Conformational changes accompany phosphorylation of the epidermal growth factor receptor C-terminal domain

Protein Sci. 2005 Nov;14(11):2793-803. doi: 10.1110/ps.051630305. Epub 2005 Sep 30.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / chemistry
  • Catalytic Domain
  • ErbB Receptors / chemistry*
  • ErbB Receptors / metabolism
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes / chemistry
  • Light
  • Luminescent Proteins / chemistry
  • Phosphorylation
  • Protein Structure, Tertiary
  • Scattering, Radiation


  • 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate
  • Fluorescent Dyes
  • Luminescent Proteins
  • blue fluorescent protein, Aequorea victoria
  • Adenosine Triphosphate
  • ErbB Receptors