Allosteric regulation of epidermal growth factor (EGF) receptor ligand binding by tyrosine kinase inhibitors

Abstract

The epidermal growth factor (EGF) receptor is a classical receptor tyrosine kinase with an extracellular ligand-binding domain and an intracellular kinase domain. Mutations in the EGF receptor have been shown to drive uncontrolled cell growth and are associated with a number of different tumors. Two different types of ATP-competitive EGF receptor tyrosine kinase inhibitors have been identified that bind to either the active (type I) or inactive (type II) conformation of the kinase domain. Despite the fact that both types of inhibitors block tyrosine kinase activity, they exhibit differential efficacies in different tumor types. Here, we show that in addition to inhibiting kinase activity, these inhibitors allosterically modulate ligand binding. Our data suggest that the conformations of the EGF receptor extracellular domain and intracellular kinase domain are coupled and that these conformations exist in equilibrium. Allosteric regulators, such as the small-molecule tyrosine kinase inhibitors, as well as mutations in the EGF receptor itself, shift the conformational equilibrium among the active and inactive species, leading to changes in EGF receptor-binding affinity. Our studies also reveal unexpected positive cooperativity between EGF receptor subunits in dimers formed in the presence of type II inhibitors. These findings indicate that there is strong functional coupling between the intracellular and extracellular domains of this receptor. Such coupling may impact the therapeutic synergy between small-molecule tyrosine kinase inhibitors and monoclonal antibodies in vivo.

Keywords: Ligand binding; epidermal growth factor (EGF); epidermal growth factor receptor (EGFR); erlotinib; growth factor; inhibitor; lapatinib; tyrosine-protein kinase (tyrosine kinase).

Figure 1.

Effect of EGF receptor tyrosine kinase inhibitors on EGF affinity. CHO cells expressing the WT EGF receptor were treated for 30 min at 37 °C without or with the indicated inhibitor prior to binding of ¹²⁵I-EGF overnight at 4 °C. The indicated inhibitor was also included in the binding incubation medium. Points represent the mean ± S.D. of triplicate determinations in a single experiment, which was repeated a minimum of three times.

Figure 2.

Effect of erlotinib and lapatinib on EGF affinity in EGF receptor mutants. CHO cells expressing the D813N-EGF receptor (A and B), the K721A-EGF receptor (C and D), the c′973-EGF receptor (E and F), or the Y9F-EGF receptor (G and H) were treated for 30 min at 37 °C without (black lines) or with erlotinib (green lines) or lapatinib (red lines) prior to binding of ¹²⁵I-EGF overnight at 4 °C. The indicated inhibitor was also included in the binding incubation medium. Points represent the mean ± S.D. of triplicate determinations in a single experiment, which was repeated a minimum of three times.

Figure 3.

¹²⁵I-EGF competition-binding curves of WT and mutant EGF receptors treated without or with erlotinib or lapatinib. CHO cells expressing the indicated EGF receptors were treated without (black lines) or with erlotinib (green lines) or lapatinib (red lines) prior to and during incubation with labeled EGF. Increasing concentrations of unlabeled EGF were added to different wells. The data are normalized to control binding. Control binding represents the number of counts of ¹²⁵I-EGF bound by the untreated cells in the absence of any additional unlabeled EGF. Points represent the mean ± S.D. of triplicate determinations in a single experiment, which was repeated a minimum of three times.

Figure 4.

Effect of erlotinib and lapatinib on EGF receptors with mutations in subunit–subunit interfaces. CHO cells expressing the Y246D-EGF receptor (A and B), the D563A,H566A,K585A-EGF receptor (C and D), the T548R,N554R-EGF receptor (E and F), or the V924R-EGF receptor (G and H) were treated for 30 min at 37 °C without (black lines) or with erlotinib (green lines) or lapatinib (red lines) prior to binding of ¹²⁵I-EGF overnight at 4 °C. The indicated inhibitor was also included in the binding incubation medium. Points represent the mean ± S.D. of triplicate determinations in a single experiment, which was repeated a minimum of two times.

Figure 5.

¹²⁵I-EGF competition-binding curves of EGF receptors with mutations in subunit–subunit interfaces treated without or with erlotinib or lapatinib. CHO cells expressing the indicated EGF receptors were treated without (black lines) or with erlotinib (green lines) or lapatinib (red lines) prior to and during incubation with labeled EGF. Increasing concentrations of unlabeled EGF were added to different wells. The data are normalized to control binding. Control binding represents the number of counts of ¹²⁵I-EGF bound by the untreated cells in the absence of any additional unlabeled EGF. Points represent the mean ± S.D. of triplicate determinations in a single experiment, which was repeated a minimum of two times.

Figure 6.

Saturation-binding curves and ¹²⁵I-EGF competition-binding curves of mutationally activated EGF receptors. CHO cells expressing the L834R-EGF receptor (A–D) or the V665M-EGF receptor (E–H) were treated without (black lines) or with erlotinib (green lines) or lapatinib (red lines) prior to and during incubation with increasing concentrations of ¹²⁵I-EGF. Data are shown as saturation-binding curves (A, B, E, and F) or competition-binding curves (C, D, G, and H). Points represent the mean ± S.D. of triplicate determinations in a single experiment, which was repeated a minimum of three times.

Figure 7.

Schematic diagram of the proposed effects of erlotinib and lapatinib on the conformation and dimerization of the EGF receptor. The ordered, active conformation (green) and ordered, inactive conformation (red) of the kinase domain are in equilibrium with an intermediate conformation of the kinase domain in which the α-C helix is disordered (yellow). Erlotinib stabilizes the active conformation of the kinase domain, which can form asymmetric dimers and which is linked to the open conformation of the extracellular domain. Lapatinib stabilizes the inactive conformation of the kinase domain, which forms symmetric dimers, which are linked to the tethered conformation of the extracellular domain.