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OX1 orexin receptors couple to adenylyl cyclase regulation via multiple mechanisms
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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2005 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 8, 6570-6579 p.Article in journal (Refereed) Published
Abstract [en]

In this study, the mechanism of OX(1) orexin receptors to regulate adenylyl cyclase activity when recombinantly expressed in Chinese hamster ovary cells was investigated. In intact cells, stimulation with orexin-A led to two responses, a weak (21%), high potency (EC(50) approximately 1 nm) inhibition and a strong (4-fold), low potency (EC(50) = approximately 300 nm) stimulation. The inhibition was reversed by pertussis toxin, suggesting the involvement of G(i/o) proteins. Orexin-B was, surprisingly, almost equally as potent as orexin-A in elevating cAMP (pEC(50) = approximately 500 nm). cAMP elevation was not caused by Ca(2+) elevation or by Gbetagamma. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKCdelta, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of G(s) activity, orexins still elevated cAMP; however, the potencies were greatly increased (EC(50) of orexin-A = approximately 10 nm and EC(50) of orexin-B = approximately 100 nm), and the response was fully dependent on PKCdelta. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-Galpha(s) antibodies. We conclude that OX(1) receptors stimulate adenylyl cyclase via a low potency G(s) coupling and a high potency phospholipase C --> PKC coupling. The former or some exogenous G activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX(1) receptors couple to G(s) almost as efficiently as the orexin-A-activated receptors, in contrast to Ca(2+) elevation and phospholipase C activation, for which orexin-A is 10-fold more potent.

Place, publisher, year, edition, pages
2005. Vol. 280, no 8, 6570-6579 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-93308DOI: 10.1074/jbc.M407397200PubMedID: 15611118OAI: oai:DiVA.org:uu-93308DiVA: diva2:166749
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
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Supervisors
Available from: 2005-09-01 Created: 2005-09-01Bibliographically approved

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