Stoichiometry, kinetic and binding analysis of the interaction between epidermal growth factor (EGF) and the extracellular domain of the EGF receptor

Teresa Domagala, Nicky Konstantopoulos, Fiona Smyth, Robert N. Jorissen, Louis Fabri, Detlef Geleick, Irit Lax, Joseph Schlessinger, William Sawyer, Geoffrey J. Howlett, Antony W. Burgess, Edouard C. Nice

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Abstract

The kinetics, binding equilibria and stoichiometry of the interaction between epidermal growth factor and the soluble extracellular domain of the epidermal growth factor receptor (sEGFR), produced in CHO cells using a bioreactor, have been studied by three methods: analytical ultracentrifugation, biosensor analysis using surface plasmon resonance detection (BIAcore 2000) and fluorescence anisotropy. These studies were performed with an sEGFR preparation purified in the absence of detergent using a mild two step chromatographic procedure employing anion exchange and size exclusion HPLC. The fluorescence anisotropy and analytical ultracentrifugation data indicated a 1:1 molar binding ratio between EGF and the sEGFR. Analytical ultracentrifugation further indicated that the complex comprised 2EGF:2sEGFR, consistent with the model proposed recently by Lemmon et al. (1997). Global analysis of the BIAcore binding data showed that a simple Langmuirian interaction does not adequately describe the EGF:sEGPR interaction and that more complex interaction mechanisms are operative. Furthermore, analysis of solution binding data using either fluorescence anisotropy or the biosensor, to determine directly the concentration of free sEGFR in solution competition experiments, yielded Scatchard plots which were biphasic and Hill coefficients of less than unity, Taken together our data indicate that in solution there are two sEGFR populations; one which binds EGF with a KD of 2-20 nM and the other with a K(D) of 400-550 nM.

Original languageEnglish
Pages (from-to)11-29
Number of pages19
JournalGrowth Factors
Volume18
Issue number1
DOIs
Publication statusPublished - 1 Jan 2000
Externally publishedYes

Keywords

  • BIAcore
  • Binding interaction
  • EGF
  • EGFR
  • Fluorescence anisotropy
  • Kinetic analysis

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