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

Diplôme Thèse Sensing and modulating G protein-coupled receptor function using novel nanobody tools eng 2024 2024 author Valbret Zoé Suzanne 931498 director Stoeber Miriam Carolin 930635 Département de physiologie cellulaire et métabolisme (PHYME) 10.13097/archive-ouverte/unige:176885 urn:nbn:ch:unige-1768854 Sc. Vie - Bioméd. 277 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. Dewey 612 134 Département de physiologie cellulaire et métabolisme 21C false 1283 Contrôle subcellulaire des récepteurs de signalisation 1005 90b9711e-fc08-4329-a66c-d0d36be40388 Thesis Restricted Zoe_Valbret_PhD_Thesis_AfterDefence.pdf 8074549 application/pdf Private 2026-03-27 153e3b6465ac4811add0541ebf2d533c46e8ac546071bae3c90f70080bef4c66 60f50223-5b93-4472-860c-e09bd73b693e Imprimatur Public IMPRIMATUR Zoé VALBRET signé par to.pdf 164273 application/pdf 149a6b58c7fcf861f3cfbcc53838048fd6b7eda291332499136a3a820cd0f628 embargoedAccess Faculté de médecine / Section de médecine fondamentale / Département de physiologie cellulaire et métabolisme

Title	Published in	Year	Views	Downloads

Sensing and modulating G protein-coupled receptor function using novel nanobody tools		2024	278	1
Optical tools to study the subcellular organization of GPCR neuromodulation	Journal of neuroscience methods	2021	284	320

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Valbret, Zoé Suzanne

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