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Keen, Alastair C. (2021) Kinetic studies of dopamine D2 receptor molecular pharmacology. PhD thesis, University of Nottingham.

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Abstract

The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is the primary target of drugs treating the symptoms of Parkinson’s disease and schizophrenia. However, drugs acting at the D2R to manage these diseases often display efficacy for only a subset of their symptoms and have poor side effect profiles. Therefore, it is desirable to rationally design drugs that better manage disease symptoms and reduce side effects. This would be greatly aided by gaining a detailed understanding of the kinetic aspects of D2R ligand binding, signalling, regulation and trafficking.

Differences in binding kinetics at the D2R results in varying side effect profiles between antipsychotics. In chapter 2, a time resolved-fluorescence resonance energy transfer competition kinetic ligand binding assay is optimised at the D2R. The assay is used in combination with D2R mutants to determine the contribution of selected residues in the extracellular regions of the D2R in modulating binding kinetic association and dissociation rates. Findings showed that different residues in this region are important determinants of binding kinetics in a ligand-dependent manner.

Some agonists with slow dissociation rates have been shown to display apparent biased agonism at the D2R. In chapter 3, it is investigated whether the length of time an agonist binds the D2R influences observations of biased agonism. Within the selected panel of ligands, for which both binding kinetic rates and functional effects were determined, no clear relationship between agonist dissociation rate and apparent biased agonism could be established.

D2R G protein signalling is regulated through phosphorylation by G protein receptor kinases (GRKs). In chapter 4, antibodies specific for GRK2/3 phosphorylation sites on the D2R were generated and characterised. A GRK2/3 phosphorylation site within intracellular loop 3 was identified that is phosphorylated on agonist activation of the D2R. Phosphorylation of this site predicts arrestin recruitment. Measurements of D2R phosphorylation were included with other measurements of G protein activation and receptor regulation to profile selected D2R agonists.

The D2R can couple pleiotropically to G proteins of the Gαio subfamily. In chapter 5 the kinetics of D2R mediated activation of individual Gαi/o protein subtypes was investigated. Increases in agonist potency were observed when the D2R activated Gαz. This was shown to be dependent on the slow guanosine triphosphate (GTP) hydrolysis rate of Gαz by either mutation of serine 42 within the GTP binding site or co-expression with regulator of G protein signalling 20.

Investigating GPCR and D2R biased agonism in the relevant cell type has been challenging due to the lack of molecular tools. A useful method for interrogating GPCR signalling functions is using bacterially derived toxins, such as pertussis toxin, to inhibit their coupling and then evaluate the downstream changes. In chapter 6 we developed a new pertussis toxin-like protein tool that can inhibit all of the Gαi/o subfamily, including Gαz. Ga subunits that are insensitive to the toxin were characterised to serve as tools in combination with the toxin.

Finally, chapter 7 discusses the key implications of the findings in the context of the current literature and future research recommendations.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Scott, Daniel J. Lane, J. Robert Charlton, Steven J.
Subjects:	Q Science > QP Physiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	64380
Depositing User:	Keen, Alastair
Date Deposited:	31 Jul 2021 04:40
Last Modified:	31 Jul 2021 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/64380

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