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Distinct Recognition of OX1 and OX2 Receptors by Orexin Peptides
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
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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2003 (English)In: Journal of Pharmacology and Experimental Therapeutics, ISSN 0022-3565, E-ISSN 1521-0103, Vol. 305, no 2, 507-514 p.Article in journal (Refereed) Published
Abstract [en]

In this study, we have compared the abilities of orexin-A and orexin-B and variants of orexin-A to activate different Ca(2+) responses (influx and release) in human OX(1) and OX(2) receptor- expressing Chinese hamster ovary cells. Responses mediated by activation of both receptor subtypes with either orexin-A or -B were primarily dependent on extracellular Ca(2+), suggesting similar activation of Ca(2+) influx as we have previously shown for orexin-A and OX(1) receptors. Amino acid-wise truncation of orexin-A reduced its ability to activate OX(1) and OX(2) receptors, but the response mediated by the OX(2) receptor was more resistant to truncation than the response mediated by the OX(1) receptor. We also performed a sequential replacement of amino acids 14 to 26 with alanine in the truncated orexin-A variant orexin-A(14-33). Replacement of the same amino acids produced a fall in the potency for each receptor subtype, but the reduction was less prominent for the OX(2) receptor. The most marked reduction was produced by the replacement of Leu20, Asp25, and His26 with alanine. Interestingly, extracellular Ca(2+) dependence of responses to some of the mutated peptides was different from those of orexin-A and -B. The mutagenesis also suggests that although the determinants required from orexin-A for binding to and activation of the receptor are highly conserved between the orexin receptor subtypes, the OX(2) receptor requires fewer determinants. This might in part explain why orexin-B has the affinity and potency equal to orexin-A for this subtype, although it has 10- to 100-fold lower affinity and potency for the OX(1) receptor.

Place, publisher, year, edition, pages
2003. Vol. 305, no 2, 507-514 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-93307DOI: 10.1124/jpet.102.048025PubMedID: 12606634OAI: oai:DiVA.org:uu-93307DiVA: diva2:166748
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. Signaling via Orexin Receptors: A Pharmacological Study
Open this publication in new window or tab >>Signaling via Orexin Receptors: A Pharmacological Study
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The orexin receptors are a pair of newly discovered G-protein coupled receptors which are activated by the neuropeptides orexins and play a role in sleep/vigilance, apetite/metabolism and neuroendocrine regulation. On a cellular level receptor activation results in, to name but a few effects, elevation of intracellular calcium and depolarisation. All cellular effects display an uncommon dependence of extracellular Ca2+, which has been shown to be due to influx of extracellular Ca2+ as a primary response.

Here we provide evidence for a high specificity of orexin receptors for orexin peptides over other neuropeptides, despite previous reports of the opposite. Other neuropeptides could neither displace orexin-A from orexin receptors, nor affect functional responses induced by orexin peptides via orexin receptors. In an effort to assess the determinants of orexin-A binding to orexin receptors orexin-A was truncated/mutated and tested for functional responses. It was found that alterations in the orexin-A sequence had more prominent effects on the activation of OX1 than on OX2 receptors.

When the signaling of orexin receptors was investigated in neuron-like cells it was found that they couple to Ca2+-metabolism and PLC activation in a manner similar to that in non-neuronal cells. Investigations of OX1 receptor regulation of adenylyl cyclases showed orexin receptors to have a dual effect on the production of cAMP. A high-affinity inhibitory coupling and a low-affinity stimulatory coupling. The stimulatory coupling was determined to consist of two components, a low potency GS-coupling and a high-potency PKC coupling.

In conclusion we have shown that orexin receptors are preferentially activated by orexin peptides and the receptors couple to Ca2+-metabolism in a similar way in different contexts. Orexin receptors couple to both the phospholipase C and the adenylyl cyclase pathway and to some extent these pathways converge in the production of cAMP.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 40 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1376
Keyword
Physiology, orexin, receptors, calcium, cAMP, adenylyl cyclases, Fysiologi
National Category
Physiology
Identifiers
urn:nbn:se:uu:diva-4570 (URN)91-554-6049-6 (ISBN)
Public defence
2004-12-18, B21, BMC, Husargatan 3, Uppsala, 09:15
Opponent
Supervisors
Available from: 2004-11-25 Created: 2004-11-25 Last updated: 2013-09-20Bibliographically approved

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