A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes.
Epidermal growth factor (EGF) receptor plays a pivotal role in a variety of cellular functions, such as proliferation, differentiation, and migration. To monitor the EGF receptor (EGFR) activity in living cells, we developed a probe for EGFR activity based on the principle of fluorescence resonance energy transfer (FRET). Previously, we developed a probe designated as Picchu (Phosphorylation indicator of the CrkII chimeric unit), which detects the tyrosine phosphorylation of the CrkII adaptor protein. We used a pair of synthetic amphipathic helixes, WinZipA2 and WinZipB1, to bind Picchu non-covalently to the carboxyl-terminus of the EGFR. Using this modified probe named Picchu-Z, the activity of EGFR was followed in EGF-stimulated Cos7 cells. We found that a high level of tyrosine phosphorylation of Picchu-Z probe remained after endocytosis until the point when the EGFR was translocated to the perinuclear region. These findings are in agreement with the previously reported "signaling endosome" model. Furthermore, by pulse stimulation with EGF and by acute ablation of EGFR activity with AG1478, it was suggested that the phosphorylation of Picchu-Z probe, and probably the phosphorylation of EGFR also, underwent a rapid equilibrium (tau(1/2) < 2 min) between the phosphorylated and dephosphorylated states in the presence of EGF.
Itoh
Reina E
RE
Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita-shi, Osaka 565-0871, Japan.
Kurokawa
Kazuo
K
Fujioka
Aki
A
Sharma
Alok
A
Mayer
Bruce J
BJ
Matsuda
Michiyuki
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D018715:Microscopy, Video; D015335:Molecular Probes; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D050737:Proto-Oncogene Proteins c-crk; D011799:Quinazolines; D014443:Tyrosine / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D015335:Molecular Probes; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011799:Quinazolines; D020032:Tyrphostins; C101044:RTKI cpd; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D017027:Protein Tyrosine Phosphatases