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Oligomerization-Enhanced Receptor–Ligand Binding Revealed by Dual-Color Simultaneous Tracking on Living Cell Membranes

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journal contribution
posted on 19.08.2021, 18:33 by Jiqiang Li, Yanzhi Ding, Hengheng Liu, Hua He, Daoyong Yu, Xiaoqiang Wang, Xiaojuan Wang, Xiaoxi Yu, Baosheng Ge, Fang Huang
GPCR oligomerization plays a critical role in cellular signaling, yet the stoichiometry of the interactions between oligomers and binding ligands in living cells remains a longstanding challenge. Here, by developing a dual-color simultaneous tracking system based on a total internal reflection fluorescence microscope (TIRFM), the CCR5-CCL5 interactions are visualized and quantitatively assessed in real time. Results show that each oligomeric state of CCR5 could bind with CCL5 but with different binding affinities; CCR5 dimers have a 3.5-fold higher binding affinity than the monomers. The dimerization may cause an asymmetric conformational change which makes the first binding pocket have a 3.5-fold higher binding affinity and the second have only a half compared with the monomeric CCR5. This study is the first example to directly scrutinize the CCR5-CCL5 interactions at the single-molecule level on living cell membranes and will offer great potential for the interaction stoichiometry study of diverse surface proteins.