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. 2008 Jan 8;105(1):112-7.
doi: 10.1073/pnas.0707080105. Epub 2007 Dec 28.

Heterogeneity in EGF-binding affinities arises from negative cooperativity in an aggregating system

Jennifer L Macdonald  1 , Linda J Pike
Affiliations

Heterogeneity in EGF-binding affinities arises from negative cooperativity in an aggregating system

Jennifer L Macdonald et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A. 2008 Jul 1;105(26):9129

Abstract

Scatchard analysis of the binding of EGF to its receptor yields concave up plots that indicate the presence of two classes of binding sites. However, how two independent classes of sites arise from the expression of a single EGF receptor protein has never been adequately explained. Using a new analytical approach involving the simultaneous fitting of binding isotherms from cells expressing increasing levels of EGF receptors, we show that (125)I-EGF-binding data can be completely explained by a model involving negative cooperativity in an aggregating system. This approach provides an experimentally determined value for the monomer-dimer equilibrium constant, which, for wild-type EGF receptors, corresponds to approximately 50,000 receptors per cell. Therefore, changes in receptor expression within the physiological range can modulate the outcome of a signaling stimulus. Analysis of the L680N-EGF receptor mutant, in which the formation of asymmetric kinase domain dimers is blocked, indicates that the kinase dimers make a substantial energetic contribution to the ligand-independent association of EGF receptor monomers, but are not necessary for negative cooperativity. The model accurately predicts the behavior of receptor mutants, such as the dimerization-defective Y246D-EGF receptor, which exhibit a single class of binding sites and provides a framework for understanding secondary dimer formation and lateral signaling in the EGF receptor family.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Model of EGF binding in an aggregating system. Circles indicate receptor subunits. The equilibrium association constants are written above or beside the reaction to which they apply. E, EGF molecule.
Fig. 2.
Fig. 2.
125I-EGF-binding isotherms generated from two different models. 125I-EGF saturation-binding data were fit to a binding isotherm equation invoking two independent classes of sites (solid black line) or to the aggregating system shown in Fig. 1 (dashed line). The solid gray line shows the shape of a binding isotherm corresponding to a single class of sites. (Inset) Scatchard plots corresponding to the one-site (gray line) and two-site (black line) fits.
Fig. 3.
Fig. 3.
Binding of EGF to cells expressing increasing levels of wild-type EGF receptors. CHO-K1 tet-on EGFR cells were induced to express EGF receptors by using increasing doses of doxycycline. 125I-EGF-binding isotherms were generated from each set of cells, and all six isotherms were globally fit to Eq. 1, with only the value of R0 varying among curves. Data points represent the mean ± SD of triplicate determinations. Solid lines represent the fitted curve through the data points of the same color.
Fig. 4.
Fig. 4.
Dissociation of 125I-EGF from CHO cells expressing EGF receptors. CHO cells were incubated overnight with 10 nM 125I-EGF. Binding medium was removed, cells were washed, and the medium was replaced with Hams' F12 containing 25 mM Hepes (pH 7.0) and 2 mg/ml BSA in the absence (open circles) or presence (filled circles) of 10 nM unlabeled EGF. Cultures were incubated for the indicated times at 4°C, and residual EGF binding was determined as described in Materials and Methods. Data points represent the mean ± SD of triplicate determinations. Solid lines show the fit to a double exponential model by using GraphPad Prism 4.0.
Fig. 5.
Fig. 5.
Binding of 125I-EGF to EGF receptor mutants. (A) 125I-EGF-binding isotherms for cells expressing three different levels of Y246D-EGF receptors. All curves fit to a single-site model with a shared K1 of 5.8 ± 0.1 × 108 with r2 = 0.99. (B) 125I-EGF-binding isotherms for cells expressing six different levels of L680N-EGF receptors. K11, 3.6 ± 0.2 × 108; K21, 4.8 ± 2.3 × 1010; K22, 7.9 ± 1.1 × 108; L20, 6.1 ± 3.3 × 108. The r2 value for the global fit was 0.99. Points represent the mean ± SD of triplicate determinations.
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