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Doctoral thesis
English

Sensing and modulating G protein-coupled receptor function using novel nanobody tools

Imprimatur date2024
Defense date2024
Abstract

G protein-coupled receptors (GPCRs) represent a vast superfamily of transmembrane proteins that play critical roles in cellular signalling, regulate diverse physiological processes, and serve as key therapeutic targets. Opioid receptors (ORs), a subfamily of GPCRs, play a pivotal role as targets for pain medication, but their activation by opioid drugs can also trigger undesirable effects such as tolerance, dependence, and respiratory depression. Investigating the mechanisms underlying GPCR signalling and regulation, such as receptor activation, engagement of signalling partners, location-biased signalling and receptor phosphorylation/dephosphorylation, is essential for understanding the role of GPCRs in physiology and disease and requires suitable research tools.

Nanobodies (Nbs), the recombinant antigen-binding fragments of camelid heavy-chain-only antibodies, have emerged as innovative tools for studying GPCRs. Recent Nb-based biosensors have enabled real-time monitoring of GPCR activation at subcellular resolution, providing valuable insights into cellular GPCR actions. However, existing fluorescent sensors rely on single-cell confocal fluorescence microscopy for visualizing ligand-dependent biosensor re-localisation from the cytosol to the site of receptor activation, limiting their applicability to higher throughput assays. Beyond sensing GPCR activity, Nbs can also be employed to inhibit receptor function. While intracellular OR-blocking Nbs would pave the way for dissecting receptor function at the cellular and subcellular level, they have not yet been developed. My PhD research aimed to generate Nb-based tools to study ORs, and more broadly, GPCR function on a cellular and subcellular level, and to investigate the regulation of ligand-driven OR phosphorylation by phosphatases.

Overall, my PhD thesis work shows the successful development and characterization of high-affinity Nbs that effectively block OR signalling. As genetically encoded tools, the Nbs hold promise for dissecting OR signalling in individual cells and subcellular compartments. Expanding the toolkit further, we developed novel dark-to-light GPCR reporters that offer subcellular spatial and seconds temporal resolution and can be readily redesigned to probe a wide range of GPCRs. Furthermore, we make inroads into understanding OR dephosphorylation, holding implications for understanding receptor resensitisation.

eng
Citation (ISO format)
VALBRET, Zoé Suzanne. Sensing and modulating G protein-coupled receptor function using novel nanobody tools. 2024. doi: 10.13097/archive-ouverte/unige:176885
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Creation04/30/2024 8:35:25 AM
First validation05/07/2024 11:57:16 AM
Update time05/07/2024 11:57:16 AM
Status update05/07/2024 11:57:16 AM
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