Orexin-1 receptor-cannabinoid CB1 receptor heterodimerization results in both ligand-dependent and -independent coordinated alterations of receptor localization and function

Following inducible expression in HEK293 cells, the human orexin-1 receptor was targeted to the cell surface but became internalized following exposure to the peptide agonist orexin A. By contrast, constitutive expression of the human cannabinoid CB1 receptor resulted in a predominantly punctate, intracellular distribution pattern consistent with spontaneous, agonistindependent internalization. Expression of the orexin-1 receptor in the presence of the CB1 receptor resulted in both receptors displaying the spontaneous internalization phenotype. Single cell fluorescence resonance energy transfer imaging indicated the two receptors were present as heterodimers/oligomers in intracellular vesicles. Addition of the CB1 receptor antagonist SR-141716A to cells expressing only the CB1 receptor resulted in re-localization of the receptor to the cell surface. Although SR-141716A has no significant affinity for the orexin-1 receptor, in cells co-expressing the CB1 receptor, the orexin-1 receptor was also re-localized to the cell surface by treatment with SR-141716A. Treatment of cells co-expressing the orexin-1 and CB1 receptors with the orexin-1 receptor antagonist SB-674042 also resulted in re-localization of both receptors to the cell surface. Treatment with SR-141716A resulted in decreased potency of orexin A to activate the mitogen-activated protein kinases ERK1/2 only in cells co-expressing the two receptors. Treatment with SB-674042 also reduced the potency of a CB1 receptor agonist to phosphorylate ERK1/2 only when the two receptors were co-expressed. These studies introduce an entirely novel pharmacological paradigm, whereby ligands modulate the function of receptors for which they have no significant inherent affinity by acting as regulators of receptor heterodimers

Regulation of the orexin 1 receptor by beta-arrestins

The orexin 1 receptor was identified as an orphan G protein-coupled receptor (GPCR) in 1998 (Sakurai et ah, 1998). There is great interest in the orexin receptor system since it is involved in the control of feeding and energy metabolism (Sakurai et al, 1998), the modulation of neuroendocrine function (van den Pol et al, 1998; Smart, 1999) and the regulation of the sleep -wake cycle (Smart, 1999). However not much is known about the regulation of the orexin 1 receptor following stimulation. beta-arrestins bind agonist- activated, phosphorylated GPCRs and mediate their desensitisation and internalisation. They may also function as GPCR signal transducers. The aim of this thesis was to investigate internalization and signalling of the orexin 1 receptor and the involvement of beta- arrestins in these processes. In HEK293T cells expressing wild type orexin 1 receptor, orexin A stimulation triggered beta-arrestin 2 binding to the receptor and co-internalisation of receptor beta-arrestin complexes via clathrin-coated vesicles into acidic endosomes, in a dynamin-dependent manner. Moreover, studies of receptor internalisation in wild type, beta-arrestin-, Src family kinase-or Gq/n-deficient mouse embryo fibroblasts revealed sequestration of the orexin 1 receptor to be beta-arrestin-dependent, but G protein-and Src-independent. Mutational analysis of the orexin 1 receptor demonstrated that high affinity binding between the receptor and beta-arrestin 2 was conferred by a single cluster of Ser/Thr residues at the extreme C-terminus. Although this mutant form of the receptor was no longer able to co-intemalise with beta -arrestin 2, the pathway and time course of receptor internalisation was unaltered. In CHO cells, orexin A challenge induced rapid receptor phosphorylation which was partly mediated by protein kinase A (PKA) and protein kinase C (PKC). Surprisingly the levels of phosphorylation were similar for the cluster Cl mutant indicating the principal phosphorylation site to be distinct from the cluster of Ser/Thr residues essential for agonist-induced recruitment of beta-arrestins. To investigate the signalling pathways elicited by addition of orexin A, mutant forms of the orexin 1 receptor unable to stimulate G protein signalling were generated. Activation of the orexin 1 receptor caused an increase in ERKl/2 activity by a process depending on an intact endocytic pathway since inhibition of endocytosis by concanavalin A or dominant negative dynamin resulted in attenuated ERKl/2 phosphorylation. However hyperosmolar levels of sucrose had no effect on ERKl/2 activation. In addition, orexin A challenge of Xiv Src family kinase knock out MEF cells expressing the orexin 1 receptor resulted in ERKl/2 stimulation. There was significant difference in the time course of ERKl/2 phosphorylation upon stimulation of the wild type receptor and the cluster Cl mutant. On the other hand, no increase in ERKl/2 phosphorylation could be observed for the mutants unable to activate G proteins. Agonist challenge of the wild type receptor also caused stimulation of the JNK MAPK pathway. In contrast to the ERKl/2 MAPK pathway stimulation of all mutants tested resulted in increased JNK activity. p38 another member of the MAPK family was not activated after agonist challenge of the orexin 1 receptor excluding an involvement of this MAPK in orexin 1 receptor signalling. Taken together these results show that a single cluster of hydroxy amino acids within the C-terminus of the orexin 1 receptor determines the affinity of the interaction with beta- arrestin 2. They also indicate a key role of beta-arrestin scaffolding in fine tuning the kinetics of orexin 1 receptor-mediated, G protein-dependent ERKl/2 activation

Identifizierung von Barrieren und Handlungsoptionen zur Förderung der Wohneigentumsbildung in der Schweiz

Die vorliegende Masterthesis identifiziert die Hindernisse bei der Bildung von Wohneigentum in der Deutschschweiz und erforscht mögliche Massnahmen zur Überwindung dieser Barrieren. Die Thematik besitzt eine hohe Relevanz für die Schweiz, da das Land trotz politischer Bemühungen zur Steigerung weiterhin die niedrigste Wohneigentumsquote in Europa besitzt. Die Hauptbarrieren für den Zugang zum Wohneigentum wurden in 4 Cluster unterteilt: finanzielle und marktbezogene, funktionale und infrastrukturelle, persönliche und soziale sowie rechtliche und institutionelle Barrieren. Der wichtigste identifizierte Faktor war der hohe Preis für Wohneigentum. Die Befragten gaben auch an, dass der begrenzte Bestand an Wohnungen am gewünschten Standort, fehlendes Eigenkapital und komplizierte Verfahren bei der Beantragung von Baugenehmigungen weitere wichtige Hindernisse darstellen. Die vorgeschlagenen Massnahmen zur Überwindung dieser Hindernisse wurden ebenfalls in die 4 genannten Cluster eingeteilt. Die wirkungsvollste Massnahme wäre die Einführung von Steuervergünstigungen oder -abzügen für Erstwohnungskäufer. Weitere hilfreiche Massnahmen wären die Förderung von nachhaltigem Bauen und die Vereinfachung von Genehmigungsverfahren für Bauvorhaben. Als Handlungsempfehlung schlägt die Arbeit vor, den Fokus auf finanzielle und marktbezogene Massnahmen zu legen, um die Wohneigentumsquote zu erhöhen, vorausgesetzt, dies bleibt ein politisches Ziel der Schweiz. Gleichzeitig sollten weitere Massnahmen nicht vernachlässigt werden, die ausserhalb des finanziellen und marktbezogenen Clusters liegen. Beispielsweise wären hier die Vereinfachung von Genehmigungsverfahren und die Förderung nachhaltigen Bauens zu nennen. Die Arbeit weist darauf hin, dass eine hohe Wohneigentumsquote nicht nur Vorteile, sondern auch gesamtwirtschaftliche Nachteile mit sich bringen kann und weitere Forschung notwendig ist, um die Auswirkungen und die Umsetzbarkeit der vorgeschlagenen Massnahmen zu bewerten

Widespread mitochondrial depletion via mitophagy does not compromise necroptosis

Programmed necrosis (or necroptosis) is a form of cell death triggered by the activation of receptor interacting protein kinase-3 (RIPK3). Several reports have implicated mitochondria and mitochondrial reactive oxygen species (ROS) generation as effectors of RIPK3-dependent cell death. Here, we directly test this idea by employing a method for the specific removal of mitochondria via mitophagy. Mitochondria-deficient cells were resistant to the mitochondrial pathway of apoptosis, but efficiently died via tumor necrosis factor (TNF)-induced, RIPK3-dependent programmed necrosis or as a result of direct oligomerization of RIPK3. Although the ROS scavenger butylated hydroxyanisole (BHA) delayed TNF-induced necroptosis, it had no effect on necroptosis induced by RIPK3 oligomerization. Furthermore, although TNF-induced ROS production was dependent on mitochondria, the inhibition of TNF-induced necroptosis by BHA was observed in mitochondria-depleted cells. Our data indicate that mitochondrial ROS production accompanies, but does not cause, RIPK3-dependent necroptotic cell death

CXCR2 chemokine receptor antagonism enhances DOP opioid receptor function via allosteric regulation of the CXCR2–DOP receptor heterodimer

Opioid agonists have a broad range of effects on cells of the immune system, including modulation of the inflammatory response, and opioid and chemokine receptors are co-expressed by many white cells. Hetero-oligomerization of the human DOP opioid and chemokine CXCR2 receptors could be detected following their co-expression by each of co-immunoprecipitation, three different resonance energy transfer techniques and the construction of pairs of individually inactive but potentially complementary receptor G-protein α subunit fusion proteins. Although DOP receptor agonists and a CXCR2 antagonist had no inherent affinity for the alternative receptor when either receptor was expressed individually, use of cells that expressed a DOP opioid receptor construct constitutively, and in which expression of a CXCR2 receptor construct could be regulated, demonstrated that the CXCR2 antagonist enhanced the function of DOP receptor agonists only in the presence of CXCR2. This effect was observed for both enkephalin- and alkaloid-based opioid agonists, and the effective concentrations of the CXCR2 antagonist reflected CXCR2 receptor occupancy. Entirely equivalent results were obtained in cells in which the native DOP opioid receptor was expressed constitutively and in which expression of the isolated CXCR2 receptor could be induced. These results indicate that a CXCR2 receptor antagonist can enhance the function of agonists at a receptor for which it has no inherent direct affinity by acting as an allosteric regulator of a receptor that is a heterodimer partner for the CXCR2 receptor. These results have novel and important implications for the development and use of small-molecule therapeutics

Targeting Histone Demethylases in MYC-Driven Neuroblastomas with Ciclopirox

Histone lysine demethylases facilitate the activity of oncogenic transcription factors, including possibly MYC. Here we show that multiple histone demethylases influence the viability and poor prognosis of neuroblastoma cells, where MYC is often overexpressed. We also identified the approved small-molecule antifungal agent ciclopirox as a novel pan-histone demethylase inhibitor. Ciclopirox targeted several histone demethylases, including KDM4B implicated in MYC function. Accordingly, ciclopirox inhibited Myc signaling in parallel with mitochondrial oxidative phosphorylation, resulting in suppression of neuroblastoma cell viability and inhibition of tumor growth associated with an induction of differentiation. Our findings provide new insights into epigenetic regulation of MYC function and suggest a novel pharmacologic basis to target histone demethylases as an indirect MYC-targeting approach for cancer therapy

The sustainability of interactions between the orexin-1 receptor and β-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation

The orexin-1 receptor interacts with β-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-internalized into acidic endosomes. Truncations from the C-terminal tail did not prevent agonist-induced internalization of the orexin-1 receptor or alter the pathway of internalization, although such mutants failed to interact with β-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of β-arrestin-2–GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with β-arrestin-2, studies in wild-type and β-arrestin-deficient mouse embryo fibroblasts confirmed that agonist-induced internalization of this mutant required expression of a β-arrestin. Although without effect on agonist-mediated elevation of intracellular Ca(2+) levels, the C-terminally mutated form of the orexin-1 receptor was unable to sustain phosphorylation of the MAPKs (mitogen-activated protein kinases) ERK1 and ERK2 (extracellular-signal-regulated kinases 1 and 2) to the same extent as the wild-type receptor. These studies indicate that a single cluster of hydroxy amino acids within the C-terminal seven amino acids of the orexin-1 receptor determine the sustainability of interaction with β-arrestin-2, and indicate an important role of β-arrestin scaffolding in defining the kinetics of orexin-1 receptor-mediated ERK MAPK activation

Multiple roles for the C-terminal tail of the chemokine scavenger D6

D6 is a heptahelical receptor that suppresses inflammation and tumorigenesis by scavenging extracellular pro-inflammatory CC chemokines. Previous studies suggested this is dependent on constitutive trafficking of stable D6 protein to and from the cell surface via recycling endosomes. By internalizing chemokine each time it transits the cell surface, D6 can, over time, remove large quantities of these inflammatory mediators. We have investigated the role of the conserved 58-amino acid C terminus of human D6, which, unlike the rest of the protein, shows no clear homology to other heptahelical receptors. We show that, in human HEK293 cells, a serine cluster in this region controls the constitutive phosphorylation, high stability, and intracellular trafficking itinerary of the receptor and drives green fluorescent protein-tagged beta-arrestins to membranes at, and near, the cell surface. Unexpectedly, however, these properties, and the last 44 amino acids of the C terminus, are dispensable for D6 internalization and effective scavenging of the chemokine CCL3. Even in the absence of the last 58 amino acids, D6 still initially internalizes CCL3 but, surprisingly, exposure to ligand inhibits subsequent CCL3 uptake by this mutant. Progressive scavenging is therefore abrogated. We conclude that the heptahelical body of D6 on its own can engage the endocytotic machinery of HEK293 cells but that the C terminus is indispensable for scavenging because it prevents initial chemokine engagement of D6 from inhibiting subsequent chemokine uptake