G-protein coupled receptors (GPCRs) are seven-transmembrane domain membrane proteins that are drug targets for a number of conditions including pain, hypertension, and schizophrenia. The adenosine A2A receptor (A2AR) is a class A GPCR that is expressed widely throughout the body and is a therapeutic target for many diseases including neurodegenerative, inflammatory, and heart diseases. A2AR is well characterized and has been crystallized in many active conformations to better understand the conformational changes that occur during ligand binding to the receptor. Presently, all crystal structures of A2AR have used a truncated variant, A2AΔ316R, that lacks 96 amino acids of the flexible C-terminus to facilitate crystallization. Our lab has shown that the full-length C-terminus was critical for activating downstream signaling cascades, indicating that A2AΔ316R does not maintain all active receptor functions of the full-length receptor. Here, we use ligand binding to further characterize full-length A2AR, A2AΔ316R, and Rag23, a thermostable A2AΔ316R variant with five point mutations designed to favor agonist binding. FITC-APEC, a fluorescent agonist, was used to quantify ligand binding via fluorescence anisotropy. We used ligand binding to determine equilibrium binding, stability at increasing temperatures, competition with agonists and antagonists, and kinetic rates and find that the C-terminus plays a significant role in providing stability to the native receptor.

Contribution of the C-terminus to A2AR activity and stability

255th National Meeting (2018)

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