NanoBRET to probe ligand-receptor and receptor-receptor interactions in living cells

Carvalheira Alcobia, Diana (2019) NanoBRET to probe ligand-receptor and receptor-receptor interactions in living cells. PhD thesis, University of Nottingham.

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Increasing evidence has revealed the role of G protein-coupled receptors (GPCRs) in the regulation of signalling responses via complex cross-talk mechanisms with the receptor tyrosine kinase (RTK) family of transmembrane receptors. Vascular endothelial growth factor-a (VEGFa) mediates cancer angiogenesis via binding to its cognate VEGF receptor 2 (VEGFR2). The two class A GPCRs, adenosine A2A receptors and β2-adrenoceptors, have also been intimately involved in cancer angiogenesis, and their activation can contribute to cancer progression and invasion. However, the molecular mechanisms involved in the cross-talk between these receptor families are not well understood.

Using a NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) methodology, we reveal a novel mechanism in which VEGFR2 can associate with either of these Gs-coupled GPCRs to form oligomeric complexes in living cells. We also demonstrated that VEGF-stimulated VEGFR2 can induce a switch from a transient to a more stable interaction between β2AR and the adaptor protein β-arrestin-2, which may have an impact in endosomal signalling.

The pharmacological inhibition of β2-adrenoceptor (β2AR) signalling, using selective β2AR antagonists (or β-blockers), represents a potential therapeutic approach for the treatment of triple-negative breast cancer variant, which has limited treatment options. This study demonstrated the novel application of NanoBRET technology to probe specific β-blocker-β2-adrenoceptor engagement in a pre-clinical in vivo model of triple-negative breast cancer. This novel methodology will allow a strong correlation between drug-target engagement and mediated physiological response.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Hill, Stephen J.
Woolard, Jeanette
Sloan, Erika K.
Keywords: NanoBRET, Pharmacology, Breast cancer
Subjects: Q Science > QP Physiology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 56087
Depositing User: Carvalheira Alcobia, Diana
Date Deposited: 19 Jul 2019 04:40
Last Modified: 19 Jul 2021 04:30

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