Visualizing Receptor Internalization using a New Fluorescence Labeling Method

Abstract

The specific labeling of proteins in living cells using a genetically encodable tag and a small synthetic probe targeted to the tag has several advantages over widely used larger fluorescent proteins. We recently developed a quick labeling method using a high-affinity heterodimeric coiled-coil formation between the E3 tag (EIAALEK)3 attached to the target protein and the Kn probes (KIAALKE)n (n = 3 or 4) labeled with a fluorophore [1]. The size of the heterodimer (5-6 kDa) is significantly smaller than that of fluorescent proteins (∼27 kDa), minimizing perturbation to receptor function. The labeling is cell-surface specific therefore suitable to detect receptor internalization. The agonist-induced internalization of β2-adrenergic receptor (β2AR) and EGF receptor transiently expressed in Chinese hamster ovary (CHO) cells can be clearly visualized using the K4 probe. Taking the advantage of reversible labeling of the K3 probe, cell-surface and internalized β2ARs can be labeled with different fluorophores. In this study, fluorescence ratiometric detection of receptor internalization for a high throughput screening was examined using β2AR stably expressed in CHO cells. The receptors were doubly labeled with pH-sensitive fluorescein (FL) (pKa∼6.5) and pH-insensitive tetramethylrhodamine (TMR). FL fluorescence was attenuated following internalization after agonist stimulation (isoproterenol, 30 min) because of a lower pH in endosomes (pH 5-6), whereas TMR fluorescence was unchanged. An increase in the intensity ratio TMR/FL from 1 to ∼3 was observed in endosomes. Thus, the fluorescence ratio-imaging method was found to be useful for evaluation of receptor internalization.[1] Yano, Y., Yano, A., Oishi, S., Sugimoto, Y., Tsujimoto, G., Fujii, N., Matsuzaki, K. (2008). ACS. Chem. Biol. 3, 341-345.