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Department of Pharmacology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242-1109, USA
(RECEIVED June 4, 2005; FINAL REVISION August 6, 2005; ACCEPTED August 6, 2005)
The precise regulation of epidermal growth factor receptor (EGFR) signaling is crucial to its function in cellular growth control. Various studies have suggested that the C-terminal phosphorylation domain, itself a substrate for the EGFR kinase activity, exerts a regulatory influence upon it, although the molecular mechanism for this regulation is unknown. The fluorescence resonance energy transfer (FRET) technique was employed to examine how C-terminal domain conformational changes in the context of receptor activation and autophosphorylation might regulate EGFR enzymatic activity. A novel FRET reporter system was devised in which recombinant purified EGFR intracellular domain (ICD) proteins of varying C-terminal lengths were site-specifically labeled at their extreme C termini with blue fluorescent protein (BFP) and a fluorescent nucleotide analog, 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP), binding at their active sites. This novel BFP/TNP-ATP FRET pair demonstrated efficient energy transfer as evidenced by appreciable BFP-donor quenching by bound TNP-ATP. In particular, a marked reduction in energy transfer was observed for the full-length BFP-labeled EGFR-ICD protein upon phosphorylation, likely reflecting its movement away from the active site. The estimated distances from the BFP module to the TNP-ATP-occupied active site for the full-length and C-terminally truncated proteins also reveal the possible folding geometry of this domain with respect to the kinase core. The present studies demonstrate the first use of BFP/TNP-ATP as a FRET reporter system. Furthermore, the results described here provide biophysical evidence for phosphorylation-dependent conformational changes in the C-terminal phosphorylation domain and its likely interaction with the kinase core.
Keywords: protein tyrosine kinase; ErbB; HER; FRET
Abbreviations: PTK, protein tyrosine kinase • EGFR, epidermal growth factor receptor • SH2, Src homology-2 • A-loop, activation loop • ICD, intracellular domain • FRET, fluorescence resonance energy transfer • 
976, C-terminal truncation of EGFR at residue 976 • 1022, C-terminal truncation of EGFR at residue 1022 • TNP-ATP, 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate • BFP, blue fluorescence protein • EGFR-CT, C-terminal domain of EGFR
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051630305.
Reprint requests to: John G. Koland, Department of Pharmacology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109, USA; e-mail: john-koland{at}uiowa.edu; fax: (319) 335-8930.
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