G protein-coupled receptors (GPCRs) comprise a family of integral membrane proteins that mediate eukaryotic cells' responses to a wide array of extracellular signals. As a result of their ligand specificity, sensitivity, and capacity for signal transduction, GPCRs have great potential as biosensors for toxic chemical and biological reagents in microbes. Despite their inherent advantages, many GPCRs are difficult to express functionally and in high numbers within heterologous hosts such as yeast. Thus, there is a need to engineer functionally expressed GPCRs to bind ligands of interest with high affinities and specificities. Towards this end, we engineered variants of the human adenosine A2A receptor (hA2AR) with improved binding properties towards a target ligand. Protein design strategies were guided by high-resolution crystal structures of hA2AR bound to various agonists and antagonists. A fluorescent ligand-binding assay was used in concert with fluorescence-activated cell sorting (FACS) as a high-throughput screening methodology to isolate yeast cells expressing desirable hA2AR mutants. A 3.5-fold increase in cellular mean fluorescence intensity and convergence of mutated residues towards a consensus sequence were observed after four rounds of sorting. The use of this platform to further evolve and isolate improved hA2AR variants is expected to generate mutants with even greater binding affinity and specificity towards ligands of interest. Additionally, the choice of expression vector and concomitant copy number has been shown to influence the yield of hA2AR in yeast. These results are envisioned to improve screening strategies by increasing the dynamic range of signal obtained in activity assays.

Developing strategies to engineer G protein-coupled receptor binding properties

253rd National Meeting (2017)

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