A single ligand is sufficient to activate EGFR dimers

Abstract

Crystal structures of human epidermal growth factor receptor (EGFR) with bound ligand revealed symmetric, doubly ligated receptor dimers thought to represent physiologically active states. Such complexes fail to rationalize negative cooperativity of epidermal growth factor (EGF) binding to EGFR and the behavior of the ligandless EGFR homolog ErbB2/HER2, however. We report cellbased assays that provide evidence for active, singly ligated dimers of human EGFR and its homolog, ErbB4/HER4. We also report crystal structures of the ErbB4/HER4 extracellular region complexed with its ligand Neuregulin-1β that resolve two types of ErbB dimer when compared to EGFR:Ligand complexes. One type resembles the recently reported asymmetric dimer of Drosophila EGFR with a single high-affinity ligand bound and provides a model for singly ligated human ErbB dimers. These results unify models of vertebrate and invertebrate EGFR/ErbB signaling, imply that the tethered conformation of unliganded ErbBs evolved to prevent crosstalk among ErbBs, and establish a molecular basis for both negative cooperativity of ligand binding to vertebrate ErbBs and the absence of active ErbB2/HER2 homodimers in normal conditions. H uman epidermal growth factor receptor (EGFR) and its homologs, known as ErbBs or HERs, are essential receptor tyrosine kinases that mediate cell proliferation and differentiation during animal development and are the targets of multiple cancer therapies (1). EGFR is the archetype of single-pass membrane-spanning receptors thought to transmit signals by ligandinduced dimerization (2, 3), and structural studies show that ligand binding to human EGFR promotes rearrangement of its four extracellular domains from a tethered to an extended conformation in which a loop, termed the dimerization arm, becomes exposed and mediates formation of symmetric receptor dimers (4) (Fig. 1A). At odds with a ligand-induced dimerization model of EGFR signaling, however, are recent studies showing dimers of human EGFR in the absence of ligand (5-8) as well as negative cooperativity when epidermal growth factor (EGF) binds to EGFR (9). Curiously, the single Drosophila EGFR homolog adopts an extended conformation in the absence of ligand and forms asymmetric receptor dimers with a single high-affinity ligand bound (10, 11), suggesting different mechanisms may regulate EGFR activation in Drosophila and humans.We report here evidence for active, singly ligated homodimers of human EGFR and its homolog, ErbB4. We also report the crystal structure of the ErbB4 extracellular region bound to its ligand Neuregulin-1β, which allows resolution of two types of human EGFR/ErbB dimers, one of which resembles the asymmetric Drosophila EGFR dimer and appears to reflect a singly ligated ErbB dimer state. These results compel reappraisal of canonical views of ligand-induced dimerization and show that several previously anomalous properties of human EGFR and its homologs represent vertebrate innovations on a core signaling mechanism present in invertebrates. Results a...

Quantitation of the Effect of ErbB2 on Epidermal Growth Factor Receptor Binding and Dimerization

“…This would allow the formation of an asymmetric dimer of the EGF receptor and ErbB2 extracellular domains that optimizes the affinity of the lone EGF recep- tor subunit for EGF. This is consistent with the observations of Liu et al (39) who noted that the relative orientation of subdomains II and IV in the extracellular domain of ErbB2 allow it to align optimally with a liganded partner and serve as the unliganded subunit in singly ligated ErbB heterodimers.…”

Inhibition of ErbB3 by a monoclonal antibody that locks the extracellular domain in an inactive configuration

“…The key distinguishing feature of KTN3379 is its ability to inhibit the first step in a chain of events that leads to both ligand-dependent and ligand-independent ErbB3 activation. Superimposing the activated forms of soluble EGFR extracellular domain (sEGFR) (27), sErbB4 (26), or sErbB2 (28) on domain 3 of Fab3379-bound sErbB3 shows that Fab3379 clashes dramatically with a significant portion of domain 2 ( Fig. 4B and Fig.…”

“…Most reported therapeutic antibodies against EGFR and ErbB3 function mainly by competing directly with ligand-receptor interactions (15,16,25). Because the structure of NRG in complex with sErbB3 has not been solved yet, the structure of domain 3 of sErbB3 was superimposed on the corresponding structure of soluble ErbB4 extracellular domain (sErbB4) in complex with NRG (26) to demonstrate that the ligand-binding footprint of the two receptors is nearly identical. This result is not surprising, because NRG is a high-affinity ligand for both ErbB3 and ErbB4.…”

Asymmetric perturbations of signalling oligomers

“…Furthermore, MD simulations show that in ligand-bound EGFR dimers, the N-and the C-terminal TM dimers represent an active and an inactive conformation, respectively, in which the N-terminal portions of the juxtamembrane segments form antiparallel helix dimers leading to an asymmetric transition state . Comparative XRC structures can explain why the mono-liganded asymmetric form may be more common to Drosophila than to the human EGFRs (Liu et al, 2012a;Perilla et al, 2013). Importantly, asymmetric kinase dimer formation is essential for activation of wild-type and oncogenic mutant EGFRs but not for kinase phosphorylation (Zhang et al, 2006;Kancha et al, 2013).…”