Conformational changes accompany phosphorylation of the epidermal growth factor receptor C-terminal domain.
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.
Lee
Nam Y
NY
Department of Pharmacology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109, USA.
Koland
John G
JG
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D020134:Catalytic Domain; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation
C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C409504:blue fluorescent protein, Aequorea victoria; D000255:Adenosine Triphosphate; D066246:ErbB Receptors