High-throughput analysis of ligand-induced internalization of β2-adrenoceptors using the coiled-coil tag-probe method.

Receptor internalization is a useful indicator of the activity of ligands. The N-terminus of the β(2)-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.