ca influx followed by the activation of the transcription factor NFAT. HRH1

43 CHAPTER 4. RESULTS

Figure 4.3: Co-stimulation of T1AM and 5-HT does not enhance Gαs signaling at TAAR1 The cAMP content was determined via AlphaScreen™ technology to measure Gαs and Gαi/o. HEK293 were used to over-express

TAAR1. An empty vector was used for mock transfection, which showed no endogenous effect of 5-HT or T1AM

(Suppl. Fig. C.1 A, B). (A) TAAR1 was stimulated with either 5-HT, T1AM or both in a concentration of 10 µM, n = 7 measured in triplicates. (B) TAAR1 was stimulated with forskolin and either 5-HT or T1AM or both in a concentration of 10 µM. Results of n = 4 independent experiments performed in triplicates are shown. For statistics, a one-way ANOVA was performed, and the mean of each column compared with the mean of all other columns. Statistical significance was defined as **p ≤ 0.01, ****p ≤ 0.0001. was activated by its endogenous ligand (10 µM) up to 1513000 ± 222771 RLU, while co-stimulation of HIS and T1AM reduced the Gαq signal to 750377 ± 120300, which is a reduction of nearly 49.6 % (Fig. 4.4). T1AM stimulation alone was not different from the basal RLU, which makes T1AM an antagonist for HRH1.

Conclusion: T1AM was an antagonist of the HRH1.

4.1.3T 1AM is a biased ligand of the serotonin 1b receptor (5-HT1b)

4.1.3.1T 1AM activates Gαi/o signaling at the 5-HT1b

5-HT1b over-expressing cells were treated with the endogenous agonist 5-HT as well as with T1AM at a concentration of 10 µM to investigate GPCR signaling (Fig. 4.5 A). 5-HT1b is known to predom- inantly signal via Gαi/o (Bouhelal et al., 1988). Activation of Gαs is not observed due to stimulation by either 5-HT or by T1AM (Fig. 4.5 A). 5-HT induces a strong activation of Gαi/o determined as a 50 % reduction of forskolin-activated adenylyl cyclase (Fig. 4.5 B). Most remarkably, T1AM also activates 5-HT1b by reducing adenylyl cyclase activity by 30 % (Fig. 4.5 B). It should be noted that no synergistic effect of both ligands is observed and the reduction of cAMP accumulation is com- parable to stimulation with 5-HT alone (Figure 4.5 B).5-HT1b transfected cells were incubated with pertussis toxin (PTX), inhibiting Gαi/o signaling to ensure that T1AM indeed activates Gαi/o. After PTX-treatment, signaling mediated by 5-HT1b is completely abolished for both ligands (Suppl. Fig. C.2 A).

Conclusion : T1AM induced a PTX-sensitive Gαi/o signaling through the 5-HT1b.

44 4.1 Part I: T1AM and the aminergic GPCRs HRH1 and 5-HT1b

Figure 4.4: T1AM (10 µM) reduces histamine signaling at the HRH1 To measure Gαq PLC activation was measured via NFAT luciferase reporter. HEK293 were used to over-express HRH1. An empty vector was used for mock transfection, which showed no endogenous effect of HIS or T1AM (Suppl. Fig. C.1 C). HRH1 was stim- ulated with either HIS, T1AM or both in a concentration of 10 µM, n = 3 independent experiments performed in triplicates. Co-stimulation reduced the signaling of the endogenous ligand significantly. For statistics, a one-way ANOVA was performed, and the mean of each column compared with the mean of all other columns. Statistical significance was defined as ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 4.5: The TAAR1 agonist T1AM activates Gαi/o signaling through 5-HT1b. The cAMP content was determined via the AlphaScreen™ kit to measure Gαs and Gαi/o. HEK293 were used to over-express 5-HT1b.

An empty vector was used for mock transfection, which showed no endogenous effect of 5-HT or T1AM (see

Suppl. Fig. C.1 A+B). (A) 5-HT1b was stimulated with either 5-HT, T1AM or both in a concentration of 10 µM, n = 5 measured in triplicates. (B) 5-HT1b was stimulated with forskolin and either 5-HT, T1AM or both in a concentration of 10 µM, n = 13 measured in triplicates. For statistics, a one-way ANOVA was performed, and the mean of each column was compared with the mean of all other columns. Statistical significance was defined as ***p ≤ 0.001, ****p ≤ 0.0001.

45 CHAPTER 4. RESULTS

Figure 4.6: 5-HT, but not T1AM, activates PLC and MAPK signaling at the 5-HT1b For the luciferase assays, cells were stimulated with either 5-HT, T1AM or both in a concentration of 10 µM for 6 h in DMEM without

FCS. An empty vector was used for mock transfection, which showed no endogenous effect of 5-HT or T1AM on PLC or MAPK signaling (Suppl. Fig. C.1 C+D) As positive control HEK293 were transfected with TSHR and stimulated with TSH (100 mU/mL) for 6 h (Suppl. Fig C.2 D+E). (A) Activation of PLC was measured by NFAT reporter gene assay. HEK293 were used to overexpress 5-HT1b, n = 14 measured in triplicates. For statistics, a one-way ANOVA was performed, and the mean of each column was compared with the mean of all other columns. Statistical significance was defined as **p ≤ 0.001, ****p ≤ 0.0001.

4.1.3.2 Different signaling properties of 5-HT and T1AM at the 5-HT1b The 5-HT1b receptor is further described to activate MAPK and PLC signaling. Therefore I used luciferase response elements to track these signaling pathways. Mock-transfected cells show no activation for the two pathways upon 5-HT or T1AM challenge (Suppl. Fig. C.1 C+D).

4.1.3.3 PLC activation

5-HT1b can activate PLC signaling pleiotropically as a secondary pathway via Gβ/γ of Gαi/o (Dick- enson and Hill, 1998; Zhu and Birnbaumer, 1996). For this reason, I tested PLC signaling of 5-HT and T1AM at 5-HT1b and could confirm that 5-HT induced a 3.7-fold PTX-sensitive PLC activation (5530 ± 603 RLU for basal stimulation and 20221 ± 2509 RLU for 5-HT stimulation, Fig. 4.6 A and

Suppl. Fig. D.2 B+C). However, no PLC signaling was detected for T1AM (3332 ± 283 RLU for T1AM stimulation). Co-stimulation showed a reduced activation by a fold compared to 5-HT-stimulation alone (12282 ± 1462 RLU for co-stimulation).

4.1.3.4 MAPK signaling

It is known, that the Gβγ of Gi/o is capable of activating the MAPK pathway (Dickenson and Hill, 1998; Zhu and Birnbaumer, 1996). 5-HT-stimulation (10 µM) activated the SRE through the 5-HT1b about 1.8 fold (837622 ± 102545 RLU). However, it was not activated by T1AM (344934 ± 40262 RLU) compared to basal (459727 ± 48858 RLU). Again, co-stimulation showed a reduced luciferase activity compared to 5-HT stimulation (528292 ± 94357 RLU), which differed not significantly from the basal value.

Conclusion : In contrast to 5-HT, T1AM induced neither PLC activation nor MAPK signaling at the 5- HT1b receptor. This finding classifies T1AM as a biased ligand for the 5-HT1b. Co-stimulation with

46 4.1 Part I: T1AM and the aminergic GPCRs HRH1 and 5-HT1b

Figure 4.7: TAAR1 and 5-HT1b can constitute heteromeric complexes (A) To determine FRET efficiency, HEK293 over-expressed receptor pairs, one being CFP and the other YFP tagged and vice versa. The het- erodimer GHSR/MC3R was used as positive control, CB1R/MC3R as the negative control. Rat CHRM3 was used as non-interacting protein (NIP) for TAAR1. Compared to GHSR/MC3R dimers, the negative control and TAAR1/NIP showed no protein-protein-interactions, whereas TAAR1/5-HT1b did. Data were pooled from 11 to 19 cells, and a one-way ANOVA was performed, and the mean of each column was compared with the mean of all other columns. Statistical significance was defined as *p ≤ 0.001, **p ≤ 0.001, ****p ≤ 0.0001. (B) Photobleaching curve of CFP and YFP, pooled from TAAR1 and HTR1b vice versa experiments; after 10 basal cycles, photobleaching was started for 6 s intervals for another 20 cycles (start marked by arrow), n = 11 cells.

5-HT and T1AM led for both signaling pathway to a reduced activation indicating the competition of the both ligands for the binding pocket.

4.1.3.5 Confirmation of protein-protein-interaction of TAAR1 and 5-HT1b by FRET

A potential TAAR1/5-HT1b interaction or close spatial distance between the receptors was investi- gated by fluorescence energy resonance transfer (FRET). HEK293 were transfected with CFP-tagged TAAR1 and YFP-tagged 5-HT1b or vice versa. After initial basal measurement, YFP was bleached for 6 s for 20 cycles. After every bleaching step, YFP and CFP intensities were measured to calculate the FRET efficiency at the end. The ghrelin receptor (GHSR) and the receptor 3 (MC3R) are already described to form heterodimers (Tarnow et al., 2008) and therefore were used as positive control. On the other hand, GHSR and the 1 (CB1R) did not interact with each other and served as negative control. To further exclude false positive FRET results due to protein over-expression I transfected TAAR1 together with the rat muscarinic receptor 3 (rCHRM3) as non- interacting protein (NIP). All used receptors were further individually tested for FRET artifacts and bleaching efficiencies (Suppl. Fig. D.1). FRET efficiencies showed that the interaction between TAAR1 and 5-HT1b with an efficiency of 12.3 ± 1.8 % equals that of the already described heteromer GHSR/MC3R with an efficiency of 18.9 ± 1.8 %. The negative control and TAAR1 combined NIP have significantly lower FRET efficiencies (6.0 ± 1.5 % for negative control, 2.0 ± 2.0 % for TAAR1+NIP, Figure 4.7 A). Upon starting photobleach- ing of TAAR1/5-HT1b expressing cells, YFP intensity decreased to 42.7 ± 8.7 %, while CFP intensity increases from 85.9 ± 2.7 % to 100 % (Fig. 4.7 B). Photobleaching curves of the positive and negative controls are shown in Suppl. Fig D.2.

47 CHAPTER 4. RESULTS

Conclusion : FRET data suggested a protein-protein-interaction between TAAR1 and 5-HT1b.

4.1.3.6 Co-expression of TAAR1 and 5-HT1b results in the disruption of Gαi/o signaling during T1AM stimulation

The final experiment should clarify whether a heteromeric complex of TAAR1 and 5-HT1b influences

T1AM induced signaling. Here I compared TAAR1/NIP expressing HEK293 with TAAR1/5-HT1b co- expressing cells. Gαs signaling was similar in both groups (Figure 4.8 A). In both cases, T1AM induced through TAAR1 an increase in cAMP amounts. Additionally, combined stimulation with T1AM and 5-HT in cells with co-expressed receptors led to reduced levels of Gαs signaling caused by Gαi/o activation through 5-HT and T1AM at the 5-HT1b. Interestingly, when TAAR1 and 5-HT1b were co-expressed, T1AM only exerts Gαs activation, as was indicated by comparable signaling activity of T1AM for TAAR1 + NIP and TAAR1 + 5-HT1b (Figure 4.8 B). Hence, the Gαi/o signaling of T1AM at the 5-HT1b was lost in the heteromeric constellation. Furthermore, the effect of 5-HT at the heteromer was not as strong for 5-HT1b alone. FSK induced cAMP amounts were reduced by 40% for TAAR1/5-HT1b and by 50% for 5-HT1b. PLC activation or MAPK signaling was not altered through co-expression of TAAR1 and 5-HT1b (Supplemental Figure D.2 D).

Conclusion : In a heterodimeric constellation of TAAR1 and 5-HT1b, TAAR1 mediated Gαs domi-

nates 5-HT1b signaling, which might lead to apparent uncoupling of 5-HT1b to Gαi/o siganling.

4.1.4 Summary

I could demonstrate, that T1AM is a ligand for HRH1, as well as for 5-HT1b (Fig. 4.9). T1AM had antagonist capacities at the HRH1 by blocking the endogenous ligand HIS from inducing Gαq signal- ing. One may regard T1AM as a biased ligand at the 5-HT1b since it activated only Gαi/o signaling, but not PLC and MAPK signaling. 5-HT1b and TAAR1 form dimers, which uncouples T1AM-induced signaling at 5-HT1b. In the next part, T1AM sensitive brain loci are to be identified to assess if HRH1 and 5-HT1b are potential mediating T1AM-induced anapyrexia.

4.2 Part II: T1AM activates the PVN and induces Gαs signaling in hypothalamic cell lines

T1AM sensitive brain areas were to be identified to get one step closer to solving the mechanism of T1AM mediated anapyrexia. Previous studies demonstrated accumulation and stimulatory effects of T1AM in selected brain nuclei such as locus coeruleus and paraventricular nucleus (PVN) of the hypothalamus (Gompf et al., 2010; Chiellini et al., 2012). However, the exact role of this thyroid hormone metabolite in the hypothalamus remains unclear. Afterward, cell lines resembling the

T1AM sensitive brain areas were used to identify the underlying signaling cascades.

4.2.1T 1AM induces neuronal activity in the PVN

To investigate whether T1AM is capable of activating hypothalamic neurons in vivo, Carolin Höfig i.p. injected C57BL/6J mice with 50 mg/kg body weight of T1AM or DMSO/PBS as control, perfused the mice after 60 min and isolated the brains.

48 4.2 Part II: T1AM activates the PVN and induces Gαs signaling in hypothalamic cell lines

Figure 4.8: Co-expression and co-stimulation of TAAR1 and 5-HT1b leads to the abrogation of Gαi/o To measure Gαs and Gαi/o activation, cAMP accumulation was measured via an AlphaScreen technology. For con- trol, HEK293 expressing TAAR1 and a non-interacting protein (MC3R) were used (striped bars) and compared with TAAR1 and 5-HT1b co-expression (solid bars). T1AM does not induce Gαi/o activation in the heteromeric constellation, and the effect of 5-HT is lessened. Data were pooled from n,= 6-10 independent assays and mea- sured in triplicates. For statistics, a one-way ANOVA was performed to compare the ligands; a two-way ANOVA was used to compare TAAR+NIP and TAAR+5-HT1b and considered significant with *p ≤ 0.001, **p ≤ 0.001,

***p ≤ 0.001, ****p ≤ 0.0001. (A) Cells were stimulated with either 5-HT, T1AM or both in a concentration of

10 µM. (B) For Gαi/o, cells were additionally co-stimulated with forskolin and the ligands.

49 CHAPTER 4. RESULTS

Figure 4.9: T1AM and the aminergic GPCRs HRH1 and 5-HT1b

50 4.2 Part II: T1AM activates the PVN and induces Gαs signaling in hypothalamic cell lines

Figure 4.10: Theoretical background to detect neuronal activity and signaling in hypothalamic cell lines

Overview shows the experimental set up to identified FOS positive brain areas upon T1AM stimulation and to elucidate this mechanism in matching cell lines.

To monitor T1AM-induced neuronal activity, I used FOS as a marker relative to DMSO/PBS treated mice. One hour after T1AM injection, increased FOS staining of distinct neurons was visi- ble in the PVN, while DMSO/PBS treated mice showed only a few FOS positive neurons (Fig. 4.11

A). A statistical evaluation showed that T1AM-treated animals had more FOS positive cells in the PVN (Fig. 4.11 B). In average 16 ± 4 FOS positive cells were counted per brain slide of the PVN in

DMSO/PBS-treated mice, while T1AM-stimulated mice showed 60 ± 13 FOS stained cells per brain slide. T1AM had no stimulatory effect on the medial preoptic area (MPO), the supraoptic nucleus (SON), the dorsomedial nucleus of the hypothalamus (DMH), the periaqueductal gray (PAG) and the ventral tegmental segment (VTA, Fig 4.11 B and Suppl. Fig. E.1/E.2). FOS positive cells ranged from 4 to up to 25 per brain slide in these brain loci.

Conclusion : I.p. injection of 50 mg/kg bodyweight T1AM led either directly or indirectly to the activation of neurons in the PVN. The following question is now: how were the neurons probably activated?

4.2.2 Expression profile of GPCRs and TRP channels in murine hypothalamic cell lines

With the knowledge that T1AM activated PVN neurons, the next step was to elucidate possible signaling pathways, that could result in FOS activation in vivo. Therefore, I continued working in different cell models that partly match the cellular in vivo environment of the hypothalamus. Three different murine hypothalamic cell lines were chosen: the mouse hypothalamic gonadotropin- releasing-hormone neuronal cell line GT1-7 and two embryonic mouse hypothalamic cell lines mHypoE-N39 (N39) and mHypoE-N41 (N41). These cell lines are established models to study neu- roendocrine mechanisms and are known to express PVN-like markers (Mellon et al., 1990; Wetsel

51 CHAPTER 4. RESULTS

Figure 4.11: Staining of FOS positive neurons after 1 h of i.p. injection of 50 mg/kg T1AM. After C57BL/6J mice were i.p. injected with either T1AM or solvent (60 % DMSO/ 40 % PBS), brains were frozen and cryosec- tioned and stained against FOS (red) and DAPI (blue). (A) In comparison to the control mice, T1AM-treated mice showed a strong FOS staining in the PVN. All pictures were taken with a 20X objective. (B) FOS positives cells were counted in the respective nuclei. Only T1AM treated animals (n = 3) showed an increase in FOS activity in the PVN. For statistics, an unpaired t-test per row was performed. Statistics were set to **p 0.01. ≤

52 4.2 Part II: T1AM activates the PVN and induces Gαs signaling in hypothalamic cell lines et al., 1991; Mayer et al., 2009). At least two of them, N39 and N41, showed a slight increase in FOS activation after T1AM stimulation similar to the murine brain activation (N39: from 1.6 % to 5.0 % FOS positive cells, N41: from 1.2 % to 7.6 % FOS positive cells, Fig. 4.12).

Figure 4.12: 3-T1AM only induces c-FOS activation in N39 and N41 cell lines. Cells were seeded on poly- L-lysine coated glass slides. After 48 h of standard cultivation, cells were incubated with AdvancedMEM and 0.1% DMSO or 10-5 M 3-T1AM for 1 h. After acetone fixation, cells were stained with c-FOS (red) and DAPI (blue) and counted by the ImageJ software.

Besides the already known expression pattern of the three cell lines, it was crucial to elucidate the expression of potential T1AM targets.

GPCRs As GPCRs are the primary targets of T1AM, the GPCR expression profiles of the three cell lines were investigated. Here, the relative expression are shown of the trace amine associated re- ceptor (Taar1 ), the serotonin 1b receptor (5-Ht1b ), the histamine 1 receptor (Hrh1 ), the dopamine 2 receptor (Drd2 ), the α2a adrenoreceptor (Adra2a), the β1 adrenoreceptor (Adrb1 ) and the β2 adrenoreceptor (Adrb2, Fig. 4.13). When compared against the phosphoglycerate kinase 1 (Pgk1 ) as a reference gene, Adrb1 noticeably had the highest expression rate among these receptors in all three cell lines with a ratio of 94 ± 38 for GT1-7, 566 ± 312 for N39 and 1046 ± 456 for N41. The second highest expression was detected for Adrb2 with a ratio of 1.54 ± 0.34 for GT1-7, 2.77 ± 1.04 for N39 and 6.06 ± 2.34 for N41. The other receptors Taar1, 5-Ht1b, Hrh1, Drd2, and Adra2a all displayed similar low-level expression profiles. The mRNA content in all three cell lines was lower than the reference gene Pgk1 (Fig. 4.13), with ratios between 0.37 ± 0.14 for 5-Ht1b in GT1-7 and up to 0.72 ± 0.08 for Taar1 in N41.

53 CHAPTER 4. RESULTS

Figure 4.13: Expression pattern of GPCRs and TRP channels in GT1-7, N39, and N41. Results of a SYBR Green-based qPCR. Graphs show the ratios between the reference gene Pgk1 and the GPCRs or the TRP chan- nels. Data were pooled from n = 3 measured in duplicates, while n refers to different passages. The three hypothalamic cell lines show a similar expression pattern. Besides Trpm5 all genes of interest are expressed. (A) GT1-7, (B) N39, (C) N41.

54 4.2 Part II: T1AM activates the PVN and induces Gαs signaling in hypothalamic cell lines

TRP channels Besides GPCRs, previous studies identified the transient receptor potential channel

(TRP) M8 as a target for T1AM (Khajavi et al., 2015; Lucius et al., 2016). Here, I measured the gene expression levels of the TRPM subfamily and TRPV1 in three hypothalamic cell lines. The qPCR data show that none of the hypothalamic cell lines expressed Trpm5 (Fig. 4.13). In GT1-7 cells, Trpm4 was the highest expressed TRP channel with a ratio of 159 ± 89 compared to Pgk1, followed by Trpm7 with a ratio of 11.7 ± 10.1 to Pgk1. Trpv1 had a ratio of 2.96 ± 2.91 and Trpm8 of 0.03 ± 0.02 to Pgk1. Trpm1 had the lowest expression with a ratio of 0.00000064 ± 0.00000031 compared to Pgk1. Trpm6 had the lowest expression with a ratio of 0.00000064 ± 0.00000031 com- pared to Pgk1 (Fig. 4.13 A). In N39 cells, Trpm4 was also the highest expressed TRP channel with a ratio of 831 ± 617 to Pgk1, followed by Trpv1 with a ratio of 79 ± 38 to Pgk1. Trpm7 had a ratio of 59 ± 29 and Trpm8 a ratio of 2.19 ± 0.72. Trpm1, Trpm2 and Trpm3 expression ratios laid be- tween a ratio of 0.02 ± 0.025 to Pgk1 for Trpm2 and Trpm3 with a ratio of 0.000089 ± 0.000076 to Pgk1. mRNA content was comparable to GT1-7 cells, with Trpm6 having the lowest expression with a ratio of 4.95 ± 3.48 compared to the reference gene (Fig. 4.13 B). N41 cells exhibited a similar expression pattern of TRP channels as GT1-7 and N39 cell lines (Fig. 4.13C). Trpm4 (ratio to Pgk1 759 ± 674) and Trpv1 (ratio to Pgk1 514 ± 226) were the highest expressed genes, followed by Trpm7 (ratio to Pgk1 29 ± 23) and Trpm8 (24 ± 18). Trpm2 and Trpm1 were lower expressed with ratios of 0.029 ± 0.025 and 0.0014 ± 0.0011 compared to the reference gene. Trpm3 and Trpm6 were least expressed in N41 cells with ratios to Pgk1 of 0.000089 ± 0.000076 and 0.0000023 ±0.0000013.

Conclusion : GT1-7, N39 and N41 endogenously expreseds the aminergic GPCRs Taar1, 5-Ht1b, Hrh1, Drd2, Adra2a, Adrb1 and Adrb2. Furthermore, these cell lines also expressed the TRP chan-

nels Trpm1, Trpm2, Trpm3, Trpm4, Trpm6, Trpm7, Trpm8 and Trpv1. Further analysis of T1AM- induced signaling cascades in the hypothalamic cell lines could help to exclude some of these membrane proteins.

4.2.3T 1AM induces FSK-amplified Gαs signaling in mHypoE-N39 and mHypoE- N41 cell lines

Various signaling pathways can result in FOS activation. To investigate which of these pathways con- tributes to T1AM actions in the hypothalamus, I tested two major signaling cascades downstream of T1AM GPCR targets, Gαs and Gαi/o. To measure endogenous Gαs signaling, I determined the cAMP enhancement. Additionally, the cAMP content was normalized to protein concentrations to com- pare the different cell types. N41 and GT1-7 cells had a higher basal cAMP content with 3.2 ± 0.36 nM cAMP / g/L protein for N41 and 3.41 ± 0.26 nM cAMP / g/L protein for GT1-7, compared to N39 with 0.92 ± 0.13 nM cAMP / g/L protein (Fig. 4.14A, n = 4 in triplicates). In all cell lines, T1AM stimu- lation (10 µM) did not increase cAMP concentration compared to the basal cAMP content (Fig. 4.14

A). Only norepinephrine (NorEpi) and isoproterenol (ISOP) activated an endogenous Gαs signal in GT1-7 (1.6 fold for NorEpi and 1.7 fold for ISOP), N41 (1.8 fold for NorEpi and 2.1 fold for ISOP) and N39 cells (3.5 fold for NorEpi and 5.9 fold for ISOP, Suppl. Fig. B.1). Serotonin (5-HT) and phenethylamine (PEA), endogenous ligands for 5-HT1b and TAAR1, did not increase cAMP content (Suppl. Fig. B.1). The stimulation with histamine (HIS) and dopamine (DA) did not affect cellular cAMP concentrations (Suppl. Fig. B.1). Cells were incubated with forskolin (FSK), an unspecific activator of the adenylyl cyclase, which increases the basal cellular cAMP content to determine Gαi/o signaling. For all three cell lines, Gαi/o activation was not detected after stimulation with 10 µM T1AM (Fig. 4.14 B). Furthermore, FSK can

55 CHAPTER 4. RESULTS

Figure 4.14: T1AM induces a FSK-enhanced Gαs signal in murine hypothalamic cell lines. For Gαs and

Gαi/o, the cAMP content was measured via the AlphaScreen™ kit. (A) Cells were stimulated with stimulation -5 buffer or T1AM in a concentration of 10 M for 45 min. (B) Cells were co-stimulated with 50 µM FSK and either stimulation buffer or T1AM (10 µM) for 45 min. Data are shown ± SEM and are pooled from four independent assays measured in triplicates (n = 4). For statistics a two-way ANOVA was performed, followed by a Sidak correction. Statistics were set to *p≤ 0.05.

potentiate weak Gαs signaling. Dessauer et al.(1997) showed that FSK in the presence of G αs has a higher affinity to the adenylyl cyclase, yielding in higher cAMP accumulation. Here, this phe- nomenon emerges for N39 and N41, when T1AM stimulation significantly increases cAMP content in FSK-treated cells (144.2 ± 16.15 % for N39 and 160.77 ± 21.17 % for N41, n = 4 in triplicates, pN39 = 0.0128, pN41 = 0.0019). The specific ligands for TAAR1, 5-HT1b, HRH1, DRD2, ADRA2A, ADRB1, and ADRB2 did not activate Gαi/o signaling (Suppl. Fig. B.2). However, DA induces also a FSK-stimulated Gαs signal in N39 cells (Suppl. Fig. B.2 B).

Conclusion : In N39 and N41 cells, 10 µM T1AM induced a FSK-amplified cAMP increase indicating a vague response of the hypothalamic cell lines probably due to low expressed T1AM target GPCRs. Taken together, this was probably mediated by one or several Gαs coupled GPCR, like TAAR1 and ADRA2A.

4.2.4 Summary

In the murine brain, i.p. injection of T1AM (50 mg/kg body weight) led to the activation of PVN neurons (Fig. 4.15). Hypothalamic cell lines expressed several GPCRs and TRP channels that could be targets of T1AM. In N39 and N41 cells, a FSK-amplified Gαs was detected due to T1AM-stimulation (10 µM). None of the other endogenous ligands, besides DA in N39 cells, could evoke a similar signaling response. Either two Gαs-coupled receptors were activated, like TAAR1 and ADRA2A or an unknown GPCR target of T1AM is responsible for the Gαs. During this study also TRP channel activation was investigated by Noushafarin Khajavi (Fig. 5.4). She could show that in the cell line

N41 TRPM8 is activated upon T1AM stimulation (Braunig et al., 2018a).

56 4.2 Part II: T1AM activates the PVN and induces Gαs signaling in hypothalamic cell lines

Figure 4.15: T1AM and neuromodulation I.p. injection of T1AM increased FOS staining in the PVN. Hypotha- lamic cell lines expressed several GPCR and TRP targets of T1AM. N39 and N41 cells showed an increase in

FSK-amplified Gαs signaling upon a T1AM challenge.

57 Chapter 5

Discussion

T1AM has intriguing effects after the application of pharmacological doses such as anapyrexia. In general, anapyrexia is the regulatory shift of basal body temperature to a lower setpoint, which can be achieved by either vasodilation, reduced energy consumption by smooth muscles, and in case of rodents a decreased brown adipose tissue activity (Osamu and Naotoshi, 2011). T1AM in- duces vasodilation of the tail vein centrally (Gachkar et al., 2017). The body temperature of these animals decreases without a shivering response. In contrast, muscle relaxants have to counter the severe shivering of patients in the clinical induction of hypothermia. Therefore, a drug inducing anapyrexia in humans without severe side effects, such as T1AM does in rodents, would be ben- eficial to treat medical conditions such as stroke. The first step to a new drug is to understand its complete functional effects and to evaluate possible side effects for future patients. In my dis- sertation, I investigated whether GPCRs that are involved in thermoregulation are receptor targets for T1AM-induced thermoregulation. Secondly, I wanted to identify brain areas stimulated directly or indirectly by i.p. given T1AM. I used neuronal cell lines to study their activation through T1AM further.

5.1 Part I: T1AM and the aminergic histamine 1 receptor (HRH1) and serotonin 1b receptor (5-HT1b)

I was interested in identifying new aminergic receptors that might be targets for T1AM activation. The HRH1 and 5-HT1b are interesting candidates as they are involved thermoregulation. So far,

T1AM has not been tested for its ability to activate or inhibit serotonergic and histaminergic GPCRs (Brezenoff and Lomax, 1970; Oerther and Ahlenius, 2001).

5.1.1 β-TAAR1 signaling induced by different ligands

Both, T1AM and its first described receptor TAAR1 are in the focus of research (Hoefig et al., 2016; Rutigliano et al., 2018). T1AM is interesting for its anapyrexic and metabolic effects, while TAAR1 might be a new target for psychotropic drugs against schizophrenia, bipolar disorders, depression, and Parkinson‘s disease (Rutigliano et al., 2018). TAAR1 is known as neuromodulator fine tuning DRD2 signaling in the striatum (Espinoza et al., 2015, 2011). In the first study to characterize TAAR1, the GFP-tagged rat receptor was overexpressed in HEK293 cells (Bunzow et al., 2001). The fluorescence signals were detected in the cytosol, which

58 5.1 Part I: T1AM and the aminergic histamine 1 receptor (HRH1) and serotonin 1b receptor (5-HT1b) led to the assumption that TAAR1 is located intracellularly. However, protein misfolding leads to similar morphology by inclusion bodies and degradation. TAAR1 is difficult to overexpress in vitro. Reasons could be the signaling peptide and missing glycosylation site at the N-terminus to promote translation and processing in the endoplasmic reticulum (Bunzow et al., 2001; Barak et al., 2008). However, other GPCRs, like the MC4R also do not have a glycosylation site and a signaling peptide and do not cause this problem. It could further be, that cell lines, such as HEK293 cells, do not express the right set of co-factors, e.g., chaperones, for optimal TAAR1 folding. Nevertheless, to successfully perform further experiments, TAAR1 expression needed to be improved. A sufficient cell surface expression is needed for adequate signal detection, especially for protein interaction assays like FRET. Therefore, I used the first nine amino acids of ADRB2 as an N-terminally tag for TAAR1 as re- ported by Barak et al.(2008) for the following assays. Besides GPCR tra fficking, the N-terminus can be relevant for ligand binding, signal transduction, and even receptor interaction. Point mu- tations and deletions in this GPCR section predominantly lead to reduced receptor functionality

(Uddin et al., 2012; Belmer et al., 2014; Coleman et al., 2017). Measuring Gαs signaling induced by the endogenous ligand PEA, I did not observe that an extension with the first nine amino acids of

ADRB2 led to different signaling of TAAR1 or a change in the EC50 value (Fig. 4.2). The β-tag of the β-adrenergic receptor led to a higher cell surface expression and higher cAMP accumulation. T1AM stimulation achieved similar results (Fig. 4.2 A), which supports the second theory that TAAR1 is eas- ily misfolded. Presumably, HEK293 cells lack essential co-factors for a sufficient TAAR1 expression machinery. It is known, that 5-HT activates the rhesus monkey and murine TAAR1, but with a decreased signaling compared to the endogenous ligand PEA. In case of the murine TAAR1, PEA has an EC50 of 43 nM, while the EC50 for 5-HT is about 4500 nM (ligands ordered from lowest EC50 value to highest PEA > tyramine > tryptamine > DA > > 5-HT, Wolinsky et al. 2007). 5-HT activated the human TAAR1, but at a higher concentration than PEA or T1AM (Fig. 4.3). Recent reports sug- gest that 5-HT may activate other aminergic receptors differently to serotonin receptors, like the octopamine receptor 2, which again confirms the principle possibility of amine ligand promiscuity (Qi et al., 2017).

5.1.2T 1AM as an antagonist of the HRH1

My aim in this study was to elucidate whether further aminergic receptor families, namely his- tamine and serotonin receptors, are targets of T1AM.

In a first step, T1AM action at the histamine 1 receptor (HRH1) was tested. T1AM is involved in the modulation of the histaminergic system (Laurino et al., 2015). When the histamine synthesis is abolished in decarboxylase KO mice, the T1AM induced hyperalgesia was blocked. Histaminergic neurons are only found in the tuberomammillary neurons of the hypothalamus in rats (Panula et al., 1984). These are scattered throughout the posterior hypothalamus. Sev- eral brain loci express the receptors HRH1, HRH2, and HRH3. Together the histaminergic system regulates sleep, food intake, thermoregulation, and locomotor activity. It has been reported to pro- mote hyperthermic condition by changing the basal set point of the body temperature in rodents (Tabarean, 2016; Green et al., 1975). However, direct injection of histamine into the POA leads to hypothermia in rats, while the HRH1 antagonist can block this effect (Brezenoff and Lomax, 1970). On the other hand, the administration of an HRH1 agonist into the murine median

59 CHAPTER 5. DISCUSSION preoptic area induces hyperthermia, indicating that the role of the histaminic system in thermoreg- ulation is not entirely understood (Lundius et al., 2010).

Here I showed, that even though T1AM did not activate the human HRH1, it blocked the binding pocket competitively when co-administered in equimolar concentrations with histamine (HIS) in

HEK293, which reduced the Gαq signaling to about 50 % in comparison to administration of HIS alone (Fig. 4.4). In conclusion, T1AM is an antagonist of the HRH1 in the in vitro system. As T1AM concentrations in the brain are not clear, one can only speculate on the physiological impact of

T1AM on histaminic neurons. HRH1 is involved in the vasoconstriction of the big vessels and the lungs and induces vasodila- tion in the capillaries in the case of rashes. Therefore, HRH1 antagonists are used to treat allergic responses to improve breathing and to subside swelling of the skin. By depleting the histamine storages using a inhibitor in the PVN, mice started eating (Ookuma et al., 1993). Injecting an HRH1 specific agonist into this brain area inhibits feeding (Masaki et al., 2004).

I.c.v. injection of T1AM influenced food intake in mice dose-dependently (Manni et al., 2012). How- ever, the T1AM metabolite TA1 mediates most of these effects. Therefore, it has to be investigated if TA1 could also be a ligand for HRH1 in future experiments. As HRH1 is involved in both hyper- and hypothermia, it is difficult to say, if it mediates the temperature effects of T1AM. Several specific agonist and antagonist of HRH1 exist, which could be tested to elucidate its T1AM related effects.

5.1.3T 1AM induced biased signaling at the 5-HT1b

Next, I investigated the possible interaction between T1AM and 5-HT1b. The 5-HT related hormone system is evolutionarily ancient and has essential functions in lower animals, even as it is involved in behavioral tasks such as egg laying (Nichols and Sanders-Bush, 2001). In higher animals, 5-HT is involved in a variety of complex behaviors and processes such as aggression, sleep, appetite and mood (Nichols and Sanders-Bush, 2001). 5-HT-induced effects are usually mediated by 14 sero- tonin receptors (Hoyer et al., 1994). Serotonin GPCRs are targets for various pathophysiological diseases like depression, schizophrenia, migraine among others. Therefore, the functional selectiv- ity of serotonergic ligands at these receptors comes into play (Bohn and Schmid, 2010). The 5-HT1b is presynaptically and localized at the terminal axon (Boschert et al., 1994). It sup- presses aggression (Olivier and van Oorschot, 2005; Ramboz et al., 1995; Bouwknecht et al., 2001), is involved in learning and memory (Ahlander-Luttgen et al., 2003) and could be a new target for de- pression medication (Dawson et al., 2006; Nautiyal and Hen, 2017). 5-HT1b has been reported to be involved in hypothermia in guinea pigs and rats (Hagan et al., 1997; Oerther and Ahlenius, 2001). It was reported that for various serotonin receptor ligands, the thermic effect is concentration depen- dent, e.g., high concentrations of 5-methoxy-N,N-dimethyltryptamine induces hyperthermia, while lower doses result in anapyrexia, mediated by 5-HT1b and HTR2a (Gudelsky et al., 1986).

Here I show, that T1AM significantly induced Gαi/o signaling at the 5-HT1b (Fig. 4.5) but failed to induce PLC and MAPK activation by the Gβγ. This is a case of biased signaling, as T1AM only evokes Gαi/o signaling, but not Gβγ, even though the receptor is capable of signaling through both pathways at the same time with 5-HT as a ligand (Smith et al., 2018). It is known that the heterotrimeric

Gαi/o protein remains as a complex of Gα and Gβγ after activation (Bunemann et al., 2003; Frank et al., 2005). Using several FRET sensors in living cells, Bunemann et al.(2003) showed that upon activation the G i/o subunits rearrange their spatial orientation and do not separate.

60 5.1 Part I: T1AM and the aminergic histamine 1 receptor (HRH1) and serotonin 1b receptor (5-HT1b)

Additionally, in our group docking studies were performed to investigate the binding mode of

T1AM at the 5-HT1b in comparison with its endogenous ligand 5-HT with computational modeling methods (Fig. 5.1). In brief, 5-HT was placed at a receptor homology model spatially between ex- perimentally confirmed receptor/ligand interaction sites, namely between aspartate 129 (Asp129) in the transmembrane 3 (TM3), as well as Thr213 in TM5 (Bavencoffe et al., 2010; Huang, 2003).

The same starting point was used for docking T1AM into a 5-HT1b homology model. These sys- tems were modified by dynamic simulations (nanoseconds) and energetically minimized (Braunig et al., 2018a). In the binding pocket of 5-HT1b, 5-HT and T1AM share, besides the hydrogen bonds at Asp129 and Thr213, hydrophobic contacts within the TM3 (Ile130) and TM5 (Ser212). However,

T1AM, in contrast to 5-HT, is not in contact with residues at the TM6, Phe330, and Phe331, as ob- served for the endogenous ligand 5-HT. These binding differences may also lead to differences in the signaling set of T1AM at the 5-HT1b compared to the 5-HT1b/5-HT system. In 2012, Blättermann et al. were the first to describe a similarly biased ligand. Gue1654 selec- tively activates Gα and not Gβγ of Gi/o at the chemoattractant receptor OXE-R (Blättermann et al., 2012). To elucidate this mechanism they used a bioluminescence energy transfer (BRET) assay with a luciferase tag at the C-terminus of OXE-R and a GFP attached to the N-terminus of either Gβ1 or Gγ2. Upon stimulation with its endogenous ligand, the C-terminus of the GPCR and the Gβγ are spa- tially close. Adding Gue1654, the distance between the receptor C-terminus and the Gγ2 becomes larger indicated by a 40 % decrease in the BRET signal (Blättermann et al., 2012). In conclusion,

Gue1654 stabilizes a receptor conformation that only favors Gα and Gβ recruitment, which leads to the complete loss of function for Gβγ signaling by blocking the rearrangement of the trimeric G- protein. Whether this might also be applied to T1AM at the 5-HT1b, a similar BRET approach should be tested.

5.1.4 Co-expression of TAAR1 and 5-HT1b in interplay with T1AM and 5-HT modulated the Gαi/o signaling profile

Dimerization can modify the signaling properties of the individual GPCR (Rozenfeld and Devi, 2011). TAAR1 is a neuromodulator for DRD2 expression and signaling in the striatum (Espinoza et al., 2011; Navailles and De Deurwaerdere, 2011), but has also been shown to interact with ADRA2A (Dinter et al., 2015b). There are also hints, that TAAR1 influences the serotonergic system. The TAAR1 selective agonist RO5166017 inhibits the firing of the serotonergic dorsal raphe nucleus, while the partial agonist RO5203648 increases firing (Reese et al., 2007; Revel et al., 2012). FRET data of this study showed a possible interaction between TAAR1 and 5-HT1b (Fig 4.7). Sand- wich ELISA data confirm these results (Braunig et al., 2018a). This finding extends the physiologi- cally possible interactome of TAAR1 and the 5-HT1b as they are both expressed in the hypothalamus (Pazos and Palacios, 1985; Borowsky et al., 2001; Lindemann et al., 2008), in the striatum (Espinoza et al., 2011; Navailles and De Deurwaerdere, 2011) and the dorsal raphe nucleus (Lindemann et al., 2008; Beliveau et al., 2017). It is of further interest, how this potential interaction may impact the functionality of these receptors. A complete TAAR1 knock out increases the neuronal firing signifi- cantly in the serotonergic dorsal raphe nucleus in mice (Revel et al., 2011). So, it is not far off, that TAAR1 influences serotonergic GPCR expression and sensitivity through dimerization, just like it is a neuromodulator for the DRD2 (Espinoza et al., 2011, 2015). The aspect of dimerization becomes even more relevant if both receptors have the same ligands. Therefore, I tested the potential het- eromeric complex for signaling effects by an individual or by co-stimulated ligand application. The

61 CHAPTER 5. DISCUSSION

Figure 5.1: Binding of T1AM and 5-HT at the 5-HT1b (A) 3D model of the 5-HT1b binding pocket with the direct comparison of T1AM and 5-HT. (B) Schematic interaction plots of T1AM and 5-HT at the 5-HT1b. Fig by Braunig et al.(2018a)

62 5.2 Part II: The influence of T1AM on the murine brain and hypothalamic cell lines

Figure 5.2: Signaling induced by T1AM at aminergic receptors In the family of aminergic receptors, T1AM can interact with adrenergic, muscarinic, serotonergic and histaminergic receptors, as well as trace amine associated receptors.

5-HT and T1AM mediated activation of Gαi/o via the 5-HT1b was modified under co-expression con- ditions (Fig. 4.8). Notably, the action of T1AM at the heteromer did not show any Gαi/o activation at 5-HT1b, and this reduced the potency of 5-HT. In principle, uncoupling of a TAAR1 interaction partner from its signaling pathway is observed for norepinephrine-induced ADRA2A signaling in a TAAR1/ADRA2A heteromer (Dinter et al., 2015b). The here observed effect for TAAR1/5-HT1b dif- fers. The Gαs activation through T1AM at TAAR1 overrides the Gαi/o mediated effect of T1AM and 5-HT1b, while 5-HT1b and its endogenous ligand 5-HT still inhibit the adenylate cyclase. This GPCR uncoupling also suggests that under co-expressed conditions (like for TAAR1 and the 5-HT1b in hy- pothalamus, striatum and dorsal raphe nucleus), the availability and number of ligands (and their concentrations) are regulating elements for the resulting signaling output.

In summary, I showed, that T1AM is an antagonist at HRH1 and a biased ligand at 5-HT1b. Fur- ther studies will have to show if these receptors are responsible for the T1AM induced anapyrexic effect seen in rodents or if other thermoregulatory GPCRs are involved. This work also underlines the complex role of TAAR1 concerning modulating signal transduction of various aminergic GPCRs and different promiscuous ligands (Fig. 5.2). Targeting of receptors in this signaling network for therapeutically purposes might result in pleiotropic effects depending on ligand concentration and expression profile of interacting partners in different tissues.

5.2 Part II: The influence of T1AM on the murine brain and hy- pothalamic cell lines

As many GPCRs, but also ion channels like TRPs are involved in thermoregulation, I wanted to min- imize the group of candidates that might be responsible for T1AM effects. Since it is still unknown

63 CHAPTER 5. DISCUSSION

Figure 5.3: Central regulation of vasodilation and vasoconstriction One thermoregulatory mechanism is vasodilation and vasoconstriction. Several brain loci play a role in this regulation

how T1AM induces anapyrexia, I intended to shed more light into this. Until today, several ques- tions remain: If T1AM evokes the anapyrexia centrally, which brain loci are involved? How are the neurons activated? By a GPCRs or a TRP channel? Therefore, I designed the study as follows: First, I tested if activated neurons can be found after an i.p. injection of 50 mg/kg body weight of

T1AM for 60 min in the murine brain. I used FOS staining to identify active neurons. FOS staining is not a marker for general neuronal firing. Then nearly all neurons would be FOS positive. FOS is expressed and activated if there is a general switch from one mode to another with a significant change in gene expression (Bullitt, 1990). In particular, brain loci involved in thermoregulation were examined. The different brain loci are highly specialized and possess distinct neuronal subtypes.

Secondly, after T1AM sensitive brain areas were identified, resembling cell lines were used to enlighten the activating signaling cascades, and analyze if a GPCR or ion channels are involved. To investigate whether GPCRs are involved in the neuronal activation, cAMP concentrations were measured in murine cell lines, as a second messenger for Gαs and Gαi/o. Noushafarin Khajavi tested TRP channel activation (Braunig et al., 2018b).

5.2.1T 1AM-induced signalosome activated PVN neurons of C57BL/6 mice

It is known that the direct injection of T1AM into the lateral ventricle of male mice leads to the activation of neurons in the anterior commissural nucleus of the hypothalamus (Dhillo et al., 2009).

Here, I observed that i.p. injection of T1AM resulted in the activation of PVN neurons in C57BL/6 mice (Fig. 4.11). Within the hypothalamus, the paraventricular nucleus (PVN) is one of the most extensively studied nuclei and plays a pivotal role in the control of fluid homeostasis, lactation, cardiovascular regulation, feeding behavior, nociception and response to stress (Ferguson et al.,

2008). The activation in the PVN due to T1AM stimulation also regulates the central T1AM effects on glucose metabolism (Klieverik et al., 2009).

Gachkar et al.(2017) suggested that T 1AM induces the vasodilation of the tail vein centrally in male mice. The preoptic area (POA), the periaqueductal gray (PAG) and the ventral tegmental area (VTA) centrally mediate vasodilation as a response to increased body temperature. These nuclei project into the raphe pallidus (RPA) and rostral ventrolateral medulla (RVLM, Fig. 5.3). The POA is the primary regulator of the vasomotor response. Upon inhibition or cooling, the PAO induces vasoconstriction (Osborne and Kurosawa, 1994), whereas excitation and warming lead to vasodilation (Carlisle and Laudenslager, 1979; Tanaka et al., 2002). However, besides the PVN, these nuclei and further loci that are known to be involved in the shivering thermogenesis (PAG and

DMH, Tan and Knight 2018) were not affected by T1AM administration. There were no differences observed in FOS activation in these areas (Fig. 4.11 B).

DIO3 metabolizes T1AM to thyronamine (T0AM), while an amine oxidase and an aldehyde dehy-

64 5.2 Part II: The influence of T1AM on the murine brain and hypothalamic cell lines

drogenase lead to the formation of TA1 (Pietsch et al., 2007; Wood et al., 2009). Hence, the activation of PVN neurons is presumably a direct effect of pharmacological doses of T1AM or its metabolites, T0AM and TA1. The first publication of T1AM mentions that also T0AM induces anapyrexia (Scanlan et al., 2004). Although several groups have proven the hypothermic effect of T1AM, it is still debat- able to what extent this is the case, as T1AM can only be dissolved in ethanol and DMSO, substances that both lower body temperature themselves. The breakthrough publication by Scanlan et al. 2004 does not state the dilution and volume of DMSO injections for the animals. A later study by Gachkar et al.(2017) with a detailed injection protocol could show a hypothermic effect, though to a lesser degree than the first publication by Scanlan et al.(2004). In concordance, newer publications show that neither T0AM nor TA1 can induce anapyrexia (Hoefig et al., 2015a; Harder et al., 2018). Whether the PVN neurons are directly activated or activated through neuronal projections from other nuclei is uncertain in the carried out experiments. Moreover, the exact cellular mechanism initiated by T1AM once it reaches in the hypothalamic nuclei is undiscovered. The activated neurons should be further characterized using antibodies of specific subpopulation markers.

5.2.2T 1AM slightly stimulated Gαs signaling in murine hypothalamic cell lines

To elucidate the underlying mechanism behind the stimulatory effect of T1AM in the hypothalamus, I used three murine hypothalamic cell lines, GT1-7, mHypoE-N39 (N39), and mHypoE-N41 (N41). These cell lines are established models to study neuroendocrine mechanisms and known to express

PVN-like markers (Mellon et al., 1990; Wetsel et al., 1991; Mayer et al., 2009). T1AM significantly increased the FOS activation in N41 and N39 cells (Fig. 4.12).

5.2.2.1 Expression pattern of GPCRs and TRPs in hypothalamic cell lines

FOS activation can be detected downstream of GPCRs and TRPs (Bullitt, 1990). Aminergic GPCRs are known T1AM targets, and previous studies assumed that pharmacological effects of this thyroid hormone metabolite are attributable to aminergic receptor signaling (Gompf et al., 2010; Dinter et al., 2015a,b,c; Laurino et al., 2016). Also, TRPM8 activation, another target of T1AM, leads to a Ca2+ influx. Increased intracellular Ca2+ concentrations are necessary to start ERK/MAPK signaling which subsequently initiate FOS activation (Bullitt, 1990). Therefore, it might be that GPCRs or TRPs are involved in this neuronal signal activation. Since the hypothalamus expresses broadly aminergic GPCRs and TRPs, the expression profile of all three cell lines was assessed via qPCR. All hypothalamic cell lines expressed already identified T1AM targets (Adra2a, Adrb1, Adrb2, Taar1, and Trpm8 ), as well as the aminergic GPCR Drd2, 5-Ht1b, and Hrh1. Further, these cells expresse all Trpms besides Trpm5 and the Trpv1. N41 showed higher expression levels than N39 followed by GT1-7 with the lowest GPCR and TRP expression pattern. In vivo qPCR of immunohistology data of the PVN demonstrated the expression of 5-HT1b, DRD2, HRH1, ADRA2A, ADRB1, TRPM4, TRPM5, and TRPV1 (Hazell et al., 2012; Feetham et al., 2018; Williams and Morilak, 1996). Some hypothalamic neurons express ADRB2, as well as TRPM8, but not neurons of the PVN (Paeger et al., 2017; Voronova et al., 2014). In conclusion, the hypothalamic cell lines GT1-7, N39 and N41 only partially resemble PVN neurons. It is a general issue in cell culture, that cell lines can only mimic in part complex tissues.

65 CHAPTER 5. DISCUSSION

5.2.2.2T 1AM slightly stimulated Gαs signaling in murine hypothalamic cell lines

In this study, I did not detect the activation of Gαi/o in response to T1AM in murine hypothalamic cell lines (Fig. 4.14 B). Candidates for Gαi/o should have been the DRD2 and the 5-HT1b. Nevertheless, N39 and N41 showed an FSK-amplified Gαs signal due to T1AM stimulation (Fig. 4.14). Among the expressed GPCRs, TAAR1 and the ADRBs are Gαs coupled. The low GPCR expression rate might be the reason for the lack of strong Gαs in these cells (Fig. 4.13). The FSK stimulated N39, and N41 cells showed a significant increase in cAMP concentration after T1AM treatment (Fig. 4.14 B). Besides its adenylyl cyclase activating property, FSK stimulation additionally favors activation of adenylyl cyclase through Gαs (Daly et al., 1982; Darfler et al., 1982; Dessauer et al., 1997). Gαs and FSK do not compete for the adenylate cyclase activation, as they recognize different binding domains. In the presence of Gαs, FSK binds with a higher affinity to the adenylate cyclase compared to FSK stimulation without G-protein involvement (Dessauer et al., 1997). This stronger binding of FSK leads to an increase in adenylate cyclase activity and subsequently to a higher cAMP accumulation.

5.2.3T 1AM activates TRPM8 in hypothalamic cell lines N39 and N41

Besides GPCR signaling, our group also investigated TRP channel involvement in the stimulatory effects of T1AM in hypothalamic cell lines. In our group, patch clamp recordings were conducted 2+ in N39 and N41 cells which demonstrated that T1AM triggers Ca influx through activation of the TRPM8, as the specific TRPM8 inhibitors (BCTC and AMTB) diminish this effect partially (Fig.

5.4, Braunig et al. 2018b). Previous studies detected the stimulatory effect of T1AM on TRPM8 in different cell lines (IOBA-NHC, HCjEC, human uveal melanoma cells, and HCK, Khajavi et al. 2015; Lucius et al. 2016; Walcher et al. 2018). However, since the Ca2+ influx was not completely abolished in the presence of TRPM8 inhibitors in N41 cells (Fig. 5.4), further Ca2+-channels might be involved in stimulatory effect T1AM. A recent study reported that activation of the warmth-sensing TRPM2 leads to a similar thermoregulatory response, as the one observed after systemic administration of

T1AM in mice (Song et al., 2016). Therefore, TRPM2 was suggested as another T1AM target (Gachkar et al., 2017). This hypothesis could be easily tested by repeating the patch clamp recordings in the hypothalamic cell lines in the presence of a specific TRPM2 antagonist such as clotrimazole (Hill et al., 2004). GPCR signaling can also activate TRPs, e.g., via protein kinases or PLC signaling (Yekkirala, 2013;

Veldhuis et al., 2015). A similar signaling cascade has been shown for TRPM8, ADRB2, and T1AM (Dinter et al., 2015a). The TAM enhances only isoprenaline signaling at the ADRB2 but cannot induce 2+ signaling itself. However, an ADRB2 blocker completely blocked T1AM-induced Ca influx in IOBA- NHC cells (human conjunctival epithelial cells, Dinter et al. 2015a). In the cell lines N39 and N41,

T1AM might also activate a Gαs coupled GPCR, such as TAAR1 and ADRB2, and this might lead to the activation of TRMP8. Despite the expression of TAAR1 and ADRB2 in the cell line GT1-7, 2+ FOS activation, Gαs signaling or a Ca influx were detected after T1AM stimulation. Either the expression level of GPCRs and TRPs are too low to evoke signaling responses, or in N41 and N39 cells, the TRPM8 activation is mediated by a so far unknown GPCR which is not expressed in GT1-7 cells. TRPM8 is responsible for sensing cold in vivo, while its counterpart the TRPV1 senses warmth (Tan and Knight, 2018). The body responds to icilin or methanol (TRPM8 agonists) by inducing hy- perthermia (Ding et al., 2008). Further, these agonists are used as pain treatments as it is sug- gested that TRPM8 neurons upon activation secrete glutamate and thereby block pain sensory

66 5.2 Part II: The influence of T1AM on the murine brain and hypothalamic cell lines

2+ Figure 5.4: T1AM induces Ca influx via the TRPM8 In N39 (A) and N41 (B) cells, T1AM stimulation (10 µM) induces a Ca2+ influx.TRPM8 blockers, BCTC (C) or AMTB (D) partially decrease the intracellular Ca2+ concen- tration (Braunig et al., 2018b)

67 CHAPTER 5. DISCUSSION

nerves (Proudfoot et al., 2006). However, Manni et al.(2013) showed that T 1AM in low doses (0.13 - 4 µg/kg) improved memory and lowered the pain threshold, which was blocked by the MAO in- hibitor clorgyline. In conclusion, the memory and pain sensory effects of T1AM are a direct result of its metabolite TA1. Eventually, it should be clarified if TA1 is also a ligand for TRPM8 or the patch clamp recordings should be conducted using MAO inhibitors to block T1AM conversion to TA1 to solely investigate T1AM mediated ion flux. The hyperthermic effect of TRMP8 contradicts T1AM effects in rodents. Probably, an unknown thermoregulatory mechanism overrides the peripheral signaling of TRPM8 in the brain. It is also feasible that an interactome of TRPM8 and further membrane proteins in the brain can lead to anapyrexia. Moreover, the PVN does not express TRPM8 (Feetham et al., 2018). As mentioned earlier, neuronal projections from other regions of the brain or even within the hypothalamus could also induce the FOS activation of the PVN. TRPM8 activation leads to the inhibition of TRPV1, if they are co-expressed, which is the case for all used hypothalamic cell lines (Fig. 4.13,(Takaishi et al., 2016; Walcher et al., 2018)). TRPM8 and TRPV1 possess similar binding pockets and also share ligands, e.g., the antagonist BCTC (Khajavi et al., 2015). Therefore, it could be, that T1AM also activates the TRPV1. However, the TRPM8 activation in the hypothalamic cell lines N39 and N41 might disguise TRPV1 actions. T1AM could be tested in TRPM8 KO mice. Further, patch clamp experiments of brain sections of the here identified

PVN can elucidate the role of TRPM8 of the in vivo T1AM.

68 Chapter 6

Conclusion and final remarks

In this dissertation, I wanted to answer the questions if T1AM is a ligand for serotonin and histamine receptors. Besides, I wanted to elucidate if T1AM stimulates certain brain areas and if T1AM induces GPCR-mediated signaling cascades in hypothalamic cell lines?

T1AM is a ligand for HRH1 and 5-HT1b I could identify two new aminergic GPCRs as T1AM tar- gets: HRH1 and 5-HT1b. This makes aminergic T1AM signaling even more complicated (Fig. 5.2). Both GPCRs might be responsible for the anapyrexic effect, while HRH1 could also play a role in

T1AM-influenced eating behavior in rodents. To elucidate which receptor contributes to which ef- fect, HRH1 and 5-HT1b knockout mice exist to test for the known pharmacological effects of T1AM. If these GPCRs are not involved in T1AM-induced anapyrexia, the list of thermoregulatory GPCRs is still long: HRH3, 5-HT1a, 5-HT2c, 5-HT7, DRD1, and DRD2 (Lundius et al., 2010; Heisler et al., 1998; Gudelsky et al., 1986; Hedlund et al., 2003; Chaperon et al., 2003).

T1AM stimulation of 5-HT1b only leads to the activation of the α-subunit of the G-protein and not to PLC-signaling through the βγ-subunit. This unique biased signaling might be utilized to study the binding and rearrangement of Gi/o at the 5-HT1b using protein-interaction experiments. Biased signaling and biased ligands are one of the newest tools in drug development to target precisely specific signaling pathways to treat patients effectively and with minimal side effects. Nowadays, 5- HT1b agonists are used in migraine treatment to induce vasoconstriction of the intracranial blood vessels. Typical side effects of these drugs are fatigue, dizziness, paresthesias, warm sensations and neck, chest and throat tightness (Tepper et al., 2002). Unraveling if the vasoconstrictive effect of 5-HT1b is αi/o or Gβγ mediated could lead to new drugs with fewer side effects. HRH1 antagonist given as allergy-treatment show sleepiness as most severe side effects. Neither HRH antagonist nor 5-HT1b agonists have been described to induce hypothermia or anapyrexia in humans. Therefore, it would be highly unlikely for 5-HT1b and HRH1 to mediate T1AM-induced anapyrexia in humans, even if these GPCRs should be responsible for the temperature effects in rodents. However, mouse models would help to identify neuronal cell populations responsible for anapyrexia. With this knowledge, it might be possible to develop an anayprexia-inducing drug for humans.

T1AM activates neurons in the PVN The systemic injection of T1AM activated neurons in the murine PVN. One may speculate, that this activation can contribute to the anapyrexic effect and the influence on lipid and glucose metabolism of T1AM. However, it should not be left out of con- sideration, that T1AM metabolites, TA1 and T0AM, also show pharmacological effects. Therefore,

69 CHAPTER 6. CONCLUSION AND FINAL REMARKS

it should be investigated to what extent the PVN neurons are activated by T1AM or its metabolites in further studies. In hindsight, the mouse experiment should have included control groups with

T0AM, and TA1 injected mice. Thereby, one could tell which molecule activates PVN neurons.

T1AM induces a slight Gs signaling and TRPM8 activation in hypothalamic cell lines The en- dogenous signaling of hypothalamic cell lines showed that T1AM induced a slight increase in cAMP 2+ accumulation by GPCR signaling and Ca influx through the TRPM8 in vitro. It is feasible that T1AM induces a similar signaling cascade by first activating a GPCR, which leads to the activation of an ion channel subsequently leading to the induction of FOS in vivo in the murine PVN. However, TRPM8 activation leads to hyperthermia in mouse models and not anapyrexia. Further, PVN neurons do not express TRPM8. These two points speak against TRPM8 as a mediator of the anapyrexic ef- fect of T1AM. Nevertheless, it is not sure that the FOS activation in the PVN is the reason for the anapyrexic response. Moreover, several membrane proteins, like the HRH1, can induce hypo- and hyperthermia depending on the signaling constellation (Brezenoff and Lomax, 1970; Lundius et al.,

2010). Therefore, it still might be that TRPM8 induces anapyrexia upon T1AM stimulation in a spe- cific neuronal subtype.

Can the T1AM knowledge be transferred to patients? T1AM data were mainly generated in ro- dents. It is very questionable if its pharmacological effects are transferable to humans. Concerning temperature regulation, there are distinct differences in species. In contrast to rodents, humans are more inert to fluctuations of the environmental temperature. Only ethanol and hypoxia have been described to induce true anapyrexia in humans, while several compounds are known to cause anapyrexia in rodents (Osamu and Naotoshi, 2011). Furthermore, the thermoregulatory system is structured differently depending on the order of the mammals. Serotonin stimulation leads to an increase in body temperature in cats and dogs, whereas the temperature decreases in rabbits and sheep (Sheard and Aghajanian, 1967). For nore- pinephrine it is the other way around, stimulation leads to a decrease in cats and dogs and a rise in body temperature in sheep and rabbits. These results speak furthermore against the theory that a conserved ligand and receptor means that its physiological functions overlap for different species. GPCR expression pattern and receptor-ligand binding differ between species, even for highly conserved GPCRs. Taking TAAR1 as an example, several publications compare TAAR1 expression, basal activity, and ligand binding (Lindemann et al., 2005; Simmler et al., 2014; Coster et al., 2015; Simmler et al., 2016). In CHO-K1, cells rat TAAR1 has the highest expression, followed by chicken, crocodile, frog and elephant TAAR1(Coster et al., 2015). The lowest expression exhibit kangaroo and human receptors. The rat TAAR1 also has a distinct basal activity and an EC50 of 0.09 µM upon T1AM stimulation, while the human demonstrate nearly no basal signaling (EC50 of 1.69 µM upon T1AM stimulation, Coster et al. 2015). A pharmacological profile study for TAAR1 revealed that for most TAAR1 activating substances, the rat receptor has the lowest EC50, followed by the murine TAAR1 and lastly the human TAAR1 (Simmler et al., 2014). In conclusion, TAAR1 signaling is presumably more pronounced in rodents than in humans if using similar ligand concentrations. Another example is TAAR5. It is the most highly conserved TAAR subtype among all mammalian species, and still, T1AM activates only the human receptor, but not the murine (Dinter et al., 2015c).

T1AM is a multi-target ligand (Hoefig et al., 2016), now also including 5-HT1b and HRH1 as tar- gets. Furthermore, dimerization, like GPCRs and TRPs, might alter signal transduction, as shown here for TAAR1 and 5-HT1b. Presumably, every tissue and even cell type express a unique set of

70 T1AM targets. Therefore, these targets all together will mediate T1AM signaling pathways. This concept of in vivo signaling complexity illustrates the importance of in vitro studies as mine to first decipher in part T1AM signal transduction to get a complete picture of the intricacy of its signaling networks at the end.

71 Abbreviations

5-HT serotonin 5-HT1b serotonin 1b receptor ADRA2A α-adrenergix receptor 2A ADRB1 β-adrenergic receptor 1 ADRB2 β-adrenergic receptor 2 APOB100 apo-lipoprotein B100 CamKII calmodulin-dependent protein kinase II cAMP cyclic adenosin monophosphate CB1R canabinoid receptor 1 CFP cyano fluorescent protein CHRM3 muscarinergic receptor 3 CIP calf intestine phosphatase DA dopamine DIO deiodinase DIT diiodotyrosine DMEM Dulbecco’s Modified Eagle Medium DMH dorsolmedail nucleus DMSO dimethyl sulfoxide DRD dopamin receptor ECL extracellular loop FBS fetal bovine serum FOS proto-oncogene c-FOS FRET fluorescence resonance energy transfere FSK forskolin GHSR grehlin receptor GnRH gonadotrophin-releasing hormone GPCR g-protein coupled receptor GT1-7 GnRH-secreting neurons of the hypothalamus HPT axis hypothalamic–pituitary–thyroid axis HRH histamin receptor i.p. intraperitoneal ICL intracellular loop ISOP isoproterenol LC liquid chromatography MAO monoamine oxidase MAPK mitogen-activated protein kinase

72 MC3R 3 MIT monoiodotyrosine MPO medial preoptic area MS mass spectrometry N39 mHypoE-N39 N41 mHypoE-N41 NFAT nuclear factor of activated T-cells NIP non interacting protein NIS sodium-iodine transporter NorEpi L-Norepinephrine hydrochloride OCT octopamine ODC ornithine decarboxylase PAG periaqueductal gray PBS saline buffered PCR polymerase chain reaction PEA β-phenylethylamine PGK1 phosphoglycerate kinase 1 PKA protein kinase A PLC phopholipase C PVN paraventricular nucleus rCHRM3 rat muscarinergic receptor Rho rhodopsin SLUT sulfotransferases SON supra optic nucleus SRE serum response element

T0AM thyronamine T1AM 3-iodothyronamine T2AM 3,5-diiodothyronamine T3 3,5,3‘-triiodothyronine T3AM 3,5,3‘-triiodothyronamine T4 3,5,3‘,5‘-tetraiodothyrosine TA1 thyronacetic acid TAAR trace-amine-associated receptor TBS tris-buffered saline TM transmembrane TR thyroid hormone receptor TRE TR response element TRH thyrotropin-releasing hormone TRP transient receptor potential channel TRPM8 transient receptor potential cation channel family M 8 TRPV1 transient receptor potential cation channel family V 1 TSH thyrotropin/thyroid stimulating hormone TSHR thyroid stimulating hormone receptor VTA ventral tegemental segment YFP yellow fluorescent protein

73 CHAPTER 6. CONCLUSION AND FINAL REMARKS

74 List of Figures

1.1 Synthesis of thyroid hormones...... 7 1.2 Structures of THs, TAMs and aminergic ligands...... 9 1.3 Overview of GPCR Signaling...... 11 1.4 Structure of GPCRs...... 13 1.5 Thermoregulation...... 17

1.6 Overview of aminergic GPCRs as T1AM interaction partners...... 18 1.7 Experimental overview...... 21

3.1 Cloning workflow...... 29 3.2 qPCR workflow...... 33 3.3 Workflow of an AlphaScreen™ Technology based cAMP assay...... 36 3.4 Workflow for FRET experiments...... 38

4.1 Hypothesis and experimental steps in the elucidation of the role of T1AM at HRH1 and 5-HT1b...... 41

4.2 Comparison between Rho-TAAR1 and βTAAR1 regarding PEA and T1AM...... 42

4.3 Co-stimulation of T1AM and 5-HT does not enhance Gαs signaling at TAAR1...... 44

4.4 T1AM (10 µM) reduces histamine signaling at the HRH1...... 45

4.5 The TAAR1 agonist T1AM activates Gαi/o signaling through 5-HT1b...... 45

4.6 5-HT, but not T1AM, activates PLC and MAPK signaling at the 5-HT1b...... 46 4.7 TAAR1 and 5-HT1b form heterodimers...... 47 4.8 Signalosome of co-expressed TAAR1 and 5-HT1b...... 49

4.9 Summary:T1AM and the aminergic GPCRs HRH1 and 5-HT1b...... 50 4.10 Theoretical background to detect neuronal activity and signaling in hypothalamic cell lines...... 51

4.11 FOS positive PVN neurons after 1 h of i.p. injection of 50 mg/kg T1AM...... 52 4.12 cFOS staining of hypothalamic cell lines...... 53 4.13 Expression profile of GPCRs and TRP channels in GT1-7, N39, and N41...... 54

4.14 T1AM actions on hypothalamic Gαs and Gαi/o signaling...... 56

4.15 Summary: T1AM and neuromodulation...... 57

5.1 Binding of T1AM and 5-HT at the 5-HT1b...... 62

5.2 Signaling overview of new aminergic interaction partners of T1AM...... 63 5.3 Central regulation of vasodilation and vasoconstriction...... 64 2+ 5.4 T1AM induces Ca influx via the TRPM8...... 67

75 LIST OF FIGURES

A.1 Plasmid map of pcDps...... 99 A.2 Plasmid map of pEYFP-N1...... 100 A.3 Plasmid map of pEYFP-N1...... 100 A.4 Plasmid map of pGL4.3 (luc2/NFAT-RE/Hygro)...... 101 A.5 Plasmid map of pGL4.33 (luc2/SRE/Hygro)...... 101

B.1 Endogenous Gs signaling of hypothalamic cell lines...... 110

B.2 Endogenous Gi/o signaling of hypothalamic cell lines...... 111

C.1 Endogenous signaling of HEK293T...... 113 C.2 Adenylyl cyclase and PLC activity of 5-HT1b are pertussis toxin (PTX) sensitive..... 114 C.3 Influence of co-expression TAAR1 and HT1b on PLC and MAPK signaling...... 115

D.1 Controls of single transfected CFP and YFP constructs...... 116 D.2 Bleaching curves of GHSR/MC3R, CB1R/MC3R and TAAR1/rCHRM3...... 117

E.1 FOS staining of different murine brain areas (part 1)...... 118 E.2 FOS staining of different murine brain areas (part 2)...... 119

76 List of Tables

2.1 General chemicals and buffers...... 22 2.2 Protocols for culture media, agar plates and buffers...... 23 2.3 Consumables for cloning...... 23 2.4 Enzymes for cloning...... 23 2.5 Kits for cloning and plasmid purification...... 24 2.6 Consumables for cell culture...... 24 2.7 Consumables for transfection and functional assays...... 25 2.8 Reagents for immunohistochemistry...... 26 2.9 Antibodies...... 26

3.1 Cell lines...... 28 3.2 Transfection...... 32

A.1 Plasmid constructs...... 98 A.2 Primer for cloning...... 108 A.3 Sequencing primer...... 108 A.4 Murine qPCR primer...... 109

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97 Appendix A

Plasmid maps and gene sequences

Overview of used plasmids

Table A.1: Plasmid constructs

Gene Plasmid TAG resistance empty pcDps ampicilin TAAR1 pcDps beta ampicilin 5-HT1b pcDps NHA ampicilin DRD2 pcDps NHA ampicilin HRH1 pcDps ampicilin MC3R pcDps NHA ampicilin TSHR pcDps cFlag ampicilin ADRA2A pcDps NHA ampicilin empty pEYFP-N kanamycin TAAR1 pEYFP-N beta kanamycin 5-HT1b pEYFP-N kanamycin MC3R pEYFP-N kanamycin GHSR pEYFP-N kanamycin CB1R pEYFP-N kanamycin rM3 pEYFP-N kanamycin empty pECFP-N kanamycin TAAR1 pECFP-N beta kanamycin 5-HT1b pECFP-N kanamycin MC3R pECFP-N kanamycin GHSR pECFP-N kanamycin CB1R pECFP-N kanamycin rM3 pECFP-N kanamycin luc2 firefly luciferase pGL4.3 NFAT-RE hygromycin / ampicilin luc2 firefly luciferase pGL4.33 SRE hygromycin / ampicilin

98 Maps of vectores and their multiple cloning site

Plasmid sequences were gained from Addgene (www.addgene.com) and verfied via sequencing. Plasmid maps were generated with SnapGene Viewer (http://www.snapgene.com/products/snapgene_ viewer/)

pcDps

Figure A.1: Vector map of pcDps

99 APPENDIX A. PLASMID MAPS AND GENE SEQUENCES pEYFP-N1

Figure A.2: Vector map of pEYFP-N1 pECFP-N1

Figure A.3: Vector map of pECFP-N1

100 pGL4.3 [luc2/NFAT-RE/Hygro]

Figure A.4: Vector map of pGK4.3 by Promega including an NFAT response element

pGL4.33 [luc2/SRE/Hygro]

Figure A.5: Vector map of pGK4.33 by Promega including a serum response element

101 APPENDIX A. PLASMID MAPS AND GENE SEQUENCES

Gene sequences

Gene segences were gained from NCBI (https://www.ncbi.nlm.nih.gov/) and the UCSC Genome Browser (https://genome.ucsc.edu/).

TAAR1, trace amine associated receptor [Homo sapiens]

NCBI Reference Sequence: NM_138327.2 length: 1019 bp Yellow marks the added nine amino acids of the β-adrenergic receptor.

1 ATGGGGCAAC CCGGGAACGG CAGCGCCATG ATGCCCTTTT GCCACAATAT AATTAATATT TCCTGTGTGA 71 AAAACAACTG GTCAAATGAT GTCCGTGCTT CCCTGTACAG TTTAATGGTG CTCATAATTC TGACCACACT 141 CGTTGGCAAT CTGATAGTTA TTGTTTCTAT ATCACACTTC AAACAACTTC ATACCCCAAC AAATTGGCTC 211 ATTCATTCCA TGGCCACTGT GGACTTTCTT CTGGGGTGTC TGGTCATGCC TTACAGTATG GTGAGATCTG 281 CTGAGCACTG TTGGTATTTT GGAGAAGTCT TCTGTAAAAT TCACACAAGC ACCGACATTA TGCTGAGCTC 351 AGCCTCCATT TTCCATTTGT CTTTCATCTC CATTGACCGC TACTATGCTG TGTGTGATCC ACTGAGATAT 421 AAAGCCAAGA TGAATATCTT GGTTATTTGT GTGATGATCT TCATTAGTTG GAGTGTCCCT GCTGTTTTTG 491 CATTTGGAAT GATCTTTCTG GAGCTAAACT TCAAAGGCGC TGAAGAGATA TATTACAAAC ATGTTCACTG 561 CAGAGGAGGT TGCTCTGTCT TCTTTAGCAA AATATCTGGG GTACTGACCT TTATGACTTC TTTTTATATA 631 CCTGGATCTA TTATGTTATG TGTCTATTAC AGAATATATC TTATCGCTAA AGAACAGGCA AGATTAATTA 701 GTGATGCCAA TCAGAAGCTC CAAATTGGAT TGGAAATGAA AAATGGAATT TCACAAAGCA AAGAAAGGAA 771 AGCTGTGAAG ACATTGGGGA TTGTGATGGG AGTTTTCCTA ATATGCTGGT GCCCTTTCTT TATCTGTACA 841 GTCATGGACC CTTTTCTTCA CTACATTATT CCACCTACTT TGAATGATGT ATTGATTTGG TTTGGCTACT 911 TGAACTCTAC ATTTAATCCA ATGGTTTATG CATTTTTCTA TCCTTGGTTT AGAAAAGCAC TGAAGATGAT 981 GCTGTTTGGT AAAATTTTCC AAAAAGATTC ATCCAGGTGT AAATTATTTT TGGAATTGAG TTCA

5-HT1b, serotonin receptor 1b [Homo sapiens]

NCBI Reference Sequence: NM_000863.2 length: 1173 bp

1 ATGGAGGAAC CGGGTGCTCA GTGCGCTCCA CCGCCGCCCG CGGGCTCCGA GACCTGGGTT CCTCAAGCCA 71 ACTTATCCTC TGCTCCCTCC CAAAACTGCA GCGCCAAGGA CTACATTTAC CAGGACTCCA TCTCCCTACC 141 CTGGAAAGTA CTGCTGGTTA TGCTATTGGC GCTCATCACC TTGGCCACCA CGCTCTCCAA TGCCTTTGTG 211 ATTGCCACAG TGTACCGGAC CCGGAAACTG CACACCCCGG CTAACTACCT GATCGCCTCT CTGGCGGTCA 281 CCGACCTGCT TGTGTCCATC CTGGTGATGC CCATCAGCAC CATGTACACT GTCACCGGCC GCTGGACACT 351 GGGCCAGGTG GTCTGTGACT TCTGGCTGTC GTCGGACATC ACTTGTTGCA CTGCCTCCAT CCTGCACCTC 421 TGTGTCATCG CCCTGGACCG CTACTGGGCC ATCACGGACG CCGTGGAGTA CTCAGCTAAA AGGACTCCCA 491 AGAGGGCGGC GGTCATGATC GCGCTGGTGT GGGTCTTCTC CATCTCTATC TCGCTGCCGC CCTTCTTCTG 561 GCGTCAGGCT AAGGCCGAAG AGGAGGTGTC GGAATGCGTG GTGAACACCG ACCACATCCT CTACACGGTC 631 TACTCCACGG TGGGTGCTTT CTACTTCCCC ACCCTGCTCC TCATCGCCCT CTATGGCCGC ATCTACGTAG 701 AAGCCCGCTC CCGGATTTTG AAACAGACGC CCAACAGGAC CGGCAAGCGC TTGACCCGAG CCCAGCTGAT 771 AACCGACTCC CCCGGGTCCA CGTCCTCGGT CACCTCTATT AACTCGCGGG TTCCCGACGT GCCCAGCGAA 841 TCCGGATCTC CTGTGTATGT GAACCAAGTC AAAGTGCGAG TCTCCGACGC CCTGCTGGAA AAGAAGAAAC 911 TCATGGCCGC TAGGGAGCGC AAAGCCACCA AGACCCTAGG GATCATTTTG GGAGCCTTTA TTGTGTGTTG

102 981 GCTACCCTTC TTCATCATCT CCCTAGTGAT GCCTATCTGC AAAGATGCCT GCTGGTTCCA CCTAGCCATC 1051 TTTGACTTCT TCACATGGCT GGGCTATCTC AACTCCCTCA TCAACCCCAT AATCTATACC ATGTCCAATG 1121 AGGACTTTAA ACAAGCATTC CATAAACTGA TACGTTTTAA GTGCACAAGT TGA

DRD2, dopamin receptor 2, transcript variant 1 [Homo sapiens]

NCBI Reference Sequence: NM_000795.3 length: 1332 bp

1 ATGGTGAACC TGAGGAATGC GGTGCATTCA TTCCTTGTGC ACCTAATTGG CCTATTGGTT TGGCAATgTG 71 ATATTTCTGT GAGCCCAGTA GCAGCTATAG TAACTGACAT TTTCAATACC TCCGATGGTG GACGCTTCAA 141 ATTCCCAGAC GGGGTACAAA ACTGGCCAGC ACTTTCAATC GTCATCATAA TAATCATGAC AATAGGTGGC 211 AACATCCTTG TGATCATGGC AGTAAGCATG GAAAAGAAAC TGCACAATGC CACCAATTAC TTCTTAATGT 281 CCCTAGCCAT TGCTGATATG CTAGTGGGAC TACTTGTCAT GCCCCTGTCT CTCCTGGCAA TCCTTTATGA 351 TTATGTCTGG CCACTACCTA GATATTTGTG CCCCGTCTGG ATTTCTTTAG ATGTTTTATT TTCAACAGCG 421 TCCATCATGC ACCTCTGCGC TATATCGCTG GATCGGTATG TAGCAATACG TAATCCTATT GAGCATAGCC 491 GTTTCAATTC GCGGACTAAG GCCATCATGA AGATTGCTAT TGTTTGGGCA ATTTCTATAG GTGTATCAGT 561 TCCTATCCCT GTGATTGGAC TGAGGGACGA AGAAAAGGTG TTCGTGAACA ACACGACGTG CGTGCTCAAC 631 GACCCAAATT TCGTTCTTAT TGGGTCCTTC GTAGCTTTCT TCATACCGCT GACGATTATG GTGATTACGT 701 ATTGCCTGAC CATCTACGTT CTGCGCCGAC AAGCTTTGAT GTTACTGCAC GGCCACACCG AGGAACCGCC 771 TGGACTAAGT CTGGATTTCC TGAAGTGCTG CAAGAGGAAT ACGGCCGAGG AAGAGAACTC TGCAAACCCT 841 AACCAAGACC AGAACGCACG CCGAAGAAAG AAGAAGGAGA GACGTCCTAG GGGCACCATG CAGGCTATCA 911 ACAATGAAAG AAAAGCTTCG AAAGTCCTTG GGATTGTTTT CTTTGTGTTT CTGATCATGT GGTGCCCATT 981 TTTCATTACC AATATTCTGT CTGTTCTTTG TGAGAAGTCC TGTAACCAAA AGCTCATGGA AAAGCTTCTG 1051 AATGTGTTTG TTTGGATTGG CTATGTTTGT TCAGGAATCA ATCCTCTGGT GTATACTCTG TTCAACAAAA 1121 TTTACCGAAG GGCATTCTCC AACTATTTGC GTTGCAATTA TAAGGTAGAG AAAAAGCCTC CTGTCAGGCA 1191 GATTCCAAGA GTTGCCGCCA CTGCTTTGTC TGGGAGGGAG CTTAATGTTA ACATTTATCG GCATACCAAT 1261 GAACCGGTGA TCGAGAAAGC CAGTGACAAT GAGCCCGGTA TAGAGATGCA AGTTGAGAAT TTAGAGTTAC 1331 CAGTAAATCC CTCCAGTGTG GTTAGCGAAA GGATTAGCAG TGTGTGA

HRH1, histamin receptor 1, transcript variant 1 [Homo sapiens]

NCBI Reference Sequence: NM_001098213.1 length: 1464 bp

1 ATGAGCCTCC CCAATTCCTC CTGCCTCTTA GAAGACAAGA TGTGTGAGGG CAACAAGACC ACTATGGCCA 71 GCCCCCAGCT GATGCCCCTG GTGGTGGTCC TGAGCACTAT CTGCTTGGTC ACAGTAGGGC TCAACCTGCT 141 GGTGCTGTAT GCCGTACGGA GTGAGCGGAA GCTCCACACT GTGGGGAACC TGTACATCGT CAGCCTCTCG 211 GTGGCGGACT TGATCGTGGG TGCCGTCGTC ATGCCTATGA ACATCCTCTA CCTGCTCATG TCCAAGTGGT 281 CACTGGGCCG TCCTCTCTGC CTCTTTTGGC TTTCCATGGA CTATGTGGCC AGCACAGCGT CCATTTTCAG 351 TGTCTTCATC CTGTGCATTG ATCGCTACCG CTCTGTCCAG CAGCCCCTCA GGTACCTTAA GTATCGTACC 421 AAGACCCGAG CCTCGGCCAC CATTCTGGGG GCCTGGTTTC TCTCTTTTCT GTGGGTTATT CCCATTCTAG 491 GCTGGAATCA CTTCATGCAG CAGACCTCGG TGCGCCGAGA GGACAAGTGT GAGACAGACT TCTATGATGT 561 CACCTGGTTC AAGGTCATGA CTGCCATCAT CAACTTCTAC CTGCCCACCT TGCTCATGCT CTGGTTCTAT 631 GCCAAGATCT ACAAGGCCGT ACGACAACAC TGCCAGCACC GGGAGCTCAT CAATAGGTCC CTCCCTTCCT 701 TCTCAGAAAT TAAGCTGAGG CCAGAGAACC CCAAGGGGGA TGCCAAGAAA CCAGGGAAGG AGTCTCCCTG

103 APPENDIX A. PLASMID MAPS AND GENE SEQUENCES

771 GGAGGTTCTG AAAAGGAAGC CAAAAGATGC TGGTGGTGGA TCTGTCTTGA AGTCACCATC CCAAACCCCC 841 AAGGAGATGA AATCCCCAGT TGTCTTCAGC CAAGAGGATG ATAGAGAAGT AGACAAACTC TACTGCTTTC 911 CACTTGATAT TGTGCACATG CAGGCTGCGG CAGAGGGGAG TAGCAGGGAC TATGTAGCCG TCAACCGGAG 981 CCATGGCCAG CTCAAGACAG ATGAGCAGGG CCTGAACACA CATGGGGCCA GCGAGATATC AGAGGATCAG 1051 ATGTTAGGTG ATAGCCAATC CTTCTCTCGA ACGGACTCAG ATACCACCAC AGAGACAGCA CCAGGCAAAG 1121 GCAAATTGAG GAGTGGGTCT AACACAGGCC TGGATTACAT CAAGTTTACT TGGAAGAGGC TCCGCTCGCA 1191 TTCAAGACAG TATGTATCTG GGTTGCACAT GAACCGCGAA AGGAAGGCCG CCAAACAGTT GGGTTTTATC 1261 ATGGCAGCCT TCATCCTCTG CTGGATCCCT TATTTCATCT TCTTCATGGT CATTGCCTTC TGCAAGAACT 1331 GTTGCAATGA ACATTTGCAC ATGTTCACCA TCTGGCTGGG CTACATCAAC TCCACACTGA ACCCCCTCAT 1401 CTACCCCTTG TGCAATGAGA ACTTCAAGAA GACATTCAAG AGAATTCTGC ATATTCGCTC CTAA

MC3R, molanocortin 3 receptor [Homo sapiens]

NCBI Reference Sequence: NM_019888.3 length: 972 bp

1 ATGAATGCTT CGTGCTGCCT GCCCTCTGTT CAGCCAACAC TGCCTAATGG CTCGGAGCAC CTCCAAGCCC 71 CTTTCTTCAG CAACCAGAGC AGCAGCGCCT TCTGTGAGCA GGTCTTCATC AAGCCCGAGG TTTTCCTGTC 141 TCTGGGCATC GTCAGTCTGC TGGAAAACAT CCTGGTTATC CTGGCCGTGG TCAGGAACGG CAACCTGCAC 211 TCCCCGATGT ACTTCTTTCT CTGCAGCCTG GCGGTGGCCG ACATGCTGGT AAGTGTGTCC AATGCCCTGG 281 AGACCATCAT GATCGCCATC GTCCACAGCG ACTACCTGAC CTTCGAGGAC CAGTTTATCC AGCACATGGA 351 CAACATCTTC GACTCCATGA TCTGCATCTC CCTGGTGGCC TCCATCTGCA ACCTCCTGGC CATCGCCGTC 421 GACAGGTACG TCACCATCTT TTACGCGCTC CGCTACCACA GCATCATGAC CGTGAGGAAG GCCCTCACCT 491 TGATCGTGGC CATCTGGGTC TGCTGCGGCG TCTGTGGCGT GGTGTTCATC GTCTACTCGG AGAGCAAAAT 561 GGTCATTGTG TGCCTCATCA CCATGTTCTT CGCCATGATG CTCCTCATGG GCACCCTCTA CGTGCACATG 631 TTCCTCTTTG CGCGGCTGCA CGTCAAGCGC ATAGCAGCAC TGCCACCTGC CGACGGGGTG GCCCCACAGC 701 AACACTCATG CATGAAGGGG GCAGTCACCA TCACCATTCT CCTGGGCGTG TTCATCTTCT GCTGGGCCCC 771 CTTCTTCCTC CACCTGGTCC TCATCATCAC CTGCCCCACC AACCCCTACT GCATCTGCTA CACTGCCCAC 841 TTCAACACCT ACCTGGTCCT CATCATGTGC AACTCCGTCA TCGACCCACT CATCTACGCT TTCCGGAGCC 911 TGGAATTGCG CAACACCTTT AGGGAGATTC TCTGTGGCTG CAACGGCATG AACTTGGGAT AG

GHSR, growth hormone secretagogue receptor or grehlin receptor, transcript variant 1a [Homo sapiens]

NCBI Reference Sequence: NM_198407.2 length: 1101 bp

1 ATGTGGAACG CGACGCCCAG CGAAGAGCCG GGGTTCAACC TCACACTGGC CGACCTGGAC TGGGATGCTT 71 CCCCCGGCAA CGACTCGCTG GGCGACGAGC TGCTGCAGCT CTTCCCCGCG CCGCTGCTGG CGGGCGTCAC 141 AGCCACCTGC GTGGCACTCT TCGTGGTGGG CATCGCTGGC AACCTGCTCA CCATGCTGGT GGTGTCGCGC 211 TTCCGCGAGC TGCGCACCAC CACCAACCTC TACCTGTCCA GCATGGCCTT CTCCGATCTG CTCATCTTCC 281 TCTGCATGCC CCTGGACCTC GTTCGCCTCT GGCAGTACCG GCCCTGGAAC TTCGGCGACC TCCTCTGCAA 351 ACTCTTCCAA TTCGTCAGTG AGAGCTGCAC CTACGCCACG GTGCTCACCA TCACAGCGCT GAGCGTCGAG 421 CGCTACTTCG CCATCTGCTT CCCACTCCGG GCCAAGGTGG TGGTCACCAA GGGGCGGGTG AAGCTGGTCA 491 TCTTCGTCAT CTGGGCCGTG GCCTTCTGCA GCGCCGGGCC CATCTTCGTG CTAGTCGGGG TGGAGCACGA 561 GAACGGCACC GACCCTTGGG ACACCAACGA GTGCCGCCCC ACCGAGTTTG CGGTGCGCTC TGGACTGCTC

104 631 ACGGTCATGG TGTGGGTGTC CAGCATCTTC TTCTTCCTTC CTGTCTTCTG TCTCACGGTC CTCTACAGTC 701 TCATCGGCAG GAAGCTGTGG CGGAGGAGGC GCGGCGATGC TGTCGTGGGT GCCTCGCTCA GGGACCAGAA 771 CCACAAGCAA ACCGTGAAAA TGCTGGCTGT AGTGGTGTTT GCCTTCATCC TCTGCTGGCT CCCCTTCCAC 841 GTAGGGCGAT ATTTATTTTC CAAATCCTTT GAGCCTGGCT CCTTGGAGAT TGCTCAGATC AGCCAGTACT 911 GCAACCTCGT GTCCTTTGTC CTCTTCTACC TCAGTGCTGC CATCAACCCC ATTCTGTACA ACATCATGTC 981 CAAGAAGTAC CGGGTGGCAG TGTTCAGACT TCTGGGATTC GAACCCTTCT CCCAGAGAAA GCTCTCCACT 1051 CTGAAAGATG AAAGTTCTCG GGCCTGGACA GAATCTAGTA TTAATACATG A

CB1R, cannabinoid receptor 1, transcript variant 4 [Homo sapiens]

NCBI Reference Sequence: NM_001160258.1 length: 1437 bp

1 ATGAAGTCGA TCCTAGATGG CCTTGCAGAT ACCACCTTCC GCACCATCAC CACTGACCTC CTGTACGTGG 71 GCTCAAATGA CATTCAGTAC GAAGACATCA AAGGTGACAT GGCATCCAAA TTAGGGTACT TCCCACAGAA 141 ATTCCCTTTA ACTTCCTTTA GGGGAAGTCC CTTCCAAGAG AAGATGACTG CGGGAGACAA CCCCCAGCTA 211 GTCCCAGCAG ACCAGGTGAA CATTACAGAA TTTTACAACA AGTCTCTCTC GTCCTTCAAG GAGAATGAGG 281 AGAACATCCA GTGTGGGGAG AACTTCATGG ACATAGAGTG TTTCATGGTC CTGAACCCCA GCCAGCAGCT 351 GGCCATTGCA GTCCTGTCCC TCACGCTGGG CACCTTCACG GTCCTGGAGA ACCTCCTGGT GCTGTGCGTC 421 ATCCTCCACT CCCGCAGCCT CCGCTGCAGG CCTTCCTACC ACTTCATCGG CAGCCTGGCG GTGGCAGACC 491 TCCTGGGGAG TGTCATTTTT GTCTACAGCT TCATTGACTT CCACGTGTTC CACCGCAAAG ATAGCCGCAA 561 CGTGTTTCTG TTCAAACTGG GTGGGGTCAC GGCCTCCTTC ACTGCCTCCG TGGGCAGCCT GTTCCTCACA 631 GCCATCGACA GGTACATATC CATTCACAGG CCCCTGGCCT ATAAGAGGAT TGTCACCAGG CCCAAGGCCG 701 TGGTGGCGTT TTGCCTGATG TGGACCATAG CCATTGTGAT CGCCGTGCTG CCTCTCCTGG GCTGGAACTG 771 CGAGAAACTG CAATCTGTTT GCTCAGACAT TTTCCCACAC ATTGATGAAA CCTACCTGAT GTTCTGGATC 841 GGGGTCACCA GCGTACTGCT TCTGTTCATC GTGTATGCGT ACATGTATAT TCTCTGGAAG GCTCACAGCC 911 ACGCCGTCCG CATGATTCAG CGTGGCACCC AGAAGAGCAT CATCATCCAC ACGTCTGAGG ATGGGAAGGT 981 ACAGGTGACC CGGCCAGACC AAGCCCGCAT GGACATTAGG TTAGCCAAGA CCCTGGTCCT GATCCTGGTG 1051 GTGTTGATCA TCTGCTGGGG CCCTCTGCTT GCAATCATGG TGTATGATGT CTTTGGGAAG ATGAACAAGC 1121 TCATTAAGAC GGTGTTTGCA TTCTGCAGTA TGCTCTGCCT GCTGAACTCC ACCGTGAACC CCATCATCTA 1191 TGCTCTGAGG AGTAAGGACC TGCGACACGC TTTCCGGAGC ATGTTTCCCT CTTGTGAAGG CACTGCGCAG 1261 CCTCTGGATA ACAGCATGGG GGACTCGGAC TGCCTGCACA AACACGCAAA CAATGCAGCC AGTGTTCACA 1331 GGGCCGCAGA AAGCTGCATC AAGAGCACGG TCAAGATTGC CAAGGTAACC ATGTCTGTGT CCACAGACAC 1401 GTCTGCCGAG GCTCTGTGA

CHRM3, cholinergic receptor, muscarinic 3 [Rattus norvegicus]

NCBI Reference Sequence: NM_012527.2 length: 1770 bp

1 ATGACCTTGC ACAGTAACAG TACAACCTCG CCTTTGTTTC CCAACATCAG CTCTTCCTGG GTGCACAGTC 71 CCTCGGAGGC AGGGCTGCCC TTGGGGACAG TCACTCAGTT GGGCAGCTAC AACATTTCAC AAGAAACTGG 141 GAATTTCTCC TCAAACGACA CCTCCAGCGA CCCTCTCGGG GGTCACACCA TCTGGCAAGT GGTCTTCATT 211 GCCTTCTTAA CCGGCTTCCT GGCATTGGTG ACCATCATTG GCAACATCCT TGTCATTGTG GCCTTCAAGG 281 TCAACAAACA GCTGAAGACA GTCAACAACT ACTTCCTCTT AAGCCTGGCC TGTGCAGACC TGATCATCGG 351 GGTCATTTCC ATGAACCTGT TCACTACCTA CATCATTATG AACCGTTGGG CACTGGGGAA CTTAGCCTGC

105 APPENDIX A. PLASMID MAPS AND GENE SEQUENCES

421 GACCTCTGGC TTTCCATTGA CTATGTGGCC AGCAATGCCT CTGTCATGAA TCTGCTGGTC ATCAGCTTTG 491 ACAGGTACTT TTCCATCACT AGACCACTCA CCTACCGAGC CAAAAGAACA ACAAAACGAG CTGGTGTGAT 561 GATTGGTCTG GCTTGGGTCA TCTCCTTTGT CCTATGGGCT CCTGCCATCT TGTTCTGGCA ATACTTTGTA 631 GGGAAGAGAA CTGTGCCCCC AGGAGAATGT TTCATTCAGT TTCTGAGTGA GCCCACCATC ACCTTCGGCA 701 CGGCGATCGC TGCCTTTTAC ATGCCTGTCA CCATCATGAC TATTTTATAC TGGAGGATCT ATAAGGAAAC 771 TGAGAAGCGT ACCAAAGAGC TGGCTGGCCT ACAGGCCTCT GGGACAGAAG CGGAGGCAGA AAACTTTGTC 841 CACCCCACAG GCAGTTCTCG AAGCTGTAGC AGCTATGAAC TGCAACAGCA AGGCGTGAAA CGATCATCCA 911 GGAGGAAGTA CGGTCGCTGT CACTTCTGGT TCACCACCAA GAGCTGGAAG CCCAGTGCCG AGCAGATGGA 981 CCAAGACCAC AGCAGCAGCG ACAGTTGGAA CAACAACGAT GCTGCTGCCT CCCTGGAAAA CTCTGCTTCC 1051 TCCGATGAAG AGGACATTGG CTCAGAGACC AGGGCCATCT ATTCCATTGT CCTCAAGCTT CCAGGCCATA 1121 GCTCCATCCT CAACTCTACC AAGCTACCGT CCTCAGATAA CCTGCAGGTG TCCAACGAGG ACCTGGGGAC 1191 TGTGGATGTG GAGAGAAATG CTCACAAGCT TCAGGCCCAG AAGAGCATGG GTGATGGTGA CAACTGTCAG 1261 AAGGATTTCA CCAAGCTTCC CATCCAGTTA GAGTCTGCCG TGGACACAGG CAAGACCTCT GACACCAACT 1331 CCTCGGCAGA CAAGACCACG GCTACTCTAC CTCTGTCCTT CAAGGAGGCC ACGCTGGCTA AGAGGTTTGC 1401 TCTCAAGACC AGAAGTCAGA TCACCAAGCG GAAGAGGATG TCGCTCATCA AGGAGAAGAA GGCCGCCCAG 1471 ACGCTCAGTG CCATCTTGCT AGCCTTCATC ATCACGTGGA CCCCCTACAA CATCATGGTC CTGGTGAACA 1541 CCTTCTGTGA CAGCTGCATA CCCAAAACCT ATTGGAATCT GGGCTACTGG CTGTGCTATA TCAACAGCAC 1611 CGTGAACCCT GTGTGCTATG CCCTGTGCAA CAAAACATTC AGAACCACCT TCAAGACGCT CCTCTTGTGC 1681 CAGTGTGACA AAAGGAAGAG GCGCAAACAG CAGTACCAGC AGAGACAGTC GGTCATTTTT CACAAGCGAG 1751 TGCCGGAACA GGCCTTGTAG

TSHR, thyroid stimulating hormone receptor, transcript variant 1 [Homo sapi- ens]

NCBI Reference Sequence: NM_000369.2 length: 2316 bp

1 ATGAGGCCGG CGGACTTGCT GCAGCTGGTG CTGCTGCTCG ACCTGCCCAG GGACCTGGGC GGAATGGGGT 71 GTTCGTCTCC ACCCTGCGAG TGCCATCAGG AGGAGGACTT CAGAGTCACC TGCAAGGATA TTCAACGCAT 141 CCCCAGCTTA CCGCCCAGTA CGCAGACTCT GAAGCTTATT GAGACTCACC TGAGAACTAT TCCAAGTCAT 211 GCATTTTCTA ATCTGCCCAA TATTTCCAGA ATCTACGTAT CTATAGATGT GACTCTGCAG CAGCTGGAAT 281 CACACTCCTT CTACAATTTG AGTAAAGTGA CTCACATAGA AATTCGGAAT ACCAGGAACT TAACTTACAT 351 AGACCCTGAT GCCCTCAAAG AGCTCCCCCT CCTAAAGTTC CTTGGCATTT TCAACACTGG ACTTAAAATG 421 TTCCCTGACC TGACCAAAGT TTATTCCACT GATATATTCT TTATACTTGA AATTACAGAC AACCCTTACA 491 TGACGTCAAT CCCTGTGAAT GCTTTTCAGG GACTATGCAA TGAAACCTTG ACACTGAAGC TGTACAACAA 561 TGGCTTTACT TCAGTCCAAG GATATGCTTT CAATGGGACA AAGCTGGATG CTGTTTACCT AAACAAGAAT 631 AAATACCTGA CAGTTATTGA CAAAGATGCA TTTGGAGGAG TATACAGTGG ACCAAGCTTG CTGGACGTGT 701 CTCAAACCAG TGTCACTGCC CTTCCATCCA AAGGCCTGGA GCACCTGAAG GAACTGATAG CAAGAAACAC 771 CTGGACTCTT AAGAAACTTC CACTTTCCTT GAGTTTCCTT CACCTCACAC GGGCTGACCT TTCTTACCCA 841 AGCCACTGCT GTGCTTTTAA GAATCAGAAG AAAATCAGAG GAATCCTTGA GTCCTTGATG TGTAATGAGA 911 GCAGTATGCA GAGCTTGCGC CAGAGAAAAT CTGTGAATGC CTTGAATAGC CCCCTCCACC AGGAATATGA 981 AGAGAATCTG GGTGACAGCA TTGTTGGGTA CAAGGAAAAG TCCAAGTTCC AGGATACTCA TAACAACGCT 1051 CATTATTACG TCTTCTTTGA AGAACAAGAG GATGAGATCA TTGGTTTTGG CCAGGAGCTC AAAAACCCCC 1121 AGGAAGAGAC TCTACAAGCT TTTGACAGCC ATTATGACTA CACCATATGT GGGGACAGTG AAGACATGGT 1191 GTGTACCCCC AAGTCCGATG AGTTCAACCC GTGTGAAGAC ATAATGGGCT ACAAGTTCCT GAGAATTGTG

106 1261 GTGTGGTTCG TTAGTCTGCT GGCTCTCCTG GGCAATGTCT TTGTCCTGCT TATTCTCCTC ACCAGCCACT 1331 ACAAACTGAA CGTCCCCCGC TTTCTCATGT GCAACCTGGC CTTTGCGGAT TTCTGCATGG GGATGTACCT 1401 GCTCCTCATC GCCTCTGTAG ACCTCTACAC TCACTCTGAG TACTACAACC ATGCCATCGA CTGGCAGACA 1471 GGCCCTGGGT GCAACACGGC TGGTTTCTTC ACTGTCTTTG CAAGCGAGTT ATCGGTGTAT ACGCTGACGG 1541 TCATCACCCT GGAGCGCTGG TATGCCATCA CCTTCGCCAT GCGCCTGGAC CGGAAGATCC GCCTCAGGCA 1611 CGCATGTGCC ATCATGGTTG GGGGCTGGGT TTGCTGCTTC CTTCTCGCCC TGCTTCCTTT GGTGGGAATA 1681 AGTAGCTATG CCAAAGTCAG TATCTGCCTG CCCATGGACA CCGAGACCCC TCTTGCTCTG GCATATATTG 1751 TTTTTGTTCT GACGCTCAAC ATAGTTGCCT TCGTCATCGT CTGCTGCTGT TATGTGAAGA TCTACATCAC 1821 AGTCCGAAAT CCGCAGTACA ACCCAGGGGA CAAAGATACC AAAATTGCCA AGAGGATGGC TGTGTTGATC 1891 TTCACCGACT TCATATGCAT GGCCCCAATC TCATTCTATG CTCTGTCAGC AATTCTGAAC AAGCCTCTCA 1961 TCACTGTTAG CAACTCCAAA ATCTTGCTGG TACTCTTCTA TCCACTTAAC TCCTGTGCCA ATCCATTCCT 2031 CTATGCTATT TTCACCAAGG CCTTCCAGAG GGATGTGTTC ATCCTACTCA GCAAGTTTGG CATCTGTAAA 2101 CGCCAGGCTC AGGCATACCG GGGGCAGAGG GTTCCTCCAA AGAACAGCAC TGATATTCAG GTTCAAAAGG 2171 TTACCCACGA GATGAGGCAG GGTCTCCACA ACATGGAAGA TGTCTATGAA CTGATTGAAA ACTCCCATCT 2241 AACCCCAAAG AAGCAAGGCC AAATCTCAGA AGAGTATATG CAAACGGTTT TGTAA

ADRA2A, alpha-2a adrenoreceptor [Homo sapiens]

NCBI Reference Sequence: NM_000681.3 length: 1398 bp

1 ATGTTCCGCC AGGAGCAGCC GTTGGCCGAG GGCAGCTTTG CGCCCATGGG CTCCCTGCAG CCGGACGCGG 71 GCAACGCGAG CTGGAACGGG ACCGAGGCGC CGGGGGGCGG CGCCCGGGCC ACCCCTTACT CCCTGCAGGT 141 GACGCTGACG CTGGTGTGCC TGGCCGGCCT GCTCATGCTG CTCACCGTGT TCGGCAACGT GCTCGTCATC 211 ATCGCCGTGT TCACGAGCCG CGCGCTCAAG GCGCCCCAAA ACCTCTTCCT GGTGTCTCTG GCCTCGGCCG 281 ACATCCTGGT GGCCACGCTC GTCATCCCTT TCTCGCTGGC CAACGAGGTC ATGGGCTACT GGTACTTCGG 351 CAAGGCTTGG TGCGAGATCT ACCTGGCGCT CGACGTGCTC TTCTGCACGT CGTCCATCGT GCACCTGTGC 421 GCCATCAGCC TGGACCGCTA CTGGTCCATC ACACAGGCCA TCGAGTACAA CCTGAAGCGC ACGCCGCGCC 491 GCATCAAGGC CATCATCATC ACCGTGTGGG TCATCTCGGC CGTCATCTCC TTCCCGCCGC TCATCTCCAT 561 CGAGAAGAAG GGCGGCGGCG GCGGCCCGCA GCCGGCCGAG CCGCGCTGCG AGATCAACGA CCAGAAGTGG 631 TACGTCATCT CGTCGTGCAT CGGCTCCTTC TTCGCTCCCT GCCTCATCAT GATCCTGGTC TACGTGCGCA 701 TCTACCAGAT CGCCAAGCGT CGCACCCGCG TGCCACCCAG CCGCCGGGGT CCGGACGCCG TCGCCGCGCC 771 GCCGGGGGGC ACCGAGCGCA GGCCCAACGG TCTGGGCCCC GAGCGCAGCG CGGGCCCGGG GGGCGCAGAG 841 GCCGAACCGC TGCCCACCCA GCTCAACGGC GCCCCTGGCG AGCCCGCGCC GGCCGGGCCG CGCGACACCG 911 ACGCGCTGGA CCTGGAGGAG AGCTCGTCTT CCGACCACGC CGAGCGGCCT CCAGGGCCCC GCAGACCCGA 981 GCGCGGTCCC CGGGGCAAAG GCAAGGCCCG AGCGAGCCAG GTGAAGCCGG GCGACAGCCT GCCGCGGCGC 1051 GGGCCGGGGG CGACGGGGAT CGGGACGCCG GCTGCAGGGC CGGGGGAGGA GCGCGTCGGG GCTGCCAAGG 1121 CGTCGCGCTG GCGCGGGCGG CAGAACCGCG AGAAGCGCTT CACGTTCGTG CTGGCCGTGG TCATCGGAGT 1191 GTTCGTGGTG TGCTGGTTCC CCTTCTTCTT CACCTACACG CTCACGGCCG TCGGGTGCTC CGTGCCACGC 1261 ACGCTCTTCA AATTCTTCTT CTGGTTCGGC TACTGCAACA GCTCGTTGAA CCCGGTCATC TACACCATCT 1331 TCAACCACGA TTTCCGCCGC GCCTTCAAGA AGATCCTCTG TCGGGGGGAC AGGAAGCGGA TCGTGTGA

107 APPENDIX A. PLASMID MAPS AND GENE SEQUENCES

Primer

Cloning primer

Table A.2: Primer for cloning cloning βTAAR into pEYFP-N1 and pECFP-N1 with EcoR I/KpnI fw 5’-GCTTTGAATTCGCCACCATGGGGCAACCCGGGAACGGCAGCGCCATGATGCCCTTTTGCCACAA rv 5’-AATGGTACCGTCGATGAACTCAA cloning 5-HT1b into pEYFP-N1 and pECFP-N1 with XhoI/BamH I fw 5’- AAAACTCGAGCGGGAGATCTGGTACCGC rv 5’- TTTGGATCCCAACTTGTGCACTTAAAACGTATCAGT cloning βTAAR into pcDps with KpnI/SpeI fw’- AACTAAGGTACCGCCACCATGGGGCAACC rv’- TACTCTACTAGTCTATGAACTCAATTCCAAAAATAATTTACACC cloning HRH1 into pcDps with SmaI/SpeI fw 5’- CCCGGGGCCACCATGAGCCTCCCCAATTCC rv 5’- CCACTAGTTCAGGAGCGAATATGCAGAATTCTCTTGAATG

Sequencing primer

Table A.3: Sequencing primer

Sequencing primer

CD-F 5’-TAGGCCTGTACGGAAGTG CD-R 5’-GTCCAAACTCATCAATGTATC EYFP-F 5’-GTCGTAACAACTCCGCCCCATTG ECFP-R 5’-TCCACGTTCTTTAATAGTGG

108 Murine qPCR primer

Table A.4: Murine qPCR primer

forward reverse efficiency

Pgk1 5’-TCGTGATGAGGGTGGACTTC 5’-CCAGGTGGCTCATAAGGACA 1.70 Taar1 5’-TGTGCTGTGTGTGACCCTTT 5’-ACGCCAGTACCCCAGATACT 1.50 5-Ht1b 5’-GCTTTGTGAACACCGACCAC 5’-GGTGACCGAGGATGTGGATC 1.85 HRH1 5’-GGCCTGGTTTCTCTCCTTCC 5’-CATGAGCAAAGTGGGGAGGT 1.95 DRD2 5’-CTATCTGGAGGTGGTGGGTG 5’-TGAAGGACAGGACCCAGACA 1.95 Adra2a 5’-GACCGCTACTGGTCCATCAC 5’-TGGTCGTTGATCTTGCAGCT 1.66 Adrb1 5’-GACGCTCACCAACCTCTTCA 5’-AACTCTGGTAGCGAAAGGGC 1.44 Adrb2 5’-CTTCTTACGAACCAGGCCT 5’-AGGTTTTGGGCGTGGAATCT 1.66 Trpm1 5’-GTGAGCACTGGTGTCGTCA 5’-CTCAGAGGGTTGGACATGGT 1.95 Trpm2 5’-CTTGGACCCGGAGAAGAACTG 5’-TCGGGAATCCATGAGCTAAGG 1.80 Trpm3 5’-GAACTCCAGCCCAAACTCAAG 5’-GGGGCGATACCTATGGTACATAT 1.95 Trpm4 5’-AGCACAGCAACTTTCTCCGG 5’-CACCGACACCACCAAGTTTG 1.40 Trpm5 5’-ACATCCACCAAGATCCGTGT 5’-TCCCTGAATGTTGCCCTCAT 1.39 Trpm6 5’-GACCGTCAAGAACAAGGAGC 5’-CGTAGAATCCCTCCATCCTCC 1.97 Trpm7 5’-GAGTTCCTGTGGTGGCTTTG 5’-CACAACAACTGGAACTGGGG 1.45 Trpm8 5’-GAGCAAGACAAGGACAACTGG 5’-GTCCTTATGAGAGCCGTGAAC 1.95 Trpv1 5’-CTGAAGTGCATGAGGAAGGC 5’-AGTTCACCTCATCCACCCTG 1.93

109 Appendix B

Response of hypothalamic cell lines to aminergic ligands

Figure B.1: NorEpi and ISOP induce cAMP accumulation in GT1-7, N39 and N41 cells. For Gs, the cAMP content was measured via an AlphaScreen Kit. (A) GT1-7, (B) N39 and (C) N41 cells were co-stimulated with either stimulation buffer, PEA, 5-HT, HIS, DA, NorEpi or ISOP in a concentration of 10-5 M for 45 min. For statistics, a two-way ANOVA was performed, followed by a Sidak correction. Data are the mean ± SEM of n = 3 - 4 independent experiments measured in triplicates; *p≤ 0.05, **p≤ 0.01, ***p≤ 0.001, ****p≤ 0.0001.

110 Figure B.2: Stimulation with aminergic ligands had no effect on Gi/o signaling. For Gi/o , the cAMP content was measured via an AlphaScreen Kit. (A) GT1-7, (B) N39 and (C) N41 cells were co-stimulated with forskolin and either stimulation buffer, PEA, 5-HT, HIS, DA, NorEpi or ISOP in a concentration of 10-5 M for 45 min. For statistics, a two-way ANOVA was performed, followed by a Sidak correction. Data are the mean ± SEM of n = 3 - 4 independent experiments measured in triplicates; *p≤ 0.05, **p≤ 0.01, ***p≤ 0.001, ****p≤ 0.0001.

111 Appendix C

Control assays for cAMP, PLC and MAPK signaling

112 Figure C.1: 5-HT and 3-T1AM do not influence the endogenous signaling of HEK293. For Gs and Gi/o the cAMP content was measured via AlphaScreen technology (A+B). PLC and MAPK activity were determined via luciferase assays (C+E). HEK293T were transfected with an empty vector as mock control (A-D). In all assays, cells were stimulated with either 5HT, 3-T1AM or both in a concentration of 10 µM. A one-way ANOVA was performed as a statistical test. (A+D) Data are pooled from n = 3 measured in triplicates. (B) Data are pooled n = 13 measured in triplicates. (C) Data are assessed from n = 8 measured in triplicates.

113 APPENDIX C. CONTROL ASSAYS FOR CAMP, PLC AND MAPK SIGNALING

Figure C.2: Adenylyl cyclase and PLC activity of 5-HT1b are pertussis toxin (PTX) sensitive. Data are indicated as mean ± SEM. A one-way ANOVA was performed as a statistical test. (A) For Gi/o, the cAMP content was measured via AlphaScreen technology. Samples were stimulated with forskolin and either 5-HT, 3-T1AM or both in a concentration of 10 µM. Data are pooled from n =4˙ measured in triplicates. (B+C) For PLC activity, an NFAT luciferase assay was performed. 5-HT1b and ADRA2A were pre-incubated with PTX for 16 hours. (B) Samples were stimulated with either 5-HT, 3-T1AM or both in a concentration of 10 µM for six hours. Data are pooled from n = 3 measured in triplicates. (C) ADRA2A was used as PTX sensitive positive control for PLC activity. Samples were stimulated with 10 µM NorEpi for six hours with or without PTX. Data are pooled from n = 4 measured in triplicates. (D+E) HEK293T were transfected with the TSHR as a positive control and with either the NFAT (D) or the SRF (E) response vector. Cells were incubated with 100 mU/mL TSH for six hours. Data are pooled from n =4.

114 Figure C.3: The co-expression of TAAR1 and 5-HT1b did not affect 5-HT1b mediated PLC or MPAK signal- ing. Samples were stimulated with either 5-HT, 3-T1AM or both in a concentration of 10 µM for six hours. Data are indicated as mean ± SEM are pooled from n = 4. A one-way ANOVA was performed as a statistical test. (A) For PLC activity, an NFAT luciferase assay was performed. (B) A luciferase assay with the SRE response element was done for MAPK signaling.

115 Appendix D

FRET - controls

Figure D.1: HEK293T transfected with either a CFP or a YFP construct showed no FRET signal. All FRET constructs were tested if they showed a sufficient fluorescence signal and therefore the intended bleaching curves. After a stable fluorescence signal of CFP and YFP was confirmed for ten cycles, YFP was bleached at 610 nm for 20 cycles (indicated by the arrow). All constructed worked efficiently and showed no false positive FRET signal.

116 Figure D.2: GHSR/MC3R dimer, as positive control, show a CFP increase of nearly 20 % after acceptor belaching. After a stable fluorescence signal of CFP and YFP was confirmed for ten cycles , YFP was bleached at 610 nm for 20 cycles (indicated by the arrow). (A) MC3R and GSHR samples showed an CFP increase of 18.9 ± 1.8 %, n = 19. (B) CB1R and MC3R samples only increased to 6.0 ± 1.5 %, n = 15. (C) TAAR1 combined with the rat CHRM3 also showed no FRET signal, CFP signal incresed by 2.0 ± 2.0 %, n = 12; n refers to measured single cells with sufficient CFP and YFP transfection rates.

117 Appendix E cFOS staining of murine brain loci and hypothalamic cell lines

Figure E.1: 3-T1AM stimulation had no effect on FOS staining in the medial preoptic area (MPO), the supraoptic nucleus (SON) and the dorsolmedial nucleus of the hypothalamus (DMH). After intraperi- toneal injection of the C57BL/6J mice with either 3-T1AM or solvent (60 % DMSO/ 40 % PBS), brains were frozen, cryosectioned and stained against FOS and DAPI (n = 3). All pictures were taken with a 20X objective. The scale bar indicates 200 µm.

118 Figure E.2: 3-T1AM stimulation had no effect on FOS staining in the periaqueductal gray (PAG) and the ventral tegemental segment (VTA). After intraperitoneal injection of the C57BL/6J mice with either 3-T1AM or solvent (60 % DMSO/ 40 % PBS), brains were frozen, cryosectioned and stained against FOS and DAPI (n = 3). All pictures were taken with a 20X objective. The scale bar indicates 200 µm.

119 Acknowledgment

Ich möchte mich an dieser Stelle bei allen Menschen bedanken, die mich in den letzten drei Jahren bei der Anfertigung dieser Dissertation unterstütz haben. Mein besonderer Dank gilt zunächst Heike Biebermann, die mir die Möglichkeit gab im Institut für Experimentelle Pädiatrische Endokrinologie meine Dissertation zu machen. Ich möchte mich für die fortwährende Unterstützung bei der Experimentplanung oder dem Manuskriptschreiben bedanken, und natürlich auch für die Korrektur dieser Arbeit. Für die Begutachtung meiner Arbeit möchte ich Prof. Roland Lauster und Prof. Juri Rappsilber danken, sowie Prof. Jens Kurreck für den Vorsitz meines Promotionsverfahrens. Den ehemaligen Gruppenmitglieder Noushin Khajavi und Gunnar Kleinau danke ich für die pro- duktive Zusammenarbeit bei den beiden Publikationen. Aus unserer Arbeitsgruppe möchte ich mich bei unseren TAs Sabine und Cigdem bedanken, die immer hilfreich und schnell zu Stelle waren, wenn es Schwierigkeiten im Labor gab. Der ganzen Ar- beitsgruppe des IEPEs danke ich für die vielen erstklassigen Journal Clubs und Laborbesprechungen mit den ausschweifenden Diskussionen, die immer vielschichtig waren und viele neue Dankanstöße gaben. Ganz besonders danke ich Sarah Paisdzior für ihre Freundschaft, die vielen Mitttagessen, aber natürlich auch für ihre fachlichen Ratschläge und Diskussionen. Grazia Rutigliano danke ich für die erfolgreiche Kooperation. Auch ein herzlicher Dank an alle fleißigen Korrekturleser: Anna, Henning, Jonathan, Lena, Sarah und Sandro.