Orexin/hypocretin receptor signalling cascades

Abstract

Orexin (hypocretin) peptides and their two known G-protein-coupled receptors play essential roles in sleep-wake control and powerfully influence other systems regulating appetite/metabolism, stress and reward. Consequently, drugs that influence signalling by these receptors may provide novel therapeutic opportunities for treating sleep disorders, obesity and addiction. It is therefore critical to understand how these receptors operate, the nature of the signalling cascades they engage and their physiological targets. In this review, we evaluate what is currently known about orexin receptor signalling cascades, while a sister review (Leonard & Kukkonen, this issue) focuses on tissue-specific responses. The evidence suggests that orexin receptor signalling is multifaceted and is substantially more diverse than originally thought. Indeed, orexin receptors are able to couple to members of at least three G-protein families and possibly other proteins, through which they regulate non-selective cation channels, phospholipases, adenylyl cyclase, and protein and lipid kinases. In the central nervous system, orexin receptors produce neuroexcitation by postsynaptic depolarization via activation of non-selective cation channels, inhibition of K⁺ channels and activation of Na⁺/Ca²⁺ exchange, but they also can stimulate the release of neurotransmitters by presynaptic actions and modulate synaptic plasticity. Ca²⁺ signalling is also prominently influenced by these receptors, both via the classical phospholipase C-Ca²⁺ release pathway and via Ca²⁺ influx, mediated by several pathways. Upon longer-lasting stimulation, plastic effects are observed in some cell types, while others, especially cancer cells, are stimulated to die. Thus, orexin receptor signals appear highly tunable, depending on the milieu in which they are operating.

Keywords: G-protein-coupled receptor; K+ channel; Na+/K+ exchanger; adenylyl cyclase; cell death; endocannabinoid; intracellular Ca2+; non-selective cation channel; phospholipase; plasticity.

Figure 1

Comparison of the C-terminals of human and mouse OX₁ receptors with respect to the suggested protein–protein interactions. The sequences are aligned to ease the comparison. The wild-type sequences are presented first for each species, and the mutant sequences (truncated and point-mutated) underneath. The human receptor data are obtained from investigations of the interaction of the receptor with β-arrestin-2 by assessment of the co-localization in HEK-293 cells upon endocytosis (Milasta et al., 2005). The mouse receptor data depict the assessment of the interaction of the receptor C-terminus with Dynlt1 with the Y2H method (Duguay et al., 2011). The strength of the interaction is indicated by the symbols after each sequence.

Figure 2

Ca²⁺ influx in recombinant human OX₁ receptor-expressing cells. (A) Ca²⁺ influx is required for orexin responses in CHO-hOX₁ cells. (A₁) 0.3 nM orexin-A produces Ca²⁺ elevation (top trace) and a simultaneous inward current (lower trace). Removal of extracellular Ca²⁺ (‘−Ca²⁺’) attenuates both responses. The current is also effectively attenuated by substituting tetraethylammonium chloride for NaCl (TEA, 70 mM [70 mM NaCl replaced]; A₂). This research (A₁₋₂) was originally published in the Journal of Biological Chemistry. Larsson KP, Peltonen HM, Bart G, Louhivuori LM, Penttonen A, Antikainen M, Kukkonen JP, Åkerman, KE (2005). Orexin-A-induced Ca²⁺ entry: evidence for involvement of TRPC channels and protein kinase C regulation. J Biol Chem. 2005; 280: 1771–1781. © the American Society for Biochemistry and Molecular Biology. (A₃) Removal of the driving force for Ca²⁺ entry by strong depolarization (see the voltage trace; top) abolishes the Ca²⁺ response (bottom) to 10 nM orexin-A (presence indicated by vertical bars under the Ca²⁺ trace). This research was originally published in the Journal of Biological Chemistry. Lund PE, Shariatmadari R, Uustare A, Detheux M, Parmentier M, Kukkonen JP, Åkerman, KEO. The orexin OX₁ receptor activates a novel Ca²⁺ influx pathway necessary for coupling to phospholipase C. J Biol Chem. 2005; 275: 30806–30812. © the American Society for Biochemistry and Molecular Biology. (B) IMR-32 neuroblastoma cells transduced with hOX₁ baculovirus show Ca²⁺ responses regulated by extracellular Na⁺ likely via NCX. (B₁) Removal of extracellular Na⁺ (all NaCl replaced with N-methyl-d-glucamine [NMDG]) inhibits orexin-A (1 nM) responses in some cells (upper trace), while in other cells the response is stimulated (lower trace) or not affected (middle trace). 10 mM MgCl₂ blocks the response in all three types of cells. (B₂) The NCX blockers KB-R7943 (10 μM; lower trace) and SN-6 (1 μM; not shown) block most of the Ca²⁺ elevation in the cells showing Na⁺-dependent elevation whereas the cells with Na⁺-independent Ca²⁺ elevation are not affected (upper trace). The Na⁺-dependence of the responses is not shown in the figure. Both KB-R7943 and SN-6 are more potent inhibitors of the reverse mode of NCX. The effect of 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) is also shown in B₂. (B₃) Expression of the dominant-negative TRPC6 subunit abolishes the Na⁺-dependent component of the response. Average traces are shown in B₃. Reprinted from Cell Calcium, 48(2−3), Louhivuori LM, Jansson L, Nordström T, Bart G, Näsman J, Åkerman KEO, Selective interference with TRPC3/6 channels disrupts OX₁ receptor signalling via NCX and reveals a distinct calcium influx pathway, pp. 114–123, © 2010, with permission from Elsevier. Figures adapted from Lund et al. (2000), Larsson et al. (2005), and Louhivuori et al. (2010) as indicated. The figures are reproduced with permission.

Figure 3

Ca²⁺ influx in native orexin receptor-expressing neurons. (A) Cultured rat hypothalamic neurons loaded with fura-2 AM. (A₁) Response to orexin-B (‘HCRT’) is concentration-dependent. Ca²⁺ influx rather than release from intracellular stores is likely required for the response since it is blocked by removal of extracellular Ca²⁺ (‘0 Ca²⁺/EGTA’; (A₂) but not by 2 μM thapsigargin (A₃). TTX, tetrodotoxin (1 μM). The response was also blocked by 100 μM Cd²⁺ and 1 μM bisindolylmaleimide I, a PKC inhibitor (not shown). (B) Mouse dorsal raphé neurons recorded in acute brain slices. (B₁) Upper right, a single neuron loaded with the Ca²⁺ indicator bis-fura-2 via the patch pipette. Upper left, fluorescence (dF/F) traces (indicating changes in intracellular [Ca²⁺]) from the same cell. Bottom trace is membrane potential. Middle and uppermost traces are simultaneous Ca²⁺-dependent fluorescence (average dF/F) from the soma (black box on the cell image) and from a proximal dendrite (white box on the cell image) respectively. The somatic Ca²⁺ trace and the current clamp recording are magnified in the right-hand bottom corner, demonstrating that the depolarization and somatic Ca²⁺ elevation occur before the action potential firing is triggered. The calibration bars indicate 10% dF/F and 10 mV for fluorescence and voltage trace, respectively, and 20 s. Orx, 300 nM orexin-A. (B₂) Ca²⁺-dependent fluorescence (dF/F) from neurons loaded with fura-2 AM. Orexin-A (300 nM; ‘Orx’) produces a Ca²⁺ influx that is attenuated by the L-type VGCC blocker, nifedipine (left), and the PKC inhibitor bisindolylmaleimide I (1 μM; right). (B₃) Ca²⁺ -dependent bis-fura-2 fluorescence (dF/F) recorded as in B₁ under voltage-clamp conditions. Orexin-A (300 nM; ‘Orx’) reversibly enhances the somatic Ca²⁺ transient (left column, top trace) produced by a voltage jump from −60 to −30 mV (bottom trace) without changing the total membrane current (left column, middle trace). Nifedipine (‘Nif’, 1 μM; middle column) and bisindolylmaleimide (‘bis’, 1 μM; right column) fully attenuate the orexin-enhancement of the Ca²⁺ transient. These data indicate that orexin-A stimulates Ca²⁺ influx both by depolarising these neurons followed by opening of VGCCs and by a PKC-dependent enhancement of Ca²⁺ influx via L-type VGCCs. A₁₋₃ adapted from van den Pol et al. (1998); B₁ and B₃ adapted from Kohlmeier et al. (2008); B₂ adapted from Kohlmeier et al. (2004). The figures are reproduced with permission.

Figure 4

Schematic representation of possible orexinergic mechanisms in synaptic signalling. (A) Postsynaptic orexin signalling mechanisms. Ion fluxes are not shown. The PLC pathway may be active as suggested by some PKC-dependent effects as well as release of the endocannabinoid 2-AG, although there is little direct evidence for IP₃-mediated Ca²⁺ release from ER in neurons. Inhibition of leak/inward rectifier/voltage-gated K⁺ channels (2P/Kir/Kv) (‘KC’) at least sometimes utilizes the G_q−PLCβ−PKC pathway. PKC could also be involved in the activation of NCX, but NCX may also act passively, driven by Ca²⁺ elevation (influx or release from ER; forward mode) or Na⁺ elevation (reverse mode). There also is some evidence for PKC-dependent activation of postsynaptic L-type VGCCs. The putative role of PIP₂ is not depicted in the figure: PIP₂ is required by NCX, some K⁺ and other channels, and PLD and cPLAα, and thus hydrolysis of PIP₂ (by PLC) would lead to inhibition of these while elevated PIP₂ (via, e.g. the PLD pathway) would stimulate these. Different NSCCs may be positively or negatively regulated by PIP₂. (B) Presynaptic orexinergic modulation of glutamate- or GABAergic terminal signalling by orexinergic enhancement of VGCC-mediated Ca²⁺ influx. Indirect evidence suggests that orexin stimulation may be able to trigger transmitter release by depolarization or other means (depicted as NSCC, although not directly identified in this response).

Figure 5

The OX₁ receptor activation-mediated cell death pathways mapped in CHO cells. (A) the SHP-2-dependent cascade (Voisin et al., ; El Firar et al., 2009); (B) the p38-mediated cascade (see Ammoun et al., 2006b). Please note that the site of action of caspases in the cascade has not been determined. (C) The altered cell death response upon caspase inhibition (Ammoun et al., 2006b).

Figure 6

Suggested interaction schemes in ERK signalling upon OX₁ orexin receptor (blue) and CB₁ cannabinoid receptor (orange) co-expression in the same cells. (A) Dimerization of OX₁ and CB₁ receptors enhances the signalling/ligand potency (Hilairet et al., ; Ellis et al., 2006). (B) Enhanced signalling is obtained by OX₁ receptor-mediated production of the CB₁ ligand 2-AG and subsequent co-signalling of these two receptors (Turunen et al., ; Jäntti et al., 2013). See Orexins and endocannabinoids for details.