A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes
- Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita-shi, Osaka 565-0871 (Japan)
- Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030-3301 (United States)
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} {sub 1/2} < 2 min) between the phosphorylated and dephosphorylated states in the presence of EGF.
- OSTI ID:
- 20717620
- Journal Information:
- Experimental Cell Research, Vol. 307, Issue 1; Other Information: DOI: 10.1016/j.yexcr.2005.02.026; PII: S0014-4827(05)00083-2; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); ISSN 0014-4827
- Country of Publication:
- United States
- Language:
- English
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