A single ligand is sufficient to activate EGFR dimers

A single ligand is sufficient to activate EGFR dimers Ping Liu, Thomas E. Cleveland IV, Samuel Bouyain1, Patrick O. Byrne, Patti A. Longo, and Daniel J. Leahy2 Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205 Edited by Wayne A. Hendrickson, Columbia University, New York, NY, and approved May 7, 2012 (received for review January 19, 2012) Crystal structures of human epidermal growth factor receptor (EGFR) with bound ligand revealed symmetric, doubly ligated re- ceptor dimers thought to represent physiologically active states. Such complexes fail to rationalize negative cooperativity of epider- mal 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 crys- tal 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 verte- brate and invertebrate EGFR/ErbB signaling, imply that the teth- ered conformation of unliganded ErbBs evolved to prevent cross- talk among ErbBs, and establish amolecular basis for both negative cooperativity of ligand binding to vertebrate ErbBs and the ab- sence of active ErbB2/HER2 homodimers in normal conditions. Human epidermal growth factor receptor (EGFR) and itshomologs, known as ErbBs or HERs, are essential receptor tyrosine kinases that mediate cell proliferation and differentia- tion during animal development and are the targets of multiple cancer therapies (1). EGFR is the archetype of single-pass mem- brane-spanning receptors thought to transmit signals by ligand- induced 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 con- formation 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 reg- ulate 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 hu- man 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 pre- viously anomalous properties of human EGFR and its homologs represent vertebrate innovations on a core signaling mechanism present in invertebrates. Results and Discussion We reasoned that if singly ligated dimers of human EGFR exist as implied by negative cooperativity (9), an EGFR variant incapable of binding ligand may remain able to participate in signaling dimers. To test this idea, we introduced debilitating amino-acid substitutions into the ligand-binding site of one EGFR variant and the kinase active site of another. These variants show negli- gible ligand-dependent phosphorylation when expressed indivi- dually in CHO cells, but coexpression restores phosphorylation in response to ligand as judged by both general and specfic anti- phosphotyrosine antibodies (Fig. 1B and SI Appendix, Fig. S1). The simplest explanation for this observation is that ligand-bind- ing deficient receptors are able to pair with kinase-deficient receptors to form active, singly ligated EGFR dimers. Similar re- sults were obtained for ErbB4/HER4 (Fig. 1C). Amino-acid sub- stitutions in the ErbB4 dimerization arm in the context of either ligand-binding or kinase-activity deficient ErbB4 variants elimi- nates responsiveness when cotransfected (SI Appendix, Fig. S2), implicating dimerization arms from both partners in formation of singly ligated ErbB4 dimers. Participation of unliganded ErbBs in a signaling dimer despite burial of the dimerization arm in the tethered conformation likely reflects favorable energetics of the interreceptor dimer interface relative to the tethered state within a preformed dimer. An essential feature of EGFR activation is an asymmetric di- mer of EGFR kinase domains in which the C-terminal region of a “donor” kinase contacts the N-terminal region of an “acceptor” kinase and stimulates it (12) (Fig. 1A), and the question arises whether the extracellular asymmetry of singly ligated EGFR dimers is coupled to this intracellular asymmetry. The ability of ligand-binding deficient EGFR to be activated by kinase-dead EGFR demonstrates that unliganded EGFRs can function as the acceptor kinase (Fig. 1B). To determine if ligand-binding defi- cient EGFR can function as a donor kinase, debilitating ami- no-acid substitutions were simultaneously introduced into the ligand-binding and kinase active sites of one EGFR and into the kinase donor site of another EGFR. Neither variant showed ligand-dependent phosphorylation when expressed on its own, but weak, ligand-dependent phosphorylation was observed when coexpressed (Fig. 1B). Similar results were obtained for ErbB4 (Fig. 1C). This observation suggests that unliganded EGFRs can serve as both a donor and an acceptor kinase and that extracel- lular asymmetry is not absolutely coupled to intracellular asym- metry, consistent with studies suggesting a loose linkage between ligand binding and kinase activation (13). A recent report using a luciferase fragment complementation assay showed that normal activation of EGFR/ErbB2 heterodimers required the EGFR kinase to be active, suggesting that the liganded partner (EGFR) could initially only function as an acceptor kinase and that extra- and intracellular asymmetry are coupled (14). In this case the in- tracellular kinases differ (vs. EGFR or ErbB4 homodimers), which may contribute to additional stabilization of the EGFR kinase in the acceptor role in the absence of phosphorylation. Author contributions: P.L., T.E.C., S.B., P.O.B., P.A.L., and D.J.L. designed research; P.L., T.E.C., S.B., P.O.B., and P.A.L. performed research; P.L., T.E.C., S.B., P.O.B., P.A.L., and D.J.L. analyzed data; and D.J.L. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3U7U). 1Present address: Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri—Kansas City, 5100 Rockhill Road, Kansas City, MO 64110. 2To whom correspondence should be addressed. Email: dleahy@jhmi.edu. This article contains supporting information online at www.pnas.org/lookup/suppl/ doi:10.1073/pnas.1201114109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1201114109 PNAS ∣ July 3, 2012 ∣ vol. 109 ∣ no. 27 ∣ 10861–10866 BI O PH YS IC S A N D CO M PU TA TI O N A L BI O LO G Y It will be interesting to determine if this is indeed the case or whether other factors underlie this apparent difference. The sites of all tested amino-acid substitutions are listed in SI Appendix, Table S1. None of these sites impaired cell surface expression as judged by cell-surface biotinylation, and EGFR expression levels were estimated by Western blot (SI Appendix, Fig. S3 and Table S2). Curiously, an original ligand-binding mutation intro- duced in EGFR, D355R, failed to express on the cell surface un- less cotransfected with kinase-deficient EGFR. This observation suggests that EGFRmolecules interact early in biogenesis and that this interaction can rescue otherwise nonviable forms of EGFR. The presence of active, singly ligated EGFR dimers on the cell surface raises the question of why crystal structures of the human EGFR extracellular region with ligand bound reveal symmetric, doubly ligated EGFR dimers (15, 16). To address this issue, we sought additional crystal structures of liganded ErbB extracellu- lar regions and report here the 3.0 Å crystal structure of the human ErbB4 extracellular region (sErbB4) complexed with Neuregulin-1β (Nrg) Table 1. Three independent 2∶2 sErbB4: Nrg dimers are present in the crystallographic asymmetric unit (Fig. 2). Domains I-III of the six sErbB4 subunits superimpose well among themselves (pairwise rmsds of 0.2–0.5 Å for Cαs) and with homologous regions of high-affinity ligand complexes of both human and Drosophila EGFR extracellular regions (sEGFR) (pairwise rmsds of 1.1–1.9 Å for Cαs) (Fig. 2 and SI Appendix, Fig. S4, and Table S3) (11, 13, 15, 16). The C-terminal juxtamembrane regions of domain IV are closely apposed when well ordered, suggesting interactions between transmembrane re- gions in active receptor dimers, but regions of domain IV homo- logous to those involved in direct dimer contacts in EGFR:EGF complexes (13) are mostly disordered and few specific intersubu- nit domain IV contacts are resolvable. Although consisting of nearly symmetric 2∶2 ligand:receptor complexes in each case, comparison of sErbB4:Nrg1β and human sEGFR:ligand dimers reveals two distinct dimer interfaces Fig. 1. Evidence for singly ligated ErbB signaling dimers. (A) Schematic dia- gram 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 bear- ing a mutation in its ligand-binding site (Lig−) was tagged with Flag. Muta- tion 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. Table 1. X-ray data collection and refinement statistics ErbB4-Nrg1β Data collection Wavelength 0.98 Space group P21 Unit cell dimensions a (Å) 85.7 b (Å) 223.5 c (Å) 146.9 β (°) 99.7 No. of unique reflections 103,807 Completeness (%) 98.9 (87.0) 8.6 (1.3) Rmerge ð%Þ 11.6 (68.5) Redundancy 3.7 (2.7) Refinement Resolution (Å) 50–3.0 Rwork ð%Þ 19.0 Rfree ð%Þ 22.7 rmsd Bond angle (Å) 0.01 Bond angle (°) 1.26 Values in parentheses are for the highest- resolution shell. Fig. 2. The sErbB4:Nrg1β structure. Orthogonal views of a worm diagram of the three independent sErbB4:Nrg1β dimers in the crystallographic asym- metric 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. 10862 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1201114109 Liu et al. (Fig. 3). Dimers of sErbB4:Nrg1β and sEGFR:EGF (13, 16) are similar to one another but differ from TGFα−bound dimers of a truncated form of EGFR (tEGFR) comprising the N-terminal 3 extracellular domains (15) (Fig. 3). Superposition of a single re- ceptor subunit of the tEGFR:TGFα dimer with a single subunit of either the sErbB4:Nrg1β or sEGFR:EGF dimers reveals the op- posite ErbB subunits to differ by a 29° scissor-like rotation about the dimerization arms. This rotation disrupts dimer contacts made by the N-terminal regions of domain II in the tEGFR: TGFα complex, notably those mediated by two contiguous loops formed by residues 190–208 (187–205 in ErbB4). These loops are flush across the tEGFR:TGFα dimer interface but staggered in the sErbB4:Nrg1β and sEGFR:EGF dimers (Figs. 3 and 4). The different ErbB dimers can be explained by truncation of domain IV in the tEGFR:TGFα complex (15). The orientation of domain IV relative to domain III is conserved in EGFR struc- tures whether liganded, unliganded, Drosophila, or human (SI Appendix, Fig. S5), and modeling domain IV onto each subunit of the tEGFR:TGFα dimer shows that its presence would result in severe intersubunit clashes (Fig. 3). Accommodating domain IV in sErbB4:Nrg1β and sEGFR:EGF complexes while maintain- ing interreceptor dimerization arm contacts necessitates the scis- sor-like rotation of receptor subunits relative to their orientation in the tEGFR:TGFα dimer. A flush, tEGFR:TGFα-like arrange- ment of domain II loops is also observed in the asymmetric dimer of Drosophila sEGFR, in which only one receptor subunit has high affinity ligand bound (Fig. 4) (11). In this case, the absence of a high-affinity ligand in one subunit effectively uncouples the relative orientation of the domain I/II and III/IV pairs and allows domain I and the N-terminal region of domain II of this subunit to shift and form the flush domain II interface without requiring domain IV clashes (Fig. 4). These results highlight the impor- tance of the relative position of the distinct dimer contact regions in domains II and IV in forming optimal ErbB dimers (Fig. 1A, Fig. 2) as their relative position, and thus the nature of possible dimer contacts, changes when high-affinity ligand is bound. Spontaneous formation of the flush dimer contact in human tEGFR dimers indicates that it is almost certainly more stable than the staggered contact observed in dimers of sErbB4 and sEGFR. The flush dimer would thus preferentially form in singly ligated dimers of EGFR in which the relative positions of do- mains II and IV are uncoupled in the unliganded subunit. This observation, coupled with the results of our cell-based assays, strongly implies that asymmetric Drosophila sEGFR-like dimers are conserved in human ErbBs (SI Appendix, Fig. S6). This con- servation is satisfying from an evolutionary perspective and pro- vides a structural rationale for negative cooperativity of ligand binding to EGFR (9). Binding of ligand to the unliganded subunit of singly ligated ErbB dimers requires conversion from a flush interface to the less stable staggered dimer interface, which ne- cessarily reduces the apparent affinity of the second receptor for ligand relative to the first and results in negative cooperativity and a weaker receptor dimer (9). The fact that intracellular re- gions are required for negative cooperativity likely reflects the importance of these regions for stabilizing receptor dimers in the absence of ligand (17). A theoretical study recently suggested interactions with the cell membrane may also induce aDrosophila EGFR-like dimer in human EGFR (18). Fig. 3. Two types of vertebrate ErbB dimer interaction. (Top) Orthogonal views of worm diagrams of sErbB4:Nrg1β and sEGFR:EGF (13) dimers follow- ing superposition of domains I, II, and III (see Fig. 1A for domain nomencla- ture). 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). Themod- eled domain IV of one subunit is colored black and the other gray. The mod- eled 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 heterodi- mers. (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 superim- posed 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 rela- tive to sErbB4 subunit domains, which in the case of tEGFR, Drosophila EGFR, and ErbB2 align the domain I/II interface regions directly opposite the corre- sponding 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. Liu et al. PNAS ∣ July 3, 2012 ∣ vol. 109 ∣ no. 27 ∣ 10863 BI O PH YS IC S A N D CO M PU TA TI O N A L BI O LO G Y Our cell-based assays imply that singly ligated EGFR dimers are signaling competent (Fig. 1). The fact that the Q194A muta- tion, which preferentially targets dimer contacts in the tEGFR: TGFα complex vs. the 2∶2 sEGFR:EGF complex (SI Appendix, Fig. S7), does not impair EGFR signaling (19) suggests that dou- bly ligated EGFR dimers are also signaling competent. Addition- ally, the actual and modeled positions of domain IV are similar in 2∶2 ErbB:ligand complexes and the asymmetric Drosophila sEGFR dimer, which we take to be an approximate model of sin- gly ligated human EGFR dimers (11, 13) (SI Appendix, Fig. S8), suggesting equivalent arrangements of transmembrane and intra- cellular regions in both vertebrate dimer types. Why, then, are asymmetric Drosophila sEGFR-like dimers not observed in crystals of human sEGFR or sErbB4 complexed with ligand? The presence of asymmetric dimers in crystals of Droso- phila sEGFR indicates that the extra stability of the asymmetric dimer interface more than compensates for the extra stability available from converting the low-affinity ligand interaction to a high-affinity interaction. That vertebrate ErbBs proceed to a weaker, symmetric dimer interface with two high-affinity ligand- receptor interactions implies that the energetic balance between high- and