The presence of the juxtamembrane segment dimerizes the isolated intracellular module of EGFR in solution, suggesting that the principal function of ligand binding to the intact receptor is to change the structure of the extracellular module such that it does not impede the intrinsic ability of the intracellular module to activate (Jura et al., 2009
). Unexpectedly, we find that ligand binding must also play a role in altering the conformation of the transmembrane helix and the JM-A segment to promote receptor activation, because the intracellular module is monomeric and inhibited when it is localized to the plasma membrane on its own.
Our NMR analysis shows that the formation of the JM-A helix is coupled to the configuration of the transmembrane helices, and occurs outside of the membrane. Molecular dynamics simulations suggest that when EGFR is in an inactive conformation, the LRRLL motif within the JM-A segment is buried in the membrane (Arkhipov et al.
), consistent with NMR data for the isolated juxtamembrane segment of EGFR in detergent micelles (Choowongkomon et al., 2005
). These observations suggest that the nature of the interaction between the transmembrane helices toggles the configuration and membrane association of the JM-A portion of the juxtamembrane segments ().
A recent cell-based study clearly links an antiparallel interaction between the JM-A helices to EGFR activation and not just dimerization (Scheck et al., 2012
). We propose that the JM-A segments need to be pulled off the membrane in order to promote the asymmetric interaction between kinase domains. The latch formed by the JM-B segment of the receiver on the C-lobe of the activator kinase domain positions the sidechain of Glu 666 of the receiver near Arg 949 of the activator, providing an anchor point for the C-terminal end of the receiver JM-A segment (Red Brewer et al., 2009
; Wood et al., 2008
). In a crystal structure of the EGFR intracellular module with an intact juxtamembrane segment, the JM-A helix is anchored at this point, but is directed away from the surface of the activator kinase by crystal lattice contacts (Red Brewer et al., 2009
). We believe, instead, that the polar face of the JM-A helix of the receiver interacts with the surface of the kinase domain, as modeled earlier (Jura et al., 2009
) and seen consistently in the molecular dynamics simulations. If the receiver JM-A helix is docked in this way, then the leucine residues of the LRRLL motif point up from the surface of the kinase domain and are available for interaction with the activator JM-A helix, as shown schematically in . Our NMR data suggest that dimerization of the transmembrane helices through the N-terminal interface facilitates this arrangement.
Molecular dynamics simulations indicate that the extracellular module of EGFR prevents the close approach of the transmembrane helices that would be required for interaction through the N-terminal dimerization motif (Arkhipov et al.
), even when the receptor is dimerized in the absence of ligand. Consistent with this idea, ligand-independent activation of the receptor can be increased when flexible linkers are inserted between the extracellular module and the transmembrane helix. Thus, we believe that an essential role for EGF in receptor activation is to cause a specific conformational change in the extracellular modules that allows the transmembrane helices to interact through their N-terminal interface.
A striking feature of the activation mechanism is the strong cooperativity between the external, internal and transmembrane segments of EGFR. We speculate that because the EGFR dimer interface is mediated entirely by the receptor itself, selective pressure for inhibitory mechanisms to prevent ligand-independent activation has been particularly strong. At the same time, because EGFR signaling requires a specific dimeric configuration of kinase domains, there must also be selective pressure to stabilize that configuration. The balance of these competing requirements may have driven the evolution of counter-balanced activating and inhibiting mechanisms that minimize ligand-independent activation and facilitate the formation of appropriate heterodimers in response to external cues.