Investigating cooperativity across adenosine A3 receptor homo- and hetero-oligomers

Groenewoud, N.J.A. (2019) Investigating cooperativity across adenosine A3 receptor homo- and hetero-oligomers. PhD thesis, University of Nottingham.

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Oligomerisation of receptors is the process whereby two or more receptors associate to form a new functional unit which can have distinct characteristics. These characteristics include changes in trafficking of the receptor, changes in signalling, and changes in ligand binding. The adenosine and β-adrenoceptor families are two class A GPCR families and some receptors from this family are reported to form oligomers both within their family as well as with other class A GPCRs.

In this thesis, the dimerisation of adenosine and β-adrenoceptors was investigated using bimolecular fluorescence complementation (BiFC). BiFC is used to examine protein-protein interactions by splitting a fluorescent protein into two non-fluorescent protein fragments. Using molecular biology techniques, the two fragments were attached to adenosine and β-adrenoceptors (Chapter 3). When the two receptors associate, the two fragments reconstitute and become fluorescent. Using transient transfections in CHO cell lines, BiFC showed dimerisation between all the adenosine receptors with varying degrees of intensity. The A2A-A3 heteromer was further investigated and confirmed using bioluminescence resonance energy transfer (BRET). This dimer has not before been recorded in the literature.

CHO cell lines stably transfected with the A3R and a BiFC tag were subsequently generated and characterised (Chapter 4). No differences were seen in ligand binding of various antagonists between the three A3R expressing cell lines (A3, A3-vYC, or A3-vYnL). No differences in functional responses were seen between the wild type A3R and the A3-vYC expressing cell line. The A3-vYnL cell line appeared to be functional but had a low expression.

Negative cooperativity has previously been seen between protomers of the A3R homo-oligomer by observing increased dissociation of a fluorescent agonist from the A3R in the presence of an orthosteric antagonist. Using perfusion microscopy, the ligand binding kinetics of two fluorescent antagonists, CA200645 and AV039, were probed on the three A3 expressing cell lines (Chapter 5). The BiFC tag did not affect the ligand binding kinetics of the fluorescent ligands on the A3-vYC and A3-vYnL cell lines when compared to the WT cell line. Negative cooperativity was observed in all three cell lines after the addition of an orthosteric antagonist, showing that the negative cooperativity is not exclusive to fluorescent agonists.

The effects of BiFC constrained dimerisation on the dissociation kinetics of AV039 from the A3R were subsequently investigated (Chapter 6). In cells expressing A3-A2A and A3-A3 BiFC dimers, the dissociation rate of AV039 decreased under infinite dilution, suggesting BiFC-induced positive cooperativity. A2AR-selective unlabelled ligands did not induce allosteric effects at the A3R while A3R-selective unlabelled ligands still induced negative allosteric effects in all conditions. The effects of co-expressing A¬3 and A2A receptors were also investigated by probing the association of AV039 at the A3R using BRET and no significant effects were observed. It appears that while the A3R and A2AR co-express in various cell types and were shown to dimerise this did not appear to affect the ligand binding kinetics of the A3R in these experiments.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Hill, S.J.
Briddon, S.J.
Keywords: GPCRs, Dimerisation, Adenosine receptors, Ligand binding kinetics, A3 Adenosine receptor, B-adrenoceptors, BiFC, Perfusion Microscopy, BRET
Subjects: Q Science > QP Physiology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 56636
Depositing User: Groenewoud, Nickolaj
Date Deposited: 04 Sep 2019 13:41
Last Modified: 19 Jul 2021 04:30

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