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Title: Investigating the molecular signatures of β1-adrenergic receptor activation
Author: Solt, Andras Sandor
ISNI:       0000 0004 7968 5996
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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In this thesis I have investigated the molecular signatures of receptor activation, using the β1-adrenergic receptor (β1AR) as a prototypical class A G-protein coupled receptor (GPCR). I have used a minimally thermostabilised turkey β1AR and expressed it functionally in insect cells using a baculovirus system. The work described here established the labelling, expression, purification and sample preparation of the receptor in LMNG detergent micelles for use in nuclear magnetic resonance (NMR) spectroscopy. GPCRs are highly dynamic molecular systems, and the use of solution NMR is highly suited to the study of fine structural changes that take place within the receptor as a consequence of receptor activation. To this end the receptor was selectively labelled with 13C at the methyl position of methionine residues. The labelling was carried out during insect cell expression, by supplementing methionine deficient media with the labelled amino acid. In this way the methionines throughout the receptor served as reporters. The radio frequency signals emitted by the nuclei of these labelled residues were monitored. NMR experiments were recorded on the receptor in the presence of ligands of various efficacies together with and without G-protein mimetic nanobodies, and changes in the signal were recorded. This allowed for a pattern of molecular signatures to be established, reporting on the effect ligands and G-protein mimetics have on the receptor. This identified two conformational equilibria, between an inactive and a ligand bound-pre-activated state and between a more and a less active ternary state when bound to a G-protein mimetic. Furthermore, it was also observed that ligand binding to the G-protein mimetic saturated basal active state elicits further changes on the receptor cytoplasmic side, demonstrating that ligand efficacy modulates the nature of receptor interaction with G-proteins, which may underpin partial agonism. It was also observed that ligand binding affects the dynamics and rigidity of the receptor, with a full agonist bound receptor exhibiting extensive µs to ms timescale dynamics, compared to a more rigid nanobody bound state. The increased dynamics suggest that full agonist binding primes the receptor for interaction with various downstream signalling partners. Once this coupling takes place, ligand efficacy determines the quality of interaction in this rigidified system. In addition to activation by ligands, certain proteins, such as antibodies can cause receptor agonism in the absence of a small molecule agonist. An example of this takes place in chronic Chagas' heart disease, where anti-Trypanosoma cruzi antibodies inappropriately cross-react to β1AR, leading to chronic cardiac overstimulation and heart failure. In this thesis, the production of a published monoclonal antibody fragment was explored, in order to generate a tool for the study of this activation mechanism.
Supervisor: Nietlispach, Daniel Sponsor: MRC ; NIBR ; Wolfson College ; Biochemical Society ; Philosophical Society
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Beta-1 adrenergic receptor ; GPCR ; NMR ; Membrane proteins ; Adrenergic receptors