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Abstract:
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The 5-hydroxytryptamine (5-HT) system modulates numerous physiological and behavioural processes, and dysfunction within this system underlies many behavioural disorders, such as major depression. The 5-HT1A receptor is the primary somatodendritic autoreceptor that controls the firing rate of 5-HT neurons, but is also coupled to numerous signalling pathways. An understanding of 5-HT1A receptor signalling may lead to the development of antidepressant drugs that selectively target therapeutic pathways in treating depression. The 5-HT1A receptor is coupled to inhibitory G-proteins via its intracellular loops 2 and 3. Point mutations within these loops selectively uncouple receptor signalling pathways. In this thesis, I addressed whether mutant receptors’ uncoupling from signalling pathways is associated with alteration in G-protein interaction and coupling. Using bioluminescence resonance energy transfer (BRET) to monitor receptor-G-protein interactions, we show that both wild-type and mutant receptors demonstrate a saturable interaction with Gαi protein in unstimulated conditions. Addition of 5-HT increased the BRET signal for the wild-type 5-HT1A receptor, and this increase was blocked by a 5-HT1A receptor antagonist and G-protein blocker (pertussis toxin). Mutant receptors that were deficient in Gαi signalling, but not those that still signalled to Gαi, failed to respond to receptor activation with increased receptor-Gαi interaction. Pull down studies verified the basal and agonist-induced interaction of 5-HT1A receptors with Gαi proteins. In conclusion, we have shown that the 5-HT1A receptor interacts with Gαi consistent with a pre-coupled model and that 5-HT-induced activation enhances this interaction and requires specific residues in the intracellular loops. |
