Coiled-coil tag - Probe system for quick labeling of membrane receptors in living cells

The specific labeling of proteins in living cells using a genetically encodable tag and a small synthetic probe targeting the tag has been craved as an alternative to widely used larger fluorescent proteins. We describe a rapid method with a small tag (21 amino acids) for the fluorescence labeling of cell-surface receptors using a high affinity coiled-coil formation without metals or enzymes. The peptide probes K3 (KIAALKE)(3) and K4 (KIAALKE)(4) labeled with a fluorophore specifically stained the surface-exposed tag sequence E3 (EIAALEK)(3) attached to the N-terminus of the mouse-derived prostaglandin EP3 beta receptor in living cells (K-d = 64 and 6 nM for K3 and K4, respectively). The labeling was quick (< 1 min), nontoxic, and available even in culture medium without affecting receptor function. As an application of this tractable method, the agonist-induced internalization of the human-derived beta(2)-adrenergic receptor and epidermal growth factor receptor was successfully visualized.