Fluorescence ratiometric detection of ligand-induced receptor internalization using extracellular coiled-coil
Yoshiaki Yano1, Katsumi Matsuzaki
1Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.
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.
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