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List of Papers
This thesis is based on the following papers, which are referred to in the text by their Roman numerals.
I Regulation of OX1 orexin/hypocretin receptor-coupling to phospholipase C by Ca2+ influx. Lisa Johansson, Marie E. Ekholm and Jyrki P. Kukkonen British Journal of Pharmacology, 150(1):97-104, 2007
II Multiple phopholipase activation by OX1 orexin/hypocretin receptors. Lisa Johansson, Marie E. Ekholm and Jyrki P. Kukkonen Cellular and Molecular Life Sciences, 65(12):1948-56, 2008
III IP3-independent signaling of OX1 orexin/hypocretin receptors to Ca2+ influx and ERK Marie E. Ekholm, Lisa Johansson and Jyrki P. Kukkonen Biochemical and Biophysical Research Communications, 353(2):475-80, 2007
IV OX1 orexin receptor-mediated arachidonic acid release Pauli M. Turunen, Marie E. Ekholm, Pentti Somerharju and Jyrki Kukkonen British Journal of Pharmacology, In Press, 2009
V Rapid and easy semi-quantitative evaluation method for diacylglycerol and inositol-1,4,5-trisphophate generation in orexin receptor signaling Marie E. Ekholm, Lisa Johansson and Jyrki P. Kukkonen Acta Physiologica, In Press, 2009
Reprints were made with permission from the respective publishers.
Contents
Introduction ...... 11 G protein-coupled receptors ...... 11 GPCR classification ...... 13 G proteins ...... 14 Modulation of GPCR activity ...... 17 GPCR dimerisation/oligomerisation ...... 18 GPCRs and ion channels ...... 19 Second messengers ...... 21 IP3-metabolising enzymes ...... 26 IP3-3-kinase ...... 26 IP3-5-phosphatase ...... 26 Orexins and orexin receptors ...... 27 Systemic effects of orexin ...... 28 Aims ...... 30 Methods ...... 31 Cell cultures...... 31 Transfection ...... 31 Expression vectors ...... 32 Measurement of total inositol phosphate accumulation ...... 32 Binding-protein measurement of IP3 ...... 33 Ion exchange separation of inositol phosphates ...... 33 Microfluorometric "real-time" imaging ...... 34 Ca2+ measurements ...... 34 GFP-probe translocation ...... 34 Cell fixation and cell counting ...... 34 Western blotting ...... 35 Arachidonic and oleic acid release ...... 36 Results and Discussion ...... 37 PLC regulation ...... 37 Ca2+ influx-dependence of PLC activation ...... 37 PIP2 is not the only substrate for PLC ...... 38 PLD-dependent DAG generation ...... 38 Blocking IP3-dependent signalling ...... 39 IP3-independent ERK signalling ...... 40
Arachidonic acid release ...... 40 Ca2+-dependence of the AA release ...... 41 ERK-dependence of the AA release ...... 41 AA-dependent ROC influx ...... 42 Optical detection of DAG generation and PLC activity ...... 42 Conclusions ...... 44 Acknowledgements ...... 46 References ...... 50
Abbreviations
AA Arachidonic acid AC Adenylyl cyclase cAMP Cyclic adenosine monophosphate CHO cells Chinese hamster ovary cells CNS Central nervous system cPLA2 Cytosolic PLA2 DAG Diacylglycerol ER Endoplasmic reticulum ERK Extracellular signal-regulated kinase GAP GTPase-activating proteins GEF Guanine nucleotide exchange factor GFP Green fluorescent protein GIRK channel G protein-regulated inward rectifier K+ channel GPCR G protein-coupled receptor GRK G protein-coupled receptor kinases GTPase Guanosine triphosphatase 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 2+ iPLA2 Intracellular (Ca -independent) PLA2 JNK c-Jun N-terminal kinase Lp-PLA2 Lipoprotein-associated PLA2 LPA Lysophosphatidic acid MAPK Mitogen-activated protein kinase PA Phosphatic acid PBS Phosphate-buffered saline PC Phosphatidylcholine PC-PLC Phosphatidylcholine-specific PLC PH Pleckstrin homology PIP2 Phosphatidylinositol-4,5- bisphosphate
PI-PLC Phosphoinositide-specific PLC PKA Protein kinase A PKC Protein kinase C PLA2 Phospholipase A2 PLC Phospholipase C PLD Phospholipase D PMCA Plasma membrane Ca2+ ATPase RGS Regulator of G protein signalling ROC Receptor-operated channel SERCA Sarco-endoplasmic reticulum Ca2+ ATPase SOC Store-operated channel sPLA2 Secreted small PLA2 SR Sarcoplasmic reticulum TBM TES-buffered medium TRP Transient receptor potential VOC Voltage-operated channel
Introduction
G protein-coupled receptors G protein-coupled receptors (GPCRs) constitute the largest known gene family in the human genome. There are over 1200 human GPCRs and they account for about 2% of the human genome (Fredriksson et al., 2003) and at least 30% of all marked prescription drugs act on GPCRs (Hopkins and Groom, 2002). The GPCRs are fairly large ranging from approximately 40 kDa to 200 kDa, and they have seven transmembrane helices. The N- terminal domain is located on the extracellular side of the plasma membrane and the C-terminal domain on the cytroplasmic side. Intracellular loops 2 and 3 and the proximal region of the C-terminus are key regions for the activation of G proteins (Hermans, 2003; Wong, 2003). GPCRs are believed to share similar overall secondary structure but still they vary widely in amino acid sequence and signalling properties. The GPCR family of receptors are activated by a remarkable variety of extracellular messengers as diverse as biogenic amines, purines and nucleic acid derivates, lipids, peptides and proteins, odorants, pheromones, tastants, ions such as Ca2+ and protons, and even photons (Ellis, 2004), and they are involved in a vast variety of physiological functions, including neurotransmission, function of exocrine and endocrine glands, smell, taste, vision, chemotaxis, embryogenesis, cell growth and differentiation (Hall et al., 1999). Activation of GPCRs generates second messengers that act next to the plasma membrane or deep within the cell. A GPCR signalling complex includes the receptor, a heterotrimeric G protein composed of a Gα-GDP and a Gβγ subunits, an effector and a regulator of G protein signalling (RGS) protein (Ross and Wilkie, 2000). When activated by an agonist, the GPCRs show movement of the third intracellular loop away from the C-terminus. This movement catalyses GDP/GTP exchange on Gα, leading to the dissociation of the heterotrimer (Greasley et al., 2002; Greasley et al., 2001; Lohse et al., 2003). Both Gα-GTP and Gβγ are then free to interact and modulate the activity of proximal downstream elements of the signalling cascades, such as adenylyl cyclase (AC), phospholipases or ion channels. The activated subunits affect their target until the intrinsic GTPase activity of Gα hydrolyses the GTP to inactivate the Gα and causes it to re-associate with Gβγ thus completing the cycle.
11
Figure 1. The GDP/GTP cycle governing the activation of heterotrimeric GPCR signalling pathways and the effector molecules regulated by Gα-GTP and Gβγ
GTP hydrolysis is further accelerated by RGS proteins. The RGS proteins and the effector proteins themselves function as GTPase-activating proteins (GAPs) to accelerate the GTPase activity of Gα (see figure 1) (Ross and Wilkie, 2000). Further signalling specificity is achieved by G protein- coupled receptor kinases (GRKs) that work at the receptor level by phosphorylating receptors in their active, ligand-bound form, and in that way uncouple receptors from G proteins (Premont et al., 1995). In addition, several GPCRs have recently been demonstrated to signal via G protein-independent mechanisms both in vitro and in vivo (Rajagopal et al., 2005).
GPCR classification The human GPCRs are regularly divided into three main families, A, B and C. A still separate group is constituted by the receptors of the frizzled family. For the frizzled family direct coupling to G proteins is yet to be demonstrated. The 11 human frizzled and smoothened receptors control cell development and proliferation mediated by the secreted glycoproteins wnt and hedgehog, respectively (Huang and Klein, 2004). The human GPCRs have recently been alternatively classified into five different groups named GRAFS, which is the acronym for the groups glutamate, rhodopsin, adhesion, frizzled/taste2 and secretin (Fredriksson et al., 2003).
Class A receptors The rhodopsin-like family A is the largest family of GPCRs (Foord et al., 2005). The family is characterised by several highly conserved amino acids in the seven transmembrane helix bundle, and disulfide bridges connect the first and second extracellular loops of the receptors (George et al., 2002). Class A receptors contain receptors related to the rhodopsin and are hence also called rhodopsin-like receptors. Class A receptors bind ligands as diverse as biogenic amines (such as histamine and serotonin), peptides (e.g. opioid and somatostatin), (glyco)proteins (e.g. follicle-stimulating hormone, viral proteins), lipids (e.g. cannabinoids) and olfactants.
Class B receptors Secretin-like receptor family B comprises 50 GPCRs for peptides such as secretin, glucagon, calcitonin, parathyroid hormone, gastric inhibitory peptide and vasoactive intestinal peptide (VIP). The B family is characterised by the presence of a large N-terminus containing several cysteines that form a network of disulfide bridges (George et al., 2002). The G s-AC pathway predominates in the signalling of class B receptors (Ulrich et al., 1998).
13 Class C receptors Family C GPCRs contain receptors related to the metabotropic receptors including the metabotropic glutamate receptors, the calcium-sensing receptors (CaR), and -aminobutyric acid (GABA)B receptors. This group has 17 members in the human genome (Huang and Klein, 2004) and is characterised by long N- and C-termini, of which the N-terminus contains the ligand-binding domain (George et al., 2002).
G proteins Heterotrimeric G proteins are the intracellular partners of GPCRs. Membrane-bound heterotrimers, composed of Gα, Gβ and Gγ subunits, are closely associated with the intracellular faces of GPCRs. GDP-bound Gα subunits bind tightly to the heterodimer Gβγ. This association aids localisation of the trimer to the plasma membrane, which occurs through lipid links in Gα and Gγ (Evanko et al., 2001), and is essential for the functional coupling to GPCRs (Robillard et al., 2000). Agonist bound GPCRs act as guanine nucleotide exchange factors (GEFs), promoting the release of bound GDP by Gα. Nucleotide-free Gα then binds GTP, which is present in a significant excess to GDP in cells. The binding of GTP results in conformational changes within the three flexible switch regions of Gα (Wall et al., 1998), resulting in the dissociation of Gβγ. Both GTP-bound Gα and free Gβγ are capable of initiating signals by interacting with downstream effector proteins (see figure 1). The intrinsic guanosine triphosphatase (GTPase) activity of the Gα subunit causes the hydrolysis of GTP to GDP, returning the Gα subunit to its inactive state. Re-association of Gβγ with Gα-GDP terminates all effector interactions (Ford et al., 1998; Li et al., 1998). There have been recent studies implicating that not all G subunits dissociate from their G complex after activation but rather the subunits are rearranged (Bunemann et al., 2003; Frank et al., 2005). For a long time it was assumed that a given GPCR only interacts with a particular G protein or a given family of G proteins. This has later been refuted. Accumulating evidence demonstrates that a single GPCR species can interact with G proteins belonging to different families activating different signalling cascades, sometimes with opposing effects (Hermans, 2003). The actual coupling of a GPCR to a given heterotrimeric G protein may vary among cell types depending on which subunits are expressed and their localisation in the cell.
14 G subunits There are 16 Gα genes in the human genome, which encode 23 known Gα proteins. These genes/proteins can be divided into four major classes based on sequence similarity: Gαs, Gαi/o, Gαq and Gα12/13 (Simon et al., 1991). All four classes of Gα subunits have well-established cellular targets.
G s family The first recognised Gα effector was AC, first described by Sutherland and Rall (Sutherland and Rall, 1958). Nearly 20 years after the identification of AC as an important component of intracellular signalling, a GTP-binding protein that stimulated AC was isolated; it has since been termed Gαs (Ross and Gilman, 1977). The G s family contains G s-long, G s-short and G olf. All members of the G s family are sensitive to cholera toxin and they interact with and stimulate AC to increase cyclic adenosine monophosphate (cAMP) levels (Weinstein et al., 2002).
G i/o family
Shortly after the discovery of G s, Gαi, which inhibits AC and thus opposes the action of Gαs, was identified (Hildebrandt et al., 1983). The G i/o family includes G i1, G i2, G i3, G o1, G o2, G z, G gust, G t1 and G t2. This family, with the exception of G z, is pertussis toxin-sensitive. The most abundant G protein in the human brain is G o. Some candidates for direct effectors of G o include a GAP for the small G protein Rap (RapGAP), a GAP for G z (G zGAP), RGS17, and the G protein-regulated inducer of neurite outgrowth (GRIN) (Chen et al., 1999; Jordan et al., 1999). G i1, G i2, G i3 and Gz mediate inhibition of various AC (Sadana and Dessauer, 2009). The last three members of the G i family are involved in sensory systems. G gust, is mainly expressed in taste cells and is responsible for transducing the bitter and sweet taste sensations (Wong et al., 1996), while rod transducin (G t1) and cone transducin (G t2) are the G proteins involved in visual transduction.
G q/11 family
The G q/11 family includes G q, G 11, G 14, G 15 and G 16. G q and G 11 (which are 88% identical in amino acid sequence) are widely distributed in mammalian tissues. G 14 is found in spleen, lung, kidney and testis (Wilkie et al., 1991). The human G 16 protein and its mouse G 15 homolog are expressed in hematopoietic cells (Amatruda et al., 1991; Wilkie et al., 1991). G proteins of the Gαq class activate phosphoinositide-specific phospholipase C (PI-PLC) isozymes of the β-subfamily (PLC 1-4). PI-PLCs hydrolyse the phosphoester bond of the plasma membrane lipid PIP2 generating the ubiquitous second messengers inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (Rhee, 2001). Although members of the
15 G q/11 family are indistinguishable regarding the PLC isozymes they activate, some GPCRs can discriminate among them.
G 12/13 family
The G 12/13 family includes G 12 and G 13. This family of G proteins shares less than 45% sequence identity to other -subunits and 67% homology to each other (Milligan et al., 1992; Strathmann and Simon, 1991). Gα12/13 proteins can regulate the small G protein RhoA via GEFs that possesses Dbl- homology (DH) and pleckstrin-homology (PH) domains (Worthylake et al., 2000). Additionally, studies using activated forms of G 12/13 have demonstrated numerous proteins downstream of G 12/13 signalling. For example, c-Jun N-terminal kinase (JNK), Na+/H+-exchanger (NHE) and phopholipase D (PLD) have been shown to be activated in the presence of these mutant proteins (Collins et al., 1996; Hooley et al., 1996; Lin et al., 1996; Plonk et al., 1998; Voyno-Yasenetskaya et al., 1996)
G subunits There are five known human Gβ subunit genes, of which two have splice variants (Clapham and Neer, 1997; Fletcher et al., 1998), leading in total to G 1, G 2, G 3, G 3S, G 4, G 5 and G 5L (Clapham and Neer, 1997; Gautam et al., 1998; Schwindinger and Robishaw, 2001), and twelve human Gγ subunit genes (Huang et al., 1999; Ray et al., 1995; Simon et al., 1991). This results in a large number of potential combinations of Gβγ dimers. Gβγ is a functional heterodimer that forms a stable structural unit. Unlike the conformationally flexible Gα subunit, the Gβγ dimer does not change conformation when it dissociates from the G protein heterotrimer (Sondek et al., 1996). In addition, Gβγ association with Gα prevents Gβγ from activating its effectors. The Gβγ dimer was once thought only to facilitate coupling of Gαβγ heterotrimers to GPCRs and act as a Gα inhibitor. It is now known that Gβγ is free to activate a large number of effectors on its own following its dissociation from Gα-GTP (see figure 1) (Clapham and Neer, 1997). The first Gβγ effectors identified were GIRK channels (Logothetis et al., 1987). Gβγ can also regulate kinases and small G protein GEFs. Activation of certain GPCRs results in Gβγ-mediated activation of extracellular signal- regulated kinase 1/2 (ERK1/2), JNK and p38 mitogen-activated protein kinase (MAPK) cascades (Maier et al., 2000; Maier et al., 1999; Stephens et al., 1994; Stephens et al., 1997). Gβγ has been shown to both positively and negatively regulate various AC isoforms, activate PLCβ, PLCε and PLD and localise G protein-coupled receptor kinase (GRK) -2 and -3 to the plasma membrane (McCudden et al., 2005). The mechanism of Gβγ interaction with its effectors is not entirely clear. The signalling from G is usually associated with Gi proteins.
16 Modulation of GPCR activity GPCR signalling is tightly regulated by posttranslational modifications. Receptor phophorylation by GPCR-specific and -nonspecific kinases modulates subsequent interactions with several intracellular proteins involved in receptor internalisation and downregulation (Kohout and Lefkowitz, 2003; Tsao et al., 2001).
Receptor phosphorylation Phophorylation is a very common mechanism for the regulation of signalling. Studies of rhodopsin and the 2-adrenergic receptor have revealed a highly conserved mechanism that regulates the activity of many GPCRs (Lefkowitz et al., 1998). This mechanism involves phosphorylation of the receptor by G protein-coupled receptor kinases (GRKs), followed by interaction of the phosphorylated receptor with cytoplasmic accessory proteins called arrestins. The binding of arrestins to the receptor hinders the interaction with heterotrimeric G proteins, thus disrupting the pathway of GPCR-mediated signal transduction at the earliest stage. This constitutes a way of mediating rapid desensitisation of a ligand-activated GPCR (Sibley et al., 1985). Agonists regulate phophorylation of GPCRs by GRKs and they also regulate the affinity with which the phosphorylated receptor binds to arrestins (Gurevich and Benovic, 1997). This control assures that only receptors activated by agonist are desensitised (Lefkowitz et al., 1998). Most GPCRs are also phosphorylated by for example protein kinase A (PKA) and PKC (Lefkowitz, 2004; Lefkowitz et al., 1998).
Agonist-induced endocytosis Studies on the process of sequestration led to the observation that certain GPCRs are removed from the plasma membrane within minutes after activation (Staehelin and Simons, 1982; Toews and Perkins, 1984). This finding was later verified in cultured cells and native tissues (Keith et al., 1998; Kurz and Perkins, 1992; von Zastrow and Kobilka, 1992). GRKs and arrestins play an important role in regulating this endocytosis of GPCRs. By acting as adapters, -arrestins promote the concentration of phosphorylated receptors in coated pits (Goodman et al., 1996; Laporte et al., 2000). Many but not all GPCRs endocytose rapidly, and some GPCRs endocytose by a different mechanism (von Zastrow et al., 1993). Endocytosis is not believed to play a primary role in mediating rapid desensitisation but rather in mediating the distinct process of receptor resensitisation (Pippig et al., 1993; Yu et al., 1993). Endocytosis is believed to bring receptors in close proximity to an endosome-associated phosphatase, which mediates dephosphorylation of receptors previously phosphorylated at the cell surface. Dephosphorylated receptors are then recycled back to the plasma membrane in a resensitised state, which is fully functional to mediate subsequent rounds
17 of signal transduction upon re-exposure to agonist (Lefkowitz et al., 1998; Pippig et al., 1995). There has also been the suggestion of endocytosis playing an important role in mediating downregulation of many GPCRs by promoting proteolysis of receptors. Downregulation of GPCRs can occur via multiple mechanisms (Tsao and von Zastrow, 2000a) and one mechanism involves endocytosis of receptors followed by membrane trafficking to lysosomes (Koenig and Edwardson, 1997; Tsao and von Zastrow, 2001). The sorting of GPCRs to plasma membranes contra lysosomes following endocytosis (Gagnon et al., 1998; Tsao and von Zastrow, 2000b) is important because it determines whether agonist-induced endocytosis promotes the distinct functional consequences of receptor resensitisation or downregulation, respectively. Furthermore, the internalisation of GPCRs by -arrestins can be a crucial step in the signalling by certain GPCRs. -arrestins works as adapters for Src-family tyrosine kinases, and as receptor-regulated scaffolds for several MAPK modules. For instant, stimulation of class B GPCRs leads to GRK- mediated phosphorylation and the recruitment of -arrestins. Working as a scaffold, -arrestin gathers members of MAPK cascades together to form a complex with the receptor. This complex formation leads to GPCR- dependent MAPK signalling by the internalised receptor (Shenoy and Lefkowitz, 2003).
GPCR dimerisation/oligomerisation It was earlier believed that GPCRs existed and functioned as single monomeric units that only interacted with G proteins to produce intracellular signals. However, this view has been revised during the recent years. It is now believed that they interact with other GPCRs and intracellular regulatory proteins to form homo- or hetero-oligomeric signalling units (Angers et al., 2002; Gomes et al., 2001; Rios et al., 2001). For instance, interaction of the GABABr1 and GABABr2 gene products is needed to generate a functional GABAB receptor (Marshall et al., 1999; Mohler and Fritschy, 1999). The μ, and opiod receptors can form heterodimers with each other and this can lead to changes in the pharmacology of the receptors (George et al., 2000; Jordan and Devi, 1999). Also, the heterodimerisation of μ and receptors changes the coupling selectivity of the receptors from G i/o to what is belived to be the pertussis toxin-insensitive G Z (George et al., 2000). It has been demonstrated for the 2A- and 2C-adrenergic receptor subtypes that heterodimerisation may also cause a reduction in -arrestin recruitment (Small et al., 2006). The possibility of GPCRs to form homo- and heterodimes/oligomes adds yet another dimension to the regulation of GPCR function in cells co-expressing a multitude of different GPCRs.
18 GPCRs and ion channels Several ion channels are affected by G protein activation; to mention a few, GIRKs, voltage-gated Ca2+ channels, cardiac and epithelial chloride channels and cardiac and epithelial sodium channels are directly affected by G proteins. Of these the GIRK (or Kir3) family is the best characterised. Direct activation of GIRK1-4 by G proteins leads to rapid decrease of membrane excitability. The plasma membrane Ca2+ channels can be divided into four different types, according to their activation mechanism: voltage-operated channels (VOCs), receptor-operated channels (ROCs), mechanically-activated channels and the so-called “store-operated channels” (SOCs), which are opened following the depletion of internal Ca2+ stores. The VOCs are located near the vesicle docking site and are responsible for the influx of Ca2+ into the presynaptic neuron which allows release of neurotransmitters from the synaptic neuron. Four families of VOCs, N-type, L-type, P/Q-type, and T-type, are regulated by G proteins. G i1/i2/z and G 2+ have been shown to inhibit the N-type Ca channels, whereas G s and G can stimulate the L-type Ca2+ channels (Herlitze et al., 1996; Ikeda and Dunlap, 1999; Kaneko et al., 1999; Viard et al., 2001; Zhong et al., 2001). Of these, the G -complex has been shown to directly interact with and modulate N-type Ca2+ channel activity (Kaneko et al., 1999). Of all the plasma-membrane Ca2+ channels, ROCs are the most poorly understood. This may be due to the fact that, based on phenomenological characterisation, there seems to exist a large number of different ROCs. In 1989, Montell and Rubin first reported a protein in Drosophila involved in the inositol lipid signalling system (Montell and Rubin, 1989). The protein termed the transient receptor potential (TRP) was later discovered to constitute the first member of a family of ROCs. Whether the phenomenologically described ROCs correspond to the TRP channel members, remains to be demonstrated in most cases.
TRP channels TRP channels constitute a superfamily of cation channels which display diverse mechanisms of regulation and physiological function. These channels can be activated by a myriad of ways such as sensory signals, chemical ligands and in response to stimulation of GPCRs and tyrosine kinase-coupled receptors. The TRP superfamily is divided into seven subfamilies of TRPC, TRPV, TRPM, TRPML, TRPN, TRPP and the TRPA (Vassort and Alvarez, 2009). Even though the different families have very different modes of action and function, they all have six membrane-spanning domains and a pore region between the fifth and sixth transmembrane domain (Vassort and Alvarez, 2009). Although extensive research has focused on the TRP channels during
19 the last few years, conclusive data on the exact physiological function of these channels are still lacking. TRP proteins generate channels by homomeric and heteromeric interactions between members of the same subfamily (Vassort and Alvarez, 2009). They also interact with accessory proteins that determine their localisation and plasma membrane expression, and regulate the gating of the channel. The functional organisation of TRP channel complexes decides not only their regulation by extracellular stimuli but also serves as a platform to coordinate specific downstream cellular functions (Ambudkar, 2006; Montell, 2005). Several studies have demonstrated activation of the TRPC family by G- protein coupled receptors. The mechanism by which this occurs has not been clarified. They associate with a number of proteins involved in signal transduction (Venkatachalam and Montell, 2007) and the TRPC subfamily is believed to be activated downstream of agonist-stimulated PIP2 hydrolysis (Ambudkar, 2006; Putney, 2007; Venkatachalam et al., 2001) but there is a considerable conflict regarding their exact mode of activation. There are still no conclusive data on their physiological function in the various tissues where they are expressed. The TRPC subfamily was established following the identification and cloning of TRPC1 (Zitt et al., 1996), the first recognised mammalian TRP channel, and it is divided into three groups on the basis of sequence alignments and functional comparisons: TRPC1/4/5, TRPC3/6/7 and TRPC2 (Clapham, 2003). Studies have shown that TRPC1 associates with Orai1, a 33 kDa sarcolemmal protein that is responsible for capacitive Ca2+ entry, and in this way contributes to Ca2+ entry through SOCs. Furthermore, TRPC1 also associates with the stromal interaction molecule 1 (STIM1), a 77 kDa protein of the ER, and also in this way contributes to the SOC influx (Ambudkar et al., 2007). The other two members of the group, TRPC4 and TRPC5 are activated by G proteins and receptor tyrosine kinases. The activation of these two channels seems to require PLC enzymatic activity, but neither IP3 nor DAG itself is sufficient to activate the channels (Okada et al., 1998; Plant and Schaefer, 2003). TRPC3, TRPC6 and TRPC7 have been demonstrated to generate non-selective cation currents when activated by Gq/11-coupled receptors or DAG (Hofmann et al., 1999; Okada et al., 1999). The information on human TRPC2 is limited since it is a pseudogene in humans (Liman and Innan, 2003; Yildirim et al., 2003), but it has been demonsrated to be activated by pheromone vomeronasal receptors in rodent (Touhara and Vosshall, 2009).
20 Second messengers cAMP was the first characterised second messenger of the GPCRs (Sutherland, 1972). Calcium ion and some inositol phosphatases were next characterised as second messengers, and phospholipases, kinases and ion channels emerged as important effector systems downstream of GPCR activation (Hamm, 1998; Marinissen and Gutkind, 2001). Since then many different second messengers have been found for the GPCRs.
Phospholipases Many of the responses seen upon GPCR activation are mediated by phospholipid signalling cascades. Phospholipases are enzymes that selectively hydrolyse phospholipids and they are divided into four families, phopholipase A (PLA), phospholipase B (PLB), phospholipase C (PLC) and phospholipase D (PLD), according to the ester bond(s) that is cleaved.
Phospholipase A2
PLA2 activity was first studied in cobra venom as early as the 1890s (Burke and Dennis, 2009a). The PLA2 enzymes are characterised by their ability to specifically hydrolyse the sn-2 ester bond of phospholipid substrates. There are currently 15 separate groups and numerous subgroups of PLA2 (Schaloske and Dennis, 2006; Six and Dennis, 2000) and there are more than 20 mammalian PLA2s. The PLA2 enzymes are grouped based on their structure, enzymatic characteristics, molecular weight, disulfide bonds, subcellular localisation, requirement for Ca2+ and their cellular function. They are divided into four main categories, the secreted small sPLA2s, the 2+ larger cytosolic cPLA2s, the Ca -independent iPLA2s and the lipoprotein- associated Lp-PLA2. PLA2 enzymes signal from a large number of plasma membrane-associated receptors and extracellular signals (Farooqui and Horrocks, 2006; Schaloske and Dennis, 2006). Distinct PLA2 subgroups are localized to different cellular pools of phospholipids and can be regulated by different second messengers (Schaloske and Dennis, 2006). The products after activation of PLA2 and the resultant hydrolysis of the sn-2 ester bond of glycerophospholipids are free fatty acids and lysophospholipids (Burke and Dennis, 2009a). The free fatty acids produced include the signalling molecules arachidonic acid (AA), docosahexaenoic acid (DHA) and oleic acid and other saturated and unsaturated fatty acids. Lysophospholipids produced include lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophatidylinositol. The free fatty acids and the lysophospholipids or their downstream products have multiple roles in Ca2+ signalling such as activation of PKC, TRP channels and AA-regulated Ca2+ channels (ARCs), and they also alter membrane fluidity and stability (Bhalla and Iyengar, 1999; Goni and Alonso, 1999; Shuttleworth et al., 2004; Soboloff et al., 2007).
21 ARC channels are specific for AA and it has been demonstrated that the effect on ARC by AA is exerted from the intracellular side of the membrane (Mignen et al., 2005; Shuttleworth et al., 2004). AA can modulate the Ca2+ network by various mechanisms including direct binding to various ion channels, activation of nitric oxide synthase (NOS), PKA, PKC and DAG kinase and stimulation of calcium store discharge (Farooqui and Horrocks, 2004). It has been suggested that both cPLA2 and iPLA2 are receptor-regulated (Ghosh et al., 2006; Schaloske and Dennis, 2006), and also that both cPLA2 2+ and sPLA2 are regulated by Ca , sPLA2 by being stimulated by an increase 2+ in Ca concentration and cPLA2 by being brought to its lipid substrates by 2+ Ca elevations. iPLA2 on the other hand is either not affected by elevated Ca2+ concentrations (Canaan et al., 2002), or is inhibited by Ca2+ (Akiba and Sato, 2004; Jenkins et al., 2001). Both iPLA2 and cPLA2 members have been demonstrated to be regulated by PKC, Ca2+-calmodulin kinase II (CaMKII) and MAPK signalling pathways (Akiba and Sato, 2004; Ghosh et al., 2006; Leslie, 2004; Six and Dennis, 2000).
Phospholipase C The PLC family of enzymes contains two different forms, the PC- (phosphatidylcholine-) and the PI-specific type. PC-PLC hydrolyses PC into phosphocoline and DAG (Luberto and Hannun, 1998; Murthy and Makhlouf, 1995). The information on PC-PLC is very limited and most of the research on PC-PLC has been done in bacteria. For this reason the classical mammalian PI-PLC is henceforth referred to as simply PLC. Upon activation, PLC enzymes hydrolyse phosphatidylinositol-4,5- bisphosphate (PIP2), residing at the inner face of the plasma membrane, resulting in the production of the two cellular messengers, IP3 and DAG. These two messengers lead to the release of Ca2+ and the activation of PKC, respectively. The PLC family of enzymes incorporates PLC , PLC , PLC , PLC , PLC and PLC subfamilies. These families of enzymes exhibit relatively low sequence homology within the family but all of them contain conserved catalytic core regions called the X and Y domains. All of the PLC enzymes except PLC also contain a pleckstrin homology (PH) domain which binds membrane phosphoinositides or regulatory proteins. The PLC family consist of four isoforms, PLC 1-4. This family has, together with PLC isoforms, a unique extension in the C-terminus, which gives the enzyme membrane association. The G q family of proteins can activate PLC isoforms but not PLC , PLC nor the PLC isoforms (Lopez et al., 2001; Rebecchi and Pentyala, 2000; Rhee, 2001). In addition, PLC can be activated by G complexes liberated from G i family proteins (Rebecchi and Pentyala, 2000; Rhee, 2001; Wing et al., 2001) and Rho- GTPases (rac1/2, cdc42) via its PH domain (Harden and Sondek, 2006).
22 The PLC family has two members, PLC 1 and PLC 2. This family has two SH2 domains and one SH3 domain in between the X and Y domain. The SH2 domain enables PLC to bind to phosphotyrosine motifs which in turn leads to the binding of tyrosine kinases and therby phosphorylation and activation of the PLC enzyme. The PLC family is also activated by phosphatidylinositol-3,4,5-trisphosphate (PIP3) via binding to the SH3 domain (Harden and Sondek, 2006). The PLC family consist of three members, PLC 1, PLC 3 and PLC 4. This family has high sensitivity for Ca2+ as compared to PLC , PLC and PLC . The PH domain of the PLC family of enzymes exclusively binds to IP3 and PIP2 (Garcia et al., 1995). The PLC isoform is sperm-specific and is central in the Ca2+ mobilisation required for fertilisation. This isoform is the only one of the PLC isoforms that lacks a PH domain and it has the highest sensitivity for Ca2+ of all PLC isoforms (Kouchi et al., 2005). The PLC isoform has two Ras-binding domains similar to those found in other known effectors of the Ras family. It also has a RasGEF domain that catalyses the transition of GDP to GTP on Ras and Rap proteins. PLC is under the control of Ras-family GTPases (in particular H-Ras and also Rap1A and Rap2B) as well as G 12, G 13 and G subunits (Lopez et al., 2001; Wing et al., 2001). The PLC has two members, PLC 1 and PLC 2, of which PLC 2 has been demonstrated to be neuron-specific. This family seems to be activated by most of the G and G subunits and also by some of the Ras and Rho family members (Katan, 2005). An increase in the intracellular Ca2+ concentration afflicted by PLC, either by Ca2+ release from the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) or Ca2+ influx, amplifies the activity of either the same or another PLC isoform. This amplification leads to further Ca2+ increase or Ca2+ oscillations (Kim et al., 1999; Thore et al., 2005). Also, PKA and PKC have been demonstrated to stimulate or inhibit PLC signalling dependent on the cellular setting, and it is believed that this mechanism together with Ca2+ feedback is involved in the control of amplitude, frequency and duration of PLC signalling (Nash et al., 2001; Young et al., 2003).
Phospholipase D PLD is a phospholipid-specific phosphodiesterase that hydrolyses PC to phosphatic acid (PA) and choline. There are two mammalian PLD genes, with two splice variants each, PLD1 (PLD1a and PLD1b) and PLD2 (PLD2b and PLD2c) (Colley et al., 1997; Hammond et al., 1995; Hammond et al., 1997; Steed et al., 1998). Both PLD1 and PLD2 seem to respond to GPCR activation (Du et al., 2000; Parmentier et al., 2006; Wang et al., 2003; Zhang et al., 1999). PLD1 and PLD2 are regulated by PKC and GTPases of the ARF family and, in addition, PLD1 is regulated by Rho family GTPases
23 (Chen and Exton, 2004; Exton, 2002; Powner and Wakelam, 2002). It was earlier considered that all PLD activation seen upon GPCR activation was a direct effect of the PLC-dependent Ca2+ increase and activation of PKC, since almost all of the receptors known to activate PLC also showed PLD activation. Today it is known that PLD can be activated independently of PLC activation (Balboa and Insel, 1998; Muthalif et al., 2000; Rumenapp et al., 1997; Schmidt et al., 1994). PLD1 is found throughout the cell, but in particular in the intracellular compartments (Brown et al., 1998; Freyberg et al., 2001; Hughes and Parker, 2001). In contrast, PLD2 is almost exclusively found in the plasma membrane in lipid raft sections (Czarny et al., 1999). PLD1 is also found in lipid rafts. Both enzymes have been demonstrated to associate with membrane receptors, and PIP2 affects the subcellular localisation and the activation of PLD. In addition PIP3 can also activate the enzymes. PA is considered to transmit many of the biological functions affected by PLD. PA is further metabolised to the GPCR agonist lysophosphatidic acid (LPA) by PLA2 and to DAG by phosphatidic acid phosphohydrolase. Yet most of the DAG produced upon GPCR activation is from PIP2 breakdown by PLC (Nilssen et al., 2005). PA directly binds to and affects the cellular localisation and activity of cellular proteins such as kinases Raf-1 and mTOR and protein phosphatase 1, and the activation of PLD by GPCRs affects a vide variety of cellular responses such as calcium mobilisation, secretion, endocytosis, exocytosis, mitogenesis and apoptosis (Jenkins and Frohman, 2005; Liscovitch et al., 2000).
Ca2+ An central second messenger regulated by GPCRs is Ca2+. Many hormones and neurotransmitters transmit their message by causing an increase in the 2+ 2+ 2+ concentration of free cytoplasmic Ca ([Ca ]i). Elevated [Ca ]i drives gene expression, initiates proliferation, stimulates fluid and electrolyte secretion, controls muscle contraction, mediates neurotransmitter release, triggers fertilisation and initiates apoptosis. Ca2+ signals generally results from the opening of Ca2+ channels or the activity of Ca2+ transporters. These channels are located either on the plasma membrane, or inside the cell on the ER or SR. The binding of many hormones and growth factors to specific receptors on the plasma membrane leads to the activation of PLC, which catalyses the hydrolysis of PIP2 to produce the intracellular messengers IP3 and DAG. IP3 is highly mobile in the cytoplasm and diffuses into the cell interior where it encounters specific receptors, IP3 receptors (IP3Rs), on the ER/SR. The binding of IP3 changes the conformation of IP3Rs such that a channel is opened, thus allowing the Ca2+ stored at high concentrations in the ER/SR to enter the cytoplasm (Berridge, 1993; Irvine, 1990).
24 2+ The [Ca ]i increase is followed by activation of the plasma membrane Ca2+ ATPase (PMCA) pumps and Na+/Ca2+-exchanger that extrude Ca2+ to the outside, and the sarco-endoplasmic reticulum ATPase (SERCA) pumps 2+ 2+ that return Ca to the internal stores. [Ca ]i then stabilises at a plateau determined by the relative activities of the Ca2+ pumps and Ca2+ channels. Under physiological conditions, the Ca2+ signal rarely takes the form of a 2+ monotonic change in [Ca ]i, Rather, it is in the form of finely regulated 2+ repetitive [Ca ]i oscillation (Kasai et al., 1993; Rooney and Thomas, 1993; Thorn et al., 1993). Importantly, activation of different GPCRs that use the same principal biochemical machinery in the same cells, evoke Ca2+ oscillations (Yule et al., 1991) and Ca2+ waves distinct for each receptor (Shin et al., 2001; Xu et al., 1996; Xu et al., 1999). A central question in cell signalling is how cells generate receptor-specific signals.
MAPK The MAPK are a superfamily of kinases with four different subgroups: ERK1/2, JNK, ERK5 and p38 MAPK. MAPK are activated by phosphorylation cascades that classically start with the activation of a small G protein via a GEF. This leads to the activation of a MAPK kinase kinase (MAPKKK) which phosphorylates a MAPK kinase (MAPKK) that in turn phosphorylates a MAPK. The family of small GTPases have several members of which the best characterised belong to the Ras, Rho and ARF families (Gutkind, 2000; Luttrell and Luttrell, 2003). The ability of GPCRs to activate p21 Ras and the ERK/MAPK signalling pathway has been acknowledged for some time. Early work, which demonstrated the sensitivity of thrombin- or LPA- mediated Ras activation to pertussis toxin pre-treatment, implicated the involvement of G i proteins (van Corven et al., 1993). However, these GPCR-mediated activations of Ras were also sensitive to genistein, thus indicating that tyrosine kinase activity was also involved. As more GPCRs were found to activate Ras, it became apparent that not all the activation mechanisms involved G i, but also G q-signalling was able to activate Ras (Chen et al., 1996; Dikic et al., 1996). Since then all G subunits (G s, G q/11, G i/o, G 12/13) and G subunits have been demonstrated to be able to project to Ras and thereby to the ERK pathway (Gutkind, 2000; Luttrell and Luttrell, 2003).
25 IP3-metabolising enzymes
Like all second messengers, IP3 has a short half-life within the cell (Michell, 1997). IP3 is rapidly metabolised through one of two pathways: i) further phosphorylation of the inositol ring by IP3-3-kinases (Irvine et al., 1986) or ii) removal of the 5-phosphate from the inositol ring by IP3-5-phophatases (Michell, 1997).
IP3-3-kinase
IP3-3-kinases are the enzymes responsible for the ATP-dependent conversion of IP3 to inositol-1,3,4,5-tetrakisphosphate (IP4). This molecule is further metabolised to many highly phosphorylated inositol phosphates, some of which show specific biological functions. Both IP3 and IP4 are involved in 2+ Ca signalling, putting IP3-3-kinase in a central position as a regulator of two Ca2+-elevating second messengers. Three mammalian isoforms of this enzyme have so far been isolated and designated the A, B and C isoforms (51, 100 and 75 kDa, respectively) (Choi et al., 1990; Dewaste et al., 2000; Takazawa et al., 1991). Mammalian IP3-3-kinases can be activated by calmodulin (CaM) in a 2+ Ca -dependent manner to different degrees. Up to 20-fold increase in IP3-3- kinases' enzymatic activity by Ca2+/CaM has been observed. All three 3- 2+ kinase isoforms are also directly regulated by [Ca ]i. The A- and B-kinases 2+ are substantially activated by increased [Ca ]i, while the C-kinase is unique in that Ca2+ alone decreases the catalytic activity of the enzyme (Communi et al., 1995; Dewaste et al., 2000; Soriano et al., 1997). This effect on the C- kinase is reversed in the presence of CaM (Dewaste et al., 2000).
IP3-5-phosphatase To date eight inositol polyphosphate 5-phophatases have been isolated and characterised (Erneux et al., 1998). They are classified into three groups according to their substrate specificity: type I enzymes hydrolyse the water- soluble substrates IP3 and IP4 (Verjans et al., 1994), type II enzymes hydrolyse PIP2 and PIP3 in addition to IP3 and IP4 (Matzaris et al., 1998) and type III enzymes can hydrolyse IP3 but preferentially hydrolyse IP4 and PIP3 (Damen et al., 1996). The inositol polyphosphate 5-phosphatases dephosphorylate the 5- position of phosphoinositide and inositol phosphate molecules resulting in the generation of PtdIns(3,4)P2, PtdIns(4)P, PtdIns(3)P, Ins(1,4)P2 and Ins(1,3,4)P3. Since the latter two no longer mobilise intracellular calcium but may lead to other signalling pathways, the 5-phosphatases may act as signal- generating or signal-terminating enzymes (Erneux et al., 1998; Majerus et al., 1999).
26 Overexpression of the 43 kDa IP3-5-phophatase I enzyme in CHO cells abolishes ATP-induced calcium oscillations (De Smedt et al., 1997). In contrast, lowered expression of the 5-phosphatase using an anti-sense strategy results in increased IP3 levels, leading to increased sensitivity and amplitude of the calcium oscillations in response to both low- and high-dose agonist stimulation (Speed et al., 1999).
Orexins and orexin receptors In 1998 two groups simultaneously reported the discovery of two hypothalamic GPCRs responding to two neuropeptides, which they named hypocretin receptors and hypocretins and orexin receptors and orexins, respectively (de Lecea et al., 1998; Sakurai et al., 1998). It soon became clear that the peptides isolated by the two groups were identical. In this thesis the receptors will be referred to as OX1 orexin receptors and OX2 orexin receptors and the neuropeptides as orexin-A and orexin B. Both orexin-A and orexin-B is cleaved from the same 130-131 amino acid-long precursor peptide named preproorexin. Orexin-A is 33 amino acids in length and has two disulfide bridges and orexin-B is a 28 amino acid-long linear peptide (see Table 1). The orexin receptors belong to the family A rhodopsin-like receptors (Fredriksson et al., 2003). The OX1 orexin receptor is 425 amino acids in length and the OX2 orexin receptor is 444 amino acids long. OX1 orexin receptors show 64% sequence identity with OX2 orexin receptors and between the cloned mammalian orthologs there are 91-98% sequence identity. Both orexin peptides bind equally well to the OX2 orexin receptor but orexin-A binds with 10 times higher affinity to the OX1 orexin receptor as compared to the binding to the OX2 orexin receptor (Sakurai et al., 1998). This is also seen when looking at Ca2+ responses: orexin-A is 10 times more potent in activating the OX1 orexin receptor in Chinese hamster ovary (CHO) cells (Ammoun et al., 2003; Holmqvist et al., 2002; Sakurai et al., 1998) Orexin neurons are predominantly found in the hypothalamus and project throughout the central nervous system (CNS) to areas involved in the control of sleep/wakefulness, feeding, neuroendocrine homeostasis and autonomic regulation (Date et al., 1999; Peyron et al., 1998; Willie et al., 2001). But the expression of orexins and their receptors are not restricted to the CNS, orexins and orexin receptors are also expressed in peripheral tissues and may control several physiological responses in peripheral tissues (Voisin et al., 2003). In the first studies, application of orexin-B was shown to increase postsynaptic current frequency in rat hypothalamic neurons in culture (de Lecea et al., 1998), and orexin-A and -B increased food intake in non-fasted
27 2+ rats and elevated Ca in CHO cells (Sakurai et al., 1998). Both OX1 orexin 2+ receptors and OX2 orexin receptors elevate Ca when recombinantly expressed in CHO cells (Holmqvist et al., 2001; Lund et al., 2000; Okumura et al., 2001; Sakurai et al., 1998; Smart et al., 1999). It has been 2+ demonstrated that at low orexin-A concentrations, the [Ca ]i increase is primarily triggered by the activation of ROCs. This Ca2+ influx somehow 2+ activates PLC leading to Ca release from the ER through cleavage of PIP2 to DAG and IP3, and subsequent opening of IP3R and additional influx through SOCs (Kukkonen and Akerman, 2001; Larsson et al., 2005; Lund et al., 2000). At high concentrations of orexin-A, PLC activation and Ca2+ release from the ER is seen even without simultaneous activation of ROCs (Kukkonen and Akerman, 2001; Lund et al., 2000). Further studies have shown that these ROCs may be TRP channels (Nasman et al., 2006). Native neurons from several sites within the CNS have been demonstrated to respond to orexins with activation of non-selective cation currents. The identity of these channels is not yet known (Kukkonen and Åkerman, 2005 ). OX1 orexin receptors heterologously expressed in CHO-cells are also capable of activating ERK phosphorylation (Ammoun et al., 2006; Hilairet et al., 2003). Multiple signalling pathways, including Rac, Src, PI3-kinase and protein kinase C, are suggested to be involved in this response. Ca2+ 2+ influx but not Ca release, plays a central role in the OX1 orexin receptor signalling to ERK (Ammoun et al., 2006).
Systemic effects of orexin The main physiological function of orexins is undoubtedly regulation of sleep/wakefulness (de Lecea and Sutcliffe, 2005; Hungs and Mignot, 2001). Reduction in the peptides precursor, their receptors, or the neurons leads to the sleep disorder narcolepsy in both animals and humans (Chemelli et al., 1999; Lin et al., 1999; Peyron et al., 1998; Willie et al., 2003). Narcolepsy is a sleep disorder that is characterised by daytime sleepiness, cataplexy and striking transition from wakefulness into rapid eye movement (REM) sleep. Orexin neurons in the hypothalamus are activated in normal awakening in rat, in sleep-deprived rats and also by drugs against narcolepsy. Orexins also excite neurons in regions involved in sleep regulation and increase wakefulness (Kukkonen et al., 2002; Sakurai, 2007). Orexin-A and preproorexin mRNA expression seem to vary in rat during the night/day- cycle and orexin neurons are a major target of projections from areas regulating wake/sleep states and the circadian rhythm (Taheri et al., 2000). Orexins have been demonstrated to have a role in arousal and the wake/sleep transition and seem to be important for the smoothening this transition (Saper, 2006; Sutcliffe and de Lecea, 2002). Orexin were earlier believed to play a crucial role in energy homeostasis and feeding behaviour (Hara et al., 2001; Sakurai et al., 1998; Yamanaka et
28 al., 2003), but the current view is that orexins may rather regulate short-term appetite, energy metabolism and feeding-associated processes. Intracerebroventricular injections of orexins induce feeding and the intraperitoneal administration of a selective OX1 orexin receptor antagonist SB-334867 reduces food consumption in rats (Haynes et al., 2000). It has been argued that the effect on feeding behaviour by the orexins is dependent on the influence of orexins on metabolic rate by an increase in arousal rather than by up-regulation of food intake per se (Sutcliffe and de Lecea, 2002). Orexins also affect hormonal secretion, the most well-demonstrated effects being those on the hormones involved in the stress response and energy metabolism (Kukkonen et al., 2002).
Table 1. Orexin peptide sequences (known or assumed )of mammalians, birds and amfibians
Orexin-A Homo sapiens Man QPLPDCCRQKTCSCRLYELLHGAGNHAAGILTL Rattus norvegicus Rat QPLPDCCRQKTCSCRLYELLHGAGNHAAGILTL Mus musculus Mouse QPLPDCCRQKTCSCRLYELLHGAGNHAAGILTL Canis familiaris Dog QPLPDCCRQKTCSCRLYELLHGAGNHAAGILTL Sus scrofa Pig QPLPDCCRQKTCSCRLYELLHGAGNHAAGILTL Bos taurus Cow QPLPDCCRQKTCSCRLYELLHGAGNHAAGILTL Ovis aries Sheep QPLPDCCRQKTCSCRLYELLHGAGNHAAGILTL Gallus gallus Chicken QSLPECCRQKTCSCRIYDLLHGMGNHAAGILTL Xenopus laevis African clawed frog APDCCRQKTCSCRIYDILRGTGNHAAGILTL
Orexin-B Homo sapiens Man RSGPPGLQGRLQRLLQASGNHAAGILTM Rattus norvegicus Rat RPGPPGLQGRLQRLLQANGNHAAGILTM Mus musculus Mouse RPGPPGLQGRLQRLLQANGNHAAGILTM Canis familiaris Dog RPGPPGLQGRLQRLLQASGNHAAGILTM Sus scrofa Pig RPGPPGLQGRLQRLLQASGNHAAGILTM Ovis aries Sheep RPGPPGLQGRLQRLLQASGNHAAGILTM Gallus gallus Chicken KSIPPAFQSRLYRLLHGSGNHAAGILTI Xenopus laevis African clawed frog RSDFQTMQSRLQRLLQGSGNHAAGILTM
29 Aims
The aim of this thesis was originally to find the signalling pathway leading 2+ to the receptor-operated Ca influx seen upon OX1 orexin receptor activation. As the project went on, our investigations diversified and we started to investigate the signalling pathway leading from the activated OX1 orexin receptor to phospholipases as well. Our investigations aimed at 2+ assessing the ability of different Ca influx pathways to amplify OX1 orexin receptor signalling to PLC and to investigate the coupling of the OX1 orexin receptor to phospholipase activation. When conducting these studies it became apparent that we needed to construct a method that would block the 2+ IP3-dependent Ca release in order to enable investigations of the ROC- influx alone. The need for a method that would speed up the gathering of concentration-response data from GFP-fused signalling probes also emerged.
30 Methods
Cell cultures
CHO cells recombinantly expressing the human OX1 orexin receptor (Lund et al., 2000) were grown in Ham's F-12 medium (Gibco, Paisley, UK) supplemented with 100 U/ml penicillin G (Sigma Chemical Co., St. Louis, MO, USA), 80 U/ml streptomycin (Sigma), 400 μg/ml geneticin (G418; Gibco) and 10% (v/v) foetal calf serum (Gibco) at 37 ºC in 5% CO2 in an air ventilated humidified incubator in 260 ml plastic culture flask (75 cm2 bottom area; Greiner Bio-One GmbH, Frickenhausen, Germany). Wild-type
(wt) CHO cells (not expressing OX1 orexin receptors), were grown under the same conditions except that there was no geneticin added in the medium. Neuro-2a-hOX1 cells, neuroblastoma cells recombinantly expressing human OX1 orexin receptors (Holmqvist et al., 2002), were grown in Dulbecco’s modified Eagle’s medium (Gibco), with supplements as described for the CHO-OX1 cells. The confluence of the cell culture, independent of the assay, was about 80% by the time of the experiments.
Transfection Cells were grown to 40-50% confluence. The cells were washed twice with PBS and the medium was changed to Opti-MEM (GIBCO) and transfected using Lipofectamin reagent (0.58 μl/cm2; Invitrogen Corp., Carlsbad, CA, USA). After five hours, the OPTI-MEM was replaced with fresh medium (Ham's F-12 medium or Dulbecco’s modified Eagle’s medium depending on cell type) with all the usual supplements. Experiments were performed 48 h after transfection. Transfection efficiency was about 60% determined by the expression of pEGFP-C1 (Clontech, Palo Alto, CA, USA). The total amount of DNA (130 ng/cm2) was kept equal in all the transfections using pUC18 (an empty plasmid).
31 Expression vectors pEGFP-C1-PLCδ-PH (fusion of enhanced GFP (green fluorescent protein) and the PH domain of PLCδ1) was from Dr. Tobias Meyer (Stanford University School of Medicine, Stanford, CA, USA) (Stauffer et al., 1998), pEGFP-N1-PKCα-C1 (here referred to as GFP-C1-PKCα) from Dr. Christer Larsson (Lund University, Lund, Sweden) (Raghunath et al., 2003), pCGN- hPLD1-K898R (dominant negative human PLD1) and pCGN-mPLD2- K758R (dominant negative mouse PLD2) from Prof. Michael A. Frohman (University Medical Center at Stony Brook, Stony Brook, NY, USA) (Sung et al., 1997), pcDNA3-InsP3-3k-A (ins-1,4,5-P3 3-kinase-A; here referred as IP3-3KA) and pcDNA3-InsP3-5-Phosphatase-I (ins-1,4,5-P3 5-phosphatase; here referred as IP3-5P1) from Dr. Christophe Erneux (Free University of Brussels, Brussels, Belgium) (De Smedt et al., 1997), pEGFP-C1-PKC wt (fusion of enhanced GFP and mouse PKC , here referred to as GFP-PKC ) from Dr Johanna Ivaska (VTT Medical Biotechnology, Turku, Finland) (Ivaska et al., 2002), pcDNA3.1-hM1 (human muscarinic M1 receptor) from UMR cDNA Resource Center (www.cdna.org), pUC18 from Invitrogen (Carlsbad, CA, USA) and pEGFP-C1 from Clontech (Palo Alto, CA, USA).
Measurement of total inositol phosphate accumulation Membrane phosphoinositides were prelabeled by incubating the cells with 3 Ci/ml [3H]-inositol for 18 h in culture medium after which the cells were washed and placed in TES-buffered medium (TBM, consisting of 137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1.2 mM MgCl2, 0.44 mM KH2PO4, 4.2 mM NaHCO3, 10 mM glucose and 20 mM TES [2-([2-hydroxy-1,1- bis(hydroxymethyl) ethyl] amino) ethane sulfonic acid]; pH was adjusted to 7.4 with NaOH) containing 10 mM LiCl to inhibit inositol monophosphatase, at 37 °C for 10 min. The cells were stimulated with orexin-A or carbamoylcholine chloride (carbachol) for 2 or 20 min and the reactions stopped by removal of the medium and addition of 0.4 M perchloric acid and freezing. After thawing, the samples were neutralised with 0.5 volumes of 0.36 M KOH with 0.3 M KHCO3. The insoluble fragments were spun down, and the supernatants were subjected to anion exchange chromatography (AG 1-X8; Bio-Rad, Hercules, CA, USA) with H2O (for inositol; discarded), 5 mM Na2- tetraborate with 60 mM NH4-formate (for glycerophosphoinositol; discarded), and 0.1 M formic acid with 1 M NH4-formate (for inositol mono-, bis- and trisphosphates; collected) (Ammoun et al., 2006; Berridge et al., 1983). The inositol phosphate fraction was dissolved in scintillation cocktail (Optiphase Hisafe 3, PerkinElmer, Boston, MA, USA) and analysed with a liquid scintillation counter.
32 Binding-protein measurement of IP3 Cells cultured on plastic culture dishes were detached using PBS containing 0.2 g/l EDTA, spun down, washed once with TBM and resuspended in TBM. The cell number was adjusted to107 cells/ml by counting in a Bürker chamber. The cells were then stimulated with orexin-A and the cell suspension rapidly mixed. The reactions were terminated by adding ice-cold perchloric acid to a final concentration of 4% (v/v), vortexing and submersion in ice. The precipitate was sedimented by centrifugation at 2000 g for 15 min at 4 °C. The supernatants were neutralised with 1.5 M KOH with 60 mM HEPES, and 0.5 M Tris-HCl was added to a final concentration of 0.1 M (final pH 8.6). The resulting KClO4 sediment was removed by centrifugation at 2000 g for 15 min at 4 °C. The IP3 concentration of the samples was determined using D-myo-inositol-1,4,5-trisphosphate – [3H] Biotrak Assay (Amersham Bioscience, Buckinghamshire, UK) (Lund et al., 2000).
Ion exchange separation of inositol phosphates The cells were prelabeled with 3 μCi/ml [3H]inositol for 20 h. The medium was replaced with TBM and the cells were incubated for 10 min, after which they were stimulated with orexin-A for 10 s. The reaction was terminated by replacement of the medium with ice-cold 0.4 M perchloric acid and immediate freezing on dry ice. The thawed supernatants were neutralised with 0.36 M KOH with 0.3 M KHCO3 and the inositol phosphate fractions with different charges were isolated with AG 1-X8-anionexchange chromatography (De Smedt et al., 1997). The elutions were as follows: H2O (for inositol; discarded), 5 mM sodium tetraborate with 60 mM ammonium formate (for glycerophosphoinositol; discarded), 5 mM sodium tetraborate with 0.15 M ammonium formate (inositol monophosphates (IP1); collected), 0.1 M formic acid with 0.4 M ammonium formate (inositol bisphosphates (IP2); collected), 0.1 M formic acid with 0.7 M ammonium formate (inositol trisphosphates (IP3); collected) and 0.1 M formic acid with 1.5 M ammonium formate (inositol tetrakisphosphates (IP4); discarded). Radioactivity was analysed in a liquid scintillation counter as above.
33 Microfluorometric "real-time" imaging Ca2+ measurements The cells on glass coverslips were loaded with 4 M fura-2 acetoxymethyl ester for 20 min at 37 °C in TBM supplemented with 0.5 mM probenecid. The coverslips were rinsed once and then mounted immediately in a pre- heated (37 °C) perfusion chamber (~160 μl), placed in a thermostat- controlled holder and perfused with TBM supplemented with 0.5 mM probenecid at 37 °C. Additions to the chamber were made by perfusion. Recordings were made using TILLvisION version 4.01 imaging system (TILL Photonics GmbH, Gräfelding, Germany) with Nikon TE200 fluorescence microscope (20×/0.5 dry objective) and a CCD camera. The cells were excited by alternating 340- and 380-nm light from a xenon lamp through a monochromator and the emission light collected through a 400-nm dichroic mirror and a 475-nm barrier filter and the collected data was analysed in Microsoft Excel. The cells were transfected with GFP together with the gene(s) of interest to be able to select for the transfected cells.
GFP-probe translocation The cells were cultivated on cover slips as above and transfected with GFP- based indicators of interest (and possible other genes). Fluorescence was measured with the imaging system as above, but with 100×/1.30 oil immersion objective. 490 nm light was used for excitation, and the emission light was collected through a 505-nm dichroic mirror and a 520-nm barrier filter. The coverslips were constantly perfused with TBM at 37 °C as above.
Cell fixation and cell counting Cover slips with cells transfected with GFP-probes were stimulated with orexin-A for 1 min after which the cells were fixed. Six different fixation protocols were tested: (1) methanol ( 20 °C, 10 min), (2) methanol and acetone (1:1 (v); 20 °C, 10 min), (3) methanol and acetic acid (19:1 (v); 70 °C, 5 min), (4) ethanol ( 20 °C, 10 min), (5) 4% paraformaldehyde (+20 °C, 20 min), (6) methanol ( 20 °C, 10 min) followed by 4% paraformaldehyde (+20 °C, 20 min). The fixed cells were washed three times with PBS and twice with H2O and then mounted on glass slides using Mowiol 4-88 containing 2.5% (w/v) DABCO (1,4- diazabicyclo[2.2.2]octane) (Kukkonen et al., 1997). The mounting medium was allowed to set for at least 48 h, after which fluorescent pictures were taken with the TILLvisION v. 4.01 imaging system. The cells were excited at 490 nm with the use of a monochromator
34 and the fluorescence was collected through a 505-nm dichroic mirror and 520-nm barrier filter with a CCD camera. The images were analysed in a semi-quantitative manner by manually counting cells showing no, intermediate or full translocation, with assigned values of 0, 0.5 and 1, respectively, or only with no translocation (0) and translocation (1).
Western blotting The cells were serum-starved in Ham's F-12 medium with all the supplements except for fetal calf serum for 24 h before stimulation. Stimulation of the cells was performed in the same medium for 10 minutes, and stopped by aspiration of the medium, followed by a wash with ice-cold PBS supplemented with 1 mM Na+-orthovanadate, 10 mM NaF, and 250 μM p-nitrophenol phosphate and an immediate lysis with lysis buffer (PBS supplemented with 50 mM HEPES and 150 mM NaCl (pH 7.5) supplemented with 10% (vol/vol) glycerol, 1% (vol/vol) Triton X-100, 1.5 + + mM MgCl2, 1 mM EDTA, 10 mM Na -pyrophosphate, 1 mM Na - orthovanadate, 10 mM NaF, 250 μM p-nitrophenol phosphate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Volumes corresponding to 15 μg protein were diluted in Laemmli buffer, boiled for 3 minutes, separated by SDS-PAGE (10% acrylamide), and transferred to methanol-soaked polyvinylidene difluoride membranes (Amersham). The membranes were blocked with 0.1% (vol/vol) Tween 20 and 5% (wt/vol) milk in PBS (TPBS-milk) before probing with antisera. Phosphorylated ERK (pERK) was detected using rabbit polyclonal anti- active-MAPK (anti pThr183-pTyr185-ERK1/2) antibody (V803A; 1:5000; Promega Corp., Madison, WI, USA). Primary antibodies were detected using horseradish peroxidase-conjugated donkey antirabbit antibody F(ab')2 fragments (1:2000; Amersham). Primary antibody incubation was done in TPBS at room temperature for 1 h. Secondary antibody incubation was performed in room temperature for 1 h after two 30-minute washes with TPBS. Peroxidase enzymatic activity was visualised using chemiluminescence detection kit EZ-ECL (Biological Industries, Kibbutz Beit Haemek, Israel). After detection of pERK, the membranes were washed for 2 h with TPBS-milk, and the total MAPK in each lane was probed with polyclonal anti-total MAPK (ERK1/2) antibody (NA 9340; 1:2000; Oncogene Research Products, San Diego, CA, USA) and the same secondary antibody as above. The same detection system was used as above. The densities of the exposed films were quantified. The controls, which the data was to be compared with, were always run on the same gel. The phospho- specific signals were correlated to the total ERK.
35 Arachidonic and oleic acid release Cells were plated on Primaria (BD bioscience, Erembodegem, Belgium) 24- well plates (20 000 cells / well) and left to grow for 24 h. 0.1 Ci [3H]-AA (or [3H]-oleic acid), was added in each well and the cells cultured for another 20 h. The incubation medium was removed and the cells were washed twice with TBM supplemented with 2.4 mg/ml bovine serum albumin (BSA), and finally left in TBM without BSA at 37 °C. The cells were then stimulated for 7 min, after which 200 l of the total volume of 250 l in each well was transferred to an Eppendorf tube on ice. These samples were spun down for 1.5 min at 4 °C and 100 l of the medium was transferred to a scintillation tube. The cells on the 24-well plates were dissolved in 0.1 M NaOH and transferred to separate scintillation vials. Radioactivity of both types of samples was analysed by liquid scintillation counting. In some cases, orexin stimulation was preceded by some inhibitor preincubation. In such cases, the inhibitor used was added to the cells after one wash with TBM (containing BSA) and the cells preincubated for 30 min in the absence of BSA. The cells were washed once again with TBM containing BSA, changed to fresh TBM without BSA but still containing the inhibitor and immediately stimulated with orexin-A for 7 min. Controls (vehicle only) were treated in the same manner. In case of inhibitors not requiring such long preincubation period, the inhibitors were added in TBM without BSA after the two washes with TBM supplemented with BSA and the cells stimulated directly in this medium after a 5 min preincubation. Controls (vehicle only) were treated in the same manner.
36 Results and Discussion
PLC regulation Ca2+ influx-dependence of PLC activation 2+ A previous study has suggested a role for Ca in OX1 orexin receptor signalling to PLC (Lund et al., 2000), and we set out to investigate the 2+ importance of Ca influx for the signalling of OX1 orexin receptors to PLC 2+ (Paper I). The results demonstrate that Ca influx is required for OX1 orexin receptor stimulation of PLC activity at low concentrations of orexin-A, whereas at higher concentrations, the response is less dependent on Ca2+ influx. We also confirmed that Ca2+ elevation alone is a weak stimulant of PLC activity in CHO cells. This infers that even though Ca2+ in itself is a weak stimulant of the activation of PLC, it can strongly enhance PLC activity induced by stimulation of the OX1 orexin receptor. Elevating the intracellular concentration of Ca2+ seems to be a general and important mechanism in orexin receptor signalling. In our recombinant system it appears as though this Ca2+ elevation is necessary for orexin receptor signalling (Ammoun et al., 2006), but the mechanistic explanation and physiological relevance in native systems is yet to be determined. To determine the role of the different influx pathways (ROCs and SOCs) in OX1 orexin receptor-stimulated PLC activity we first needed to evaluate different inhibitors for ROC and SOC influx (Paper I). Based on these results and our previous data on 2-APB (Kukkonen and Akerman, 2001), we classify the inhibitors as follows: TEA, a strong ROC inhibitor; MgCl2, a strong (though weaker than TEA) ROC inhibitor and also a weak SOC inhibitor; dextromethorphan, SKF-96365 and 2-APB, strong SOC inhibitors. Using these blockers we determined that both SOCs and ROCs are important for the PLC activation seen upon OX1 orexin receptor stimulation, but the ROC influx pathway is of greater importance at low receptor activation level while the SOC influx further amplifies the PLC activity (Paper I). To see if this was true for another GPCR, we examined whether muscarinic M1 receptor signalling to PLC was affected by SOCs. The results demonstrate that SOCs weakly amplify muscarinic receptor signalling to PLC throughout the carbachol concentration range (Paper I). This is the expected behaviour based on previous studies (Kim et al., 1999; Willars and Nahorski, 1995) and we consider the behaviour of OX1 orexin receptors as
37 unusual. Other GPCRs have been shown to give rise to different PLC activities in the same cell type (Young et al., 2003) and one explanation for this unusual signalling could be that the OX1 receptor engages different PLC isoforms at different activation levels. Another explanation is that the much higher level of PLC activity obtained with OX1 orexin receptors as compared to muscarinic receptors leads to a bottleneck at some stage of the signal cascade and that this bottleneck in turn leads to an apparent lack of Ca2+ effect at high orexin-A levels despite the actual amplification.
PIP2 is not the only substrate for PLC Our investigation (Paper II) revealed that the total inositol phosphate production occurred at 15-fold higher potency than the IP3 production. At 1 nM orexin-A, essentially only IP1 is generated whereas at 300 nM both IP1, IP2 and IP3 are generated implicating that primarily PI or PIP instead of PIP2 is used as substrate by PLC at low concentrations of orexin-A. This result again offers the possibility that different enzymes are activated at different levels of OX1 orexin receptor activation (see above). The isoforms of PLC that are activated at different levels of OX1 orexin receptor activation are yet to be resolved. Our results indicate that OX1 orexin receptors activate several different PLC isoforms. Since our earlier studies suggest that OX1 orexin receptor signalling gives rise to src and phosphoinositol-3-kinase activation, PLC could be a possible candidate (Ammoun et al., 2006). PLCβ (via Gαq/11 or Gβγ subunit form Gi/o) is classically thought to couple to GPCRs and is also a possible candidate in the signalling.
PLD-dependent DAG generation Equal amounts of DAG and inositol phosphates should be generated upon PLC activation if all of the produced DAG is the result of PIP2 breakdown. Our study (Paper II) demonstrate that this is not true after OX1 orexin receptor stimulation. DAG production follows the total inositol phosphate release at high concentrations of orexin-A while at low concentrations, DAG production is clearly more efficient than inositol phosphate release. This led us to believe that some other enzyme than PLC is activated upon orexin receptor stimulation. The most likely candidates are PLD and PC-specific PLC/sphigomyelin synthase. No effect was seen on DAG generation when using PC-specific PLC/sphigomyelin synthase inhibitor but inhibition of PLD fully reversed the high potency component of DAG generation after which the DAG generation followed the release of total inositol phosphates. This makes PLD a probable candidate in the high potency DAG generation seen upon OX1 orexin receptor stimulation. Since we inhibited both PLD1 and PLD2 at the same time, we cannot pinpoint the isoform responsible. The low concentration
38 of orexin needed to stimulate PLD activation makes involvement of the cAMP and ERK pathways unlikely, since these pathways are not activated at this low receptor activation. Gi/o proteins are activated with high potency by OX1 orexin receptors and the high potency DAG generation seen upon OX1 orexin receptor stimulation overlaps with the PKC activation (Holmqvist et al., 2005). This could suggest a connection between these pathways. The low level of receptor activation needed to stimulate PLD infers that this pathway could be of great importance for orexin receptor signalling. DAG has been demonstrated to have more targets than PKC such as TRP channels (Jung et al., 2002; Tesfai et al., 2001), and both TRP channels and DAG have been associated with orexin signalling (Larsson et al., 2005; Nasman et al., 2006). These facts give the possibility that the high potency DAG component in OX1 orexin receptor signalling is involved in the signalling to Ca2+ influx that governs the coupling to other signalling pathways. If there in fact is PLD activation upon OX1 orexin receptor activation, PA could possibly also be involved in the signalling from OX1 orexin receptors.
Blocking IP3-dependent signalling 2+ We devised a method to abolish the IP3-dependent Ca release from the ER (and thus also the SOC-influx) by overexpression of the IP3-metabolising enzymes IP3-3KA and IP3-5PI (Paper III). Our study demonstrates that the IP3-signals are strongly reduced when the different IP3-metabolising enzymes are expressed alone or together. IP3-5P1 is slightly more effective in metabolising IP3 than IP3-3KA and it may also have the advantage of not producing the possibly active metabolite IP4. We also examined how the reduced IP3 levels affected the signalling from two classical Gq/PLC-coupled receptors, endogenous P2Y purinoceptors and heterologously expressed M1 muscarinic receptors. A clear reduction 2+ (approximately 70%) in Ca response is seen in IP3-5P1- and IP3-3KA- expressing cells, demonstrating that signalling from classical Gq-coupled 2+ receptors to Ca release is effectively attenuated by a reduction in IP3 levels. 2+ The next step was to examine how Ca signalling from OX1 orexin receptors is affected by the overexpression of the IP3-metabolising enzymes. 2+ The IP3-dependent Ca elevation seen at high doses of orexin is highly sensitive to the IP3-metabolising enzymes. In the presence of extracellular Ca2+, however, the number of cells responding with Ca2+ elevation is only slightly reduced by overexpression of the IP3-metabolising enzymes, demonstrating that the receptor-operated Ca2+ influx response is independent of IP3 production. When repeating the experiments in a different cell-line (neuro-2a-hOX1) we got the same results confirming the general ability of IP3-3KA and IP3- 5P1 to “knock-down” OX1 orexin receptor signalling via IP3.
39 Non-selective cation channels may constitute novel important effectors for G protein-coupled receptors (Barritt, 1999; Clapham, 2003; Gudermann et al., 2004), mediating both electrical activity and Ca2+ signalling. In most cases the molecular identity of the channel as well as the signal pathway between the receptor and the channels are unknown. Orexin receptors make a good model system for investigations of this signalling, except for their 2+ ability to connect to PLC-dependent Ca signalling as well. The IP3- metabolising enzymes used eliminate the latter response without affecting the receptor-operated Ca2+ influx and thus allow mechanistic studies on the isolated receptor-operated pathway.
IP3-independent ERK signalling We have earlier demonstrated that Ca2+ influx is important for the ERK activation seen upon OX1 orexin receptor stimulation and that blocking of both ROC and SOC influx together attenuates ERK phosphorylation while blocking of SOC alone does not (Ammoun et al., 2006). Using the method stated above we demonstrate that it is the ROC influx that is essential in ERK signalling from OX1 orexin receptors (paper III), since there is no difference in the level of ERK phosphorylation between control cells and cells transfected with IP3-3KA, IP3-5P1 or both enzymes.
Arachidonic acid release
Our study demonstrates that stimulation of the OX1 orexin receptor with either orexin-A or orexin-B gives a robust arachidonic acid (AA) release (Paper IV). The preference for orexin-A over orexin-B was only 2-fold as compared to the expected 10- to 100-fold. The agonist concentration response curve observed is clearly biphasic and the AA release occurred in the same concentration range as the high-potency DAG production by PLD and the high potency PLC activation (Johansson et al., 2008). Since orexin receptors seem to activate both PLD and PLC, di- and monoacylglycerol lipases could be involved in the AA release as well as the more "traditional" PLA2 pathway (Leslie, 2004). By blocking iPLA2 and cPLA2 the AA release was almost fully attenuated, whereas inhibition of iPLA2 alone only inhibited a specific component of the response. This indicates that several PLA2 isoforms may be involved in the orexin-induced AA release but it should be noted that no PLA2 inhibitor is absolute selective towards PLA2 (Akiba and Sato, 2004; Burke and Dennis, 2009b), which leaves the door open for other lipid-metabolising enzymes as well. Further studies are needed before one could say for certain that PLA2 is responsible for the AA release seen upon OX1 orexin receptor stimulation.
40 Ca2+-dependence of the AA release Removal of extracellular Ca2+, or the driving force for Ca2+ influx (with + high-K medium), fully eliminated the OX1 orexin receptor-induced AA release (Paper IV). Inhibiting the ROC influx strongly, though not fully, inhibited the AA release at low orexin-A concentrations but to a lesser extent at high concentrations of orexin-A, where there is also SOC influx. Inhibition of the SOC influx only weakly inhibited AA release at both low and high concentrations of orexin-A indicating that ROC influx is of greater importance for the AA release mechanism, a result that correlates well with other signalling studies on orexin receptors (see, for example, papers I and III and Ammoun et al., 2006). 2+ Since Ca influx seemed important for AA release by OX1 orexin receptors, we next tested Ca2+-elevating agents' ability to activate AA release. The results demonstrate that Ca2+ elevation in itself strongly activates AA release in CHO cells. This indicates that OX1 orexin receptor- induced Ca2+ influx could also directly activate AA release via (likely) cPLA2.
ERK-dependence of the AA release
Phophorylation has been identified as one major mediator for cPLA2 and iPLA2 activation and PKC and ERK have often been implicated in this process. As they are also involved in the signalling from OX1 orexin receptors, we used inhibitors of PKC and inhibitors for components of the ERK pathway to investigate if PKC or ERK are involved in the AA release seen upon OX1 orexin receptor stimulation (paper IV). Inhibitors of both conventional and novel PKC did not render an effect on AA release and an activator of PKC did not stimulate release of AA, and therefore we conclude that PKC is not involved in this release. On the other hand, MEK1/2 (MAPK/ERK kinase1/2) inhibitors strongly inhibited AA release and this suggested to us that the ERK pathway is partly responsible for the AA release. ERK is strongly activated upon OX1 orexin receptor stimulation (Ammoun et al., 2006; Milasta et al., 2005) and constitutes a possible candidate mediator of Ca2+ influx-stimulated AA release. ERK is only modestly activated by Ca2+ influx alone (Ammoun et al., 2006) but the possibility still exists that the level of active ERK required for stimulation of AA release is much lower than the maximal.
41 AA-dependent ROC influx We next wanted to evaluate the interaction of Ca2+ influx and AA (paper IV). To investigate the different components of Ca2+ signalling we used the method described in paper III, i.e. we expressed the IP3-metabolising enzymes IP3-3KA and IP3-5P1. When SOC influx was blocked, blocking AA release significantly reduced the number of cells responding with Ca2+ elevation and also the average cell response. On the other hand, AA did not affect Ca2+ release from the ER. These results strongly indicate that AA or its metabolites are involved in the receptor-mediated Ca2+ influx but not in Ca2+ release. Since Ca2+ influx seems to be required for AA release and AA, or a metabolite of AA, is needed for ROC influx governed by the OX1 orexin receptor, this could constitute a positive feedback loop. The results of this study could mean that we have found one of the components of the OX1 orexin receptor-operated Ca2+ influx pathway. Studies made with HEK293 cells support this finding (Peltonen et al., 2009). But there is still the possibility that another product of the phospholipase reaction is more relevant for OX1 orexin receptor-dependent ROC influx, and this has to be further investigated.
Optical detection of DAG generation and PLC activity PLC activity can be measured using both optical and biochemical methods. We set out to determine if these methods give comparable results (Paper II). The concentration-response relationship between the live-cell imaging measurements and the measurements from the binding-protein assay for IP3 correlated rather well, which affirms that live-imaging and biochemical essays offer detection with comparable sensitivity and dynamic range. We also developed a new method that allows us to fix cells showing translocation of GFP-fused constructs (Paper V). In real-time imaging, strong receptor stimulus produces a strong fluorescence translocation response between the cytosol and the plasma membrane (Ammoun et al., 2006; Johansson et al., 2008). Fixation, even at the optimal conditions, somewhat reduces the contrast between the cytosol and the plasma membrane, but the membrane fluorescence is still clearly separated from the cytosol. We conclude that one has to perform a pilot study to decide the best fixation protocol to be used for the GFP-construct of interest. The results demonstrate that the fixation was stable, meaning that the cells showed the same degree of translocation after several days. Both real-time imaging and fixed cell counting are capable of producing concentration-response data; however, the relationships are not the same. While PKC -GFP data from cell counting correspond well with the real-time
42 imaging data, different ways of determining the IP3/PIP2 levels demonstrate some divergence from each other. This study demonstrates that cell fixation followed by cell counting offers some major advantages as compared to live-cell imaging and biochemical assays. It is an easy procedure that can be performed with a regular fluorescence microscope and it gives a rapid production of comparative concentration-response data. However, one should be aware that since the counting of cells is performed manually there is a subjective decision affecting the results and also the fixing of cells means loss of the single-cell- quantitative aspect since it only enables counting of translocated cells versus non-translocated cells. Therefore this method should be seen as a complement to live-cell imaging.
43 Conclusions
When starting my work on this thesis I could never have believed the complex role for, and the multitude of, phospholipases in OX1 orexin receptor signalling. The work presented in this thesis only represents the beginning of the discovery of the role for phospholipases in this signalling and more studies are on the way. It will be very interesting in the future to find out the physiological role for this promiscuous signalling of orexin receptors to diverse phospholipases. The following conclusions can be drawn from the work in this thesis:
2+ OX1 orexin receptor-mediated PLC activity is strongly amplified by Ca influx at low concentrations of orexin-A. Both SOCs and ROCs contribute to this amplification but ROCs appear to be more important. At least two different PLC isoforms and at least one PLD isoform are involved in this signalling, but the molecular identity of these isoforms is yet to be determined. Furthermore, the study implicates that DAG is an important messenger in orexin receptor signalling. Since orexin receptors seem to activate PLD, PA could also be a possible interactor in the signalling pathways.
The method of over-expressing the IP3-metabolising enzymes IP3-3KA and 2+ IP3-5P1 is sufficient for elimination of the release of Ca from the ER, leaving the receptor-operated Ca2+ channels in the membrane as the only source for orexin receptor-induced Ca2+ elevation. Using this method we demonstrated that ERK activation by the OX1 orexin receptor is fully supported by the ROC influx. Also, with the use of this method we were able to demonstrate that AA release seems to enhance OX1 orexin receptor- dependent Ca2+ influx. This method enables further studies on the signalling 2+ pathways leading from OX1 orexin receptors to the receptor-operated Ca influx.
Stimulation of the OX1 orexin receptor results in a strong release of AA. Both cPLA2 and iPLA2 may be involved in this release, with a reservation for the specificity of pharmacological inhibitors. Furthermore, ERK and ROCs are involved in this signal cascade.
44 Orexin-A and orexin-B should not be used in native tissues to determine involvement of OX1 or OX2 orexin receptors since our study demonstrates that the OX1 orexin receptor can mediate signals from both orexin-A and orexin-B with almost equal potency.
Counting of fixed cells showing translocation of GFP-fused signalling molecules can produce similar concentration response curves as real-time imaging and biochemical methods. This method complements traditional live-cell imaging and is an easy procedure that you do not need specific equipment for, besides a rather ordinary fluorescence microscope. The method is suitable for all GFP-based probes as long as the fixation conditions are optimised.
45 Acknowledgements
First and foremost I would like to thank my supervisor professor Jyrki Kukkonen. Without you there would never have been a thesis with my name on the front cover. Thank you for giving me the opportunity to work in your group and for your endless help. Your knowledge in the vast majority of neuroscience never ceases to amaze me. We also had a lot of fun times outside the lab and I hope that we will have a lot more in the future. I would also like to thank my co-supervisor professor Karl Åkerman for sharing your knowledge in orexin signalling with me and for all interesting discussions over the years.
During my work in the Kukkonen group I have had some great colleagues. Maybe because of it I have sometimes got a bit less done than I was supposed to, but I enjoyed every second of repose this gave me. Karin Nygren, you have given me so much help during the work that lead to this thesis. I don’t know how to let you know exactly how much you have meant for me during this time. You are always eager to help wherever you can. And maybe most important you have always listened when I needed to discuss something or just have a break from science. You have the most caring hart I have ever met, just make sure to think of your self once in a while. Lisa , we had a lot of fun times together. I think we both learned that we should not stand next to each other conducting the same experiment as this would give you an ulcer. I wish I had your well thought-out modus operandi and your ability to take on experiments with a strategic order. I loved being your office-buddy and I’m sorry for disturbing you with my endless chatter. When I first arrived in the Kukkonen group I found a troubled guy that was always thinking too much and to deeply about every part of his life. But despite this he was never far away from a laugh and had a very caring heart. Tomas you still overanalyse everything and I still love your company. There was also an extremely scattered lady that always seemed to be running to something and I think it took her a year to figure out that I was a new PhD-student in the group. But as time went by I learned that Sylwia has a very big heart that she tries to squeeze everybody she meets into. I hope that you now have slowed your pace down so that you can enjoy the moment from time to time.
46 I would also like to thank my corridor buddies. Christina Berqvist, not only do you know everything, you are also willing to share this knowledge with others. Without you I would have mist a lot of fika and forgot about lunch dozens of times. I really enjoyed our morning chats and will miss them a great deal. Helena Fällmar, it’s hard to find a more genuine caring soul. You’re a fun person to hang around and a laugh is never far away. I have missed you these last couple of months when you have been on maternity leave. If I thought Tomas was over thinking it, it was just because I hadn’t met Daniel Ocampo Daza yet. I always enjoyed our discussions even though we rarely have the same opinion, but that is what makes an interesting discussion. Görel Sundström, you say you don’t like people, but people like you! You always have time, or pretend to have time, for a chat whenever I need a break, and also, it’s nice to have another person who actually watches something besides science on TV to talk to. I thank both you and Daniel for trying to answer all the strange questions I asked you during the writing of this thesis, questions like “what are the difference between old world snakes and new world snakes” was never frown upon but lead to a intense googling for the answer. Whenever I had a computer problem I always turned to Tomas Larsson for help, and you always helped me without complaining. I really miss our discussions about “hembygden”. Helen Åkerberg I really appreciated all the discussions about the future. It was nice to have somebody who shares your values to talk to. Ingrid Lundell, you always helped me when I came to you for help. No problem was too small for you to engage yourself in. Bo Xu I enjoyed our discussions during fika and lunch, especially the discussions concerning cultural differences between China and Sweden; it’s always nice to learn about other cultures. Jenny Widmark, you always seam to have a smile left over to brighten every ones day, thank you for contributing to the nice atmosphere during fikas and lunch. Birgitta Hägerbaum thank you for help with diverse things regarding the daily activity of the lab. Last but not least I would like to thank the mastermind of the group Dan Larhammar. Thank you for all your help with questions regarding the writing of the thesis. And mostly thank you for letting me be an “adoptive-member” of your group!
My time as a member of the unit of physiology is now over and I thank all present and past members. I would especially like to thank Gunnar Flemström, Göran Sperber and Olle Nylander for help related to my teaching of students. Markus Sjöblom, John Sedin, Josefin Dahlbom, Hanna Olsén, Reneé Goldkuhl and Klas Abelsson, thank you for interesting discussions during various fikas and parties and always contributing to the nice atmosphere at “fysiologen”. I would also like to thank Gunnila Jedstedt for help with various things during my PhD studies. Former members of physiology Liselotte Pihl and Magnus Bengtsson, we had a lot of fun and laughs and I miss you.
47 Without the administration none of us would get anything done so a huge thanks to Ulla Johansson, Emma Andersson, Lena Karlsson, Gunno Nilsson, Birgitta Klang, Marita Berg and a special thanks to Maria Larsson for all fun discussions, I hope we will se more of each other in Storvreta.
I would also like to thank all you other people at the department of Neuroscience, present and former, for making my time here such a joyful period of my life. Especial thanks to Madelen Lek, Lotta Israelsson, Mikeal Corell, Nicole Neumann, Henrik Gezelius, Nadine Rabe and Hanna Wootz for all our fun discussions.
All though the people you work with are very important for your work, it is almost more important to have good friends and family around you. The following people were not involved in my work but my work depended on them being in my life. Thank you Anja and Pontus for always being there when me and Niklas needed help with anything and thank you for bringing the little bundle of joy Julia into our lifes. Sara I met you as soon as I walked through the doors at Ångström and I have enjoyed your company ever since. Thank you for dropping everything in a seconds notice and going with me to Katrineholm when my grandmother was ill, I will never forget that, and neither will my mother. Were there is Sara there is Mats. Thank you for all heated discussions during these years, I know we drive everyone else crazy but I have really enjoyed them. Eleni, we had a lot of fun at Ångstrom, and we have had a lot of fun since then, and Theo thank you for taking over the data support . I and Niklas hope to be invited to a lot more greek dinners. Anna thank you for all those nights at nationerna, “du kan kalla mig shoby doo”, and also for all interesting discussions of more serious character. Karin I miss you a lot and hope that you and Tomas won’t stay in Oslo forever. Thank you for all wine nights and for introducing me to the abuse of black pepper. Cissi, we have had a lot of fun and I miss having you here in Uppsala. Maybe you could move in with me and Niklas again, we have a basement just for you….Thank you for always being there for me. Mårten och Veronica I can’t start to describe what you mean to me, especially during this last year. Veronica, I wouldn’t have survived this last year without you. I miss our “mammadagar”. It was a lot of work but in hindsight we had al lot of fun. Thank you for always being there for us and Lucas. And a special thanks to Lova for entertaining Lucas when his parents are too tired. Those two will rule the world in a couple of years.
Åsa thank you for everything, for lending me money in the end of the month when I was a broke student and you were working, for coming to me with dinner when Per past away, for listening to my complaints over the telephone for hours at a time, and lots more. I have known you for over 20
48 years and I know we will be friends for more than 20 years more. You know I love you to bits. I only hope that one day I will be able to repay you for all that you have done for me. Magnus thank you for taking such good care of my friend and also for brightening every party with your monologues, och det är jag som ska ha mundiaré, humpf……
Last but not least I would like to thank my family. Fredrik you are the most stubborn person I have ever met and the best brother anyone could ask for. Thank you for always challenging me and always making me feel as do I can do everything I set my mind to. Anna-Sara, thank you for giving my brother a softer edge. Kerstin and Einar thank you for taking me into your family and all the love and support you have showed me over the last ten years.
Mor and Far you have always believed more in me than I ever did. I always know that I have your support and that you will be there when I need you, this means everything to me and I love you both more than I can ever express.
Niklas thank you for sticking by me all these years. Thank you for giving me a home to return to at the end of the day. Thank you for loving me even when I am having a bad day, and lets face it, when its bad its bad . And most of all thank you for giving me Lucas. I love you. Lucas thank you for showing me the world all over again, by struggling with just sitting up or looking at a spoon for hours, just fascinated over its shape and structure, you teach me to appreciate everything that I take for granted and to live a little more in the moment.
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63 / - / , * 0 1 2 3 ) 4 2 ,
, , 2 5 3 ) 4 2 , 6 / / - 46 ) ) 4 )55 4 2 )5+ 2 7 2 8 2 5 , 9 5 : 8 , + 6 8 )55 4 , +) , 4 ); 1 ; < 5 - )55 2 / 1 2 5 3 ) 4 2 , 7 = > ) 46 $ 6 8 )+ , ) , ?< 5 - )55 2 / 1 2 5 3 ) 4 2 , @7A
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