Cellular Signaling Mechanisms of Hypocretin/Orexin

Publisher Copyright: © 2021 The Author(s).Orexin receptors (OXRs) are promiscuous G-protein-coupled receptors that signal via several G-proteins and, putatively, via other proteins. On which basis the signal pathways are selected and orchestrated is largely unknown. We also have an insufficient understanding of the kind of signaling that is important for specific types of cellular responses. OXRs are able to form complexes with several other G-protein-coupled receptors in vitro, and one possibility is that the complexing partners regulate the use of certain signal transducers. In the central nervous system neurons, the main acute downstream responses of OXR activation are the inhibition of K+ channels and the activation of the Na+/Ca2+ exchanger and non-selective cation channels of unknown identity. The exact nature of the intracellular signal chain between the OXRs and these downstream targets is yet to be elucidated, but the Gq-phospholipase C (PLC) protein kinase C pathway - which is a significant signaling pathway for OXRs in recombinant cells - may be one of the players in neurons. The Gq-PLC pathway may also, under certain circumstances, take the route to diacylglycerol lipase, which leads to the production of the potent endocannabinoid (eCB), 2-arachidonoyl glycerol, and thereby connects orexins with eCB signaling. In addition, OXRs have been studied in the context of neurodegeneration and cancer cell death. Overall, OXR signaling is complex, and it can change depending on the cell type and environment.Peer reviewe

Endocannabinoid Signaling in Embryonic Neuronal Motility and Cell-Cell Contact - Role of mGluR5 and TRPC3 Channels

Cell-cell communication plays a central role in the guidance of migrating neuronal precursor cells during the development of the cerebral cortex. Endocannabinoids (eCBs) have previously been shown to be one of the central factors regulating neuronal migration. In this study the effects of eCBs on different parameters, expected to affect embryonic cortical neuronal motility have been analyzed in neurosphere-derived neuroblasts using time-lapse microscopy. Increased endogenous production of the endocannabinoid 2-arachidonyl glycerol (2-AG) causes bursts of neuroblast motility. The neuroblasts move longer distances and show a low frequency of turning, and the number of neuron-neuron contacts are reduced. Similar changes occur interfering with the function of the metabotropic glutamate receptor 5 (mGluR5) or its transducer canonical transient receptor potential channel 3 (TRPC3) or the neuregulin receptor ErbB4. Blocking of 2-AG production reverses these effects. The data suggest that eCB-regulated neuronal motility is controlled by mGluR5/TRPC3 activity possibly via NRG/ErbB4 signaling. (C) 2018 IBRO. Published by Elsevier Ltd. All rights reserved.Peer reviewe

Regulation of radial glial process growth by glutamate via mGluR5/TRPC3 and neuregulin/ErbB4

Radial glial cells play an essential role through their function as guides for neuronal migration during development. Disruption of metabotropic glutamate receptor 5 (mGluR5) function retards the growth of radial glial processes in vitro. Neuregulins (NRG) are activated by proteolytic cleavage and regulate (radial) glial maintenance via ErbB3/ErbB4 receptors. We show here that blocking ErbB4 disrupts radial process extension. Soluble NRG acting on ErbB4 receptors is able to promote radial process extension in particular where process elongation has been impeded by blockade of mGluR5, the nonselective cation channel canonical transient receptor potential 3 (TRPC3), or matrix metalloproteases (MMP). NRG does not restore retarded process growth caused by ErbB4 blockade. Stimulation of muscarinic receptors restores process elongation due to mGluR5 blockade but not that caused by TRPC3, MMP or ErbB4 blockade suggesting that muscarinic receptors can replace mGluR5 with respect to radial process extension. Additionally, NRG/ErbB4 causes Ca2+ mobilization in a population of cells through cooperation with ErbB1 receptors. Our results indicate that mGluR5 promotes radial process growth via NRG activation by a mechanism involving TRPC3 channels and MMPs. Thus neurotransmitters acting on G-protein coupled receptors could play a central role in the maintenance of the radial glial scaffold through activation of NRG/ErbB4 signaling.Peer reviewe

Orexin/hypocretin receptor chimaeras reveal structural features important for orexin peptide distinction

AbstractWe wanted to analyze the basis for the distinction between OX1 and OX2 orexin receptors by the known agonists, orexin-A, orexin-B and Ala11, d-Leu15-orexin-B, of which the latter two show some selectivity for OX2. For this, chimaeric OX1/OX2 and OX2/OX1 orexin receptors were generated. The receptors were transiently expressed in HEK-293 cells, and potencies of the agonists to elicit cytosolic Ca2+ elevation were measured. The results show that the N-terminal regions of the receptor are most important, and the exchange of the area from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptor’s ligand profile

Stapled truncated orexin peptides as orexin receptor agonists

The peptides orexin-A and -B, the endogenous agonists of the orexin receptors, have similar 19-amino-acid C-termini which retain full maximum response as truncated peptides with only marginally reduced potency, while further N-terminal truncations successively reduce the activity. The peptides have been suggested to bind in an α‐helical conformation, and truncation beyond a certain critical length is likely to disrupt the overall helical structure. In this study, we set out to stabilize the α‐helical conformation of orexin‐A15–33 via peptide stapling at four different sites. At a suggested hinge region, we varied the length of the cross-linker as well as replaced the staple with two α-aminoisobutyric acid residues. Modifications close to the peptide C‐terminus, which is crucial for activity, were not allowed. However, central and N‐terminal modifications yielded bioactive peptides, albeit with decreased potencies. This provides evidence that the orexin receptors can accommodate and be activated by α-helical peptides. The decrease in potency is likely linked to a stabilization of suboptimal peptide conformation or blocking of peptide backbone–receptor interactions at the hinge region by the helical stabilization or the modified amino acids.Peer reviewe

Autocrine Endocannabinoid Signaling through CB 1 Receptors Potentiates OX 1 Orexin Receptor Signaling s

ABSTRACT It has been proposed that OX 1 orexin receptors and CB 1 cannabinoid receptors can form heteromeric complexes, which affect the trafficking of OX 1 receptors and potentiate OX 1 receptor signaling to extracellular signal-regulated kinase (ERK). We have recently shown that OX 1 receptor activity releases high levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), suggesting an alternative route for OX 1 -CB 1 receptor interaction in signaling, for instance, in retrograde synaptic transmission. In the current study, we set out to investigate this possibility utilizing recombinant Chinese hamster ovary K1 cells. 2-AG released from OX 1 receptor-expressing cells acted as a potent paracrine messenger stimulating ERK activity in neighboring CB 1 receptor-expressing cells. When OX 1 and CB 1 receptors were expressed in the same cells, OX 1 stimulation-induced ERK phosphorylation and activity were strongly potentiated. The potentiation but not the OX 1 response as such was fully abolished by specific inhibition of CB 1 receptors or the enzyme responsible for 2-AG generation, diacylglycerol lipase (DAGL). Although the results do not exclude the previously proposed OX 1 -CB 1 heteromerization, they nevertheless unequivocally identify DAGL-dependent 2-AG generation as the pivotal determinant of the OX 1 -CB 1 synergism and thus suggest a functional rather than a molecular interaction of OX 1 and CB 1 receptors

Pharmacological characterization of the orexin/hypocretin receptor agonist Nag 26

One promising series of small-molecule orexin receptor agonists has been described, but the molecular pharmacological properties, i.e. ability and potency to activate the different orexin receptor-regulated signal pathways have not been reported for any of these ligands. We have thus here assessed these properties for the most potent ligand of the series, 4'-methoxy-N,N-dimethyl-3'4N-(3-{[2-(3-methylbenzamido)ethyl]amino}phenyl sulfamoy1]-(1,1'-biphenyl)-3-carboxamide (Nag 26). Chinese hamster ovary-K1 cells expressing human orexin receptor subtypes OX1 and OX2 were used. Ca2+ elevation and cell viability and death were assessed by fluorescent methods, the extracellular signal-regulated kinase pathway by a luminescent Elk-1 reporter assay, and phospholipase C and adenylyl cyclase activities by radioactive methods. The data suggest that for the G(q)-dependent responses, Ca2+, phospholipase C and Elk-1, Nag 26 is a full agonist for both receptors, though of much lower potency. However, saturation was not always reached for OX1, partially due to Nag 26s low solubility and partially because the response decreased at high concentrations. The latter occurs in the same range as some reduction of cell viability, which is independent of orexin receptors. Based on the EC50, Nag 26 was OX2 selective by 20-200 fold in different assays, with some indication of biased agonism (as compared to orexin-A). Nag 26 is a potent orexin receptor agonist with a largely similar pharmacological profile as orexin-A. However, its weaker potency (low-mid micromolar) and low water solubility as well as the non-specific effect in the mid-micromolar range may limit its usefulness under physiological conditions.Peer reviewe

Autocrine endocannabinoid signaling through CB 1 receptors potentiates OX 1 orexin receptor signaling MOL #80523 2 Running title: CB 1 receptor signaling potentiates OX 1 receptor signaling

Although the results do not exclude the previously proposed OX 1 -CB 1 heteromerization, they nevertheless unequivocally identify diacylglycerol lipase-dependent 2-AG generation as the pivotal determinant of the OX 1 -CB 1 synergism and thus suggest rather a functional than a molecular interaction of OX 1 and CB 1 receptors