Calcium and Phospholipases in Orexin Receptor Signaling

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 327

LISA JOHANSSON

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List of papers

I OX1 orexin receptors activate extracellular signal-regulated kinase in Chinese hamster ovary cells via multiple mecha- 2+ nisms: the role of Ca influx in OX1 receptor signaling. Sylwia Ammoun, Lisa Johansson , Marie E. Ekholm, Tomas Holmqvist, Alexander S. Danis, Laura Korhonen, Olga A. Sergeeva, Helmut L. Haas, Karl E. Åkerman and Jyrki P. Kukkonen Mol Endocrinol. 20(1):80-99, 2006

II Regulation of OX1 orexin/hypocretin receptor-coupling to phospholipase C by Ca2+ influx. Lisa Johansson , Marie E. Ekholm and Jyrki P. Kukkonen Br J Pharmacol. 150(1):97-104, 2007

III IP3-independent signalling of OX1 orexin/hypocretin receptors to Ca2+ influx and ERK. Marie E. Ekholm, Lisa Johansson and Jyrki P. Kukkonen Biochem Biophys Res Commun. 353(2):475-480, 2007

IV Diacylglycerol is generated from multiple sources upon OX1 orexin/hypocretin receptor activation of phospholipases. Lisa Johansson , Marie E. Ekholm and Jyrki P. Kukkonen Manuscript 2008

Contents

Introduction...... 11 G-protein-coupled receptors...... 11 The G-protein subunits ...... 12 Regulation of GPCR signaling ...... 13 GPCRs and the MAPK pathway...... 14 Ca2+...... 15 Phospholipases ...... 17 Phospholipase C ...... 17 Phospholipase D ...... 20 The phosphatidylinositols...... 21 Phosphatidic Acid...... 21 Orexins ...... 22 Properties of the orexin peptides and receptors...... 23 Distribution...... 23 Physiology ...... 24 Signaling...... 26 Narcolepsy ...... 28 Aims...... 29 Materials and Methods...... 30 Results and Discussion ...... 36 Paper I ...... 36 Paper II ...... 39 Paper III...... 40 Paper IV ...... 42 Conclusions...... 46 Acknowledgements...... 48 References...... 50

Abbreviations

The list is intended to aid the reader and is not covering all abbreviations used in the thesis. Only the most frequently used abbreviations or the ones needing clarification may be found.

5-HT 5-hydroxytryptamine (serotonin) AC adenylyl cyclase cAMP cyclic adenosine 3',5'-monophosphate CHO Chinese hamster ovary CNS central nervous system CSF cerebrospinal fluid DAG diacylglycerol EC50 effective concentration50 = concentration producing half-maximal response ER endoplasmic reticulum ERK extracellular signal-regulated kinase GEF guanine-nucleotide exchange factor GFP green fluorescent protein GIRK G-protein-regulated inwardly- rectifying K+ GPCR G-protein-coupled receptor M1 muscarinic M1 receptor IP1 inositol monophosphate IP2 inositol bisphosphate IP3 inositol 1,4,5-trisphosphate IP3-3KA IP3 3-kinase type A IP3-5P1 IP3 5-phosphatase type I IP3R inositol 1,4,5-trisphosphate receptor LPA lysophosphatidic acid NPY neuropeptide Y NSCC non-selective cation channel MAPK mitogen-activated protein kinase MAPKK MAPK kinase MAPKKK MAPK kinase kinase MCH melanocyte-concentrating hormone mGluR metabotropic glutamate receptor

OX1R OX1 orexin receptor OX2R OX2 orexin receptor PA phosphatidic acid PC phosphatidylcholine PDK1 3-phosphoinositide-dependent kinase 1 PH Pleckstrin homology PI phosphatidylinositol PI3K phosphoinositide 3-kinase PIP phosphatidylinositol-(4)-phosphate PIP2 phosphatidylinositol-(4,5)- bisphosphate PIP3 phosphatidylinositol-(3,4,5)- trisphosphate PIP5K phosphatidylinositol-(4)-phosphate 5- kinase PI-PLC phosphatidylinositol-specific phospholipase C PC-PLC phosphatidylcholine-specific phospholipase C PKA protein kinase A PKC protein kinase C PLA2 phospholipase A2 PLC phospholipase C PLD phospholipase D PPO preproorexin PSD post-synaptic density PTB phosphotyrosine-binding ROC receptor-operated Ca2+ channel RTK receptor tyrosine kinase RyR ryanodine receptor SERCA SR/ER Ca2+ ATPase SH2 Src homology 2 SH3 Src homology 3 SOC store-operated Ca2+ channel SR sarcoplasmic reticulum TBM TES-buffered medium TRP transient receptor potential VDCC voltage-dependent Ca2+ channel

Introduction

G-protein-coupled receptors The G-protein-coupled receptors (GPCRs) are the largest and most diverse gene family in the human genome. They regulate almost all physiological processes in mammals and are also the most commonly used drug target, either direct or indirect. In the last 30 years our knowledge about this family of receptors has grown, showing it to be subject to a complex regulation (reviewed in Pierce et al., 2002; Lefkowitz, 2004; Jacoby et al., 2006). GPCRs are, as the name implies, classically coupled to heterotrimeric G- proteins. Since it in the last years has become evident that GPCRs also can couple to signaling pathways without the help of these G-proteins (reviewed in Heuss et al., 2000; Marinissen et al., 2001), other names, based on their structure, seven transmembrane receptors, heptahelical receptors or serpen- tine receptors, have been assigned. The GPCRs have previously been classified into three major families, A, B and C, based on the sequence similarity (reviewed in Pierce et al., 2002; Jacoby et al., 2006). With the use of the bioinformatics approach, the classification of GPCRs has been revised from ABC to GRAFS, which stands for: G – Glutamate; R – Rhodopsin; A – Adhesion; F – Frizzled/taste; S – Secre- tion (Fredriksson et al., 2003; reviewed in Jacoby et al., 2006). Through genomic screens, the term orphan receptors also surfaced. This refers to putative receptors found on the basis of sequence similarity to known receptors, but for which the ligand is not yet known. The first GPCR discovered in this way was the 5-HT1A receptor, and another good example of deorphani- zation is the orexin/hypocretin system (Fargin et al., 1988; Sakurai et al., 1998; reviewed in Pierce et al., 2002; Lefkowitz, 2004). Now there are over a 1000 orphan GPCRs waiting to be coupled to a ligand; these are mainly olfactory receptors (reviewed in Lefkowitz, 2004). GPCRs have very diverse extracellular ligands, for example, ions (Ca2+ and H+), purines and other nucleotide derivatives, peptides and non-peptide neurotransmitters, lipids, hormones and other proteins. Other more diffuse categories of ligands are the odorants, pheromones, tastants and photons (reviewed in Pierce et al., 2002; Jacoby et al., 2006). It was earlier believed that GPCRs existed only as monomers. Recent studies have shown that this is not the case. There are several examples of the existence of homo- and hetero-di/oligomers (reviewed in Pierce et al.,

11 2002; Jacoby et al., 2006). Oligomerization affects the versatility and diversity of signaling and tissue/signaling specificity. Opiod receptors exist, depending on the tissue, as a homodimer or a heterodimer with differences in ligand specificity (reviewed in Jacoby et al., 2006). Heterodimerization of the GABAB receptors R1 and R2 has been shown to be crucial for membrane insertion and signaling (reviewed in Pierce et al., 2002; Jacoby et al., 2006).

The G-protein subunits Heterotrimeric G-proteins are complexes of -, - and -subunits. The function of any GPCR is to act as a guanine-nucleotide exchange factor (GEF) for these G-proteins (reviewed in Pierce et al., 2002; Lefkowitz, 2004). Thus, binding of an agonist to the receptor leads to the exchange of GDP for GTP on the -subunit. This is thought to lead to the dissociation of the - GTP and -subunit, and independent signaling of each component to effectors such as ion channels, adenylyl cyclase (AC), phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K) (reviewed in Pierce et al., 2002; Jacoby et al., 2006). The hydrolysis of GTP to GDP and dissociation of Pi then terminates the signaling; this process can be enhanced by RGS (regulators of G-protein signaling) proteins leading to a faster inactivation of the system. GPCRs may not absolutely need agonist binding to be able to activate G-proteins, a phenomenon called constitutive activity (reviewed in Pierce et al., 2002). This may cause diseases, which may be treatable with particular kinds of drugs, so called inverse agonists (reviewed in Lefkowitz, 2004; Jacoby et al., 2006). The heterotrimeric G-protein -subunits are divided in 4 families based on sequence homology and each GPCR can activate more than one type of -subunit (reviewed in Marinissen et al., 2001; McCudden et al., 2005; Jacoby et al., 2006). The Gq/11 family has four different members, Gq, G11, G14 and G15. This family couples to the enzyme PLC, which results in an elevation of intracellular Ca2+ by the second messengers inositol-1,4,5- trisphosphate (IP3) and diacylglycerol (DAG), the latter of which activates protein kinase C (PKC). The Gs-family has three members, Golf, Gs-long and Gs-short; these all stimulate ACs leading to an increase in cAMP. Gi/o has nine different members, Gi1, Gi2, Gi3, Go1, Go2, Gz, Gt1, Gt2 and Ggust. One of the classical effects of Gi proteins is to inhibit AC. G12/13 has two members, + + G12 and G13, which activate, for example, RhoGEFs and the Na /H - exchanger (reviewed in McCudden et al., 2005; Jacoby et al., 2006). The G subunit forms a tight complex and can be combined of 5 different isoforms of G and 12 different isoforms of G. There is some specificity in the interaction of different G and G with each other, G, receptors and effectors (reviewed in McCudden et al., 2005). Some examples of the effectors and pathways that G dimers signal to are G-protein-regulated inwardly-rectifying K+ channels (GIRKs), voltage-dependent Ca2+ channels

12 (VDCCs), AC, phospholipase A2 (PLA2), PLC, phospholipase D (PLD), Na+/H+-exchanger, PI3K and RasGEFs (reviewed in Marinissen et al., 2001; Jacoby et al., 2006; Oude Weernink et al., 2007a). Different subsets of G- protein subunits are expressed in different cell/tissue types, possibly leading to some tissue specificity of signaling.

Regulation of GPCR signaling Phosphorylation is a very common mechanism for the regulation of signaling. G-protein-coupled receptor kinases (GRKs) are universal regulators of the GPCR family, and most GPCRs are also phosphorylated by, for example, protein kinase A (PKA) and PKC (reviewed in Pierce et al., 2002; Lefko- witz, 2004). In contrast to PKA and PKC, GRKs interact with the activated form of the receptor and phosphorylation of the receptor leads to desensitization (reviewed in Pierce et al., 2002; Lefkowitz, 2004). Of the four arrestin genes, arrestin 2 and 3 are known as -arrestin 1 and 2, respectively. - arrestins bind to the phosphorylated receptors, which leads to i) internalization and then ii) either to degradation or recycling of the receptor (reviewed in Pierce et al., 2002; Jacoby et al., 2006). -arrestins also work as adaptor proteins linking the receptor to elements of the clathrin-coated pits machin- ery and signaling proteins (reviewed in Lefkowitz, 2004). Phosphorylation of the receptor can also switch the coupling from one G-protein to another and thereby change the signaling pathway used (reviewed in Pierce et al., 2002; Jacoby et al., 2006). Internalization and/or degradation is an example of a short-term regulatory mechanism and alteration of the transcription of the receptor gene is a more long-term regulatory mechanism for the receptors (reviewed in Pierce et al., 2002). Compartmentalization in the cell is in part accomplished via the formation of dynamic complexes by adaptor proteins (also called scaffolding proteins). Adaptors might give alternatives to the classical G-protein signaling pathways and modulate and fine-tune the existing signaling pathways via the heterotrimeric G-proteins. This also facilitates the down-stream signaling pathways by increased specificity and rate of signaling. -arrestins can tie the GPCRs to new signaling pathways, for example, via binding of Src (a non-receptor tyrosine kinase), which can lead to mitogen-activated protein kinase (MAPK) signaling. Other adaptors for GPCRs are for example Homer and PSD-95. Homer is expressed in mammals, where it links together the metabotropic glutamate receptors (mGluRs) of the family I (mGluR1 and -5 couple), other adaptors, IP3 receptors (IP3Rs) or ryanodine receptors (RyRs) and TRPC1 and -C2. PSD-95 can connect receptors, ion channels and other proteins via the PDZ-binding domain that is found in several GPCRs. G- protein-independent signaling can occur via the PDZ-binding domain (reviewed in Pierce et al., 2002; Jacoby et al., 2006). GPCRs can activate several different Ca2+ influx pathways and Ca2+ release (reviewed in Barritt,

13 1999). This activation might be through a coupling by scaffolds to the TRPs (reviewed in Clapham et al., 2001). Sometimes the GPCRs hi-jack receptor tyrosine kinases (RTKs) in a process called transactivation, possibly via binding of both receptors to scaffolding proteins (reviewed in Neve, 2005). The organization of the plasma membrane, with a more loosely packed part, the disordered part, and a liquid-ordered structure, the lipid raft, which has an even more specialized part, the caveolae, contributes to bringing the components of a signaling cascade into one place. It has been shown that the caveolae of the membrane contain many of the components needed in GPCR signaling, for example, the ACs and the G-proteins. The different parts also attract different isoforms of the Ras proteins (reviewed in Prior et al., 2001; Pierce et al., 2002).

GPCRs and the MAPK pathway There are several examples of agonists of GPCRs that affect the MAPK pathway, for example serotonin, noradrenalin, prostaglandins, sphingosine- 1-phosphate, LPA (lysophosphatidic acid), TRH (thyrotropin-releasing hormone), somatostatin, oxytocin and bombesin (reviewed in Gutkind, 2000). The crosstalk can either be by transactivation, as mentioned above, or via second messengers, and the complex regulation are coordinated by different scaffolding proteins, adaptors and special microdomains of the plasma membranes. Src has, for example, not only been shown to associate with arrestin but also with Gs, Gi and GPCRs (Luttrell et al., 1999; Cao et al., 2000; Ma et al., 2000; reviewed in Luttrell et al., 2003; Rozengurt, 2007). GPCRs also induce phosphorylation of the adaptor protein Shc and thereby the SOS-Grb2 complex is created leading to the activation of the MAPK pathway (reviewed in Rozengurt, 2007). The MAPKs are a superfamily of kinases with four different subgroups: extracellular signal-regulated kinases (ERK1 and 2), c-Jun NH2-terminal kinases (JNKs), ERK5 and p38 MAPKs. MAPK activation is a product of a phosphorylation cascade and classically starts with an activation of a GEF for a small GTPase. This leads to the activation of a MAPK kinase kinase (MAPKKK) which phosphorylates a MAPK kinase (MAPKK) that finally phosphorylates and activates a MAPK. There are several different members of MAPKKKs and MAPKKs of which, for example, the MAPKKK Raf and the MAPKKs MEK1 and MEK2 are involved in the GPCR/ERK pathway. The family of small (monomeric) GTPases has several members, of which some of the best characterized belong to the Ras, Rho and ARF families (reviewed in Gutkind, 2000; Luttrell et al., 2003). All of the Gq/11, Gi/o, Gs, G12/13 and the G subunits have been reported to activate Ras or Rho or the Raf kinase (direct or indirect via signaling complexes or second messengers) and thereby activate the ERK pathway (reviewed in Gut- kind, 2000; Luttrell et al., 2003). Gq can activate for example both Src and

14 Pyk2 (also a tyrosine kinase) that further activate Ras. Via PLC, Gq and G also activate PKC, which then activates Raf. Via the elevation of cAMP, Gs activates PKA and Epac (a GTP/GDP exchange protein), which then activates Rap which can activate Raf. Gs can also form complexes with arrestins and Src, which then results in a cAMP-independent activation of the ERK/MAPK pathway (Buchanan et al., 2006; reviewed in Gutkind, 2000; Stork et al., 2002; Luttrell et al., 2003; Rozengurt, 2007). The Gi protein activates the MAPK/ERK pathway via its G subunit (reviewed in Luttrell et al., 2003). The Ras GTPases (H-, K- and N-Ras, Ral and Rap are a few of the members) are activated by GEFs, tyrosine kinases, GPCRs, adhesion molecules and second messengers (reviewed in Cullen et al., 2002; Quilliam et al., 2002). Besides Raf, Ras proteins also activate PI3K and PLC. The ERK/MAPK pathway is sensitive to Ca2+ levels and Ca2+ influx or release can activate Ras (reviewed in Gutkind, 2000; Cullen et al., 2002). The Rho GTPases (RhoA, B and C, Rac, Cdc42) can be activated by the G12/13 proteins, the G subunits or Gq. The ARF (ARF1-6) GTPase activity is regulated by phosphatidylinositols (PIs) and both the Rho and ARF GTPases activate PLD (reviewed in Gutkind, 2000; Cullen et al., 2002; Oude Weern- ink et al., 2007b). It should be noted that some information about the pathways and their players is omitted, due to the extensive amount of members and regulators and the versatility of the interplay, and only some of the relevant aspects are covered.

Ca2+ Ca2+ is a very versatile second messenger. For example, Ca2+ is important in an organism’s development by controlling fertilization and differentiation and then later proliferation and apoptosis. Ca2+ does not only induce differentiation in neurons, but also controls the expression of specific neurotransmitters and channels and which neuronal circuits that should be created. Other responses controlled by Ca2+ signaling are contraction, secretion, metabolism and formation of learning and memory. Ca2+ also controls excitabil- ity of the membrane, cross-talk between signaling pathways and gene transcription. Moreover, Ca2+ can by itself control the expression of its signaling components to specialize the Ca2+-signaling system for a particular cell type (reviewed in Berridge et al., 2000; Berridge et al., 2003). The signaling of Ca2+ can be simplified into 4 basic steps. First there is 1) a stimulus. 2) Intracellular mechanisms make the Ca2+ concentration increase in the cytosol. Ca2+ then becomes the messenger and 3) stimulates Ca2+ sensitive processes. This is followed by the 4) mechanisms that take away the Ca2+ and the cell returns to rest. A resting cell has a Ca2+ concentration be-

15 low or around 100 nM; during Ca2+ signaling, the Ca2+ concentration can locally and temporarily reach 1 mM (reviewed in Berridge et al., 2000). Ca2+ signaling involves many protein and lipid components such as sim- ple intracellular messengers, transmembrane receptors, plasma membrane and intracellular ion channels, buffers, transporters and signaling enzymes. Even more diverse are the targets of Ca2+. The diversity can express itself as a change in speed, frequency or amplitude of the Ca2+ signal and the ability to cross-talk with other signaling pathways. Every cell type has a unique set of components, which creates Ca2+ signaling with different spatial and tem- poral properties (reviewed in Berridge et al., 2000; Berridge et al., 2003). Ca2+ elevation can come from intracellular stores, the endoplasmic/sarcoplasmic reticulum (ER/SR), or from the extracellular medium. The 2+ ER/SR stores are controlled by Ca and the second-messenger IP3, which is 2+ produced when PLC is activated. Ca affects both the IP3R and RyR, located in the ER/SR, and when the increase of Ca2+ in the cytosol activates the release of Ca2+ from the stores, it is referred to as Ca2+-induced calcium release (CICR). Other intracellular messengers that affect Ca2+ release are sphingosine 1-phosphate (S1P), cyclic ADP ribose (cADPR), nicotinic acid- adenine dinucleotide phosphate (NAADP) and phosphorylation cascades. IP3R can also be linked to signal pathways via scaffolding proteins; for example, in neurons, IP3Rs and mGluRs are connected via the Homer protein (reviewed in Berridge et al., 2000; Berridge et al., 2003). Ca2+ from the outside is driven into the cell by the electrochemical gradi- ent, entering, for instance, via VDCCs, receptor-operated Ca2+ channels (ROCs), store-operated Ca2+ channels (SOCs), thermo sensors or stretch- activated channels. VDCCs are the most well-known. They exist in excitable cells and generate a quick Ca2+ influx, controlling fast processes such as muscle contraction and exocytosis at the synaptic nerve endings. ROCs either respond to ligand binding or are somehow activated by receptor- mediated signal cascades (reviewed in Berridge et al., 2003). SOCs are channels in the plasma membrane activated by a drop in the Ca2+ concentration inside the ER/SR (Putney, 2001). The two proteins, STIM and Orai, have been proposed to be responsible for the transduction of the signal, where STIM in the ER couples to Orai channels located in the plasma membrane (reviewed in Rozengurt, 2007). Many of the ROCs, thermo sensors and stretch-activated channels may belong to the TRP ion channel family (reviewed in Berridge et al., 2003; Clapham, 2003). Most of the Ca2+ entering the cytosol is bound to buffers or effectors, which shape the amplitude and the duration of the Ca2+ signal and can help to decide whether a local or global signal is to be produced (reviewed in Berridge et al., 2000; Berridge et al., 2003). The pumps and exchangers used to restore and maintain the resting cytosolic level and keep the stores loaded are the plasma membrane Ca2+ ATPase (PMCA), the Na+/Ca2+-exchanger,

16 the SR/ER Ca2+ ATPase (SERCA) and the mitochondrial Ca2+ uniporter (reviewed in Berridge et al., 2003). Ca2+ spikes can elicit a cellular response and the spikes can also be repeated resulting in waves that are spread throughout the cell; if a wave is repeated it is referred to as an oscillation. The amplitude and frequency of the spikes, waves and oscillations are interpreted and an appropriate response is elicited. As a result, the Ca2+ signal can be different depending on the cell type and the receptor types activated. Activation by glutamate in Purkinje cells results in one single Ca2+ transient but in the hippocampal neurons the response has an oscillating pattern (Kawabata et al., 1996; reviewed in Berridge et al., 2003).

Phospholipases Phospholipases are enzymes that catalyze the hydrolysis of a lipid substrate. This group includes the families’ phospholipase A (PLA), B (PLB), C (PLC) and D (PLD). Within each family there are different types and isoforms. PLA has two main isoforms, PLA1 and PLA2, and hydrolyzes phosphatidic acid (PA) into LPA or phosphatidylcholine (PC) into arachidonic acid (reviewed in Makhlouf et al., 1997; Aoki, 2004). PLB has the ability to hydrolyze glycerophospholipids and it also displays both PLA1 or PLA2 and lysophospholipase activities (Nauze et al., 2002).The enzyme PLC has two types, the PC- and the PI-specific. PC-PLC hydrolyses PC into phosphocho- line and DAG and is probably the same enzyme as sphingomyelin synthase (Murthy et al., 1995; Luberto et al., 1998). Reports on the mammalian PC- PLC are still very limited and the main information is today based on bacte- ria. Therefore the classical mammalian PI-PLC is here simply referred to as PLC. PLC and PLD are discussed in more detail below.

Phospholipase C PLC classically refers to an enzyme that hydrolyses the phosphate-bond of PIs, as mentioned above. The PLC enzyme has been studied since the 1950's and the first isoforms to be cloned in the PLC family were the PLC, - and - . Since then, PLC, - and - have also been cloned. All PLCs are dependent on Ca2+ for catalysis and they are bound to the plasma membrane by hydrophobic residues, via PIs or via possibly other linkers. All PLC enzymes hydrolyze PIs (PI [phosphatidylinositol], PIP [phosphatidylinositol-(4)- monophosphate] and PIP2 [phosphatidylinositol-(4,5)-bisphosphate]) in the same way and produce IP3 and DAG when PIP2 is cleaved. IP3 elevation may result in an elevation of intracellular Ca2+ (se above) and DAG activates targets such as PKC. The common core of most of the PLC isoforms consists of a PH domain, EF-hands, a catalytic region (the X and Y boxes) and a C2-

17 domain; the last two are together the most conserved domains within the PLC family (reviewed in Rhee, 2001; Katan, 2005).

The structure and signaling of the PLC isoforms The PLC family consists of four isoforms, PLC1-4, and is directly regulated by the Gq family -subunits and G-subunits. PLC has, together with the PLC isoforms, in addition to the other domains of the PLCs, a unique extension in the C-terminus, which gives the enzyme membrane association. PLC2 and -3 can also be activated by proteins from the family of the Rho GTPases (Rac1/2, Cdc42) via the PH domain (reviewed in Harden et al., 2006). The crystal structure of PLC2 with bound Rac1 has recently been resolved (Jezyk et al., 2006). The PLC1 and - isoforms are special in that sense that they are the only isoforms that have two SH2-domains and one SH3-domain between the X and Y boxes. The SH2-domain makes it possible for the enzyme to bind phosphotyrosine motifs, which leads to binding to tyrosine kinases, phosphorylation of the PLC enzyme and its activation (reviewed in Rhee et al., 1997; Harden et al., 2006). These isoforms are also activated by phosphatidylinositol-3,4,5-trisphosphate (PIP3) via binding to the SH2-domain of the PLC (reviewed in Rhee et al., 1997; Harden et al., 2006). The isoenzyme for which the crystal structure was first resolved, even though only partial, belongs to the PLC family (Essen et al., 1996). This family consists of the PLC1, -3 and -4. It seems likely that PLC is the prototypic PLC and the other isoenzymes have evolved further gaining specialized functions (reviewed in Katan, 2005; Harden et al., 2006). PLC has a high sensitivity for Ca2+ compared to the PLC, - - isoforms and has a PH-domain that selectively binds PIP2 and IP3, in contrast to the other isozymes (Singh et al., 2003; reviewed in Rhee, 2001; Harden et al., 2006). The PLC isoform is sperm-specific and is required for the Ca2+ mobilization that is central for fertilization. It is the only isoform that lacks the PH- domain and it has the highest sensitivity for Ca2+ of all the isoforms (Kouchi et al., 2005; reviewed in Katan, 2005). PLC is directly regulated by small GTPases from the Ras and Rho families and by the subunits of the heterotrimeric G-proteins. PLC is the only isoform that lacks the EF-hands but has two Ras-binding domains similar to those found in other known effectors of the Ras family. It also has a RasGEF domain that can catalyze the transition from GDP to GTP for Ras and Rap proteins. G12/13 is believed to stimulate the PLC via the activation of the Rho molecules; this activation seems to be Ras-independent. Also the G subunits can activate PLC (reviewed in Wing et al., 2003; Katan, 2005; Harden et al., 2006). PLC1 and -2 are the two enzymes in the PLC family that have, between them, the largest sequence similarity and they also are the isoforms most closely related to the isoforms. Most of the G and G subunits have

18 been shown to stimulate PLC and some of the Ras and Rho family members. PLC2 is neuron-specific. This is the most recent family members discovered and based on the extensive studies of sequence similarities of the PLCs and the fact that the databases for many genomes are almost com- pleted, these might have been the last of the members in the PLC family to be discovered (reviewed in Katan, 2005). There are other newly discovered enzymes that have sequence similarities to some regions of the PLCs but lack the catalytic residues or the PLC activities. These include "PLC-like enzymes" PLC-L1 and -L2 and phospholipase- C-related catalytically inactive proteins (PRIPS) (reviewed in Katan, 2005). An observant reader notices that two PLCs are apparently missing from the list, namely PLC and PLC2. The PLC isoenzyme has been found, but was shown to be a proteolytic fragment of PLC1, and the bovine PLC2 was later shown to be the same as the mouse and human PLC4 (Taylor et al., 1992; reviewed in Rhee et al., 1989). Both the PLC and - have a PDZ-binding domain and in, at least, the PLC this domain has been shown to interact with scaffolding proteins and create a complex used in GPCR signaling (reviewed in Katan, 2005). One example is the interaction between the PLC3, Shank2 and Homer, which is important in the intracellular Ca2+ signaling of the mGluR receptor (Hwang et al., 2005).

PLC and Ca2+ It has been shown that the increase in intracellular Ca2+ by PLC, by either Ca2+ release from the ER/SR or Ca2+ influx, amplifies the activity of either the same PLC isoform or another, leading to further Ca2+ increase or Ca2+ oscillations (Kim et al., 1999; Thore et al., 2005). Moreover, the Ca2+ oscillations can differ in the same cell type depending on the GPCR activated, 2+ and oscillations of IP3 can be crucial for induction of Ca oscillations (Nash et al., 2002; Young et al., 2003). Also the cell type has importance in deter- mining what kind of a Ca2+ signal is produced, for instance, due to differences in the PLC isoforms expressed/engaged (reviewed in Berridge et al., 2003). In recent years DAG has been shown to have a lot of other targets than PKC, such as the TRP channels (Tesfai et al., 2001; Jung et al., 2002). The PLCs can also hydrolyze PI or PIP instead of PIP2 (further discussion in The phosphatidylinositols), which could lead to a generation of DAG without IP3 production. This would not result in Ca2+ release from the ER/SR, but could instead activate the TRP channels via DAG and still give an increase of intracellular Ca2+.

19 Phospholipase D PLD is an enzyme that hydrolyzes PC to form PA and free choline. It can also be artificially involved in transphosphatidylation of primary alcohols creating phosphatidylalcohol (reviewed in Houle et al., 1999; Jenkins et al., 2005). Its activity was first discovered in 1947-48 and the genes were cloned in the mid 1990s (Hanahan et al., 1948; Hammond et al., 1995; reviewed in McDermott et al., 2004). PLD activity can be found in most mammalian cell types. The mammalian PLD family consists of PLD1 and PLD2. The PLD1 isoform has two splice variants, PLD1a and -1b. PLD1 has a low basal activity and it is activated by PKC, Rho GTPases and ARF GTPases. PLD2 has a higher basal activity and it responds to ARF GTPases and PKC. They are both dependent on PIP2 for their enzymatic activity but PIP3 can also activate the enzyme. PLD itself activates mTOR (mammalian target of rapamycin) and PI(4)P 5-kinase (PIP5K), an enzyme that converts PI(4)P to PI(4,5)P2. PLD has also been shown to have a role in the activation of PI3K, Akt and ERK1/2. Synaptojanin, a lipid phosphatase, inhibits PLD activity by de- creasing PIP2 levels (reviewed in Houle et al., 1999; Jenkins et al., 2005; Oude Weernink et al., 2007b). The isoforms share a 50% amino acid sequence identity and both have a PH domain, a PX domain and a catalytic core with 4 conserved domains. The PH domain is not necessary for the enzymatic activity and the binding of PIP2 is believed to occur in the catalytic core of the enzyme. Instead, the PH domain, together with the PX domain, helps with the correct cellular localization of PLD. PIP3, and possibly also PIP5K, bind at the PX domain and this domain also shows GTPase-activating protein (GAP) activity. The small GTPases have an unknown interaction site on PLD. The subcellular localization is different for the two enzymes. PLD1 can be found everywhere in the cell but it predominantly resides in the perinu- clear endosomes and Golgi, whereas PLD2 has been found in the plasma membrane and in the cytosol. PLD1 does not seem to be statically localized and instead recycles and translocates; this is probably regulated by the binding to PIP2 (reviewed in Jenkins et al., 2005; Oude Weernink et al., 2007b). GPCRs activate PLD via G12/13, Gs and Gi/o proteins via for example, PIP5K, PKA, Epac or small GTPases (Huang et al., 2004; reviewed in Oude Weernink et al., 2007b). Gs and G subunit are also capable of directly activating PLD (reviewed in Oude Weernink et al., 2007a). It was earlier believed that activation of PLD by PKC was secondary to PLC activation but activation of PLD has also been shown to occur independently of PLC and PKC (reviewed in Cazzolli et al., 2006; Oude Weernink et al., 2007b). For example, the muscarinic M3 receptor activates PLC via Gq and PLD via G12 (independent of PLC) (Rumenapp et al., 2001). The ARF GTPases can activate PLD and PIP5K and are also found to colocalize with PIP5K and PIP2. They bind PIP2 with high affinity and can

20 thereby terminate PLD signaling, i.e. give negative feedback of PLD activation, since PIP2 activates PLD. Activation of PLD by Rho GTPases seems to occur via PIP5K regulation (reviewed in Oude Weernink et al., 2007b).

The phosphatidylinositols The substrate specificities of the PLCs are still unknown and it has been shown that PLC also can hydrolyze PIP and PI, besides PIP2. The known common substrate, PIP2, does not only work as a substrate for PLCs, but also as an important membrane constituent that affects enzyme activities, ion channel conductance and the intracellular signaling networks by changing the intracellular localization/activity of proteins that have PIP2-binding do- 2+ mains. PIP2 can affect, for example, the PIP3 3'-phosphatase PTEN and Ca , + + K and Na channels and PLD. Changing the concentration of PIP2 and other PIs, by, for example, PLC, can change the membrane association and activation of other proteins (reviewed in Katan, 2005). PIP2 can be phosphorylated by PI3K to PIP3. PIP3 can bind, for example, PH, C2 and PTB domains and coordinate the activities of many proteins by membrane association or by direct regulation (reviewed in Harden et al., 2006). One example is the downstream signaling process from PI3K that starts with the recruitment of PIP3-binding proteins to the membrane, by, for instance, 3-phosphoinositide- dependent kinase 1 (PDK1), which activates protein kinase B/Akt, PKC and/or MAPK pathways. The different subtypes of PI3K can be activated by RTKs and/or GPCRs. The signaling is deactivated by phosphatases, for example, PTEN, which hydrolyses PIP3 to PIP2 (reviewed in Wymann et al., 2003). The regeneration of PIs from higher phosphorylated forms, of either PIs or inositol phosphates, is conducted by several different kinases and phosphatases, which also have different substrate specificities. Already mentioned are synaptojanin, PTEN and PIP5K and two other examples (relevant for this thesis) are found in the regulation of the short half-life of IP3. There are two pathways that can degrade this second messenger. IP3 can be dephosphorylated by IP3 5-phosphatases where the produced Ins-(1,4)-P2 is eventually degraded to Ins-(4)-P and inositol and finally PIs are regenerated. The other pathway is performed by IP3 3-kinases, which, by adding a phosphate group, forms Ins-(1,3,4,5)-P4. This product is dephosphorylated to another form of IP3, the Ins-(1,3,4)-P3, and sequential dephosphorylation then leads to the regeneration of inositol and PIs (reviewed in Pattni et al., 2004).

Phosphatidic Acid The PA produced by PLD can be metabolized to DAG via phosphatidic acid phosphohydrolase (PAP). This form of DAG has a different fatty acyl com-

21 position then the DAG created by PLC from PIs, thus the signaling to PKC can differ (reviewed in Houle et al., 1999; Jenkins et al., 2005). PKC has several different isoforms, which are divided into the conventional isoforms (PKC, - and -, activated by DAG, Ca2+ and phospholipids), the novel isoforms (PKC, -, - and -, activated only by DAG and phospholipids) and the atypical isoforms (PKC and - / , unresponsive to both Ca2+ and DAG). PKC and its different isoforms have been shown to have different preferences for different types of DAG (Sanchez-Pinera et al., 1999; Madani et al., 2001). This is mainly based on in vitro studies which has further demonstrated that DAG derived from PA (saturated/monosaturated) seems to be a poor activator of PKC and DAG derived from PIs (polyunsaturated), is the more potent activator. Studies performed in cellular systems has somewhat confirmed this and have even suggested that DAG derived from PA is not capable of activating PKC but others have showed the opposite (reviewed in Hodgkin et al., 1998; Becker et al., 2005). DAG kinase (DGK) can convert DAG back to PA. PA can, as mentioned before, also be turned into LPA via the PLA2 enzyme and LPA can be con- verted back to PA by lysophosphatidic acid acyl transferase (LPAAT) (reviewed in Houle et al., 1999; Jenkins et al., 2005). PA can, besides working as a second messenger, also function as a lipid anchor and thereby affect cellular localization and activity of several different proteins, both by itself and via PIP2 generation since PA activates PIP5K. This leads to a feed-forward mechanism, since the produced PIP2 stimulates PLD activity and this feed-forward loop can be stopped by PLC, phosphatases or ARF and Rho GTPases (reviewed in Jenkins et al., 2005; Oude Weernink et al., 2007b). The ARF and Rho GTPases also regulate PA production by giving the correct localization and activation of PLD and PIP5K, which results in the right spatial production of PIP2 and PA (reviewed in Oude Weernink et al., 2007b).

Orexins The neuropeptides orexins and their receptors were discovered in 1998 by two independent groups and for that reason they were assigned two different names, hypocretins and orexins (de Lecea et al., 1998; Sakurai et al., 1998). The name hypocretin came from the resemblance of orexin-B to secretin (de Lecea et al., 1998). Sakurai et al. found an orphan receptor called HFGAN72, which later became the OX1 receptor. The name orexin is from the discovery that orexin increases the food intake in non-fasting rats when injected in the lateral cerebral ventricle (Sakurai et al., 1998).

22 Properties of the orexin peptides and receptors The precursor peptide preproorexin (PPO) is 130-131 amino acids (aa) long in mammals. It is cleaved into two molecules, orexin-A, 33 aa, and orexin-B, 28 aa. Orexin-A has two disulfide bridges while orexin-B is a linear peptide. Orexin-A has an identical sequence in human, pig, dog, rat and mouse but orexin-B shows sequence differences between species. Orexin-A seems to be more stable in the physiological environment and is more lipid soluble than orexin-B and therefore it may have the ability to cross the blood-brain- barrier (reviewed in Beuckmann et al., 2002; Kukkonen et al., 2002). The receptors belong to the rhodopsin-like receptors of GPCRs, independently of whether ABC- or GRAFS-classification is used (Fredriksson et al., 2003; reviewed in Kukkonen et al., 2002). There are two receptors discovered so far, the OX1 receptor (OX1R) and the OX2R receptor (OX2R), with 64% sequence identity between them. The OX1R is 425 and OX2R 444 aa long. Between the cloned mammalian orthologs (human, dog, porcine, mouse and rat) there is 91-98% sequence identity. Both orexin-A and -B bind equally well to OX2R, but orexin-A binds with 10 times higher affinity to OX1R than orexin-B (reviewed in Beuckmann et al., 2002; Kukkonen et al., 2002). Also when studying the Ca2+ response, orexin-A is 10 times more potent than orexin-B in activating OX1R in Chinese hamster ovary (CHO) cells (reviewed in Kukkonen et al., 2002). Ever since the first selective antagonist (SB-334867) for the OX1R was made, several other selective and dual antagonists have been developed and, besides SB-334867, the OX2R selective antagonist, JNJ-10397049, and the dual antagonist, ACT-078573, have been used to study the effects of the receptors (Porter et al., 2001; Smart et al., 2001; Hirose et al., 2003; Langmead et al., 2004; McAtee et al., 2004; Brisbare-Roch et al., 2007; Silveyra et al., 2007; Bergman et al., 2008).

Distribution The information of the distribution of orexinergic neurons is mainly based on rat. From the lateral hypothalamus, the orexinergic cell bodies mainly project within the hypothalamus and to the thalamic nuclei, cerebral cortex, brain stem and the spinal cord. There are approximately equal amounts of OX1R and OX2R mRNA in the whole rat brain and the distribution of the receptors shows almost the same pattern with a few exceptions (see Figure 1). The cell bodies in the hypothalamus are mixed with melanocyte- concentrating hormone (MCH)-rgic cells (reviewed in Beuckmann et al., 2002; Kukkonen et al., 2002; Mieda et al., 2002). The orexinergic neurons innervate neuronal circuits for a great number of transmitters (noradrenaline, dopamine, serotonin, histamine, acetylcholine, vasopressin, vasoactive intestinal peptide [VIP], somatostatin, corticotropin-releasing hormone [CRH],

23 neuropeptide Y [NPY], agouti-related peptide [AgRP], proopiomelanocortin [POMC], cocaine- and amphetamine-regulated transcript [CART], GABA, MCH and glutamate) and are themselves innervated by VIP, vasopressin, POMC and NPY neurons (reviewed in Kukkonen et al., 2002). Almost all orexinergic cells express the leptin receptor (reviewed in Beuckmann et al., 2002; Kukkonen et al., 2002).

Figure 1. Orexinergic pathways and orexin receptor mRNA distribution in the rat. Arcn, arcuate nucleus; CA1-3, areas of hippocampus; cC, cingulate cortex; CMn, centromedial nucleus; dR, dorsal raphe nucleus; LC, locus ceruleus; mE, median eminence; mR, median raphe nucleus; nST, nucleus of solitary tract; olfB, olfactory bulb; olfT, olfactory tubercle; PVn, paraventricular nucleus (in the thalamus and hypothalamus); Rm, nucleus raphe magnus; Ro, nucleus raphe obscurus; SCn, suprachiasmatic nucleus; SOn, supraoptic nucleus; STn, spinal trigeminal nucleus; TMn, tuberomamillary nucleus; VAMn, ventral anteromedial nucleus. The figure is from (Kukkonen et al., 2002) and used with kind permission from the American Journal of Physiology Cell Physiology.

In the periphery, orexins and their receptors are found in the gastrointestinal tract, pancreas, reproductive organs, adipose tissue, adrenal, thyroid, heart, kidney, lung, pituarity, pineal gland and sympathetic neurons (reviewed in Kukkonen et al., 2002; Heinonen et al., 2008).

Physiology Orexins main purpose is thought to be the regulation of sleep and wakefulness but they also seem to be important in regulating appetite, energy metabolism and feeding-associated processes like gastrointestinal functions. When it was discovered that dogs with the sleeping disorder narcolepsy (see below) had a mutation in the gene for OX2R, which leads to a non-

24 functional receptor, it became clear that the orexinergic system was important for normal sleep and wakefulness, and this is still the most well- demonstrated physiological effect of the orexins (Lin et al., 1999; reviewed in Sakurai, 2007). The orexinergic neurons are also shown to project to areas in the brain involved in sleep/wakefulness and both receptor subtypes are present. The orexin-positive neurons in the hypothalamus are activated in normal awakening in rat, in sleep-deprived rats and also by drugs against narcolepsy. Moreover, orexins excite neurons in regions involved in sleep regulation and increases wakefulness (reviewed in Kukkonen et al., 2002; Sakurai, 2007). Orexin-A and preproorexin mRNA also seem to vary in rat during the day and night cycle (i.e. light-dark experiments) and orexin neurons are a major target of projections from areas regulating wake-sleep states and the circadian rhythm (Taheri et al., 2000; reviewed in Sutcliffe et al., 2002; Saper, 2006). Moreover, the involvement of orexins in arousal and transition of wake-sleep has also been shown, and orexins seem necessary for a smooth transition between the states (reviewed in Saper et al., 2001; Sutcliffe et al., 2002; Saper, 2006). Recent work has also shown that pro- longed wakefulness leads to synaptic plasticity (i.e. increased amount of synapses and synaptic strength) of the orexinergic neurons (Rao et al., 2007). The hypothalamic area is regulating food intake and there are lots of con- tacts between orexins and other feeding-regulating transmitters, for example, leptin, NPY and MCH. As mentioned earlier, food intake is increased if orexin-A is injected in the lateral cerebral ventricle and this effect can be blocked by the antagonist SB-334867 and anti-orexin antibodies, supporting the idea of the involvement of orexins in feeding. Under normal conditions the increase of orexinergic activity can be a response to lowered blood glucose levels. Fasting has been shown to increase the number of orexin-A- positive neurons and to activate both central and peripheral orexinergic systems (reviewed in Beuckmann et al., 2002; Kukkonen et al., 2002; Sakurai, 2007). An interesting contradiction is that short-term fasting of rats gives rise to a downregulation of orexin receptor levels (mRNA and protein) in the gastrointestinal tract (duodenal mucosa) but an upregulation in the hypothalamus (Lu et al., 2000; Karteris et al., 2005; Bengtsson et al., 2007). The effect of orexins is believed to be in the short-time regulation of food intake and energy balance. Orexin has also a regulating effect of the water balance, since orexin increases water intake in rats and orexins are important for the food-seeking behavior that appears upon starvation i.e. orexin induces wakefulness to help the animal to stay awake to seek food. Moreover, fasting affects the variation of the orexin levels in relation to the circadian rhythm (reviewed in Beuckmann et al., 2002; Kukkonen et al., 2002; Sakurai, 2007). Orexin has been shown to be involved in autonomic/endocrine functions. This seems logical since the orexinergic neurons projects to the brain stem and the spinal cord. The autonomic effects that can be seen when orexin-A is

25 injected intracerebroventricularly are increased blood pressure, heart rate, intestinal motility, gastric acid secretion and sympathetic neuron activity; all these effects can be blocked by SB-334867. In addition, orexinergic neurons in the periphery may affect intestinal secretion and uptake and endocrine secretion. The endocrine cells in the periphery respond to orexin by stimulation or inhibition of hormone synthesis and/or secretion (reviewed in Kukk- onen et al., 2002; Heinonen et al., 2008). It is a possibility that orexins are working as hormones between the CNS and the periphery since there are orexinergic neurons projecting to endocrine releasing sites like the hypothalamus, pituitary and pancreas. Orexin can affect the release of hormones, some connected to stress response, from the hypothalamic-pituitary-adrenal (HPA) system. There is thus a possibility that orexins mediate stress responses via the corticotrophin-releasing factor (CRF) and there are several reports of orexins involvement in stress response and reward system. It is believed that orexinergic system is involved in the relapse of drug addiction and that stress induces the reinstatement of the drug-seeking behavior (de Lecea et al., 2006; Boutrel et al., 2007; reviewed in Sakurai, 2007).

Signaling The two responses that were first seen when stimulating the receptors with orexin was an elevation of intracellular Ca2+ in CHO cells and an increase in the electrical activity in neurons in the hypothalamus (de Lecea et al., 1998; Sakurai et al., 1998). It is possible that orexins increase Ca2+ influx in the presynaptic part and inhibit K+ channels in the post-synaptic part. Also the activation of non-selective cation channels (NSCCs) and the Na+/Ca2+- exchanger is suggested (reviewed in Kukkonen et al., 2002; Kukkonen et al., 2005). Ca2+ elevation upon stimulation of orexin receptors has been shown to occur in many different cell lines, CHO-K1, PC12 (rat), neuro-2a (mouse) and BIM (neuronal hybrid cells; human/rat), probably elicited via the Gq and PLC pathway. The elevation has also been observed in neurons of hypothalamus and cortex (reviewed in Kukkonen et al., 2002; Kukkonen et al., 2005). In studies performed in CHO cells the elevation of Ca2+ seems to arrive from different sources depending on the concentration of the ligand. At low orexin-A concentrations Ca2+ influx seems to be due to the opening of ROCs, but at high activation of the receptor, the Ca2+ elevation is a result of the emptying of the ER and further influx from SOCs (Lund et al., 2000; Kukkonen et al., 2001b; Larsson et al., 2005). The primary response might depend on NSCC and this influx of Ca2+ also seems to amplify the PLC activity (Lund et al., 2000). At high orexin-A concentrations, PLC is activated independently of Ca2+ influx (Lund et al., 2000; Kukkonen et al., 2001b; reviewed in Kukkonen et al., 2002; Kukkonen et al., 2005) (see Figure 2).

Figure 2. A. Low activation of OX1R (low orexin-A concentration). The ROC influx pathway is activated by OX1R and the PLC activity is amplified by the ele- 2+ vated intracellular Ca levels. The mechanism behind the OX1R activation of the ROC and how the ROC influx couples the OX1R to other signaling pathways is still not clear. The activating mechanism behind the SOC is also not established. B. High activation of OX1R (high orexin-A concentration). The ROC and SOC influx pathways are still activated but the PLC enzyme is activated independently by the influx.

Orexin receptors do not only display coupling to the Gq proteins but also to the Gs- and Gi/o-proteins and orexins may thus give rise to either an increase or decrease in cAMP via activation or inhibition of the ACs (Holmqvist et al., 2005; reviewed in Kukkonen et al., 2002; Kukkonen et al., 2005). This difference in response might be due to the fact that different cell types express different kinds of G-proteins and AC isoforms. Orexin receptors have also been shown to activate ERK and further studies have shown that they couple to the mitogen-/stress-activated protein kinase (MAPK/SAPK) signaling pathways linking orexins to cellular growth, differentiation and cell death (Ammoun et al., 2006; reviewed in Kukkonen et al., 2002). Orexin receptors seem to increase the frequency of quantal synaptic events, which indicates that orexins cause a presynaptic activation resulting in enhanced transmitter release. Orexins also excite neurons via postsynaptic depolarization in the CNS (hypothalamus, brainstem, spinal cord) (reviewed in Kukkonen et al., 2002; Kukkonen et al., 2005). This might occur by inhibition of K+ channels, possibly via PKC or the GIRK channels, or activation of NSCCs (reviewed in Kukkonen et al., 2002; Kukkonen et al., 2005). The receptor could, by depolarization-induced activation of the VDCCs, elevate the intracellular Ca2+ levels.

27 It is possible that the synaptic and the paracrine orexins use different pathways in the signaling due to the difference in orexin concentration, since the concentration in the synaptic cleft and the surrounding area could theo- retically be micromolar, rapidly dropping with distance (in the 3rd power), which gives high activation in the cleft and low activation for the paracrine orexin (reviewed in Kukkonen et al., 2002).

Narcolepsy The disorder narcolepsy is characterized by disturbed REM sleep, muscle paralysis (cataplexy), hallucinations and daytime hypersomnia. In man, narcolepsy is a sporadic disease affecting 0.03-0.1% of the population throughout the world (Sutcliffe et al., 2004). Narcolepsy can be found in dogs both as familial and sporadic variants. In the familial case, both the Labrador re- trievers and the Doberman pinchers harbor an inactivating mutation in their OX2R (Lin et al., 1999). Rat or mice, which had the PPO or orexin- producing or -responding neurons removed by different genetic or toxic methods, all display a narcoleptic phenotype (Chemelli et al., 1999; Gerashchenko et al., 2001; Hara et al., 2001). In human narcoleptics, cerebrospinal fluid (CSF) orexin-A levels are low or undetectable and post- mortem brain tissues show low numbers of orexinergic neurons (reviewed in Dauvilliers et al., 2003; Sakurai, 2007). The same is found in sporadically narcoleptic dogs (Ripley et al., 2001). An interesting finding, putatively re- lating to the dual role of orexins in wakefulness and appetite/metabolism is that the BMI (body mass index) is higher than normal in the narcoleptic pa- tients (reviewed in Beuckmann et al., 2002; Sakurai, 2007). Though the role of orexin in narcolepsy is undisputed, this finding has not yet lead to any therapeutic strategy, and the conventional therapy with, e.g., amphetamine derivatives is continued with its substantial side-effects. An- other interesting issue to tackle is the mechanistic background of narcolepsy, most centrally whether an autoimmune response lies behind the neurodegen- eration (Chabas et al., 2003).

28 Aims

The general aim of this thesis was to further investigate the previously demonstrated coupling of the orexin receptor to calcium influx and phospholipase activity.

The specific aims were:

to analyze the mechanism of ERK phosphorylation that occurs upon orexin receptor activation, including the involvement of different signal pathways (Paper I)

to analyze the importance of Ca2+ influx in general, and different 2+ Ca influx pathways, on PLC activation in response to OX1 receptor signaling (Paper II)

2+ to set up a method to block IP3-dependent Ca release to enable investigations of the mechanisms of the ROC influx activation involved in orexin receptor signaling (Paper III)

to analyze the involvement of different phospholipases in orexin receptor signaling, especially the DAG generation (Paper IV)

29 Materials and Methods

This section is a summary of material and methods of the four papers and for detailed information the reader is referred to the more descriptive part in respective paper. Most of the plasmid constructs used in all of the papers was generous gifts from other scientists, whom are gratefully acknowledged in the papers.

Cell culture

CHO cells, recombinantly expressing human OX1 (CHO-OX1) or OX2 (CHO-OX2) orexin receptors, was used in all four papers and were grown in Ham’s F-12 medium. Also neuro-2a-hOX1 cells (murine/mouse neuroblastoma cell line), recombinantly expressing human OX1, was used in paper I and III, and was grown in DMEM. The medias were supplemented with penicillin G, streptomycin, geneticin and fetal calf serum (pH 7.4). Cells were grown at 37 C with 5% CO2 in an air-ventilated humidified incubator. Cells were grown in plastic culture flasks (75 cm2 bottom area) or for experiments: on 24-well plates (1.77 cm2 well bottom area) (total inositol phosphate generation experiments in paper II and IV), on uncoated circular glass coverslips (13 or 25 mm in diameter; cell counting (III) and imaging (I and IV), respectively) and for all other experiments on circular plastic culture dishes (8.3, 21.2 or 56 cm2 well bottom area). The confluence of the cell culture, independent of the assay, was 70-90% by the time of the experiments. Primary cultures of striatal neurons (used in paper I) were prepared from newborn (day 0) Sprague Dawley rats and striati were dissected from cor- onal slices. After trypsinization and wash, the cells were dissociated by mild trituration in nutrient medium (Minimum Essential Medium supplemented with fetal calf serum, glucose, glutamine, insulin, NaHCO3 and HEPES; pH 7.4). Washed cells were resuspended in the nutrient medium and plated on polyethylenimide-coated glass coverslips (25 mm in diameter). The cells were cultured in this medium overnight in the same incubator as above. The following day, one volume of Neurobasal medium supplemented with B27 and gentamicin was added on top of the nutrient medium. On day 5, cytosine arabinoside was added to inhibit proliferation of fibroblasts and glia. The cells were studied after 7–14 days in culture.

30 Transient transfection

Cells (CHO-OX1 and neuro-2a-hOX1) were grown to 40-50% confluence and were washed with PBS (phosphate buffered saline). OPTI-MEM was then added and cells were transfected using Lipofectamine reagent (incorpo- ration of DNA into the cell). After 5 h the medium was replaced with fresh culture medium (see above). The total amount of DNA was kept equal in all the transfections using pUC18 (an empty plasmid). Experiments were performed ~48 h after transfection. In paper I, a GST-ERK construct (fusion of GST and ERK) was intro- duced together with the inhibitor plasmid to circumvent the problem of less than 100% transfection efficiency. The much higher molecular weight of the fusion protein allows separation from the endogenous ERK and separation of responses in transfected cells from those in nontransfected ones can be made. In other experiments transfection efficiency was 40–70% as determined using expression of pEGFP-C1 (plasmid with enhanced GFP [green fluorescent protein]) or some functional criteria (e.g. phosphodiesterase activity; paper I).

Western Blotting (paper I and III) For measurement of the activation of ERK (phosphorylation of ERK) cells were prior to stimulation serum starved for approximately 24 h. Stimulation of the cells was performed in the same medium at 37 C for different times (3–180 min). Stimulation was stopped by aspiration of the medium and washing of the cells with ice-cold PBS and an immediate lysis with lysis buffer, the last two both supplemented with phosphatase and protease inhibitors. The protein concentration was quantified and volumes corresponding to 10 g of protein were diluted in Laemmli sample buffer, boiled, separated by SDS-PAGE (10% acrylamide), and transferred to methanol-soaked polyvi- nylidene difluoride membranes. The membranes were blocked with 0.1% (vol/vol) Tween 20 and 5% (wt/vol) milk in PBS (TPBS-milk) before prob- ing with antisera. Primary antibody incubation was done in TPBS at 4 C overnight, and secondary antibody incubation was done in TPBS-milk at room temperature for 1 h after two 20 min washes with TPBS. Specific protein detection was performed using rabbit polyclonal antiactive-MAPK (anti- pERK1/2) antibody. Primary antibodies were detected using horseradish peroxidase-conjugated donkey anti rabbit antibody F(ab´)2 fragments and peroxidase enzymatic activity was visualized using chemiluminescence detection kits. After this, the membranes were washed for 2 h with TPBS-milk, and the total MAPK in each lane was probed, using rabbit polyclonal antito- tal-MAPK (ERK1/2) antibody and the same secondary antibody as above. The same detection system was used as above. The densities of the bands on

31 the exposed films were quantified using a homemade system and the system was calibrated using films of known densities. The controls, to which the data were to be compared, were always run on the same gel. The phos- phospecific signal (i.e. signal from anti-pERK1/2) was correlated to the total ERK.

Immunocytochemistry for pERK (paper I) Before experiments, coverslips with primary striatal neurons were serum starved for 24 h in serum-free nutrient medium. The cells were stimulated under different conditions for 10 or 30 min, after which the media were rapidly removed, the coverslips were washed with ice-cold PBS, and the cells were fixed in methanol for 10 min at -20 C. The coverslips were washed once with PBS in room temperature, three times with TBS (50 mM Tris-HCl + 150 mM NaCl, pH 7.4) supplemented with 0.1% (vol/vol) Triton X-100 and once with TBS. Block was done with 5% (wt/vol) BSA in TBS for 1 h in room temperature. Primary antibody incubation using rabbit polyclonal anti- phospho-p44/42 MAPK was performed in TBS with 3% BSA overnight at +4 C. This was followed by three washes with TBS supplemented with 0.1% Triton X-100 and one with TBS at room temperature. The cells were then incubated with the secondary antibody (Texas red-labeled goat antirab- bit IgG) in TBS with 3% BSA for 1 h at room temperature. The coverslips were washed three times with TBS and twice with water before mounting on glass slides using mounting media (Mowiol). Fluorescence was monitored and quantified.

Ca2+ measurements (paper I-III) Ca2+ measurements were performed as microfluorometric imaging of indi- vidual cells with CHO-hOX1 or neuro-2a-hOX1 cells attached on glass coverslips. Briefly, the cells were loaded with fura-2AM (fluorescent probe for Ca2+) for 20 min at 37 °C in TES-buffered medium (TBM, pH 7.4) with probenecid, rinsed once, and used immediately. The coverslips were mounted in a perfusion chamber (160 μl) and were constantly perfused with TBM at a rate of 1 ml/min and kept at 37 C. The additions into the chamber were made by perfusion. An imaging system (TILLvisION) with a fluorescence microscope (20 magnification; air objective) was used for measurements. The cells were excited with 340- and 380-nm light and the emitted light collected by a high-resolution camera. One 340 and one 380 reading were obtained after each second and the ratio was obtained after background subtraction. The cells were transfected with GFP together with the inhibitor/blocker of interest (to be able to select for the transfected cells) and the data were analyzed using the TILL software and Microsoft Excel.

32 The experimental procedure for Ca2+ measurements in cell suspension (parts of paper I) was slightly different. The cells were harvested and loaded with fura-2AM (20 min at 37 °C) and were then stored on ice as pellets (medium removed) and was for each measurement resuspended in TBM at 37 °C. The fluorescence was monitored in a stirred quartz microcuvette in a thermostated cell holder and measured using a fluorescence spectrophotome- ter. Experiments were calibrated by adding digitonin (detergent that disrupts the plasma membrane), which gives the maximum value of fluorescence, or EGTA (Ca2+ chelator), which gives the minimum value of fluorescence.

"Real-time" imaging of GFP probe translocation (paper I and IV) Microfluorometric imaging was performed to measure translocation of the GFP-probes GFP-PH-PLC (fusion of EGFP and PH domain of PLC1; paper I, III, IV), GFP-PKCwt (fusion of EGFP and PKC; paper I), and GFP-PKC-C1 (fusion of EGFP and C1 domain of PKCpaper IV). Cov- erslips with cells, transfected with the GFP-probes, were rinsed and mounted in a perfusion chamber and then essentially performed as above (Ca2+ Measurements). The same imaging system was also used for the measurements, but with a 100 magnification and oil immersion objective and cells were now excited with 490 nm light. In paper I the fluorescence was also measured using a confocal imager (same magnification as above and oil immersion objective) and cells were instead excited with the 488-nm Argon laser line.

Cell counting (paper III) Coverslips with cells transfected with the probe GFP-PH-PLC were transferred to TBM, stimulated with different concentrations of orexin-A for 3 min, after which the TBM was replaced with ice-cold methanol and then further fixated for 10 min in 20 C. The fixed cells were washed three times with PBS and twice with H2O and mounted on glass slides using mounting media (Mowiol). The mounting medium was allowed to set for 48 h after which fluorescence pictures were taken with the TILL imaging system described above (with 490 nM excitation, 40 magnification and oil immersion objective). The images were analyzed in a semiquantitative manner by manually counting fluorescent cells showing no, intermediate and full translocation. These degrees of translocations were assigned values of 0, 0.5 and 1, respectively, and the average value from different images was calculated.

Measurement of cAMP production (paper I) Cellular ATP was prelabeled by incubating the cells with [3H]adenine for 2 h in culture medium, after which the cells were detached, washed, and incu-

33 bated in TBM at 37 C containing IBMX (a phosphodiesterase inhibitor) for 10 min (some experiments in paper I were performed in the absence of IBMX). The cells were then stimulated for 10 min. The reactions were inter- rupted by centrifugation, removal of the supernatant, addition of perchloric acid, and freezing. [3H]ATP + [3H]ADP and [3H]cAMP fractions of the cell extracts were isolated by sequential Dowex/Alumina (anion-exchange) chromatography and radioactivity was determined by dissolving the fractions in an appropriate volume of scintillation cocktail and analyzed in liquid scintillation counter. The conversion of [3H]ATP to [3H]cAMP was calculated as a percentage of the total eluted [3H]ATP + [3H]ADP and normalized to the recovery of a [14C]cAMP tracer.

Measurement of total inositol phosphate generation (paper I, II and IV) Membrane phosphoinositides were prelabeled by incubating the cells with myo-[3H] inositol for 18 h in culture medium after which the cells were (in paper I: first detached with PBS-EDTA) washed and incubated in TBM at 37 C containing LiCl (to inhibit inositol monophosphatase) for 10 min. The TBM was for some of the experiments supplemented by the appropriate concentration of an inhibitor. The cells were then stimulated with orexin-A (2 or 20 min) and the reactions were stopped by, removal of TBM (only for attached cells), addition of perchloric acid and freezing to lyse the cells. Af- ter thawing, the samples were neutralized (by KOH + KHCO3) and the in- soluble fragments were spun down. The supernatant was then subjected to anion-exchange chromatography. Different buffers were used to wash out unwanted products (inositol and glycerolphosphoinositol) and to elute the inositol phosphates (mono-, bis- and trisphosphates). Radioactivity was determined using scintillation counting as described above (Measurement of cAMP production).

Ion exchange separation of inositol phosphates (paper IV) The cells were prelabeled as above (Measurement of total inositol phosphate generation). The medium was replaced with TBM and the cells incubated for 10 min after which orexin-A was added. The experiments were terminated 10 s later by replacement of the media with ice-cold perchloric acid and immediate freezing with dry ice. The thawed supernatants were neutralized (by KOH + KHCO3) and the inositol phosphate fractions with different charges were isolated with anion-exchange chromatography as above, but several different buffers were used to wash out unwanted products (inositol and glycerophosphoinositols) and to elute IP1, IP2, IP3 and IP4 separately.

34 Radioactivity was determined using scintillation counting as described above (Measurement of cAMP production).

Binding protein measurement of IP3 (paper IV) Cells were detached with PBS-EDTA and spun down, washed once with TBM and resuspended in TBM. The cell number was adjusted by counting cells in a Bürker chamber. The cells were kept in 37 C and stimulated with orexin-A (except the basal samples). The cell suspension was rapidly mixed and the reactions were terminated by adding ice-cold perchloric acid, vor- texed and put on ice. The precipitate was sedimented by centrifugation. The supernatants were neutralized (KOH + Hepes) and Tris-HCl was added to get a correct final pH (8.6) for further analyze. The resulting KClO4 sedi- ment was removed by centrifugation and the IP3 concentration of the supernatant of the samples was determined using a commercial available assay kit ([3H] Biotrak Assay). The kit basically performs a competition assay where 3 the produced IP3 in the samples competes with [ H]-labeled IP3 of binding to a binding-protein which has a high affinity for IP3.

35 Results and Discussion

Most of the experiments in this study were performed in CHO cells recombinantly expressing OX1R or OX2R. Usage of other cell or receptor types is indicated.

Paper I

Previous work has shown that activation of OX1R induces ERK phosphorylation in CHO cells and the signaling pathways between OX1R and the ERK/MAPK pathway were further examined in Paper I (Kukkonen et al., 2002). Stimulation of either OX1R or OX2R, with orexin-A, lead to a strong phosphorylation of ERK1/2 with a slow decline, and the response could, for OX1R, be blocked by its antagonist SB-334867. The activation was orexin concentration-dependent and orexin-A was more potent in activating ERK than orexin-B. Ca2+ influx has previously been shown to be of importance for the signaling of orexin receptors to other effectors (i.e. PLC) (Lund et al., 2000). The ERK response was right-shifted compared to the Ca2+ elevation response. A reduction in phosphorylation of ERK, upon orexin-A stimulation, was observed when the extracellular Ca2+ concentration was reduced. This was observed not only for CHO cells, but also in neuro-2a cells (stably expressing OX1R) and native striatal neurons, further supporting the hypothesis of 2+ the importance of Ca influx for ERK activation upon OX1R stimulation. CHO cells were used for the further studies since the native neurons show a strong heterogeneity in expressing orexin receptors. Moreover, CHO cells have previously been used in the lab and the signaling effects of orexins, in CHO cells, is well demonstrated. Elevation of Ca2+ in the cell by other means than orexin-A (by either thapsigargin or ionomycin) increased the phosphorylation of ERK but not as strongly as orexin-A did, and had a much faster decline in the response, indicating that Ca2+ alone is not sufficient for the production of the strong and long-lasting elevation of phosphorylated ERK seen upon orexin-A stimulation. When blocking Ca2+ influx via SOCs (with 2-APB) or both SOCs and ROCs (with Ni2+), the ERK phosphorylation was reduced, indicating that both types of influxes are involved in the activation of ERK by orexin-A.

36 This supports the notion that Ca2+ influx is of central importance for the activation of ERK by OX1R receptors. The possibility that ERK activation is a result of PLC or AC activation was investigated, since previous studies have shown that OX1R activate these pathways (Lund et al., 2000; Holmqvist et al., 2005). A PLC inhibitor (U-73122) was shown to reduce ERK phosphorylation indicating involvement of PLC. Further analysis of this pathway revealed the involvement of the down stream effector PKC, since stimulation of PKC (with TPA) increased phosphorylation of ERK and inhibition of conventional PKCs and/or novel PKCs blocked the ERK response. Since the magnitude of inhibition of the ERK response by PLC and conventional/novel PKC inhibitors was equal, and the conventional/novel PKCs are activated via DAG in the PLC pathway, DAG is the most likely effector to be involved in the activation of the 2+ ERK pathway via OX1R, instead of IP3 and Ca release. Further examina- tion of the AC pathway showed that it could be ruled out as a possible con- tributor. For example, the orexin-A concentrations used to obtain the maximal ERK response is well below the EC50 for the cAMP response and when applying a cAMP analog (8-Br-cAMP) or forskolin (AC activator) a very low and transient ERK response was achieved, despite a substantial elevation of cAMP. Moreover, overexpression of phosphodiesterase (metabolizes cAMP) did not, upon orexin-A stimulation (same concentration used to obtain the maximal ERK response), result in any significant decrease in cAMP and the ERK response was only slightly inhibited. Both ERK response and activation of PLC, upon OX1R activation seem to be dependent on Ca2+ influx. Thus, the hypothesis arose that Ca2+ acts like a “connecting factor” to couple OX1R to other pathways and the previously shown OX1R activation of AC and cAMP elevation was analyzed in this perspective (Holmqvist et al., 2005). Upon OX1R activation by high concentration of orexin-A (strong elevation of cAMP) in combination with reduction of the extracellular Ca2+ levels or inhibition of ROCs and SOCs (Ni2+), reduced the cAMP elevation. Ca2+ elevation alone (using thapsigargin or ionomycin) could not increase cAMP levels. Thus, the OX1R seems to use Ca2+ as a general mechanism to connect to signaling pathways (Figure 3A). The importance of the type of influx (ROC and SOC) for the coupling of OX1R to the ERK pathway was also analyzed. No potent and selective blocker of the ROC is known, but thapsigargin appears to fully block this pathway (Lund et al., 2000). However, thapsigargin simultaneously activates SOCs. Thapsigargin-treatment did not affect the ERK response upon orexin- A stimulation. Blocking of the SOC-dependent influx with 2-APB inhibited the ERK response to orexin-A stimulation in the presence of thapsigargin, whereas 2-APB did not affect this response in the absence of thapsigargin. This was interpreted to mean that SOC-mediated influx could replace the ROC-mediated influx in supporting the OX1R signaling (thapsigargin-treated cells).

37 The involvement of potential actors of the ERK/MAPK pathway was also analyzed for OX1R activation of ERK. Inhibiting Ras with dominant negative constructs (H/K/N-Ras) nearly completely blocked the orexin-A- induced ERK response. The same approach for the other Ras GTPases (RalA and Rap1) did not have any effect on the ERK response. The inhibition of Src, PI3K and atypical PKC reduced the orexin-A-induced ERK response, and all to the same extent, showing their involvement in the pathway from OX1R to ERK. Inhibition of MEK1 (MAPKK) was shown to completely inhibit the phosphorylation of ERK. OX1R has been shown to be able to activate several different G-proteins (Gq/11, Gs, Gi/o) (Randeva et al., 2001; Holmqvist et al., 2005; Magga et al., 2006; reviewed in Kukkonen et al., 2005). GPCRs can also transactivate receptor tyrosine kinases, although this has not yet been shown for OX1R. This gives many possible scenarios for activation of the ERK pathway by 2+ OX1R. The results points towards involvement of Ca , Src, Ras, PI3K, DAG (from PLC), conventional/novel and atypical PKCs and MEK1 and, thus, a few possible pathways may be proposed. Gq can activate PLC and the therewith activated DAG and PKC can activate Ras and Raf, respectively. Pyk2 can also be activated by Gq and by G, leading to Src (also directly activated by G) and Ras activation. PI3K can be activated by G- proteins and Ras and from PI3K several routes to ERK activation are possible via Src, conventional PKC and PLC (see Figure 3B). Whether PI3K can, via phosphorylation by PDK1, directly activate atypical PKCs is still a controversy, but can also be a possible pathway for ERK activation (reviewed in Eyster, 2007).

2+ Figure 3. A, Ca appears to be a general mechanism of the OX1R to couple to signaling pathways even though PLC can be activated with no Ca2+- influx present (se Introduction). B, Possible signaling pathways connecting OX1R to the activation of ERK. Some steps are omitted and not all pathways are covered in the picture.

38 The cellular context probably decides which pathway is used by supply- ing the right activators, effectors and scaffold proteins, and there are most likely several parallel or cross-talking routes that lead from the OX1R to the activation of ERK. Since ERK activation controls important functions such as cell differentiation, proliferation and death, it is likely that several pathways exists, ensuring that the signal is progressed even though one route is hindered.

Paper II In the second paper the importance of the two Ca2+ influx pathways, ROC and SOC, for the PLC activity upon OX1R activation was investigated. Orexin-A stimulation of OX1R leads to, via PLC activation, the release of inositol phosphates and this was confirmed in this study (Lund et al., 2000). When blocking or abolishing the influx of Ca2+ from the extracellular side using different methods (Ca2+-free medium [1 μM Ca2+], EGTA [Ca2+ chelator], K+-rich medium [depolarizes the cells and reduces the driving force for Ca2+ entry]) the inositol phosphate generation was markedly inhibited at the low orexin-A concentrations (i.e. at low activation level of OX1R) but not at the higher concentrations (i.e. at high activation level of OX1R). This behavior has previously been observed for PLC activated by OX1R, and it was also shown that Ca2+ alone (via elevation of intracellular Ca2+ levels with thapsigargin or ionomycin) is a weak stimulant of PLC activity (Lund et al., 2000). 2+ Altogether, the results show that Ca influx seems to be necessary for OX1R activation of PLC but not at the higher orexin-A concentrations. When extracellular Ca2+ concentration is strongly reduced with a chelator (EGTA), the intracellular Ca2+ concentrations may be affected too. Lowering of the effective intracellular Ca2+ concentration (using BAPTA-AM or Ba2+- containing medium) gave indeed the same response on inositol production as EGTA did, which lead to the conclusion that, to not interfere with the intracellular milieu, the more mild treatment (Ca2+-free medium) is to be preferred. The next step was to evaluate what impact ROC and SOC influxes has on OX1R-induced PLC activity. This was done by using a set of blockers (TEA, Mg2+, dextromethorphan, SKF-96365 and 2-APB). Their effects in our system were first confirmed by evaluating the Ca2+ response, elicited by thapsigargin (induces SOC influx) and 1 nM orexin-A (induces ROC influx). This resulted in the classification that TEA was a strong ROC blocker and the others were either strong SOC blockers (dextromethorphan, SKF-96365) or was an incomplete blocker of both influxes (Mg2+). 2-APB has previously been used in our lab and is regarded as a strong SOC blocker (Paper I and (Kukkonen et al., 2001a; Bootman et al., 2002).

39 Blocking of ROC influx (TEA) gave a strong inhibition of inositol phosphate generation at low receptor activation. None of the other blockers gave equally strong inhibition, but had among them similar effects. For the higher orexin-A concentrations, there was almost no inhibition for any of the inhibitors, except for the ROC blocker (TEA) that showed inhibition at even the second highest orexin concentration. Additionally, blocking of the ROC influx gave almost the same inhibiting effect as the nominally Ca2+-free medium or depolarization. Thus, both pathways are of importance, but the ROC influx might be of greater importance at low receptor activities and the SOC influx seems to amplify the PLC activity, and the latter effect has been observed for other receptors and cell types (further discussion below) (Wojcikiewicz et al., 1994; Kim et al., 1999; Thore et al., 2005). This supports the view of the importance of the ROC influx pathway for the signaling of the OX1R and further supports the earlier conclusion (paper I) that influx via ROCs helps the OX1R to couple to other signaling pathways. It has previously been shown that upon muscarinic cholinoreceptor (Gq- coupled GPCRs) activity, Ca2+ is important for the PLC activity and that SOC amplifies the PLC activity (Wojcikiewicz et al., 1994; Kim et al., 1999; Thore et al., 2005). These effects were compared with the response of the inositol production upon OX1R activation in our system by transiently transfecting CHO-OX1R cells with the human muscarinic M1 receptor (M1). M1-stimulated PLC activity was inhibited at all agonist concentrations by a SOC blocker (2-APB), as previously observed by others (Willars et al., 1995; Kim et al., 1999) and not just at the low receptor activation levels as for the OX1R. Thus, the behavior of the OX1R can be regarded somewhat unusual. One explanation could be that different PLC isoforms are engaged by the two receptors or that orexin receptors couple to different PLC isoforms at high and low receptor activities. GPCRs have, in the same cell type, been observed to give rise to different PLC activities (Young et al., 2003). Orexin receptors have been shown to couple to Ca2+ influx pathways in other cell types, both neurons and endocrine cells, leading to an increase in the intracellular Ca2+ concentration, which indicates that this is a general and important mechanism in orexin receptor signaling. Also, in our recombinant system it seems to be necessary for the signaling of orexin (paper I and II). In native systems it is still not known how this works and the mechanistic explanation and physiological relevance are left to be determined.

Paper III In Papers I and II, the importance of Ca2+, especially ROC, influx for OX1R signaling was established. Out of this arose the interest in the mechanism of ROC activation. Consequently, in Paper III a method was created to 2+ knock down the PLC-dependent (IP3-induced) Ca release from the ER, in

40 order to separately investigate the mechanisms of generation of ROC influx. For this purpose we chose to transiently overexpress the IP3-metabolizing enzymes, IP3-3KA and IP3-5P1 (De Smedt et al., 1997). To visualize the production (i.e. lack of production) of IP3, the cells were cotransfected with the PIP2/IP3-probe, GFP-PH-PLC (GFP attached to the PH domain of PLC) together with the enzymes. This probe has previously been used to monitor PIP2 break-down/IP3 production and has a high affinity for PIP2 (membrane) and IP3 (cytosol) (see Figure 4) (Stauffer et al., 1998; Hirose et al., 1999). In non-stimulated cells a clear membrane localization of the GFP-PH-PLC probe was observed with and without coexpression of the IP3-3KA and IP3-5P1 enzymes indicating no nonspecific effects of the enzymes. Upon stimulation of the cells with orexin-A, concentration- dependent IP3 production was observed (= translocation of the probe from the plasma membrane to the cytosol). When stimulating cells expressing IP3- 3KA and/or IP3-5P1 the response was much reduced, indicating that the level of IP3 was lowered.

Figure 4. GFP-PH-PLC1 probe A, Basal conditions i.e. no stimulation. The probe is located in the membrane, attached to PIP2. B, Stimulation with 300 nM orexin-A leads to translocation of the probe to the cytosol due to degradation of PIP2 in the membrane and generation of IP3 in the cytosol.

2+ The effects of the enzymes on the IP3-activated Ca release from the ER were then analyzed. This was done in the absence of extracellular Ca2+ to 2+ abolish ROC-dependent influx. OX1R-stimulated Ca release was markedly lowered when the IP3-metabolizing enzymes were present. This effect was then compared with two classical Gq-coupled GPCRs, the endogenously expressed P2Y receptors (purinergic receptors in CHO cells) or transiently transfected human M1 (as in paper II) and when stimulating the cells with agonists (ATP or carbachol, respectively) the same effect was observed, indicating that the classical signaling from PLC is blocked by the enzymes and is not only an effect on OX1R signaling. The effect of the enzymes was also evaluated in the presence of normal extracellular Ca2+ levels to see if removal of Ca2+ release had any effects on the ROC influx. This was done both in CHO and neuro-2a OX1R-expressing 2+ cells. The Ca elevation was only slightly reduced by the expression of IP3-

41 3KA and IP3-5P1 under these conditions, indicating that ROC influx is independent of Ca2+ release and the effect of the enzymes is not cell type- specific. Thus, the enzymes seem to effectively inhibit Ca2+ release, enabling further mechanistic studies of the importance of the ROC influx in OX1R signaling, as they leave the ROC influx as the only source for Ca2+ to affect signal pathways. The observation from Paper I, that ROC influx seems to be the primary signal for the activation of the ERK pathway by OX1R, was further analyzed using these enzymes. ERK activation was, upon orexin-A stimulation, not affected by the presence of the IP3-metabolizing enzymes, supporting the hypothesis that ROC influx is for OX1R the primary connector to the ERK pathway, and not Ca2+ release or SOC influx.

Paper IV In the final paper the focus turned to the phospholipase activity that occurs upon OX1R stimulation. Several different methods (biochemical and fluorescent) were used to measure metabolites generated upon phospholipase activity. In biochemical methods (for example ion exchange chromatography) large cell populations are often used, and therefore these methods give better and quicker statistics. Fluorescent methods (visual method by, for example, a probe tagged with GFP) can instead give information of the spatial and tem- poral profiles but for much fewer cells in each experiment. GFP-PH-PLC (from Paper III) was used as an indicator of PLC activity towards PIP2. Upon orexin-A stimulation of OX1R, a quick and reversible translocation was observed for the probe when looking at cells in real time. This probe displayed a clear concentration-response relationship, indicating a breakdown of PIP2 and generation of IP3. Another way to measure PLC activity, a binding-protein IP3 assay (biochemical method) was also used. This displayed a similar concentration-response relationship as GFP-PH- PLC, further supporting that this optical probe is a good detector of IP3 generation. PLC activity upon OX1R activation was also measured by production of total inositol phosphates (biochemical method). The concentration-response curve for orexin-A was 7-fold left-shifted compared to the IP3 generation response. As mentioned in the introduction there is no real evidence that PLCs' preferred substrate is PIP2, and a suspicion arose that PLC hydrolyzes another substrate (PI or PIP), which would explain the left-shift i.e. the lack of IP3 production at the lower orexin-A concentrations. When the inositol phosphates released upon orexin-A stimulation were separated (in combination with very short reaction times to avoid unwanted IP3 break-down), it was revealed that in fact IP1 was the dominating product at the low orexin-A concentrations. Thus, PLC may use PI or PIP as a substrate at the low

42 orexin-A concentrations and PIP2 at the higher concentrations. The most likely explanation is that there are two different enzymes that are activated at the different activation levels of the OX1R (see 2. and 3. of Figure 5).Which of the different isoforms of PLC that are responsible remains to be eluci- dated. Since OX1R can activate several different G-proteins, PLC (via Gq/11 or G subunit from Gi/o) and PLC via PI3K-produced PIP3) are plausible candidates. It is still not known if OX1R can activate G12/13 proteins but if it can, PLC is also a possible candidate.

1. Only PLD 2. PLC is 3. PLC uses PIP2 as substrate is activated activated and uses PC and uses as substrate PI and PIP as substrate DAG

Response Total IPs

IP3

Orexin-A concentration

Only DAG DAG, DAG and IP3

is generated IP1 and IP2 is generated is generated

Figure 5. 1. At the lowest orexin-A concentrations PLD is activated using PC as substrate and thereby only DAG is generated (with PA as an intermediate step; see introduction). 2. At higher concentrations, a PLC isoform with a substrate preference for PI or PIP is activated leading to DAG and inositol (mono- and bis-) phosphate (IP1 and IP2) production. 3. At the highest concentration, a PLC isoform is activated which uses PIP2 as substrate, now leading to the production of IP3 together with DAG. [Concentration-response curves are from figure 4A in Paper IV; Total inositol phosphates = Total IPs; The IP3 response is measured with the binding protein assay; Response = % of maximal response upon orexin-A stimulation.]

Another probe, GFP-PKC-C1 (GFP-tagged C1-domain of PKC) (Raghunath et al., 2003), was used to detect the generation of the other me- tabolite produced by PLC activation, DAG. The principle is the same as for the probe detecting IP3 generation (GFP-PH-PLC but in this case, the probe is located in the cytosol, and upon generation of DAG in the membrane, the probe is translocated due to its high affinity for DAG (see Figure 6). A quick and reversible translocation of the probe to the membrane could be visualized in real-time and a clear concentration-response relationship was observed. The probe apparently detected a high-potency component (~10%) and a low-potency component (~90%) of DAG generation (part 1 compared to 2 together with 3, respectively, Figure 5). Moreover, an interesting difference appeared when the concentration-response curve of the

43 probe (DAG generation) and the production of total inositol phosphates was compared. DAG and total inositol phosphate production should, if PIs are used as the substrate regardless of the type (PI, PIP, PIP2), have the same concentration-response relationship. But no total inositol production was detected at the low orexin-A concentrations where the GFP-PKC-C1 probe indicated DAG production (the high-potency component). At the higher concentrations of orexin-A, the concentration-response curve of the total inositol generation followed the curve for the DAG generation (the low- potency component) (Figure 5). The suspicion again arose of the usage of another substrate that, in this case, could generate DAG without inositol phosphate production. The apparent candidate substrate was PC and two enzymes that use this substrate, PC-PLC (probably actually sphingomyelin synthase) and PLD were investigated. The inhibitor for the PC-PLC, D609, has been shown to be able to block orexin signaling in neurons (Luberto et al., 1998; Uramura et al., 2001). However, D609 had no effect on the response of the GFP-PKC-C1 probe at low orexin-A concentrations. Thus, PC-PLC was ruled out as a candidate enzyme. Cells were then transiently transfected with dominant negative constructs for PLD (1 and 2). This almost completely blocked the response of the probe at low orexin-A concentrations. This finding together with the fact that the DAG production per- fectly follows the PI/PIP hydrolysis (total inositol phosphate production) when PLD was inhibited, makes PLD the most probable candidate for the DAG production at low activation levels of OX1R. PLD seems to be activated at concentrations of orexin-A where cAMP and ERK pathways, that could be responsible for stimulating PLD, are not activated in OX1R signaling. The Gi/o proteins are, on the other hand, activated with a high potency by OX1R and may constitute a possible pathway for the activation of PLDs by OX1R. Interestingly, the high potency component of DAG generation over- laps with the PKC activation we saw in a previous study (Holmqvist et al., 2005).

Figure 6. GFP-PKC-C1 probe A, Basal conditions, i.e. no stimulation; the probe is located in the cytosol. B, Stimulation with 30 nM orexin-A leads to the translocation of the probe due to DAG generation in the membrane.

44 Thus, DAG could to be of great importance in orexin signaling and might also be the signal of preference. The DAG produced could target PKC but in recent years DAG has been shown to also have other targets such as the TRP channels (Tesfai et al., 2001; Jung et al., 2002). TRP channels and DAG have been associated with OX1R signaling (Larsson et al., 2005; Nasman et al., 2006). DAG produced by PLD at low OX1R activation levels might acti- 2+ vate Ca influx and this could be the source for the OX1R coupling to other pathways. Moreover, several studies indicates that DAG generated from PA seems to be a poor activator of PKCs and further supports another role than activating PKC for the high potency component of DAG in OX1R signaling (reviewed in Hodgkin et al., 1998). Finally, the biochemical and fluorescent methods seem to be well correlated and may for some studies be interchangeable. Since the information that can be obtained from each method is quite different, they are probably best used combined.

45 Conclusions

The results strongly indicate that Ca2+ influx is very important for the coupling of orexin receptors to multiple signal pathways, and therewith support the hypothesis of the central role of ROC influx in orexin receptor signaling. More specific conclusions of each paper are as follows:

I Orexin receptor activation results in a strong, rapid and long- lasting ERK activation. The analyzes of signaling pathways shows involvement of Ca2+, Src, Ras, MEK1, PI3K, DAG (from PLC) and conventional/atypical PKC. The OX1R-stimulated ERK response is dependent on Ca2+ influx via ROCs in several cell types (CHO, neuro-2a and striatal cells). The ROC influx seems to be the primary connector to the ERK pathway but can be replaced by SOC influx. Moreover, the activation of the AC 2+ pathway by OX1R stimulation is dependent on Ca influx, indicating that this is a general mechanism used by the orexin receptors for connection to other pathways.

II The ROC and SOC influxes are important for the amplification of the PLC activity upon OX1R activation. The ROC influx seems to be of greater importance for the PLC activation at low orexin-A concentrations, i.e. at low receptor activity, indicating that the ROCs probably have a central role in the orexin signaling since low doses probably are more relevant in the physiological setting.

III Overexpression of the IP3-metabolizing enzymes, IP3-3KA and 2+ IP3-5P1, is an excellent technique to abolish Ca release from ER. This is not only true for orexin receptor signaling, but also for other classically Gq-coupled receptors. This enables further mechanistic studies of the effect of ROC influx on orexin receptor signaling. Moreover, this technique confirmed that Ca2+ influx via ROCs is the primary connector of orexin receptors to the ERK pathway.

IV Different PLC isoforms with different substrate specificities seem to be activated in the OX1R signaling depending on the re-

46 ceptor activity level. This assumption is based on the fact that DAG is produced at the lower orexin-A concentrations without any generation of IP3, implying that PI and PIP are hydrolyzed instead of PIP2. At higher concentrations there is a production of both metabolites. At very low orexin-A concentrations, DAG is generated via OX1R-induced PLD activation and without any inositol phosphate production. Comparisons of the biochemical and fluorescent methods used suggested a good correlation between them.

It would be intriguing to test these findings in native systems to evaluate the physiological meaning of the complex signaling patterns displayed by the orexin receptors. To identify the identity of the PLC isoforms involved in OX1R signaling is also of importance. Moreover, the signaling properties of the DAG produced at the low orexin-A concentration is of high interest, especially since it might be the key to the activation of the ROCs primarily regulated by the orexin receptors.

47 Acknowledgements

I would like to express my sincere gratitude to all colleagues, friends and family who have, in one way or another, contributed to the completion of this thesis. Especially I would like to thank (in a Swedish-English-mix):

My supervisor, Professor Jyrki Kukkonen, for being an excellent supervisor, for sharing your wide expertise in cell signaling, for teaching me critical thinking and for giving me a completely new vocabulary (“såtta”, “klåpa” just to mention a few examples)… My co-supervisor, Professor Karl Åker- man, for sharing your great knowledge in orexin and calcium signaling and creating a cozy atmosphere with the “klipp-eti-klopp” of your nice shoes…

Alla föredetta och nuvarande medlemmar av Jyrki Kukkonens grupp. Speci- ellt Marie Ekholm, för din generositet och snällhet (i ordets bästa bemärkel- se), för ditt oändliga tålamod och för att du stått ut med mig i mina mindre fina stunder och för all hjälp med oändlig läsning…och Karin Nygren för allt arbete och fixande du hjälpt mig med under åren och för din fina genuina omtanke. Stort Stort Tack till er två!

Min vapendragare i vått (ja vad säger man... 7 olika typer av lera, 6 olika typer av våtmark....eller 32 olika arter av knott) å torrt (ja ibland har det ju faktiskt varit torrt) Liselotte Pihl. Jag är så glad att just du fanns här på fysi- ologen, för du är en fantastiskt fin och rolig vän och den enda jag kan tänka mig att sova med iklädd en svart sopsäck i ett vattenfyllt tält...

Alla andra underbara medarbetare på fysilogen och försöksdjursvetenskap, nuvarande och föredettingar, som tog så fint hand om mig när jag var ny och liten. Tack också för mycket fint stöd under alla år och för alla mysiga ut- flykter och fikastunder. Speciellt vill jag nämna: Olle Nylander (valet av ordförande var så lätt), Gunnar Flemström, Markus Sjöblom, Magnus Bengtsson (elitserien here we come!!), Gunilla Jedstedt, Annika Jägare, Klas Abelson, Gabriella Persdotter-Hedlund, Reneé Goldkuhl, Hans- Eric Carlsson (speciellt för min underbara lilla Svea) och Göran Sperber. Ett speciellt tack till, Gunno Nilsson, för allt fixande och trixande och för att du gör tillvaron mycket lättare för en trött liten labbamanuens...

48 All administrationspersonal som alltid har tid att hjälpa till och med sin posi- tivitet lyser upp och underlättar undervisnings-, pappers- och UPPDOK träsket: Lena Karlsson, Emma Andersson, Birgitta Klang, Marita Berg, Maria Larsson och Ulla Jonson.

Alla föredetta och nuvarande medlemmar av Dan Larhammars grupp som det är så roligt och trivsamt att få dela arbetsyta med: Dan Larhammar, Christina Bergqvist, Helena Svahn, Helena ”Hille” Åkerberg, Görel Sundström, Susanne Dreborg, Daniel Ocampo Daza, Tomas Larsson (tusen tack för all hjälp med allt smått å gott jag kommit dragandes med...), Ingrid Lundell (stort tack för du frivilligt tog dig tid att hjälpa mig med mitt manuskript!). Det är verkligen en lyckträff att få dela labb med er!

All members of the Neuroscience department, former and present, that I have forgotten to mention and have been of great help and support. Speciellt tack till: Lotta Israelsson vars goda råd och fina lathund varit till stor hjälp, Håkan Steen för allt lånande av material och metoder och svar på frågor jag kommit med i tid och otid... Birgitta Hägerbaum för att alltid ha ett gott litet skratt på gång och för en outtröttlig insats att om och om igen autokla- vera mina e-kolvar... Speciellt tack också till min första handledare, Aneta Ringholm, som tog så fint hand om mig under mina första stapplande steg...

Mina vänner här i Uppsala. Vill speciellt nämna: Molekylärtjejerna (Åsa, Maria, Lisa KW, Anna och Elin), Greta och alla glada medarbetare på Nautilus. Mina goa grannar Johan (måste bli fler döda tanter i Lännavattnet i sommar va?), Anders och Jonatan som fyllde min lic-dag med den bästa presenten så jag orkade ett helt år till! Tack också till Olle (Daiquirins okrönta mästare!!), Henry, Pelle, Jessica och Sara för fina små uppmunt- rande tillrop och många roliga och festliga sammankomster!

Mina vänner från hemifrån som det alltid är så ”nöjäsigt” att träffa och ”sar- ja” om gamla och nya tider: Josse, Marie, Camilla, Maria och Anna. Att ni alltid funnits där betyder mer än ni kan tro. Vänner från rötterna är mer än guld värt!

Svea min söta lilla pälsmössa!

Mamma Kina och Pappa Aage, Min Syster Yster Katarina och hennes underbara familj, Christian, Alexander, Klara och Ludvig. Ni är verkligen det som betyder allra mest för mig och jag är så glad att veta att ni finns där för mig! Kommer alltid att fortsätta att längta hem...

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A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.)

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