Structural dynamics of the A 2A adenosine receptor revealed by single‐molecule FRET

Abstract

G-protein-coupled receptors (GPCRs) constitute the largest superfamily of membrane proteins in humans, which mediate critical physiological processes, such as neurotransmission, homeostasis, inflammation, reproduction, olfaction, vision, taste, and others. Over 30% of all FDA-approved drugs target GPCRs. The A2A adenosine receptor (A2AAR) is one of the best studied GPCRs. A2AAR is expressed in many organs and tissues, it regulates the cardiovascular tonus and promotes healing of inflammation-induced injuries by suppressing immune cells. In the brain, A2AAR modulates dopamine and glutamate neurotransmission. A2AAR is a promising target for drugs against insomnia, chronic pain, depression, Parkinson's disease, and cancer. More than 50 X-Ray or CryoEM structures provide high-resolution structural snapshots of A2AAR, but current understanding of the A2AAR structure-function relationships remains critically incomplete without detailed knowledge of its conformational dynamics. Here we applied single-molecule FRET (smFRET) to investigate the conformational dynamics of A2AAR in lipid nanodiscs. Using smFRET, we tracked the relative movements of two fluorescent dyes attached to the intracellular tip of the transmembrane helix TM6 and to the C-terminal intracellular helix H8 of A2AAR. Our measurements revealed sub-millisecond conformational dynamics of A2AAR. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptor's constitutive activity. For the agonist-bound A2AAR, we detected faster (390±80 µs) ligand efficacy-dependent dynamics. This work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.