Epidermal Growth Factor Receptor Monomers Research Articles

The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

Spatially organized reaction dynamics between proto‐oncogenic epidermal growth factor receptor (EGFR) and protein tyrosine phosphatases determine EGFR phosphorylation dynamics in response to growth factors and thereby cellular behavior within developing tissues. We show that the reaction dynamics of mutual inhibition between RPTPγ phosphatase and autocatalytic ligandless EGFR phosphorylation enable highly sensitive promigratory EGFR signaling responses to subnanomolar EGF levels, when < 5% receptors are occupied by EGF. EGF thereby triggers an autocatalytic phospho‐EGFR reaction by the initial production of small amounts of phospho‐EGFR through transient, asymmetric EGF‐EGFR2 dimers. Single cell RPTPγ oxidation imaging revealed that phospho‐EGFR induces activation of NADPH oxidase, which in turn inhibits RPTPγ‐mediated dephosphorylation of EGFR, tilting the autocatalytic RPTPγ/EGFR toggle switch reaction towards ligandless phosphorylated EGFR. Reversibility of this reaction to EGF is maintained by the constitutive phosphatase activity of endoplasmic reticulum‐associated TCPTP. This RPTPγ/EGFR reaction at the plasma membrane causes promigratory signaling that is separated from proliferative signaling induced by accumulated, liganded, phosphorylated EGF‐EGFR in endosomes. Accordingly, loss of RPTPγ results in constitutive promigratory signaling from phosphorylated EGFR monomers. RPTPγ is thus a suppressor of promigratory oncogenic but not of proliferative EGFR signaling.

New trend in ligand-induced EGFR trafficking: A dual-mode clathrin-mediated endocytosis model

Accumulated evidence has established that ligand-bound activated epidermal growth factor receptor (EGFR) dimers rapidly undergo endocytosis via clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE), and are then sorted to recycling and degradation pathways, respectively. On the other hand, cellular stress triggers the non-canonical CME of ligand-free EGFR monomers via the phosphorylation of serine/threonine residues by p38. By integrating these findings on ligand- and stress-induced events, we herein propose a dual-mode CME model in which physiological concentrations of EGF trigger the non-canonical CME of ligand-free monomeric EGFR in parallel with the canonical CME of ligand-bound activated dimeric EGFR. Our established understanding of the EGFR endosomal trafficking pathway needs to be reconsidered with the application of this model. SignificanceIn this review, we propose a dual-mode clathrin-mediated endocytosis (CME) model, in which physiological concentrations of EGF trigger the p38-mediated non-canonical CME of ligand-free monomeric EGFR in parallel with the canonical CME of ligand-bound activated dimeric EGFR. It has been 60 years since the discovery of EGF this year, and it has become necessary to reconsider the mechanism of EGFR activation and to analyze its new physiological functions. By adapting a new trafficking model, it is expected to be applied to anti-EGFR therapy that has been developed in this century.

Investigating the Structure of Epidermal Growth Factor Receptor (EGFR) Dimers and Oligomers in Cells

A crucial step in the evolution of multi-cellular organisms was the emergence of receptor tyrosine kinases (RTKs). Trafficked to the plasma membrane, RTKs transduce signals in response to agonist ligands. EGFR was the first RTK to be cloned and is of paramount importance to cell function and human health. EGFR mutations and gene amplification are observed in many human cancers. Structural insights from macro-molecular crystallography and nuclear magnetic resonance described the atomic resolution structure of inactive ligand-free EGFR monomers and active ligand-bound dimers, and how these might underpin receptor autophosphorylation.

Comprehensive Model for Epidermal Growth Factor Receptor Ligand Binding Involving Conformational States of the Extracellular and the Kinase Domains.

The epidermal growth factor (EGF) receptor (EGFR) undergoes ligand-dependent dimerization to initiate transmembrane signaling. Although crystallographic structures of the extracellular and kinase domains are available, ligand binding has not been quantitatively analyzed taking the influence of both domains into account. Here, we developed a model explicitly accounting for conformational changes of the kinase and extracellular domains, their dimerizations and ligand binding to monomeric and dimeric receptor species. The model was fitted to ligand binding data of suspended cells expressing receptors with active or inactive kinase conformations. Receptor dimers with inactive, symmetric configuration of the kinase domains exhibit positive cooperativity and very weak binding affinity for the first ligand, whereas dimers with active, asymmetric kinase dimers are characterized by negative cooperativity and subnanomolar binding affinity for the first ligand. The homodimerization propensity of EGFR monomers with active kinase domains is ∼100-times higher than that of dimers with inactive kinase domains. Despite this fact, constitutive, ligand-independent dimers are mainly generated from monomers with inactive kinase domains due to the excess of such monomers in the membrane. The experimental finding of increased positive cooperativity at high expression levels of EGFR was recapitulated by the model. Quantitative prediction of ligand binding to different receptor species revealed that EGF binds to receptor monomers and dimers in an expression-level dependent manner without significant recruitment of monomers to dimers upon EGF stimulation below the phase transition temperature of the membrane. Results of the fitting offer unique insight into the workings of the EGFR.

Structure and Dynamics of the EGF Receptor as Revealed by Experiments and Simulations and Its Relevance to Non-Small Cell Lung Cancer.

The epidermal growth factor receptor (EGFR) is historically the prototypical receptor tyrosine kinase, being the first cloned and the first where the importance of ligand-induced dimer activation was ascertained. However, many years of structure determination has shown that EGFR is not completely understood. One challenge is that the many structure fragments stored at the PDB only provide a partial view because full-length proteins are flexible entities and dynamics play a key role in their functionality. Another challenge is the shortage of high-resolution data on functionally important higher-order complexes. Still, the interest in the structure/function relationships of EGFR remains unabated because of the crucial role played by oncogenic EGFR mutants in driving non-small cell lung cancer (NSCLC). Despite targeted therapies against EGFR setting a milestone in the treatment of this disease, ubiquitous drug resistance inevitably emerges after one year or so of treatment. The magnitude of the challenge has inspired novel strategies. Among these, the combination of multi-disciplinary experiments and molecular dynamic (MD) simulations have been pivotal in revealing the basic nature of EGFR monomers, dimers and multimers, and the structure-function relationships that underpin the mechanisms by which EGFR dysregulation contributes to the onset of NSCLC and resistance to treatment.

A conformational sensor based on genetic code expansion reveals an autocatalytic component in EGFR activation

Epidermal growth factor receptor (EGFR) activation by growth factors (GFs) relies on dimerization and allosteric activation of its intrinsic kinase activity, resulting in trans-phosphorylation of tyrosines on its C-terminal tail. While structural and biochemical studies identified this EGF-induced allosteric activation, imaging collective EGFR activation in cells and molecular dynamics simulations pointed at additional catalytic EGFR activation mechanisms. To gain more insight into EGFR activation mechanisms in living cells, we develop a Förster resonance energy transfer (FRET)-based conformational EGFR indicator (CONEGI) using genetic code expansion that reports on conformational transitions in the EGFR activation loop. Comparing conformational transitions, self-association and auto-phosphorylation of CONEGI and its Y845F mutant reveals that Y845 phosphorylation induces a catalytically active conformation in EGFR monomers. This conformational transition depends on EGFR kinase activity and auto-phosphorylation on its C-terminal tail, generating a looped causality that leads to autocatalytic amplification of EGFR phosphorylation at low EGF dose.

Cisplatin-induced non-canonical endocytosis of EGFR via p38 phosphorylation of the C-terminal region containing Ser-1015 in non-small cell lung cancer cells.

The aberrant activation of receptor tyrosine kinases (RTKs) is associated with tumor initiation in various types of human cancer, including non-small cell lung cancers (NSCLCs). Tyrosine kinase-independent non-canonical RTK regulation has also been investigated in tumor malignant alterations, including cellular stress responses. It was recently reported that the phosphorylation of epidermal growth factor receptor (EGFR) at C-terminal Ser-1015 serves a critical role in growth factor and cytokine signaling. In the present study, the role of non-canonical EGFR regulation has been investigated in NSCLC cells treated with cisplatin, a common chemotherapeutic agent. Cisplatin-induced p38 activation triggered the Ser-1015 phosphorylation of EGFR, with similar kinetics to previously reported Ser-1047 phosphorylation, in a tyrosine kinase-independent manner. In addition, phosphorylation around Ser-1015 triggered endocytosis of a dimer deficient mutant of EGFR. The non-canonical endocytosis of EGFR monomers was primarily controlled by the region around Ser-1015 only; however, Ser-1047 on internalized EGFR was equally phosphorylated. The results of the present study provide mechanistic evidence for the cisplatin-induced non-canonical regulation of EGFR.

Ligand-activated epidermal growth factor receptor (EGFR) signaling governs endocytic trafficking of unliganded receptor monomers by non-canonical phosphorylation

The canonical description of transmembrane receptor function is initial binding of ligand, followed by initiation of intracellular signaling and then internalization en route to degradation or recycling to the cell surface. It is known that low concentrations of extracellular ligand lead to a higher proportion of receptor that is recycled and that non-canonical mechanisms of receptor activation, including phosphorylation by the kinase p38, can induce internalization and recycling. However, no connections have been made between these pathways; i.e. it has yet to be established what happens to unbound receptors following stimulation with ligand. Here we demonstrate that a minimal level of activation of epidermal growth factor receptor (EGFR) tyrosine kinase by low levels of ligand is sufficient to fully activate downstream mitogen-activated protein kinase (MAPK) pathways, with most of the remaining unbound EGFR molecules being efficiently phosphorylated at intracellular serine/threonine residues by activated mitogen-activated protein kinase. This non-canonical, p38-mediated phosphorylation of the C-tail of EGFR, near Ser-1015, induces the clathrin-mediated endocytosis of the unliganded EGFR monomers, which occurs slightly later than the canonical endocytosis of ligand-bound EGFR dimers via tyrosine autophosphorylation. EGFR endocytosed via the non-canonical pathway is largely recycled back to the plasma membrane as functional receptors, whereas p38-independent populations are mainly sorted for lysosomal degradation. Moreover, ligand concentrations balance these endocytic trafficking pathways. These results demonstrate that ligand-activated EGFR signaling controls unliganded receptors through feedback phosphorylation, identifying a dual-mode regulation of the endocytic trafficking dynamics of EGFR.

Moderate and strong static magnetic fields directly affect EGFR kinase domain orientation to inhibit cancer cell proliferation.

Static magnetic fields (SMFs) can affect cell proliferation in a cell-type and intensity-dependent way but the mechanism remains unclear. At the same time, although the diamagnetic anisotropy of proteins has been proposed decades ago, the behavior of isolated proteins in magnetic fields has not been directly observed. Here we show that SMFs can affect isolated proteins at the single molecular level in an intensity-dependent manner. We found that Epidermal Growth Factor Receptor (EGFR), a protein that is overexpressed and highly activated in multiple cancers, can be directly inhibited by SMFs. Using Liquid-phase Scanning Tunneling Microscopy (STM) to examine pure EGFR kinase domain proteins at the single molecule level in solution, we observed orientation changes of these proteins in response to SMFs. This may interrupt inter-molecular interactions between EGFR monomers, which are critical for their activation. In molecular dynamics (MD) simulations, 1-9T SMFs caused increased probability of EGFR in parallel with the magnetic field direction in an intensity-dependent manner. A superconducting ultrastrong 9T magnet reduced proliferation of CHO-EGFR cells (Chinese Hamster Ovary cells with EGFR overexpression) and EGFR-expressing cancer cell lines by ~35%, but minimally affected CHO cells. We predict that similar effects of magnetic fields can also be applied to some other proteins such as ion channels. Our paper will help clarify some dilemmas in this field and encourage further investigations in order to achieve a better understanding of the biological effects of SMFs.

EGF-dependent re-routing of vesicular recycling switches spontaneous phosphorylation suppression to EGFR signaling.

Autocatalytic activation of epidermal growth factor receptor (EGFR) coupled to dephosphorylating activity of protein tyrosine phosphatases (PTPs) ensures robust yet diverse responses to extracellular stimuli. The inevitable tradeoff of this plasticity is spontaneous receptor activation and spurious signaling. We show that a ligand-mediated switch in EGFR trafficking enables suppression of spontaneous activation while maintaining EGFR's capacity to transduce extracellular signals. Autocatalytic phosphorylation of tyrosine 845 on unliganded EGFR monomers is suppressed by vesicular recycling through perinuclear areas with high PTP1B activity. Ligand-binding results in phosphorylation of the c-Cbl docking tyrosine and ubiquitination of the receptor. This secondary signal relies on EGF-induced EGFR self-association and switches suppressive recycling to directional trafficking. The re-routing regulates EGFR signaling response by the transit-time to late endosomes where it is switched-off by high PTP1B activity. This ubiquitin-mediated switch in EGFR trafficking is a uniquely suited solution to suppress spontaneous activation while maintaining responsiveness to EGF.

Recruitment of the Adaptor Protein Grb2 to EGFR Tetramers

Adaptor protein Grb2 binds phosphotyrosines in the epidermal growth factor (EGF) receptor (EGFR) and thereby links receptor activation to intracellular signaling cascades. Here, we investigated how recruitment of Grb2 to EGFR is affected by the spatial organization and quaternary state of activated EGFR. We used the techniques of image correlation spectroscopy (ICS) and lifetime-detected Förster resonance energy transfer (also known as FLIM-based FRET or FLIM–FRET) to measure ligand-induced receptor clustering and Grb2 binding to activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected with fluorescently labeled forms of Grb2 (Grb2–mRFP) and EGFR (EGFR–eGFP). Following stimulation of the cells with EGF, we detected nanometer-scale association of Grb2–mRFP with EGFR–eGFP clusters, which contained, on average, 4 ± 1 copies of EGFR–eGFP per cluster. In contrast, the pool of EGFR–eGFP without Grb2–mRFP had an average cluster size of 1 ± 0.3 EGFR molecules per punctum. In the absence of EGF, there was no association between EGFR–eGFP and Grb2–mRFP. To interpret these data, we extended our recently developed model for EGFR activation, which considers EGFR oligomerization up to tetramers, to include recruitment of Grb2 to phosphorylated EGFR. The extended model, with adjustment of one new parameter (the ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1% of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2. Together, our experimental and modeling results further implicate tetrameric EGFR as the key signaling unit and call into question the widely held view that dimeric EGFR is the predominant signaling unit.

Gefitinib induces epidermal growth factor receptor dimers which alters the interaction characteristics with ¹²⁵I-EGF.

The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer® Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of 125I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0–3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8–2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF – EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with 125I-EGF.

Ganglioside GM3 is stably associated to tyrosine-phosphorylated ErbB2/EGFR receptor complexes and EGFR monomers, but not to ErbB2

Gangliosides are known to modulate the activation of receptor tyrosine-kinases (RTKs). Recently, we demonstrated the functional relationship between ErbB2 and ganglioside GM3 in HC11 epithelial cell line. In the present study we investigated, in the same cells, the ErbB2 activation state and its tendency to form stable molecular complexes with the epidermal growth factor receptor (EGFR) and with ganglioside GM3 upon EGF stimulation. Results from co-immunoprecipitation experiments and western blot analyses indicate that tyrosine-phosphorylated ErbB2 and EGFR monomers and stable ErbB2/EGFR high molecular complexes (heterodimers) are formed following EGF stimulation, even if the receptors co-immunoprecipitates also in the absence of the ligand; these data suggest the existence of pre-dimerization inactive receptor clusters on the cell surface. High performance-thin layer chromatography (HP-TLC) and TLC-immunostaining analyses of the ganglioside fractions extracted from the immunoprecipitates demonstrate that GM3, but not other gangliosides, is tightly associated to the tyrosine-phosphorylated receptors. Furthermore, we show that GM3 is preferentially and in a SDS-resistant manner associated to the activated ErbB2/EGFR complexes and EGFR monomer, but not to ErbB2. Altogether our data support the hypothesis that the modulating effects produced by GM3 on ErbB2 activation are mediated by EGFR.

A network model of early events in epidermal growth factor receptor signaling that accounts for combinatorial complexity

We consider a model of early events in signaling by the epidermal growth factor (EGF) receptor (EGFR). The model includes EGF, EGFR, the adapter proteins Grb2 and Shc, and the guanine nucleotide exchange factor Sos, which is activated through EGF-induced formation of EGFR–Grb2–Sos and EGFR–Shc–Grb2–Sos assemblies at the plasma membrane. The protein interactions involved in signaling can potentially generate a diversity of protein complexes and phosphoforms; however, this diversity has been largely ignored in models of EGFR signaling. Here, we develop a model that accounts more fully for potential molecular diversity by specifying rules for protein interactions and then using these rules to generate a reaction network that includes all chemical species and reactions implied by the protein interactions. We obtain a model that predicts the dynamics of 356 molecular species, which are connected through 3749 unidirectional reactions. This network model is compared with a previously developed model that includes only 18 chemical species but incorporates the same scope of protein interactions. The predictions of this model are reproduced by the network model, which also yields new predictions. For example, the network model predicts distinct temporal patterns of autophosphorylation for different tyrosine residues of EGFR. A comparison of the two models suggests experiments that could lead to mechanistic insights about competition among adapter proteins for EGFR binding sites and the role of EGFR monomers in signal transduction.

Decorin Evokes Protracted Internalization and Degradation of the Epidermal Growth Factor Receptor via Caveolar Endocytosis

Decorin inhibits the epidermal growth factor receptor (EGFR) by down-regulating its tyrosine kinase activity, thereby blocking the growth of a variety of transformed cells and tumor xenografts. In this study we provide evidence that decorin directly binds to the EGFR causing its dimerization, internalization, and ultimately its degradation. Using various pharmacological agents to disrupt clathrin-dependent and -independent endocytosis, we demonstrate that decorin evokes a protracted internalization of the EGFR primarily via caveolar-mediated endocytosis. In contrast to EGF, decorin targets the EGFR to caveolae, but not to early or recycling endosomes. Ultimately, however, both EGF- and decorin-induced pathways converge into late endosomes/lysosomes for final degradation. Thus, we have discovered a novel biological mechanism for decorin that could explain its anti-proliferative and anti-oncogenic mode of action.