title: Fluorescence ratiometric detection of ligand-induced receptor internalization using extracellular coiled-coil tag-probe labeling.
abstract: We report a new method for the detection of ligand-induced receptor internalization by fluorescence ratiometric imaging of pH in endosomes in combination with a recently developed posttranslational labeling system based on the formation of a heterodimeric coiled-coil structure. The N-terminus of the β2-adrenergic receptor expressed on the cell surface was doubly labeled with pH-sensitive fluorescein and pH-insensitive tetramethylrhodamine. A significant increase in the tetramethylrhodamine-to-fluorescein fluorescence intensity ratio was observed after incubation with agonists in a concentration-dependent manner. This simple and accurate method of detecting the agonistic activity of receptors will be useful for high-throughput screening of drug candidates.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.
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 / Q000378:metabolism; D058672:Adrenergic beta-2 Receptor Antagonists / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D008024:Ligands; D010455:Peptides; D018343:Receptors, Adrenergic, beta-2; D011956:Receptors, Cell Surface