Understanding the Conformational Dynamics of the A2A Adenosine Receptor by Single-Molecule Fluorescence

G protein-coupled receptors (GPCRs) are one of the largest transmembrane receptor families. The wide expression and diverse signal transduction functions make them an ideal target for characteristic drug design. The A_2A adenosine receptor (A_2AAR), a representative of class A GPCRs, has been extensively used to reveal the structural information of class A GPCRs. As a purinergic receptor, A_2AAR is activated by adenosine in the human body, triggering cyclic adenosine monophosphate (cAMP) and immunosuppression pathways. A_2AARs are highly expressed in the brain and are crucial in regulating glutamate and dopamine release. As a result, the A_2AAR became a potential therapeutic target for treating neurodegenerative diseases. In recent years, A_2AARs have been identified in specific cardiac cells where they regulate myocardial oxygen consumption and coronary blood flow. Accordingly, the A_2AAR-targeted drug design and optimization has become increasingly valued. However, the dynamic nature of A_2AAR structures poses challenges in drug design. Therefore, the study of structural dynamics provides valuable information for ligand identification and drug prediction. In this work, we used total internal reflection fluorescence (TIRF) imaging to visualize the real-time dynamics of ligand binding and activation of the A_2AAR at the molecular level. We observed reversible and sequential dynamic behaviors in A_2AAR transmembrane helix VII (TM VII). Furthermore, the constitutively activating mutations (CAMs) didn’t show the ligand-specific conformational dynamics like the wild type A_2AAR, but CAMs and the Mini-Gs shifted conformational equilibrium towards the intermediate state, while D52N, a constitutively inactivating mutation, had the opposite effect. We also successfully stabilized the A_2AARs in native membrane environment and incorporated them into nanodiscs using the polymers styrene maleic acid (SMA) and di-isobutylene maleic acid (DIBMA). This allowed us to observe more transition events between active-like conformational states between transmembrane helices IV and VI upon agonist and Mini-G_S binding. Overall, this work has filled the gap in understanding A_2AAR structural dynamics at the single-molecule level, emphasizing the significance of intermediate states in A_2AAR dynamics and paving the way for future A_2AAR-targeted drug design strategies.