G protein-coupled receptors (GPCRs) are an important class of drug targets due to their involvement in various signaling pathways. GPCRs transmit signals from the extracellular environment to the interior of cells, regulating numerous physiological processes. GPCRs are known to engage in protein-protein interactions (PPIs) with a variety of intracellular signaling molecules, including G proteins, β-arrestins, and other adaptor proteins. Understanding these interactions is crucial for unraveling the complex signaling networks mediated by GPCRs and developing targeted therapeutics. Assay development plays a pivotal role in studying GPCR PPIs, allowing researchers to investigate the binding partners, functional consequences, and dynamics of these interactions. Developing assays for GPCR PPIs requires careful consideration of the specific interaction being studied, the choice of experimental techniques, and the suitability for the desired readout. These assays provide valuable insights into the molecular mechanisms underlying GPCR signaling and can aid in the discovery and development of novel therapeutic strategies targeting GPCR associated diseases. Considering that PPIs are highly dynamic processes, cellular assays are often essential for their study because they closely mimic the biological complexities of cellular environments. The present thesis aimed to develop an in cellulo proteomic assay by developing an intracellular nanobody targeting the V5-peptide tag. Functionalizing this anti-V5 intrabody would allow it to be implemented in various cell-based assays with any protein carrying the V5-tag. Therefore, the NbV5:V5 tag system presents itself as a versatile tool for live-cell imaging and a befitting adaptation to existing cellular assays dedicated to probing PPIs. The NbV5:V5 tag system has been applied to interrogate G protein-coupled receptor signaling with the ultimate goal of performing cellular studies of a broader and systematic nature using the genome-wide V5-tagged ORF library.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/45872 |
| Date | 22 January 2024 |
| Creators | Matte, Kevin |
| Contributors | Giguère, Patrick M. |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
Page generated in 0.0026 seconds