Asp-960/Glu-961 Controls the Movement of the C-terminal Tail of the Epidermal Growth Factor Receptor to Regulate Asymmetric Dimer Formation

David Piwnica-Worms,Katherine S. Yang,Jennifer L. Macdonald-Obermann,Linda J. Pike

doi:10.1074/jbc.m110.103317

Abstract

The epidermal growth factor (EGF) receptor is a tyrosine kinase that dimerizes in response to ligand binding. Ligand-induced dimerization of the extracellular domain of the receptor promotes formation of an asymmetric dimer of the intracellular kinase domains, leading to stimulation of the tyrosine kinase activity of the receptor. We recently monitored ligand-promoted conformational changes within the EGF receptor in real time using luciferase fragment complementation imaging and showed that there was significant movement of the C-terminal tail of the EGF receptor following EGF binding (Yang, K. S., Ilagan, M. X. G., Piwnica-Worms, D., and Pike, L. J. (2009) J. Biol. Chem. 284, 7474-7482). To investigate the structural basis for this conformational change, we analyzed the effect of several mutations on the kinase activity and luciferase fragment complementation activity of the EGF receptor. Mutation of Asp-960 and Glu-961, two residues at the beginning of the C-terminal tail, to alanine resulted in a marked enhancement of EGF-stimulated kinase activity as well as enhanced downstream signaling. The side chain of Asp-960 interacts with that of Ser-787. Mutation of Ser-787 to Phe, which precludes this interaction, also leads to enhanced receptor kinase activity. Our data are consistent with the hypothesis that Asp-960/Glu-961 represents a hinge or fulcrum for the movement of the C-terminal tail of the EGF receptor. Mutation of these residues destabilizes this hinge, facilitating the formation of the activating asymmetric dimer and leading to enhanced receptor signaling.

Highlights

In most epidermal growth factor (EGF) receptor kinase domain structures, ϳ10 –20 residues downstream of position 961 are missing from the structure [12, 17,18,19,20]
To determine whether Asp-960 and/or Glu-961 were involved in EGF-induced conformational changes in the EGF receptor, the double point mutant, D960A/E961A-EGF receptor was constructed
D960A/E961A-EGF receptor is still subject to MAP kinase-de- Formation—Because kinase activation is dependent upon pendent desensitization and that the double point mutation asymmetric dimer formation, we examined the structure of the enhances EGF receptor kinase activity by a mechanism other EGF receptor asymmetric dimer [14] to determine whether than ablation of MAP kinase-dependent desensitization of the Asp-960 or Glu-961 could contribute to the formation of this receptor

Summary

Introduction

To compare the EGF-induced conformational changes in wild-type, T669A-, and D960A/E961A-EGF receptors, luciferase fragments were fused to the C termini of each receptor and the constructs stably expressed in CHO cells. A monomer from determine whether elevated EGF receptor autophosphoryla- the inactive, symmetric kinase dimer [15] was aligned with the tion was associated with changes in our luciferase fragment (yellow) activator kinase from the asymmetric dimer [14].

Results

Conclusion