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High-Throughput Analysis of Ligand-Induced Internalization of β2-Adrenoceptors Using the Coiled-Coil Tag–Probe Method

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journal contribution
posted on 07.02.2012, 00:00 by Yuki Takeda, Yoshiaki Yano, Katsumi Matsuzaki
Receptor internalization is a useful indicator of the activity of ligands. The N-terminus of the β2-adrenergic receptor expressed on the cell surface was labeled with fluorophores using a novel coiled-coil labeling system. Endocytosis of the receptors was automatically detected using a fluorescence image analyzer by evaluating (1) translocation of the receptor from cell-surface to intracellular regions and (2) acidification in endosomes. Both parameters increased upon agonist stimulation in a dose-dependent manner. The extent of endocytosis was significantly dependent on the agonist used, indicating the presence of a biased signaling for endocytosis. The receptor antagonists can also be screened by competitive inhibition of agonist-induced endocytosis. The image analysis approach has proven to be useful for high-throughput characterization and screening of GPCR ligands.