title: High-throughput analysis of ligand-induced internalization of β2-adrenoceptors using the coiled-coil tag-probe method.
abstract: 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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Takeda
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Yano
authors.forename: Katsumi
authors.initials: K
authors.lastname: Matsuzaki
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists / Q000737:chemistry; D058672:Adrenergic beta-2 Receptor Antagonists / Q000737:chemistry; D000818:Animals; D001331:Automation; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D057166:High-Throughput Screening Assays; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2