27.5. Signalling mechanisms used by the orexin-1 receptor

Public examination of a doctoral dissertation in the field of cell biology

Doctoral candidate: MSc Hanna Peltonen

Time and venue: 27.5.2011 at 12 noon, Medistudia Auditorium L1, Kuopio Campus

The orexin-1 receptor (OX1R) is a member of a superfamily of G-protein coupled receptors (GPCRs). Two hypothalamic neuropeptides, orexin-A/hypocretin-1 (Ox-A) and orexin-B/hypocretin-2, bind to and activate OX1R and a related GPCR, the orexin-2 receptor. The physiological effects of the orexin system include regulation of feeding, energy metabolism, endocrine and autonomic systems, and sleep/wake cycle. At the cellular level, OX1R stimulation leads to neuronal excitation, activation of the adenylyl cyclase pathway and several protein kinases, and an increase in the intracellular Ca2+ concentration ([Ca2+]i). OX1R couples to Gq/11-proteins and activates phospholipase C (PLC -pathway leading to release of Ca2+ from the intracellular Ca2+ stores and the subsequent capacitative Ca2+ entry (CCE) aiming to refill depleted stores. This is the main pathway utilized when high nanomolar Ox-A concentrations are evaluated, but at lower, and probably physiologically more relevant, concentrations there was a predominance of receptor-operated Ca2+ influx without any detectable release of stores.

The aim of this study was to elucidate the poorly characterized signalling mechanisms used by the OX1R leading to and regulating Ca2+ responses. The investigations with the patch clamp and Ca2+ imaging techniques in recombinant cell models demonstrated that even a low nanomolar Ox-A concentration was able to evoke a highly voltage-dependent depolarization of the membrane, an increased ion current, and a robust elevation of [Ca2+]i. It was found that Ca2+ responses at low Ox-A concentrations were acutely dependent on voltage and extracellular Ca2+ concentration as well as being sensitive to blockers of receptor-operated Ca2+ entry. Protein kinase C (PKC) was found to be a crucial regulator of these responses. Based on the pharmacological profile of the responses and the experiments with dominant negative canonical transient receptor potential channel (TRPC) subtypes, the most probable ion channel involved in the Ca2+ influx is the diacylglycerol-activated and PKC-regulated TRPC3. At higher Ox-A concentrations, Ca2+ responses occurred also in the absence of extracellular Ca2+ and were sensitive to inhibitors of store release and CCE, pointing to involvement of the PLC -pathway. In addition to TRPC3 channel, phospholipase A2 and protein kinase D1/3 were identified as novel targets of OX1R mediated activation and as prominent modulators of Ca2+ oscillations induced by low nanomolar Ox-A concentrations.

In conclusion, it was possible to confirm the present view of Ca2+ signalling of OX1R and to identify novel players in the pathways activated by the receptor. It is hoped that these results will be useful in constructing a more comprehensive picture of OX1R signalling and in developing efficient therapies for OX1R -related disorders.

The doctoral dissertation of Master of Science Hanna Peltonen, entitled Signalling mechanisms used by the orexin-1 receptor will be examined at the Faculty of Health Sciences. The opponent in the public examination will be Associate Professor Shahidul Islam of Karolinska Institutet and the custos will be Professor Karl Ã…kerman of the University of Helsinki.

Photo available for download at http://www.uef.fi/vaitoskuvat  

Contact: Hanna Peltonen, GSM 050-3543080, hanna.peltonen@uef.fi

 

Publishing year: 2011

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