EGFR in limbo

The epidermal growth factor receptor (EGFR) is normally activated by ligand-induced dimerization. Oncogenic mutations in EGFR promote activation in a largely ligand-independent manner. Shan et al. uncover a partially disordered state of EGFR kinase, providing evidence that oncogenic mutations counteract this intrinsic structural instability to promote dimerization and aberrant activation.     

The single transmembrane domains of ErbB receptors self-associate in cell membranes.

Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) alpha-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM alpha-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers. In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo. Using an approach for analyzing TM domain interactions in Escherichia coli cell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM equivalent to ErbB2-TM > ErbB3-TM. A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer. We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor. Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization. Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups.     

Mutational activation of ErbB family receptor tyrosine kinases: insights into mechanisms of signal transduction and tumorigenesis

Signaling by the Epidermal Growth Factor Receptor (EGFR) and related ErbB family receptor tyrosine kinases can be deregulated in human malignancies as the result of mutations in the genes that encode these receptors. The recent identification of EGFR mutations that correlate with sensitivity and resistance to EGFR tyrosine kinase inhibitors in lung and colon tumors has renewed interest in such activating mutations. Here we review current models for ligand stimulation of receptor dimerization and for activation of receptor signaling by receptor dimerization. In the context of these models, we discuss ErbB receptor mutations that affect ligand binding and those that cause constitutive receptor phosphorylation and signaling as a result of constitutive receptor dimerization. We discuss mutations in the cytoplasmic regions that affect enzymatic activity, substrate specificity and coupling to effectors and downstream signaling pathways. Finally, we discuss how emergent mechanisms of ErbB receptor mutational activation could impact the search for clinically relevant ErbB receptor mutations.     

Cholesterol depletion from the plasma membrane triggers ligand-independent activation of the epidermal growth factor receptor

We recently demonstrated that depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) caused activation of MAPK (Chen, X., and Resh, M. D. (2001) J. Biol. Chem. 276, 34617-34623). MAPK activation was phosphatidylinositol 3-kinase (PI3K)-dependent and involved increased tyrosine phosphorylation of the p85 subunit of PI3K. We next determined whether MbetaCD treatment induced tyrosine phosphorylation of other cellular proteins. Here we report that cholesterol depletion of serum-starved COS-1 cells with MbetaCD or filipin caused an increase in Tyr(P) levels of a 180-kDa protein that was identified as the epidermal growth factor receptor (EGFR). Cross-linking experiments showed that MbetaCD induced dimerization of EGFR, indicative of receptor activation. Reagents that block release of membrane-bound EGFR ligands did not affect MbetaCD-induced tyrosine phosphorylation of EGFR, indicating that MbetaCD activation of EGFR is ligand-independent. Moreover, MbetaCD treatment resulted in increased tyrosine phosphorylation of EGFR downstream targets and Ras activation. Incubation of cells with the specific EGFR inhibitor AG4178 blocked MbetaCD-induced phosphorylation of EGFR, SHC, phospholipase C-gamma, and Gab-1 as well as MAPK activation. We conclude that cholesterol depletion from the plasma membrane by MbetaCD causes ligand-independent activation of EGFR, resulting in MAPK activation by PI3K and Ras-dependent mechanisms. Moreover, these studies reveal a novel mode of action of MbetaCD, in addition to its ability to disrupt membrane rafts.     

Internalization of EGF receptor following lipid rafts disruption in keratinocytes is delayed and dependent on p38 MAPK activation

The receptor for epidermal growth factor (EGF) plays an important role in epidermal keratinocytes and is known to move out of lipid raft after cholesterol depletion, leading to ligand-independent activation. Accumulation of evidence indicates the ability of EGF receptor (EGFR) to undergo internalization without participation of the ligand under the control of p38 MAPK during stress conditions. Since cholesterol depletion using methyl-beta-cyclodextrin is known to induce ligand-independent activation of EGFR in keratinocytes, we investigated by confocal microscopy and ligand-binding tests the processing and localization of EGFR following lipid raft disruption. Here, we report the dimerization and the slow internalization of the receptor accompanied by the delayed phosphorylation of tyrosine 1068 and its degradation by the proteasome. We also demonstrate the involvement of p38 MAPK during the process of internalization, which can be considered as a protective response to stress. Moreover, cholesterol-depleted keratinocytes recover their ability to proliferate during the recovery period that follows lipid raft disruption.     

Activation of ErbB2 by 2-methyl-1,4-naphthoquinone (menadione) in human keratinocytes: role of EGFR and protein tyrosine phosphatases

Activation of ErbB receptor tyrosine kinases triggers multiple signaling pathways that regulate cellular proliferation and survival. We here demonstrate that ErbB2 is activated via the epidermal growth factor receptor (EGFR) upon exposure of cultured human keratinocytes to 2-methyl-1,4-naphthoquinone (menadione). Both ErbB2 and EGFR are shown to be regulated by protein tyrosine phosphatases that are inhibited by menadione, giving rise to the hypothesis that phosphatase inhibition by menadione may result in a net activation of EGFR and an enhanced ErbB2 phosphorylation. Isolated PTP-1B, a protein tyrosine phosphatase known to be associated with ErbB receptors, is demonstrated to be inhibited by menadione.     

Epidermal growth factor receptor (EGFR) signaling in cancer

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Evidence suggests that the EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas, and in vivo and in vitro studies have shown that these proteins are able to induce cell transformation. Amplification of the EGFR gene and mutations of the EGFR tyrosine kinase domain have been recently demonstrated to occur in carcinoma patients. Interestingly, both these genetic alterations of the EGFR are correlated with high probability to respond to anti-EGFR agents. However, ErbB proteins and their ligands form a complex system in which the interactions occurring between receptors and ligands affect the type and the duration of the intracellular signals that derive from receptor activation. In fact, proteins of the ErbB family form either homo- or hetero-dimers following ligand binding, each dimer showing different affinity for ligands and different signaling properties. In this regard, evidence suggests that cooperation of multiple ErbB receptors and cognate ligands is necessary to induce cell transformation. In particular, the growth and the survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. This phenomenon is also important for therapeutic approaches, since the response to anti-EGFR agents might depend on the total level of expression of ErbB receptors and ligands in tumor cells.     

Extracellular generation of hydrogen peroxide is responsible for activation of EGF receptor by ultraviolet A radiation

Receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) have been proposed to be activated in cells exposed to ultraviolet A (UVA) radiation (320-400 nm) and to be involved in photocarcinogenesis. Singlet oxygen and hydrogen peroxide are being discussed as mediators of the activation of signal transduction pathways by UVA. It is demonstrated here that EGFR is not activated in cells exposed to UVA in the absence of extracellular photosensitizers. Rather, UVA was capable of activating the EGFR and the related ErbB2 receptor tyrosine kinase in HeLa cells and human keratinocytes only under conditions that allowed for the extracellular photochemical generation of H(2)O(2), such as when cells were covered with cell culture medium during exposure to UVA. Pretreatment of cells with vanadate was required for UVA-induced EGFR activation, pointing to the involvement of protein tyrosine phosphatases. Unlike H(2)O(2), photochemically generated singlet oxygen did not activate EGFR but instead impaired the activation of EGFR by its ligand, EGF. In summary, extracellularly generated H(2)O(2) mediates UVA-induced activation of the EGFR and of ErbB2, whereas intracellular generation of reactive oxygen species upon exposure of cells to UVA is not sufficient for activation of the receptor.     

Inducible dimerization of RET reveals a specific AKT deregulation in oncogenic signaling

Dominant-activating mutations in the RET (rearranged during transfection) proto-oncogene, a receptor tyrosine kinase, are causally associated with the development of multiple endocrine neoplasia type 2A (MEN2A) syndrome. Such oncogenic RET mutations induce its ligand-independent constitutive activation, but whether it spreads identical signaling to ligand-induced signaling is uncertain. To address this question, we designed a cellular model in which RET can be activated either by its natural ligand, or alternatively, by controlled dimerization of the protein that mimics MEN2A dimerization. We have shown that controlled dimerization leaves proximal RET signaling intact but impacts substantially on the tuning of the distal AKT kinase activation (delayed and sustained). In marked contrast, distal activation of ERK remained unaffected. We further demonstrated that specific temporal adjustment of ligand-induced AKT activation is dependent upon a lipid-based cholesterol-sensitive environment, and this control step is bypassed by MEN2A RET mutants. Therefore, these studies revealed that MEN2A mutations propagate previously unappreciated subtle differences in signaling pathways and unravel a role for lipid rafts in the temporal regulation of AKT activation.     

Control of epidermal growth factor receptor endocytosis by receptor dimerization, rather than receptor kinase activation

Given that ligand binding is essential for the rapid internalization of epidermal growth factor receptor (EGFR), the events induced by ligand binding probably contribute to the regulation of EGFR internalization. These events include receptor dimerization, activation of intrinsic tyrosine kinase activity and autophosphorylation. Whereas the initial results are controversial regarding the role of EGFR kinase activity in EGFR internalization, more recent data suggest that EGFR kinase activation is essential for EGFR internalization. However, we have shown here that inhibition of EGFR kinase activation by mutation or by chemical inhibitors did not block EGF-induced EGFR internalization. Instead, proper EGFR dimerization is necessary and sufficient to stimulate EGFR internalization. We conclude that EGFR internalization is controlled by EGFR dimerization, rather than EGFR kinase activation. Our results also define a new role for EGFR dimerization: by itself it can drive EGFR internalization, independent of its role in the activation of EGFR kinase.     

The crystal structure of XC847 from Xanthomonas campestris: a 3'-5' oligoribonuclease of DnaQ fold family with a novel opposingly shifted helix

The mechanism by which ligand‐activated EGFR induces autophosphorylation via dimerization is not fully understood. Structural studies have revealed an extracellular loop mediated receptor dimerization. We have previously presented experimental data showing the involvement of a positive 13 amino acid peptide (R645–R657; P13⁺) from the intracellular juxtamembrane domain (JM) of EGFR important for intracellular dimerization and autophosphorylation. A model was presented that suggest that P13⁺ interacts with a negative peptide (D979–E991; P13^?) positioned distal to the tyrosine kinase domain in the opposite EGFR monomer. The present work shows additional data strengthening this model. In fact, by analyzing protein sequences of 21 annotated ErbB proteins from 9 vertebrate genomes, we reveal the high conservation of peptides P13⁺ and P13^? with regard to their sequence as well as their position relative to the tyrosine kinase (TK) domain. Moreover in silico structure modeling of these ErbB intracellular domains supports a general electrostatic P13⁺/P13^? interaction, implying that the C‐terminal of one receptor monomer is facing the TK domain of the other monomer in the receptor dimer and vice versa. This model provides new insights into the molecular mechanism of ErbB receptor activation and suggests a new strategy to pharmacologically interfering with ErbB receptor activity. Proteins 2006. © 2005 Wiley‐Liss, Inc.     

Dynamic Analysis of the Epidermal Growth Factor (EGF) Receptor-ErbB2-ErbB3 Protein Network by Luciferase Fragment Complementation Imaging

ErbB3 is a member of the ErbB family of receptor tyrosine kinases. It is unique because it is the only member of the family whose kinase domain is defective. As a result, it is obliged to form heterodimers with other ErbB receptors to signal. In this study, we characterized the interaction of ErbB3 with the EGF receptor and ErbB2 and assessed the effects of Food and Drug Administration-approved therapeutic agents on these interactions. Our findings support the concept that ErbB3 exists in preformed clusters that can be dissociated by NRG-1β and that it interacts with other ErbB receptors in a distinctly hierarchical fashion. Our study also shows that all pairings of the EGF receptor, ErbB2, and ErbB3 form ligand-independent dimers/oligomers. The small-molecule tyrosine kinase inhibitors erlotinib and lapatinib differentially enhance the dimerization of the various ErbB receptor pairings, with the EGFR/ErbB3 heterodimer being particularly sensitive to the effects of erlotinib. The data suggest that the physiological effects of these drugs may involve not only inhibition of tyrosine kinase activity but also a dynamic restructuring of the entire network of receptors.     

Structure-Function Relationships of ErbB RTKs in the Plasma Membrane of Living Cells

We review the states of the ErbB family of receptor tyrosine kinases (RTKs), primarily the EGF receptor (EGFR, ErbB1, HER1) and the orphan receptor ErbB2 as they exist in living mammalian cells, focusing on four main aspects: (1) aggregation state and distribution in the plasma membrane; (2) conformational features of the receptors situated in the plasma membrane, compared to the crystallographic structures of the isolated extracellular domains; (3) coupling of receptor disposition on filopodia with the transduction of signaling ligand gradients; and (4) ligand-independent receptor activation by application of a magnetic field.This review deals exclusively with the disposition and function of the human ErbB (HER) family of receptor tyrosine kinases (RTKs) in the plasma membrane of living cells. We have divided the material into four main topics: (1) distribution and aggregation state of ErbB family members; (2) the 3D structure of the ErbB1 and ErbB2 receptors; (3) the role of receptors located on extensions (filopodia) of the cell body; and (4) the phenomenon and implications of nonligand-dependent activation of the receptors.     

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.     

Transmembrane peptides as inhibitors of ErbB receptor signaling

Receptor tyrosine kinases have a single transmembrane (TM) segment that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, mutations within some of these receptors, and recent studies with the epidermal growth factor (EGF) and ErbB2 receptors have indicated that interactions between TM domains do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimerization and activation. One consequence of the importance of these interactions is that short hydrophobic peptides corresponding to these domains should act as specific inhibitors. To test this hypothesis, we constructed expression vectors encoding short fusion peptides encompassing native or mutated TM domains of the EGF, ErbB2, and insulin receptors. In human cell lines overexpressing the wild-type EGF receptor or ErbB2, we observed that the peptides are expressed at the cell surface and that they inhibit specifically the autophosphorylation and signaling pathway of their cognate receptor. Identical results were obtained with peptides chemically synthesized. Mechanism of action involves inhibition of dimerization of the receptors as shown by the lack of effects of mutant nondimerizing sequences, completed by density centrifugation and covalent cross-linking experiments. Our findings stress the role of TM domain interactions in ErbB receptor function, and possibly for other single-spanning membrane proteins.     

ErbB2 and ErbB3 receptors mediate inhibition of calcium-dependent chloride secretion in colonic epithelial cells

We have previously demonstrated that epidermal growth factor (EGF) inhibits calcium-dependent chloride secretion via a mechanism involving stimulation of phosphatidylinositol 3-kinase (PI3-K). The muscarinic agonist of chloride secretion, carbachol (CCh), also stimulates an antisecretory pathway that involves transactivation of the EGF receptor (EGFR) but does not involve PI3-K. Here, we have examined if ErbB receptors, other than the EGFR, have a role in regulation of colonic secretion and if differential effects on ErbB receptor activation may explain the ability of the EGFR to propagate diverse signaling pathways in response to EGF versus CCh. Basolateral, but not apical, addition of the ErbB3/ErbB4 ligand alpha-heregulin (HRG; 1-100 ng/ml) inhibited secretory responses to CCh (100 microM) across voltage-clamped T(84) epithelial cells. Immunoprecipitation/Western blot studies revealed that HRG (100 ng/ml) stimulated tyrosine phosphorylation and dimerization of ErbB3 and ErbB2, but had no effect on phosphorylation of the EGFR. HRG also stimulated recruitment of the p85 subunit of PI3-K to ErbB3/ErbB2 receptor dimers, while the PI3-K inhibitor, wortmannin (50 nM), completely reversed the inhibitory effect of HRG on CCh-stimulated secretion. Further studies revealed that, while both EGF (100 ng/ml) and CCh (100 microM) stimulated phosphorylation of the EGFR, only EGF stimulated phosphorylation of ErbB2, and neither stimulated ErbB3 phosphorylation. EGF, but not CCh, stimulated the formation of EGFR/ErbB2 receptor dimers and the recruitment of p85 to ErbB2. We conclude that ErbB2 and ErbB3 are expressed in T(84) cells and are functionally coupled to inhibition of calcium-dependent chloride secretion. Differential dimerization with other ErbB family members may underlie the ability of the EGFR to propagate diverse inhibitory signals in response to activation by EGF or transactivation by CCh.     

Parsing ERK activation reveals quantitatively equivalent contributions from epidermal growth factor receptor and HER2 in human mammary epithelial cells

HER2, a member of the epidermal growth factor receptor (EGFR) tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we applied a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2 and their downstream activation of ERK to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we could separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrated that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activate ERK were quantitatively equivalent. We found that HER2-mediated effects on EGFR dimerization and trafficking were sufficient to explain the observed HER2-mediated amplification of epidermal growth factor-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared with the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking yielding increased EGFR sparing cause the sustained HER2-mediated enhancement of ERK signaling.     

The ErbB kinase domain: structural perspectives into kinase activation and inhibition

Epidermal growth factor receptor (EGFR) and its family members, ErbB2, ErbB3 and ErbB4, are receptor tyrosine kinases which send signals into the cell to regulate many critical processes including development, tissue homeostasis, and tumorigenesis. Central to the signaling of these receptors is their intracellular kinase domain, which is activated by ligand-induced dimerization of the receptor and phosphorylates several tyrosine residues in the C-terminal tail. The phosphorylated tail then recruits other signaling molecules and relays the signal to downstream pathways. A model of the autoinhibition, activation and feedback inhibition mechanisms for the ErbB kinase domain has emerged from a number of recent structural studies. Meanwhile, recent clinical studies have revealed the relationship between specific ErbB kinase mutations and the responsiveness to kinase inhibitor drugs. We will review these regulation mechanisms of the ErbB kinase domain, and discuss the binding specificity of kinase inhibitors and the effects of kinase domain mutations found in cancer patients from a structural perspective.     

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.     

The extracellular domains of ErbB3 retain high ligand binding affinity at endosome pH and in the locked conformation

The extracellular, ligand binding regions of ErbB receptors consist of four domains that can assume at least two alternative conformations, extended and locked. The locked conformation, observed in several crystal structures, is held together by a noncovalent intramolecular tether and is incompatible with current models for receptor dimerization and ligand activation. Based on structures of ligand-receptor complexes in the extended conformation, the high affinity ligand binding pocket between domains I and III is disrupted in the locked conformation. Therefore the biological role of the locked conformation is not clear. To address the impact of the locked conformation on ligand binding, we compared extracellular domains of wild-type ErbB3, mutant domains in a constitutively locked or extended conformation and partial extracellular domain constructs. We found that the constitutively locked receptor domains and truncated constructs carrying only domains I-II or III-IV strongly bind ligand, albeit with reduced affinity compared to wild-type receptor. This suggests that the locked conformation cannot be discounted for ligand binding. The significant binding by both partial interfaces in domains I and III also suggests that "partial bivalency" may be the reason for the low nanomolar and high picomolar binding observed for ErbB3 in the respective "low" and high affinity states. In contrast to EGFR (ErbB1), ErbB3 retains high ligand binding affinity at an endosome-comparable pH in both the extended and locked conformations. Ligand affinity for the locked conformation even improves at low pH. For ErbB3, the contribution of domain I to ligand binding is strong and increases at low pH while its contribution is thought to be minimal for EGFR, regardless of pH. This shift in domain contribution and pH dependency provides a mechanistic explanation for some of the divergent properties of EGFR and ErbB3.