Simultaneous visualization of the extracellular and cytoplasmic domains of the epidermal growth factor receptor

The active EGFR kinase domain dimerizes through an asymmetrical interface, and it is even more active when an asymmetric dimer is formed. Negative-stain EM analysis of EGFR now shows that the kinase domains of a dimer can exist in three different states depending on the presence of ligand, mutations and inhibitors, whereas the receptor ectodomain remains in just one conformation. To our knowledge, no structural study to date has characterized, in an intact receptor, the coupling of conformational change in extracellular domains through a single-pass transmembrane domain to conformational change in cytoplasmic domains. Here we examine such coupling, and its unexpected complexity, using nearly full-length epidermal growth factor receptor (EGFR) and negative-stain EM. The liganded, dimeric EGFR ectodomain can couple both to putatively active, asymmetrically associated kinase dimers and to putatively inactive, symmetrically associated kinase dimers and monomers. Inhibitors that stabilize the active or inactive conformation of the kinase active site, as well as mutations in the kinase dimer interface and a juxtamembrane phosphorylation site, shift the equilibrium among the three kinase association states. This coupling of one conformation of an activated receptor ectodomain to multiple kinase-domain arrangements reveals previously unanticipated complexity in transmembrane signaling and facilitates regulation of receptor function in the juxtamembrane and cytoplasmic environments.