Ligand Binding Kinetics to Evaluate Function and Stability of A_2AR in Nanodiscs

G-Protein coupled receptors (GPCRs) represent one of the largest classes of therapeutic targets. However, developing successful therapeutics to target GPCRs is a challenging endeavor with many molecules failing during in vivo clinical trials due to a lack of efficacy. The in vitro identification of drug targeted residence time (1/k_off) has been suggested to improve prediction of in vivo success. Here, a ligand binding assay using fluorescence anisotropy was implemented to successfully determine on-rates (k_on) and off-rates (k_off) of labeled and unlabeled ligands binding to the adenosine A_2A receptor (A_2AR) purified into nanodiscs (A_2AR-NDs). The kinetic assay was used to determine the optimal storage conditions of A_2AR-NDs, where they were found to be stable for more than six months at -80^oC. The binding assay was implemented to further understand receptor function by determining the effects of charged lipids on agonist binding kinetics, how sodium levels allosterically modulate A_2AR function, and how A_2AR protonation affects agonist binding.