Engineering and Investigating Structural Features of Adenosine A2a Receptor Oligomers

G protein-coupled receptors (GPCRs) and their oligomers are promising therapeutic targets for the treatment of several diseases due to their key role in cell signaling. However, design of structurally inspired pharmaceuticals to target specific oligomers has been hindered by experimental difficulties such as obtaining sufficient quantities of functional protein for structural characterization, and applying biophysical techniques for protein structure determination. I sought to address these limitations by developing a set of tools to structurally characterize homo-oligomers formed by the human adenosine A2a receptor (A2aR), a class A GPCR that has several documented homo- and hetero-meric associations that are targets for treatment of nervous system disorders, as well as cancer. Toward this goal, I developed an expression system in the yeast Saccharomyces cerevisiae to produce and purify he full-length, wild-type human A2aR and several A2aR variants for oligomer separation by size exclusion chromatography (SEC). By combining SEC with multi-angle light scattering (MALS), I verified that two of the three separate peaks observed during chromatographic separation corresponded to the predicted molecular weight of an A2aR monomer and dimer. Efficient separation of these two oligomer species is critical for in vitro characterization, and enables the study of structure-function relationships between the monomer and dimer species.;Additionally, I engineered and screened a six-site saturation mutagenesis library of A2aR variants to identify a properly folded A2aR construct void of free cysteines to facilitate biophysical characterization by electron paramagnetic resonance (EPR). To accomplish this, fluorescence activated cell sorting (FACS) was used to screen the A2aR variant library, guided by thermodynamic predictive modeling. Three rounds of FACS sorting enriched a number of variants with most or all transmembrane cysteines in the receptor removed. Modeling was applied to selected variants to predict which variants would effectively be "EPR dark", such that no spin label attach to any endogenous cysteines in the receptor. This experimental pipeline facilitates the application electron paramagnetic resonance (EPR), that has the power elucidate structural dynamics in functionally important yet "difficult" regions of proteins by attaching a spin label to cysteines introduced at sites of interest.;One such uncharacterized region within A2aR is its long, intrinsically disordered C-terminus, for which there is very little structural information available. I investigated the role of A2aR's C-terminus on receptor structure and function by interrogating subtle changes to the structural dynamics of the distal C-terminal C394 residue in the presence of different A2aR ligands. In particular, I focused on the role that the C-terminus plays in A2aR oligomerization in vitro by determining the SEC profile for common C-terminal truncations in literature, A2a-Delta(317-412)R and A2a-Delta(335-412). These results suggested that the distal portion of the C-terminus experiences subtle changes in its physical environment upon ligand binding, which could lend insight toward the mechanisms by which the C-terminus regulates its interactions with other cellular proteins, suggesting that this region is important for helping to stabilize the dimer state in vitro..;Overall, GPCRs have historically been popular targets for commercial therapeutics. With increasing reports that GPCRs tune function by forming oligomer contacts with other receptors, there is considerable motivation to elucidate oligomer structure of pharmaceutically "interesting" targets. This work represents a robust strategy that can be applied to the production and study of other GPCRs to gain a better understanding of the role of protein oligomerization and their structure, as well as a strategy to better understand the function of intrinsically disordered, flexible protein regions.