(188do) Contributions of the C-Terminus and Mutations to a2Ar Activity and Stability

G-protein coupled receptors (GPCRs) are seven-transmembrane domain membrane proteins that bind with extracellular ligands, activating intracellular downstream signaling cascades. This interaction makes GPCRs ideal drug targets for a number of conditions including pain, hypertension, and schizophrenia. A number of GPCRs have been crystallized, including the well-characterized adenosine A_2A receptor (A_2AR), to improve our understanding of the structure-function relationship and changes that occur during ligand binding. At present, all of the A_2AR crystal structures are of an A_2AR variant, A_2AΔ316R, that lacks 96 amino acids from the C-terminus. Removal of the C-terminus facilitates crystal structure formation due to the nature of the long, flexible tail. However, our lab has shown that the full-length C-terminus is necessary to activate downstream signaling cascades, suggesting that A_2AΔ316R does not behave identically to full-length A_2AR. Here, we use fluorescence anisotropy to characterize ligand binding of full-length A_2AR, A_2AΔ316R, and Rag23, a thermostable A_2AΔ316R variant with five point mutations. Fluorescence anisotropy was used to determine equilibrium affinity, kinetic rates, competitor affinity, and receptor stability at increasing temperatures. Competition binding experiments confirm that Rag23 has an increased affinity for agonist binding when compared to A_2AR; however, these binding characteristics do not translate into a statistically significant change in the K_d of the fluorescent ligand, FITC-APEC, perhaps due to the naturally low affinity of the ligand for receptor. Additionally, the removal of the C-terminus decreases the T_m of A_2AR, but the addition of the five thermostabilizing point mutations significantly increases the T_m such that the point mutations negate the destabilizing effect of the truncation.