Dowsell, Ryan; (2025) Beyond anchoring: investigating the functions of adenylyl cyclase transmembrane domains. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cyclic adenosine 3’,5’-monophosphate (cAMP) is one of the most fundamental and universal second messengers. As the enzyme responsible for synthesizing cAMP, the adenylyl cyclase serves as the master regulator of cAMP signal transduction, playing a crucial role in regulating synaptic plasticity, cancer, metabolism and cellular growth. Nine transmembrane adenylyl cyclase isoforms have been identified in mammals, many of which are co-expressed within the same cell types with overlapping regulatory mechanisms. The large, poorly conserved transmembrane domains lack a clearly defined function beyond subcellular anchoring. In this study, I investigated two long-speculated functions of the adenylyl cyclase transmembrane domains: acting as receptors and serving as protein interaction hubs. To address the receptor hypothesis, I developed a novel assay using purified adenylyl cyclases and a cAMP FRET biosensor to screen for ligands that target the transmembrane domain. Furthermore, I established a TurboID-based proximity labelling strategy in primary hippocampal neurons to address the interactor hypothesis. Through proof-of-concept compound screening, I found that the antifungal imidazole, clotrimazole, inhibits AC8, likely through targeting of the transmembrane domain. Following a subsequent screen of 948 neuronal signalling compounds, I identified numerous novel AC8 activators, including the putative neurotransmitter agmatine, and novel inhibitors, including the serotonin modulator vortioxetine. Through hippocampal proximity labelling, I revealed that the GABAAR α2 subunit and the metabotropic glutamate receptor mGluR5 are novel membrane protein interactors of AC8, with mGluR5 likely interacting with the AC8 transmembrane domain. After expanding this in situ labelling approach, I identified various isoform-specific interactors for AC1, 2, 3, 7, 8 and 9, many of which are membrane proteins and promising candidates for follow-up structural studies. Together, this study provides new insights into the functions of the adenylyl cyclase transmembrane domains, advancing our understanding of cAMP regulation and uncovering novel therapeutic targets for the manipulation of cAMP signalling.

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