Human orexin receptor signaling : subtype differences and dimerization

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The orexin system, composed of the orexin peptides and the G-protein-coupled orexin receptors, was discovered in 1998 and immediately gained much interest for its (potential) role in the regulation of appetite–metabolism and sleep–wakefulness. To date, the research on the orexin system does not show any decline, and while there still are many open questions, we know a lot and even have some drugs acting on the orexin receptors in clinical use. Orexin receptors are not only interesting from the therapeutic point of view but also from a more scientific one. The receptors signal via at least three families of G-proteins – with apparently different ones preferred in different tissues – to multiple signal transduction systems. The studies I–III included in this thesis further scrutinize these aspects of orexin receptors by cell-level measurements with CHO-K1 Chinese hamster cell lines stably expressing human OX1 and OX2 orexin receptors. The idea was to see whether the responses observed with OX2 were similar as (the previously found) OX1 responses. The responses assessed were Ca2+ elevation, activation of the phospholipase C cascade (phospholipase C itself and diacylglycerol lipase), phospholipase D, and phospholipase A2, as well as activation and inhibition of adenylyl cyclase (utilized solely for the measurement of Gαi and Gαs activities). The studies also included characterization of the novel Gq inhibitor, UBO-QIC/FR900359, with other receptors coupling to certain G-protein families, to finally allow its use to distinguish the G-protein pathways of orexin receptors. The results, in short, suggest a) that the signaling of the human orexin receptor subtypes is largely the same (in opposition to what is often claimed) and b) that most of the responses in recombinant CHO-K1 cells (all on the list above except for the adenylyl cyclase regulation) are mediated by Gq (or the closely related G11 and G14). For a), small differences could yet be seen: Activation of phospholipase D (Gq) and adenylyl cyclase (Gαs) was weaker for OX2 than for OX1. Based on these studies, it is impossible to conclude whether these differences are due to i) distinct receptor properties or ii) just differences in the expression system, e.g., subtle differences in the receptor expression level or a result of the clonal selection needed to establish the cells lines. For b), the adenylyl cyclase inhibition by orexin receptors was unexpected since it, though being fully inhibitable by the Gi inactivator pertussis toxin, was also fully (OX1) or partially (OX2) inhibited by UBO-QIC/FR900359. This is difficult to explain, though it goes well together with other oddities of orexin receptor signaling. It nevertheless stresses that the downstream readouts, however well determined, are weak substitutes for direct measurements of G-protein activation and vice versa. The final study (IV) presents derivation of a mathematical model for an obligate G-protein-coupled receptor homodimer and some characterization of the model. The model allows the co-operativity to be separately set for receptor binding and activation. Most interestingly, it could be shown – as had been hypothesized – that antagonistic ligands (no intrinsic activity) can be both antagonists and allosteric modulators at the same time solely via the orthosteric binding site. Although this is simple and obvious, it seems to constitute a novel concept. However, no experiments were performed in this study, and thus it cannot be verified that this is actually taking place.
  • Pertovaara, Antti, Handledare
  • Turunen, Pauli, Handledare
Tryckta ISBN978-951-51-8053-7
Elektroniska ISBN978-951-51-8054-4
StatusPublicerad - 2022
MoE-publikationstypG5 Doktorsavhandling (artikel)

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