Coiled-coil tag--probe system for quick labeling of membrane receptors in living cell.
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 receptor in living cells (Kd = 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 2-adrenergic receptor and epidermal growth factor receptor was successfully visualized.
Yoshiaki
Y
Yano
Akiko
A
Yano
Shinya
S
Oishi
Yukihiko
Y
Sugimoto
Gozoh
G
Tsujimoto
Nobutaka
N
Fujii
Katsumi
K
Matsuzaki
D000818:Animals; D016466:CHO Cells / Q000166:cytology* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D066246:ErbB Receptors / Q000032:analysis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D051379:Mice; D018341:Receptors, Adrenergic, alpha-2 / Q000032:analysis / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D018078:Receptors, Prostaglandin E / Q000032:analysis / Q000378:metabolism; D058308:Receptors, Prostaglandin E, EP3 Subtype; D013194:Staining and Labeling; D013997:Time Factors
D005456:Fluorescent Dyes; C545647:PTGER3 protein, human; C545648:Ptger3 protein, mouse; D018341:Receptors, Adrenergic, alpha-2; D011956:Receptors, Cell Surface; D018078:Receptors, Prostaglandin E; D058308:Receptors, Prostaglandin E, EP3 Subtype; D066246:ErbB Receptors