Stoichiometry, kinetic and binding analysis of the interaction between epidermal growth factor (EGF) and the extracellular domain of the EGF receptor.
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:sEGFR 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 KD of 400-550 nM.
Domagala
T
T
The Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Victoria, Australia.
Konstantopoulos
N
N
Smyth
F
F
Jorissen
R N
RN
Fabri
L
L
Geleick
D
D
Lax
I
I
Schlessinger
J
J
Sawyer
W
W
Howlett
G J
GJ
Burgess
A W
AW
Nice
E C
EC
D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques; D016466:CHO Cells; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005347:Fibroblasts; D005454:Fluorescence Polarization; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D020349:Surface Plasmon Resonance; D014461:Ultracentrifugation
D004815:Epidermal Growth Factor; D066246:ErbB Receptors