Molecular Determinants of Orexin Receptor Ligand Interaction : Studies on Ligand Selectivity and Impact of Calcium
Year: 2014
Type: dissertation
Abstract: Neuropeptides orexin-A and orexin-B, and their receptors OX1 and OX2 were first found as regulators of appetite, and later several other functions have been found as well. Orexin peptides and receptors have been researched, especially in the field of drug discovery; however many relevant biochemical properties remain partly unsolved, including the factors determining orexin ligand binding properties and ligand selectivity. The study of these factors was pursued in this thesis. Ligand selectivity was studied with chimaeric orexin receptors. We mapped some of the molecular determinants of orexin receptors that are needed for the selectivity of orexin agonists and OX1-specific antagonist. The second quarter of the orexin receptors seems to be the most important area both for agonist and antagonist selectivity. However, for antagonist selectivity, the third quarter also seems to have a role. Activated orexin receptors cause a strong elevation of intracellular calcium. However, reduction of extracellular calcium attenuates that increase and other orexin receptor signalling, mediated by certain phospholipases and kinases, for instance. It remains unknown, however, how calcium causes these effects. With [125I]-orexin-A, a clear decrease in binding was observed after reduction of the extracellular calcium concentration. Also, we saw a similar reduction in the activities of phospholipase C (PLC) and adenylyl cyclase (AC). The concentration-relationship of calcium was identical for radioligand binding, PLC activation, and AC stimulation, while AC inhibition was more strongly attenuated. When the driving force for calcium influx was reduced with high-K+ medium, the orexin-A-induced PLC activity was more strongly reduced than orexin-A binding. In addition, inhibition of the orexin receptor-operated calcium channels had a more pronounced effect on the PLC activity than on the binding. It is thus suggested that reduction of extracellular calcium concentration both inhibits orexin binding and attenuates enzymatic activity. Orexin-B has higher binding affinity for OX2 than OX1 receptor and [Ala11, D-Leu15]-orexin-B, an orexin-B variant, has been reported to display even higher OX2-selectivity. We observed that [Ala11, D-Leu15]-orexin-B showed much lower OX2-selectivity than originally reported. In addition, the selectivity of both forms of orexin-B was dependent on the cell line. These findings may be caused by biased agonism of the orexin receptor, meaning that the orexin receptor can be found in multiple conformations, each of which can interact differently with an agonist. This result extends our knowledge of orexin ligand binding properties, and the phenomenon should be considered, for instance, when novel agonists for orexin receptors are screened.
Source: Työväentutkimus Vuosikirja
Author: Jaana Putula
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1500212819