Effects of the adenosine A2A receptor C-terminus on ligand binding, stability, and downstream signaling
G protein-coupled receptors (GPCRs) are the largest family of proteins in humans and are expressed widely throughout the body. GPCRs consist of seven-transmembrane helices that bind extracellular ligands to initiate intracellular downstream signaling via interaction with G proteins, and function in many short and long-term responses in the body, including taste, immune function, and sugar sensing. Extracellular binding and the coupled downstream signaling pathway means that GPCRs are ideal drug targets for many diseases, making them of great interest to the pharmaceutical industry. Some GPCRs have been crystallized in an effort to better elucidate the structure-function relationship to aid in the design of novel therapeutics. The adenosine A2A receptor (A2AR) is a GPCR that has been crystallized bound to agonist, antagonist, and G protein. Although these crystal structures are informative in regards to A2AR structure when associated with binding partners, all current crystal structures truncate nearly 100 amino acids of the C-terminus. As a crystallization strategy, this truncation makes sense considering the C-terminus is long and unstructured. However, truncating roughly 25% of the protein, as well as making other point mutations calls into question the authenticity of the crystal structures in reflecting functional receptor and thus their potential value for therapeutic design. Beyond structural studies, biophysical characterization of drug binding to receptors in vitro to predict efficacy in vivo has shifted away from measures of affinity and selectivity and towards determination of kinetic rates. Kinetic rate constants in combination with affinity and drug residence time are thought to be better predictors of drug behavior in vivo. For these reasons, this thesis focuses on experiments to characterize A2AR kinetic rate constants. Previously, our lab showed that truncating the A2AR C-terminus reduced downstream cAMP signaling in mammalian cells, although where the effect on the signaling pathway occurred was not determined. Here, we report that truncation of the C-terminus ablates receptor association to Gαs, the first step in signaling. In this work, A2AR ligand binding kinetics, stability, and association to Gαs are characterized to better delineate the importance of interactions between receptor and stimuli in a way that is impactful to drug design.