G protein-coupled receptors (GPCRs) are membrane proteins responsible for myriad physiological functions. Around a third of drugs on the market already target GPCRs. However, the emphasis on developing GPCR assays to screen compound libraries for potential GPCR-interacting drugs is still very strong. One such cell-based assay was developed at GlaxoSmithKline (GSK) by Dowell and Brown [1, 2]. This assay uses a cheap, rapidly growing unicellular organism – baker’s yeast. In this organism the pheromone signalling pathway is one of only two GPCR-activated pathways. Yeast engineering done at GSK in 2000 produced MMY strains lacking the native yeast Ste2 GPCR and other key proteins, together with the integration of chimeric yeast/mammalian G protein alpha subunits thereby allowing mammalian GPCRs to couple to the yeast signalling machinery . A significant limitation of the assay has been that some pharmacologically-important receptors cannot be tested using the current MMY strains and protocols. The aim of this study was to optimise the functional expression of adrenergic receptors that had previously been difficult to express and/or characterise pharmacologically in these yeast strains. The work described here examined both the MMY strains themselves and the expression constructs, asking the question how these features might contribute to a robust assay readout for two GPCR classes; the α- and β-adrenoceptors. The origin of the instability of some of the MMY strains was traced to the experimental strategy originally used to delete the URA3 marker gene and a stable MMY strain was generated as a proof-of-principle. A systematic comparison between different expression vector designs identified that the addition of the mating factor α (MF-α) leader sequence to the amino-terminus of the β1- and β2-adrenoceptors is required to produce a pharmacological response in the yeast assay. Notably, this permitted reproducible functional expression of the β2-adrenoceptor, which had not been previously possible at GSK, despite being published in the scientific literature. These improvements allowed for the pharmacological characterisation of a panel of interacting β-AR ligands, yielding data that has shed new light on ligand bias. Finally, a cholesterol-expressing yeast engineering strategy was designed to specifically investigate the role of plasma membrane composition in GPCR functional studies. The insights gained from the experiments described this thesis should allow for improved testing of other GPCRs in the drug development pipeline.
|Date of Award||1 Mar 2018|
|Supervisor||Roslyn Bill (Supervisor) & Simon Dowell (Supervisor)|