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OX1 orexin receptors activate extracellular signal-regulated kinase in Chinese hamster ovary cells via multiple mechanisms: the role of Ca2+ influx in OX1 receptor signaling
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
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2006 (English)In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 20, no 1, 80-99 p.Article in journal (Refereed) Published
Abstract [en]

Activation of OX1 orexin receptors heterologously expressedin Chinese hamster ovary (CHO) cells led to a rapid, strong,and long-lasting increase in ERK phosphorylation (activation).Dissection of the signal pathways to ERK using multiple inhibitorsand dominant-negative constructs indicated involvement of Ras,protein kinase C, phosphoinositide-3-kinase, and Src. Most interestingly,Ca2+ influx appeared central for the ERK response in CHO cells,and the same was indicated in recombinant neuro-2a cells andcultured rat striatal neurons. Detailed investigations in CHOcells showed that inhibition of the receptor- and store-operatedCa2+ influx pathways could fully attenuate the response, whereasinhibition of the store-operated Ca2+ influx pathway alone orthe Ca2+ release was ineffective. If the receptor-operated pathwaywas blocked, an exogenously activated store-operated pathwaycould take its place and restore the coupling of OX1 receptorsto ERK. Further experiments suggested that Ca2+ influx, as such,may not be required for ERK phosphorylation, but that Ca2+,elevated via influx, acts as a switch enabling OX1 receptorsto couple to cascades leading to ERK phosphorylation, cAMP elevation,and phospholipase C activation. In conclusion, the data suggestthat the primary coupling of orexin receptors to Ca2+ influxallows them to couple to other signal pathways; in the absenceof coupling to Ca2+ influx, orexin receptors can act as signalintegrators by taking advantage of other Ca2+ influx pathways.

Place, publisher, year, edition, pages
2006. Vol. 20, no 1, 80-99 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-93309DOI: 10.1210/me.2004-0389PubMedID: 16141359OAI: oai:DiVA.org:uu-93309DiVA: diva2:166750
Available from: 2005-09-01 Created: 2005-09-01 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Orexin Receptors in Recombinant CHO Cells: Signaling to Short- and Long-Term Cell Responses
Open this publication in new window or tab >>Orexin Receptors in Recombinant CHO Cells: Signaling to Short- and Long-Term Cell Responses
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Recently discovered neuropeptides orexins (orexin-A and -B) act as endogenous ligands for G-protein-coupled receptors called OX1 and OX2 receptors. Our previous studies have established model systems for investigation of the pharmacology and signaling of these receptors in recombinant CHO cells. OX1 receptor-expressing CHO cells were mainly utilized in this thesis.

Orexin-A and -B activate both OX1 and OX2 receptors. However, orexin-B is less potent in activating OX1 receptors than orexin-A, whereas the peptides are equipotent on OX2 receptors. We have performed mutagenesis on orexin-A to investigate the basis for this selectivity. We show that OX2 receptor is generally less affected by the mutations and thus OX2 receptor appears to have less strict requirements for ligand binding, likely explaining the lack of difference in affinity/potency between orexin-A and orexin-B on OX2 receptor.

The other studies focus on orexin receptor signaling. OX1 receptors are shown to regulate adenylyl cyclase both in positive and negative manner, activate different MAP-kinases (ERK1/2 and p38) and induce cell death after long-lasting stimulation. Adenylyl cyclase regulation occurs likely through three different G-protein families, Gi, Gs and Gq. For ERK1/2, several downstream pathways, such as Ras, Src, PI3-kinase and protein kinase C (PKC) are implicated. OX1 receptor-mediated activation of ERK is suggested to be cytoprotective whereas p38 MAP-kinase induces programmed cell death.

Three particularly interesting findings were made. Firstly, novel PKC δ (delta) is suggested to regulate adenylyl cyclase, whereas conventional and atypical PKCs are involved in activation of ERK. Secondly, adenylyl cyclase and ERK activation is fully dependent on extracellular Ca2+. Further experiments suggest that the previously discovered receptor-operated Ca2+ influx is not affecting the downstream effectors of orexin receptors but that it instead enables orexin receptors to couple to several signal cascades. Thirdly, upon inhibition of caspases, classical mediators of programmed cell death, OX1 receptor-mediated cell death is not reversed, but instead the pathways to death are altered so de novo gene transcription is no longer required for cell death.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 92 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 56
Keyword
Neurosciences, orexins, cell signaling, Neurovetenskap
National Category
Neurology
Identifiers
urn:nbn:se:uu:diva-5890 (URN)91-554-6300-2 (ISBN)
Public defence
2005-09-22, B42, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 09:00
Opponent
Supervisors
Available from: 2005-09-01 Created: 2005-09-01Bibliographically approved
2. Calcium and Phospholipases in Orexin Receptor Signaling
Open this publication in new window or tab >>Calcium and Phospholipases in Orexin Receptor Signaling
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The neuropeptides orexin-A and -B act as endogenous ligands for G-protein-coupled receptors (GPCRs) called OX1 and OX2 receptors. Previous observations have established that orexin receptors have an ability to couple to different G-proteins and signaling pathways and induce Ca2+ elevations via both receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs). This thesis further elucidates the intracellular signaling mechanisms of orexin receptors.

Orexin receptors were shown to activate ERK (extracellular signal-regulated kinase) via Ras, protein kinase C, phosphatidylinositol-3 kinase and Src. Ca2+ influx was shown to be obligatory for the activation of ERK and adenylyl cyclase, wherewith a hypothesis was formed that submembrane Ca2+ elevation is of central importance for the regulation of orexin receptors' coupling to different signaling pathways. This was further investigated with respect to OX1R-mediated activation of phospholipase C (PLC) showing that ROC influx was of more central importance for the OX1R signaling, but also SOCs amplified PLC activity. A technique to block OX1R-induced IP3 increase and subsequent Ca2+ release was devised, leaving ROC influx as the only source of Ca2+ elevation upon OX1R activation. This block had no effect on OX1R-mediated activation of ERK, showing that ROC-dependent influx is the most central Ca2+ elevating process in OX1R signaling. OX1Rs' coupling to PLC was further investigated by measuring the metabolites generated, inositol phosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities, which results in, at low orexin-A concentrations, DAG production without concomitant production of IP3. At even lower orexin-A concentrations, OX1Rs generate DAG by activating phospholipase D. In conclusion, the results strengthen the hypothesis that ROCs have a central role in orexin receptor signaling and DAG may be the signal of preference.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 58 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 327
Keyword
Physiology, orexin, receptor, calcium, phospholipase, cell signaling, GPCR, Fysiologi
Identifiers
urn:nbn:se:uu:diva-8613 (URN)978-91-554-7151-4 (ISBN)
Public defence
2008-05-02, B22, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2008-04-11 Created: 2008-04-11 Last updated: 2013-09-20Bibliographically approved

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