The ErbB4 extracellular region retains a tethered-like conformation in the absence of the tether

The epidermal growth factor receptor (EGFR) and its homologs ErbB3 and ErbB4 adopt a tethered conformation in the absence of ligand in which an extended hairpin loop from domain II contacts the juxtamembrane region of domain IV and tethers the domain I/II pair to the domain III/IV pair. By burying the hairpin loop, which is required for formation of active receptor dimers, the tether contact was thought to prevent constitutive activation of EGFR and its homologs. Amino‐acid substitutions at key sites within the tether contact region fail to result in constitutively active receptors however. We report here the 2.5 Å crystal structure of the N‐terminal three extracellular domains of ErbB4, which bind ligand but lack domain IV and thus the tether contact. This ErbB4 fragment nonetheless adopts a domain arrangement very similar to the arrangement adopted in the presence of the tether suggesting that regions in addition to the tether contribute to maintaining this conformation and inactivity in the absence of the tether contact. We suggest that the tether conformation may have evolved to prevent crosstalk between different EGFR homologs and thus allow diversification of EGFR and its homologs.     

Oligomers of ERBB3 have two distinct interfaces that differ in their sensitivity to disruption by heregulin

ErbB receptors associate in a ligand-dependent or -independent manner, and overexpression of epidermal growth factor receptor (ErbB1) or ErbB2 results in ligand-independent activation. Ligand-independent activation is poorly understood, and dimerization alone is not sufficient for activation. ErbB receptors also form higher order oligomers, but the mechanism of oligomer formation and their contribution to signaling are not known. The kinase-deficient ErbB3 as well as its extracellular domains are particularly prone to ligand-independent oligomerization, and oligomers are destabilized by binding of the ligand heregulin. In contrast, ligand binding facilitates heterodimerization with ErbB2 and is expected to stabilize an extended conformation of the ErbB3 extracellular domain (ECD) in which the dimerization interface is exposed. In the absence of ligand, ErbB3 can adopt a closed conformation that is held together by an intramolecular tether. We used a constitutively extended form of the ErbB3-ECD to analyze the conformation of the ECD in oligomers and the mechanism of oligomer disruption by heregulin. The extended conformation of the ECD forms oligomers more readily, suggesting the crystallographically defined dimer interface is one of the interfaces involved in oligomerization. Heregulin destabilizes oligomeric complexes but not dimers, which are neither stabilized nor disrupted by ligand binding, indicating a distinct second interface in oligomers of ErbB3. Cross-linking and activation studies on membrane-embedded ErbB3/ErbB2 chimeras confirm this dual effect of heregulin. Most of the ErbB3-ECD on the cell surface is apparently kept in an open conformation through oligomerization, and the resulting oligomers adopt a conformation representing a state of reduced activity.     

Ligand-induced structural transitions in ErbB receptor extracellular domains

Crystallographic studies showed that epidermal growth factor (EGF) receptor activation involves major domain rearrangements. Without bound ligand, the extracellular region of the receptor (sEGFR) adopts a "tethered" configuration with its dimerization site occluded by apparently autoinhibitory intramolecular interactions. Ligand binding causes the receptor to become "extended," breaking the tether and exposing the dimerization site. Using small-angle X-ray scattering (SAXS), we confirm that the tethered and extended conformations are also adopted in solution, and we describe low-resolution molecular envelopes for an intact sEGFR dimer. We also use SAXS to monitor directly the transition from a tethered to extended configuration in the monomeric extracellular regions of ErbB3 and a dimerization-defective EGFR mutant. Finally, we show that mutating every intramolecular tether interaction in sEGFR does not greatly alter its conformation. These findings explain why tether mutants fail to activate EGF receptor and provide new insight into regulation of ErbB receptor conformation.     

A 50-A separation of the integrin alpha v beta 3 extracellular domain C termini reveals an intermediate activation state

The integrin alpha(v)beta(3) has been shown to exist in low and high affinity conformations. Activation to the high affinity state is thought to depend on the "switchblade-like" opening, from a low affinity bent conformation with a closed headpiece to an extended form of the integrin with an open headpiece. Activation has been shown to depend on separation of the cytoplasmic domains. How cytoplasmic domain separation is related to separation of the transmembrane domains is unknown, and the distance of separation of the transmembrane domains required for activation has not been defined. A constrained secreted form of alpha(v)beta(3) was engineered that introduced a 50-A separation of the integrin C-terminal tails of the extracellular domains of the alpha(v) and beta(3) subunits. Receptor binding and recognition by ligand-induced binding state (LIBS) monoclonal antibodies demonstrated that the mutant receptor was locked into a low affinity state that was likely in a partially extended conformation but with a closed headpiece. In the presence of RGD peptide, the constrained receptor was able to fully extend, as determined by full exposure of LIBS epitopes. In the presence of the appropriate LIBS antibody, high affinity ligand binding of the constrained receptor was achieved. The results support the existence of transient intermediate activation states of secreted alpha(v)beta(3). Furthermore, these results with the secreted alpha(v)beta(3) receptor support a model for the full-length membrane-bound form of alpha(v)beta(3), whereby a 50-A lateral separation of the integrin alpha(v) and beta(3) transmembrane domains would be sufficient to enforce the switchblade-like opening to the extended conformation but insufficient for full receptor activation.     

Structural Evidence for Loose Linkage between Ligand Binding and Kinase Activation in the Epidermal Growth Factor Receptor

The mechanisms by which signals are transmitted across the plasma membrane to regulate signaling are largely unknown for receptors with single-pass transmembrane domains such as the epidermal growth factor receptor (EGFR). A crystal structure of the extracellular domain of EGFR dimerized by epidermal growth factor (EGF) reveals the extended, rod-like domain IV and a small, hydrophobic domain IV interface compatible with flexibility. The crystal structure and disulfide cross-linking suggest that the 7-residue linker between the extracellular and transmembrane domains is flexible. Disulfide cross-linking of the transmembrane domain shows that EGF stimulates only moderate association in the first two α-helical turns, in contrast to association throughout the membrane over five α-helical turns in glycophorin A and integrin. Furthermore, systematic mutagenesis to leucine and phenylalanine suggests that no specific transmembrane interfaces are required for EGFR kinase activation. These results suggest that linkage between ligand-induced dimerization and tyrosine kinase activation is much looser than was previously envisioned.Fundamental to cellular physiology is the ability to transmit extracellular signals across the cell membrane to trigger intracellular responses. Although the extracellular and intracellular portions of cell surface receptors are responsible for detecting ligands and initiating signal cascades, respectively, transmembrane (TM) domains are thought to play critical roles by specifically associating and propagating signals across the phospholipid bilayer. However, the mechanisms by which single-pass TM domains associate and conduct signals are poorly understood.The epidermal growth factor receptor (EGFR) is the prototypical type I TM receptor tyrosine kinase. EGFR and related members of the ErbB family—ErbB2, ErbB3, and ErbB4—contain a glycosylated extracellular ligand binding domain; a single-pass TM domain; and intracellular juxtamembrane, tyrosine kinase, and autophosphorylation domains. The extracellular domain of EGFR binds polypeptide growth factor ligands, such as epidermal growth factor (EGF), to stimulate an array of intracellular signaling cascades that regulate normal and oncogenic cellular growth and proliferation (3, 17, 36). In one model of growth factor-dependent EGFR activation, ligand binding promotes receptor dimerization and activation of intracellular protein tyrosine kinase activity (35); other models suggest that receptors are predimerized on the cell surface and ligand binding alters the equilibrium between inactive and active dimeric (or higher-order oligomeric) configurations (9, 29).Structural mechanisms of growth factor-mediated receptor dimerization and allosteric kinase domain activation have been proposed from recent crystal structures of isolated extracellular ligand binding domains (7) and intracellular tyrosine kinase domains (37). The orientation between the four extracellular domains is dramatically altered upon ligand binding, which frees interfaces that are masked in tethered, unliganded monomers to mediate dimer formation (7). Furthermore, an unusual asymmetric interface between two kinase domain monomers is linked to rearrangement of the kinase site to the active conformation (37). However, neither the position of the last extracellular domain, domain IV, nor association between the TM domains is well-defined experimentally in liganded receptors. The approximate location of domain IV has been suggested by models based on the orientation between domains III and IV in unliganded monomers (7, 12) and two-dimensional negative-stain electron microscopy (EM) averages (27); however, the position of domain IV in the liganded dimer has not been determined in previous crystal structures (13, 30). Thus, it is not known how the extracellular domain positions the TM domains for transmembrane signaling.Several lines of evidence suggest that the TM domain contributes directly to receptor dimerization and signaling. The neu oncogene encodes a Val → Glu substitution in the TM domain of ErbB2 that results in constitutive activation (34). Recombinant EGFR fragments consisting of the extracellular and TM domains have a 10⁵-fold higher affinity for dimerization than the isolated soluble extracellular domains (31). The TM domains of all four ErbB family members self-associate when expressed in bacterial inner membranes (26). A dimeric structure for isolated ErbB2 TM peptides in bicelles has been defined by nuclear magnetic resonance (NMR) imaging (4). However, ErbB2 does not bind ligand and does not physiologically homodimerize (17). Moreover, different ErbB family member TM domains utilize potentially distinct GxxxG sequence motifs to dimerize, as shown with fusion proteins in bacterial membranes (26). However, it is not clear how the TM domains contribute to dimerization and signaling in intact receptors on the cell surface.Here, we characterize the structural basis for EGFR transmembrane signaling. An improved crystal structure of the EGF-bound EGFR extracellular domain resolves domain IV in electron density maps and identifies a small domain IV dimerization interface, the mutation of which does not abolish signaling. The crystal structure and disulfide cross-linking demonstrate a flexible, dimeric linker between the extracellular and transmembrane domains. EGF-induced dimerization of the TM domains involves an interface far less extensive than that found in two receptors that dimerize in the absence of activation. Furthermore, mutagenesis shows that no unique interface is required for transmembrane signaling. Thus, we propose that signal transmission through the EGFR is communicated much more loosely than was previously thought.     

Locking the beta3 integrin I-like domain into high and low affinity conformations with disulfides

Although integrin alpha subunit I domains exist in multiple conformations, it is controversial whether integrin beta subunit I-like domains undergo structurally analogous movements of the alpha7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the beta(3) integrin I-like domain to lock its beta6-alpha7 loop and alpha7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor alpha(IIb)beta(3)(T329C/A347C) was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded alpha(IIb)beta(3)(V332C/M335C) was locked in a high affinity state. The results suggest that activation of the beta subunit I-like domain is analogous to that of the alpha subunit I domain, i.e. that axial movement in the C-terminal direction of the alpha7-helix is linked to rearrangement of the I-like domain metal ion-dependent adhesion site into a high affinity conformation.     

CR1/CR2 interactions modulate the functions of the cell surface epidermal growth factor receptor

Recent crystallographic data on the isolated extracellular domain of the epidermal growth factor receptor (EGFR) have suggested a model for its activation by ligand. We have tested this model in the context of the full-length EGFR displayed at the cell surface, by introducing mutations in two regions (CR1 and CR2) of the extracellular domain thought to be critical for regulation of receptor activation. Mutations in the CR1 and CR2 domains have opposing effects on ligand binding affinity, receptor dimerization, tyrosine kinase activation, and signaling competence. Tyr(246) is a critical residue in the CR1 loop, which is implicated in the positioning and stabilization of the receptor dimer interface after ligand binding; mutations of Tyr(246) impair or abolish receptor function. Mutations in CR2, which weaken the interaction that restricts the receptor to the tethered (inactive) state, enhance responsiveness to EGF by increasing affinity for the ligand. However, weakening of the CR1/CR2 interaction does not result in spontaneous activation of the receptors' kinase. We have used an antibody (mAb 806), which recognizes a transition state of the EGF receptor between the negatively constrained, tethered state and the fully active back-to-back dimer conformation, to follow conformational changes in the wild-type and mutant EGF receptors after ligand binding. Our results suggest that EGFR on the cell surface can be untethered, but this form is inactive; thus, untethering of the receptor is not sufficient for activation, and ligand binding is essential for the correct positioning of the two receptor subunits to achieve kinase activation.     

The epidermal growth factor receptor (EGFR)-S442F mutant displays increased affinity for neuregulin-2beta and agonist-independent coupling with downstream signalling events

The EGFR (epidermal growth factor receptor; ErbB1) is frequently the subject of genetic changes in human tumours which contribute to the malignant phenotype by altering EGFR signalling. Examples of such genetic changes include overexpression, extracellular domain deletions and point mutations, and small deletions in the tyrosine kinase domain. We hypothesized that a point mutation in one of the EGFR ligand-binding domains would increase the affinity of EGFR for NRG2beta (neuregulin-2beta), which is not a potent stimulus of signalling by EGFR-Wt (wild-type EGFR). This mutation would permit NRG2beta stimulation of EGFR signalling in settings in which NRG2beta does not normally do so. To test this hypothesis, we have generated and evaluated various EGFR alleles containing mutations at Val441 and Ser442. NRG2beta is a much more potent stimulus of the EGFR-S442F mutant than of EGFR-Wt. Furthermore, the affinity of NRG2beta for the EGFR-S442F mutant is greater than the affinity of NRG2beta for EGFR-Wt. Finally, the EGFR-S442F mutant constitutively suppresses apoptosis via phosphoinositide 3-kinase and Akt signalling but is not highly tyrosine phosphorylated in the absence of ligand. These results suggest that mutations in the EGFR ligand-binding domain in tumours may permit potent stimulation of EGFR signalling by ligands that are not normally potent EGFR agonists, thereby providing for a novel mechanism by which EGFR signalling may be deregulated. These results also suggest that novel EGFR mutations and signalling activities may be responsible for deregulated EGFR signalling in tumour cells.     

Molecular Determinants of Epidermal Growth Factor Binding: A Molecular Dynamics Study

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF ligands or the receptor itself.     

Energetics of ErbB1 Transmembrane Domain Dimerization in Lipid Bilayers

One of the most extensively studied receptor tyrosine kinases is EGFR/ErbB1. Although our knowledge of the role of the extracellular domains and ligands in ErbB1 activation has increased dramatically based on solved domain structures, the exact mechanism of signal transduction across the membrane remains unknown. The transmembrane domains are expected to play an important role in the dimerization process, but the contribution of ErbB1 TM domain to dimer stability is not known, with published results contradicting one another. We address this controversy by showing that ErbB1 TM domain dimerizes in lipid bilayers and by calculating its contribution to stability as −2.5 kcal/mol. The stability calculations use two different methods based on Förster resonance energy transfer, which give the same result. The ErbB1 TM domain contribution to stability exceeds the change in receptor tyrosine kinases dimerization propensities that can convert normal signaling processes into pathogenic processes, and is thus likely important for biological function.     

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

The epidermal growth factor (EGF) receptor is a member of the ErbB family of receptors that also includes ErbB2, ErbB3, and ErbB4. These receptors form homo- and heterodimers in response to ligand with ErbB2 being the preferred dimerization partner. Here we use (125)I-EGF binding to quantitate the interaction of the EGF receptor with ErbB2. We show that the EGFR/ErbB2 heterodimer binds EGF with a 7-fold higher affinity than the EGFR homodimer. Because it cannot bind a second ligand, the EGFR/ErbB2 heterodimer is not subject to ligand-induced dissociation caused by the negatively cooperative binding of EGF to the second site on the EGFR homodimer. This increases the stability of the heterodimer relative to the homodimer and is associated with enhanced and prolonged EGF receptor autophosphorylation. These effects are independent of the kinase activity of ErbB2 but require back-to-back dimerization of the EGF receptor with ErbB2. Back-to-back dimerization is also required for phosphorylation of ErbB2. These findings provide a molecular explanation for the apparent preference of the EGF receptor for dimerizing with ErbB2 and suggest that the phosphorylation of ErbB2 occurs largely in the context of the EGFR/ErbB2 heterodimer, rather than through lateral phosphorylation of isolated ErbB2 subunits.     

Trafficking of the ErbB receptors and its influence on signaling

Although members of the ErbB receptor family are found predominantly at the cell surface, these receptors undergo constant cycling between the plasma membrane and the endosomal compartment. In the absence of an activating ligand, these receptors are slowly internalized (t(1/2) approximately 30 min) but are quickly recycled. The constitutive degradation rate of the epidermal growth factor (EGF) receptor (EGFR) is slower than other ErbB family members and only the EGFR appears to alter its trafficking pattern in response to ligand binding. This altered pattern is characterized by accelerated internalization and enhanced lysosomal targeting. Ligand-regulated trafficking of the EGFR is mediated by a series of motifs distributed through the cytoplasmic domain of the receptor that are exposed by a combination of activation-mediated conformation changes and the binding of proteins such as Grb2. As a consequence of induced internalization, most EGFR signaling occurs within endosomes whereas signaling by the other members of the ErbB family appear to be generated predominantly from the cell surface. Overexpression of ErbB family members can disrupt normal receptor trafficking by driving heterodimerization of receptors with disparate trafficking patterns. Because different ErbB receptor substrates are localized in different cellular compartments, disrupted trafficking could be an important factor in the altered signaling patterns observed as a consequence of receptor overexpression.     

Rational Design and Cyclization of MIG6 Peptide to Restore its Binding Affinity for ErbB Family Receptor Tyrosine Kinases

The ErbB receptor tyrosine kinase family consists of four members: EGFR/Her1, Her2, Her3 and Her4; they have been involved in a variety of malignant tumors. The mitogen-inducible gene 6 (MIG6) is a natural tumor-suppressor protein that can inactivate ErbB signaling by directly binding to the catalytic domain of ErbB kinases. Here, a peptide segment s2p was stripped from the MIG6 interaction interface with ErbB, which exhibited a very low or no affinity to the four ErbB kinases. Structural dynamics simulations revealed that the linear peptide is highly flexible in unbound state and would incur a considerable entropy penalty upon binding to kinases. In this respect, the s2p peptide was cyclized by rational design of a disulfide bond across its two termini, resulting in a cyclic peptide s2p-c. Integration of computational analysis and experimental assay found that the cyclization can largely constrain s2p conformation, thus minimizing the entropy penalty and restoring the binding affinity of s2p-c to kinases.     

Kinase Activator-Receiver Preference in ErbB Heterodimers Is Determined by Intracellular Regions and Is Not Coupled to Extracellular Asymmetry

The EGF receptor (EGFR) family comprises four homologs in humans collectively known as the ErbB or HER proteins. ErbB proteins are receptor tyrosine kinases that become activated when ligands bind to their extracellular regions and promote formation of specific homo- and heterodimers with enhanced tyrosine kinase activity. An essential feature of ErbB activation is formation of an asymmetric kinase dimer in which the C-terminal lobe of one kinase serves as the activator or donor kinase by binding the N-terminal lobe of a receiver or acceptor kinase and stabilizing its active conformation. ErbB extracellular regions are also thought to form active asymmetric dimers in which only one subunit binds ligand. The observation that the unliganded ErbB2 kinase preferentially serves as the activator kinase when paired with EGFR/ErbB1 implied that extracellular asymmetry in ErbB proteins might be coupled to intracellular asymmetry with unliganded partners favoring the activator kinase position. Using cell-based stimulation assays and chimeric ErbB proteins, we show that extracellular asymmetry is not coupled to intracellular asymmetry and that ErbB intracellular regions are sufficient to determine relative kinase activator-receiver orientation. We further show a hierarchy of activator-receiver preferences among ErbB proteins, with EGFR/ErbB1 being the strongest receiver, followed by ErbB2 and then ErbB4, and that cis-phosphorylation of EGFR and ErbB2 appears to be negligible. This hierarchy shapes the nature of signaling responses to different ligands in cells expressing multiple ErbB proteins.     

Structural basis for ligand capture and release by the endocytic receptor ApoER2

Apolipoprotein E receptor 2 (ApoER2) is a close homologue of low‐density lipoprotein receptor (LDLR) that mediates the endocytosis of ligands, including LDL particles. LDLR family members have been presumed to explore a large conformational space to capture ligands in the extended conformation at the cell surface. Ligands are subsequently released through a pH‐titrated structural transition to a self‐docked, contracted‐closed conformation. In addition to lipoprotein uptake, ApoER2 is implicated in signal transduction during brain development through capture of the extracellular protein reelin. From crystallographic analysis, we determine that the full‐length ApoER2 ectodomain adopts an intermediate contracted‐open conformation when complexed with the signaling‐competent reelin fragment, and we identify a previously unappreciated auxiliary low‐affinity binding interface. Based on mutational analyses, we propose that the pH shift during endocytosis weakens the affinity of the auxiliary interface and destabilizes the ligand–receptor complex. Furthermore, this study elucidates that the contracted‐open conformation of ligand‐bound ApoER2 at neutral pH resembles the contracted‐closed conformation of ligand‐unbound LDLR at acidic pH in a manner suggestive of being primed for ligand release even prior to internalization.     

Cytohesins are cytoplasmic ErbB receptor activators

Signaling by ErbB receptors requires the activation of their cytoplasmic kinase domains, which is initiated by ligand binding to the receptor ectodomains. Cytoplasmic factors contributing to the activation are unknown. Here we identify members of the cytohesin protein family as such factors. Cytohesin inhibition decreased ErbB receptor autophosphorylation and signaling, whereas cytohesin overexpression stimulated receptor activation. Monitoring epidermal growth factor receptor (EGFR) conformation by anisotropy microscopy together with cell-free reconstitution of cytohesin-dependent receptor autophosphorylation indicate that cytohesins facilitate conformational rearrangements in the intracellular domains of dimerized receptors. Consistent with cytohesins playing a prominent role in ErbB receptor signaling, we found that cytohesin overexpression correlated with EGF signaling pathway activation in human lung adenocarcinomas. Chemical inhibition of cytohesins resulted in reduced proliferation of EGFR-dependent lung cancer cells in?vitro and in?vivo. Our results establish cytohesins as cytoplasmic conformational activators of ErbB receptors that are of pathophysiological relevance.     

Role of N‐glycosylation in EGFR ectodomain ligand binding

The epidermal growth factor receptor (EGFR) is a tyrosine kinase protein, overexpressed in several cancers. The extracellular domain of EGFR is known to be heavily glycosylated. Growth factor (mostly epidermal growth factor or EGF) binding activates EGFR. This occurs by inducing the transition from the autoinhibited tethered conformation to an extended conformation of the monomeric form of EGFR and by stabilizing the flexible preformed dimer. Activated EGFR adopts a back‐to‐back dimeric conformation after binding of another homologous receptor to its extracellular domain as the dimeric partner. Several antibodies inhibit EGFR by targeting the growth factor binding site or the dimeric interfaces. Glycosylation has been shown to be important for modulating the stability and function of EGFR. Here, atomistic MD simulations show that N‐glycosylation of the EGFR extracellular domain plays critical roles in the binding of growth factors, monoclonal antibodies, and the dimeric partners to the monomeric EGFR extracellular domain. N‐glycosylation results in the formation of several noncovalent interactions between the glycans and EGFR extracellular domain near the EGF binding site. This stabilizes the growth factor binding site, resulting in stronger interactions (electrostatic) between the growth factor and EGFR. N‐glycosylation also helps maintain the dimeric interface and plays distinct roles in binding of antibodies to spatially separated epitopes of the EGFR extracellular domain. Analysis of SNP data suggests the possibility of altered glycosylation with functional consequences. Proteins 2017; 85:1529–1549. © 2017 Wiley Periodicals, Inc.     

Epidermal growth factor receptor dimerization and activation require ligand-induced conformational changes in the dimer interface

Structural studies have shown that ligand-induced epidermal growth factor receptor (EGFR) dimerization involves major domain rearrangements that expose a critical dimerization arm. However, simply exposing this arm is not sufficient for receptor dimerization, suggesting that additional ligand-induced dimer contacts are required. To map these contributions to the dimer interface, we individually mutated each contact suggested by crystallographic studies and analyzed the effects on receptor dimerization, activation, and ligand binding. We find that domain II contributes >90% of the driving energy for dimerization of the extracellular region, with domain IV adding little. Within domain II, the dimerization arm forms much of the dimer interface, as expected. However, a loop from the sixth disulfide-bonded module (immediately C-terminal to the dimerization arm) also makes a critical contribution. Specific ligand-induced conformational changes in domain II are required for this loop to contribute to receptor dimerization, and we identify a set of ligand-induced intramolecular interactions that appear to be important in driving these changes, effectively "buttressing" the dimer interface. Our data also suggest that similar conformational changes may determine the specificity of ErbB receptor homo- versus heterodimerization.     

Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IV

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) has emerged as relevant target for antibody-mediated tumor therapy. Here, we describe a novel human antibody, IgG 3–43, recognizing a unique epitope formed by domain III and parts of domain IV of the extracellular region of HER3, conserved between HER3 and mouse ErbB3. An affinity of 11 nM was determined for the monovalent interaction. In the IgG format, the antibody bound recombinant bivalent HER3 with subnanomolar affinity (K_D = 220 pM) and HER3-expressing tumor cells with EC₅₀ values in the low picomolar range (27 - 83 pM). The antibody competed with binding of heregulin to HER3-expressing cells, efficiently inhibited phosphorylation of HER3 as well as downstream signaling, and induced receptor internalization and degradation. Furthermore, IgG 3–43 inhibited heregulin-dependent proliferation of several HER3-positive cancer cell lines and heregulin-independent colony formation of HER2-overexpressing tumor cell lines. Importantly, inhibition of tumor growth and prolonged survival was demonstrated in a FaDu xenograft tumor model in SCID mice. These findings demonstrate that by binding to the membrane-proximal domains III and IV involved in ligand binding and receptor dimerization, IgG 3–43 efficiently inhibits activation of HER3, thereby blocking tumor cell growth both in vitro and in vivo.     

Herstatin, an autoinhibitor of the human epidermal growth factor receptor 2 tyrosine kinase, modulates epidermal growth factor signaling pathways resulting in growth arrest

Herstatin is an autoinhibitor of the ErbB family consisting of subdomains I and II of the human epidermal growth factor receptor 2 (ErbB-2) extracellular domain and a novel C-terminal domain encoded by an intron. Herstatin binds to human epidermal growth factor receptor 2 and to the epidermal growth factor receptor (EGFR), blocking receptor oligomerization and tyrosine phosphorylation. In this study, we characterized several early steps in EGFR activation and investigated downstream signaling events induced by epidermal growth factor (EGF) and by transforming growth factor alpha (TGF-alpha) in NIH3T3 cell lines expressing EGFR with and without herstatin. Herstatin expression decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation despite receptor occupancy by ligand with normal binding affinity. Akt stimulation by EGF and TGF-alpha, but not by fibroblast growth factor 2, was almost completely blocked in the presence of herstatin. Surprisingly, EGF and TGF-alpha induced full activation of MAPK in duration and intensity and stimulated association of the EGFR with Shc and Grb2. Although MAPK was fully stimulated, herstatin expression prevented TGF-alpha-induced DNA synthesis and EGF-induced proliferation. The herstatin-mediated uncoupling of MAPK from Akt activation was also observed in Chinese hamster ovary cells co-transfected with EGFR and herstatin. These findings show that herstatin expression alters EGF and TGF-alpha signaling profiles, culminating in inhibition of proliferation.