Real-time visualization of ligand-specific conformational dynamics of GPCR C-terminal domain in living cells

The intrinsically disordered C-terminal domain (CTD) serves as a critical regulatory element in GPCR1-3. However, directly interrogating the CTD responses to different ligands is challenging due to its high flexibility, which renders it invisible to conventional structural biology techniques. To address the challenge, we developed a live-cell fluorescence imaging strategy that enables real-time visualization of CTD conformational transitions under physiological conditions. Our dual-mode approach integrates single-cell fluorescence lifetime imaging microscopy (FLIM) with single-molecule total internal reflection fluorescence microscopy (TIRFM), facilitating multi-scale analysis. Our data revealed that the dynamics of both full-length and truncated CTDs in the {beta}2-adrenergic receptors are ligand-specific. By bridging single-molecule dynamics with ensemble cellular responses, our method uncovered previously inaccessible molecular mechanisms underlying receptor activation. This advance not only elucidates how GPCRs transduce ligand binding into functional outcomes but also establishes a versatile platform for drug discovery, enabling rapid assessment of ligand efficacy and receptor activity in physiological contexts.