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 cell-based 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.

Fig. 1.

Evidence for singly ligated ErbB signaling dimers. (A) Schematic diagram showing tethered, extended, and dimeric conformations of EGFR with sites of function-targeting mutations indicated. (B) Antiphosphotyrosine and anti-EGFR Western blots of tagged full-length EGFR immunoprecipitated from stably transfected CHO cells. Wild-type (WT) EGFR was tagged with either hemagglutinin (HA) or Flag peptides, EGFR bearing an inactivating mutation in its kinase active site (Kin^-) was tagged with HA, and EGFR bearing a mutation in its ligand-binding site (Lig^-) was tagged with Flag. Mutation in the Kinase donor site (Do^-) and combination of the Kinase^- and Ligand-targeting mutations on a single EGFR (Kin^-: Lig^-) were also tested. Serum-starved cells were either untreated (−) or treated (+) with EGF for 5 minutes. Each WT or mutant EGFR was transfected singly; the Kin^- and Lig^- variant EGFRs were cotransfected as were the (Kin^-: Lig^-) and (Do^-) variants. When cotransfected, the tag used for immunoprecipitation prior to Western blotting is indicated in red. (C) Similar experiments using ErbB4 and its ligand Neuregulin 1β (Nrg) are shown. The lanes shown were run on the same gel, but rearranged electronically to match the order of experiments in (B). Bar graphs represent quantitation of bands from at least 3 independent experiments.

Fig. 2.

The sErbB4:Nrg1β structure. Orthogonal views of a worm diagram of the three independent sErbB4:Nrg1β dimers in the crystallographic asymmetric unit following superposition of domains I, II, and III of sErbB4. Domains I, II, III, and IV are colored blue, green, yellow, and red, respectively, and Nrg1β is colored magenta. Lighter hues are used for the rightmost sErbB4 subunit.

Fig. 3.

Two types of vertebrate ErbB dimer interaction. (Top) Orthogonal views of worm diagrams of sErbB4:Nrg1β and sEGFR:EGF (13) dimers following superposition of domains I, II, and III (see Fig. 1A for domain nomenclature). One receptor subunit is colored yellow, the other blue; Nrg1β is colored magenta. (Bottom) Orthogonal views of worm diagrams of the tEGFR:TGFα complex (15) colored as in the top panel. The position of domain IV has been modeled on each subunit based on the domain III/IV relationship in the sEGFR:EGF complex and apo-sEGFR structures (SI Appendix, Fig. S5). The modeled domain IV of one subunit is colored black and the other gray. The modeled regions are enclosed in a dashed red box with clashing regions indicated. Red asterisks mark the dimer interaction site mediated by the N-terminal regions of domain II, which differs in the two dimer types. Yellow and blue lines approximate the long axes of the receptor subunits to illustrate the relative scissoring of the subunits in the two dimer types.

Fig. 4.

The tEGFR:TGFα domain II dimer interface is similar to the interface in Drosophila sEGFR:Spitz complexes and modeled ErbB2-containing heterodimers. (Top row) In the leftmost panel, a “side” view of an sErbB4:Nrg1β dimer (equivalent to the Top Right image of Fig. 2) with one sErbB4 subunit colored blue, another yellow, and Nrg1β magenta is shown. Moving rightwards, one subunit of the EGFR:EGF (13), tEGFR:TGF〈 (15), or Drosophila EGFR:Spitz (11) complexes has been superposed on domains I, II, and III of the yellow sErbB4 subunit. In the far right panel, domain III of sErbB2 (22) has been superimposed on domain III of the blue sErbB4 subunit. Domains I and II of the non-ErbB4 receptors are colored red, and domains III and IV are colored light green. Colored arrows indicate shifts in unsuperposed subunit domains relative to sErbB4 subunit domains, which in the case of tEGFR, Drosophila EGFR, and ErbB2 align the domain I/II interface regions directly opposite the corresponding regions of the opposite receptor subunit. (Bottom row) “Top” views of the superpositions shown in the Top row following a 90° rotation about a horizontal axis in the plane of the page. The superposed receptor subunits are colored yellow, the unsuperposed sErbB4 subunit is colored blue, and domains I and II of the unsuperposed EGFR or ErbB2 subunits are colored red. Red and green asterisks mark the two loops encompassed by ErbB4 residues 187–205 that are staggered in ErbB4 dimers but directly opposed in tEGFR, Drosophila EGFR, and modeled ErbB2-containing dimers.

Fig. 5.

The fixed extended conformation of ErbB2 precludes formation of canonical homodimers. (A) Orthogonal views of worm diagrams of the six sErbB4:Nrg1β subunits (sErbB4 is colored blue and Nrg1β yellow) following superposition of domain III of sErbB4 with domain III of three independent sErbB2 structures (pink) (22, 23). (B) Worm diagrams of a superposition of sErbB2 (light and dark pink) on domain III of an sErbB4:Nrg1β dimer (one sErbB4 subunit colored yellow and the other blue) (Left ); only domain III of the sErbB4:Nrg1β complex is shown for clarity, and red arrows indicate the shift in position of domains I and II of the ErbB2 subunits relative to their position in the sErbB4:Nrg1β complex. The sErbB4:Nrg1β complex is shown in the middle panel. In the Right most panel, domain I and the N-terminal part of domain II of ErbB2 is superposed on the corresponding regions of each subunit of the sErbB4:Nrg1β complex; only the superposed regions of the sErbB4:Nrg1β complex are shown for clarity. Red arrows indicate the shift in position of domains III and IV of the ErbB2 subunits relative to the position of the corresponding domains in the sErbB4:Nrg1β complex. The human sErbB2 structure was used for the superposition (22).