EGF receptor


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Direct substrates of EGFR

In the case of growth factors such as the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), the steps leading to activation of MAPK require the function of the adaptor protein Grb2 (growth factor receptor binding protein 2), which can bind either directly or indirectly via its Src homology 2 domain to activated receptor tyrosine kinases. A cell-permeable mimetic of the EGF receptor Grb2 binding site has been investigated for its ability to inhibit biological responses stimulated by a variety of growth factors. Pretreatment of cells with this peptide results in the accumulation of the peptide in cells and its association with Grb2. This is associated with a complete inhibition of the mitogenic response stimulated by EGF and PDGF. In contrast, the peptide has no effect on the mitogenic response stimulated by fibroblast growth factor. The peptide could also inhibit the phosphorylation of MAPK stimulated with EGF and PDGF in the absence of an effect on the fibroblast growth factor response. These data demonstrate that cell-permeable mimetics of Src homology 2 binding sites can selectively inhibit growth factor-stimulated mitogenesis, and also directly demonstrate specificity in the coupling of activated receptor tyrosine kinases to the MAPK cascade (Williams, 1997).

The importance of Shc in coupling the EGFR to activation of Sos was investigated. EGF treatment leads to rapid tyrosine phosphorylation of Shc. Although phosphorylated EGFR can bind to both Shc and Grb2, the predominant linkage is observed between EGFR and Shc. Similarly, more Grb2 is associated with Shc than with EGFR after EGF stimulation. Immunoprecipitation of Shc from EGF-stimulated cells removes almost all EGFR-associated Grb2. Furthermore, immunodepletion of Shc proteins from membrane fractions of EGF-stimulated cells removes 93% of the Sos activity, whereas, precipitation of EGFR has only a small effect on Sos activity. These data indicate that coupling to Shc provides the major pathway linking activated EGFRs to Grb2.Sos and stimulation of the p21ras pathway (Sasaoka, 1994).

Shc is an Src homology 2 (SH2) domain protein thought to be an important component of the signaling pathway leading from cell surface receptors to Ras. A new phosphotyrosine interaction (PI) domain (also known as the phosphotyrosine binding [PTB] domain) has been described in the amino terminus of Shc. The Shc PI domain binding specificity is dependent on residues lying amino-terminal to the phosphotyrosine rather than carboxyl-terminal as is seen with SH2 domains. The Shc PTB/PI domain was mutagenized in an effort to identify residues in the domain crucial for interaction with phosphotyrosine-containing peptides. The mutants were screened for binding to the tyrosine-phosphorylated carboxyl-terminal tail of the epidermal growth factor (EGF) receptor. Most striking were mutations that alter a phenylalanine residue in block 4 of the domain severely impairing PI domain function. This phenylalanine residue is conserved in all but one subfamily of PI domains that have been identified to date. Reconstitution of this phenylalanine mutation into full-length Shc creates a protein unable to interact with the EGF receptor in living cells (Yajnik, 1996).

In response to stimulation with epidermal growth factor (EGF), the guanine nucleotide exchange factor human SOS1 (hSOS1) promotes the activation of Ras (See Drosophila Ras) by forming a complex with Grb2 and the human EGF receptor (hEGFR). hSOS1 is phosphorylated in cells stimulated with EGF or phorbol ester or following co-transfection with activated Ras or Raf. Co-transfection with dominant negative Ras resultes in a decrease of EGF-induced hSOS1 phosphorylation. The mitogen-activated protein kinase (MAPK) phosphorylates hSOS1 in vitro within the carboxyl-terminal proline-rich domain. The same region of hSOS1 is phosphorylated in vivo, in cells stimulated with EGF. Tryptic phosphopeptide mapping shows that MAPK phosphorylates hSOS1 in vitro on sites which are also phosphorylated in vivo. Phosphorylation by MAPK does not affect hSOS1 binding to Grb2 in vitro. However, reconstitution of the hSOS1-Grb2-hEGFR complex shows that phosphorylation by MAPK markedly reduces the ability of hSOS1 to associate with the hEGFR through Grb2. Similarly, phosphorylated hSOS1 is unable to form a complex with Shc through Grb2. Thus phosphorylation of hSOS1, by affecting its interaction with the hEGFR or Shc, down-regulates signal transduction from the hEGFR to the Ras pathway (Porfiri, 1996).

Rat liver parenchyma harbors equal numbers of epidermal growth factor (EGF) and insulin receptors. Following administration of a saturating dose of EGF, there was a rapid (t1/2 approximately 1.1 min) internalization of receptor coincident with its tyrosine phosphorylation at residue 1173 and receptor recruitment of the adaptor protein SHC, its tyrosine phosphorylation and its association with GRB2 and the Ras guanine nucleotide exchange factor, mSOS, largely in endosomes. This leads to a cytosolic pool of a complex of tyrosine-phosphorylated SHC, GRB2 and mSOS. These constituents are linked to Ras activation by the characteristic decrease in Raf-1 mobility on SDS-PAGE, which is maintained for 60 min. While insulin administration leads to insulin receptor beta-subunit tyrosine phosphorylation and internalization, there is little detectable tyrosine phosphorylation of SHC, recruitment of GRB2, association of a complex with mSOS or any detectable change in the mobility of Raf-1. Therefore, in normal physiological target cells in vivo, distinct signaling pathways are realized after EGF or insulin receptor activation, with regulation of this specificity most probably occurring at the locus of the endosome (Di Guglielmo, 1994).

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In C. elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. In rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signaling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells (Rozakis-Adcock, 1993).

Epidermal growth factor (EGF) receptor (EGFR) can induce cell growth and transformation in a ligand-dependent manner. To examine whether the autophosphorylation of EGFR correlates with the capacity of the activated EGFR to induce cell growth and transformation, human EGFR was truncated just after residue 1011, removing all three major autophosphorylation sites (DEL1011). Further, a point mutation was introduced at another autophosphorylation site, Tyr-992-->Phe (DEL1011+F992). The wild-type and mutant receptors are stably expressed in a NIH 3T3 variant cell line that expresses an extremely low level of endogenous EGFR and does not grow with EGF. DEL1011 and DEL1011+F992 are found to be severely impaired in EGF-induced autophosphorylation, due to the deletion of the appropriate target tyrosines. However, mutant receptors still can induce EGF-dependent DNA synthesis, morphological transformation, and anchorage-independent growth, although the extent of these is significantly reduced when compared with wild-type EGFR. EGF-induced tyrosine phosphorylation of Ras-GTPase activating protein-associated protein p62 and phospholipase C gamma 1 is dramatically reduced in the cells expressing DEL1011 and DEL1011+F992. However the tyrosine phosphorylation of Shc, the complex formation of Shc-Grb2/Ash, and the activation of microtubule-associated protein kinase are all still fully induced upon EGF stimulation without binding of Shc or Grb2/Ash to the mutant receptor. Thus, tyrosine phosphorylation of Shc may play a crucial role for activating Ras and generating mitotic signals by the activated EGFR mutant (Gotoh, 1994).

The SH3-SH3-SH3-SH2 adapter protein Nck (Drosophila homolog: Dreadlocks) links receptor tyrosine kinases, such as EGF and PDGF receptors, to downstream signaling pathways, like the p21cdc42/rac-activated kinase cascade, and to Sos-activated Ras signaling and the human Wiskott-Aldrich Syndrome protein (WASp)-mediated actin cytoskeleton changes. In EGF stimulated cells, Nck co-immunoprecipitates with a number of phosphotyrosine proteins, including the EGF receptor. To identify the phosphotyrosine protein(s) that directly interacts with Nck and to distinguish it from indirectly associated proteins, preexisting phosphoytrosine protein complexes in the cell lysate were dissociated by heat and SDS prior to the test for binding to Nck. Nck does not directly bind to the EGF receptor, instead it binds via its SH2 domain to a 62 kDa phosphotyrosine protein. The Nck-bound p62 is related to the previously identified GTPase-activating protein (GAP)-associated phosphotyrosine protein p62. Other results reported in this paper include

  1. The Nck-bound and the GAP-bound p62 proteins co-migrate with each other in SDS-PAGE.
  2. SH2 domains from Nck and GAP compete for binding to p62 in vitro.
  3. Purified GST-Nck-SH2 binds directly to the GAP-associated p62. Under these conditions, SH2 domains from PLCgamma, PI-3 kinase, SHC, and Grb2 do not bind p62.
  4. Tryptic phosphopeptide maps of the Nck- and the GAP-associated p62 proteins are identical. However, Nck and GAP do not co-immunoprecipitate with each other and apparently bind to different pools of p62.
This study suggests that the GAP-associated p62 acts as an SH2 domain docking protein and mediates the interaction between Nck and EGF receptor in response to EGF stimulation (Tang, 1997).

The murine retroviral oncogene v-cbl induces pre-B cell lymphomas and myelogenous leukemias. The protein product of the mammalian c-cbl proto-oncogene is a widely expressed cytoplasmic 120-kDa protein (p120cbl) whose normal cellular function has not been determined. Upon stimulation of human epidermal growth factor (EGF) receptor, p12ocbl becomes strongly tyrosine-phosphorylated and associates with activated EGF receptor in vivo. A GST fusion protein containing amino acids 1-486 of p120cbl, including a region highly conserved in nematodes, binds directly to the autophosphorylated carboxyl-terminal tail of the EGF receptor. Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or nerve growth factor (NGF) stimulation also results in tyrosine phosphorylation of p120cbl. Recent genetic studies in C. elegans indicate that Sli-1, a p120cbl homolog, plays a negative regulatory role in control of the Ras signaling pathway initiated by the C. elegans EGF receptor homologue. Thus p120cbl is involved in an early step in the EGF signaling pathway that is conserved from nematodes to mammals (Galisteo, 1995).

Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data reveal new potential Grb2 binding sites at Tyr-1114 [epidermal growth factor receptor (EGFR) C-tail]; Tyr-743 [platelet-derived growth factor receptor (PDGFR) insert region], Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bind Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) binds Grb2 but with medium strength. Tyr-1068 and -1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data reveal new potential binding sites at Tyr-703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. The data also identify new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirms the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bind Shc strongly, although Tyr-690 shows medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling pathways (Ward, 1996).

The interaction of activated epidermal growth factor receptor (EGFR) with the Src homology 2 (SH2) domain of the growth-factor-receptor binding protein Grb2 initiates signaling through Ras and mitogen-activated protein kinase (MAP kinase). Activation of EGFRs by ligand also triggers rapid endocytosis of EGF-receptor complexes. To analyze the spatiotemporal regulation of EGFR-Grb2 interactions in living cells, imaging microscopy has been combined with a modified method of measuring fluorescence resonance energy transfer (FRET) on a pixel-by-pixel basis using EGFR fused to cyan fluorescent protein (CFP) and Grb2 fused to yellow fluorescent protein (YFP). Efficient energy transfer between CFP and YFP should only occur if CFP and YFP are less than 50Å apart, which requires direct interaction of the EGFR and Grb2 fused to these fluorescent moieties. Stimulation by EGF results in the recruitment of Grb2-YFP to cellular compartments that contained EGFR-CFP and a large increase in FRET signal amplitude. In particular, FRET measurements indicate that activated EGFR-CFP interacts with Grb2-YFP in membrane ruffles and endosomes. These results demonstrate that signaling via EGFRs can occur in the endosomal compartment. The work also highlights the potential of FRET microscopy in the study of subcellular compartmentalization of protein-protein interactions in living cells (Sorkin, 2000).

EGFR modulates DNA synthesis and repair through Tyr phosphorylation of histone H4

Posttranslational modifications of histones play fundamental roles in many biological functions. Specifically, histone H4-K20 methylation is critical for DNA synthesis and repair. However, little is known about how these functions are regulated by the upstream stimuli. This study identified a tyrosine phosphorylation site at Y72 of histone H4, which facilitates recruitment of histone methyltransferases (HMTases), SET8 and SUV4-20H, to enhance its K20 methylation, thereby promoting DNA synthesis and repair. Phosphorylation-defective histone H4 mutant is deficient in K20 methylation, leading to reduced DNA synthesis, delayed cell cycle progression, and decreased DNA repair ability. Disrupting the interaction between epidermal growth factor receptor (EGFR) and histone H4 by Y72 peptide significantly reduced tumor growth. Furthermore, EGFR expression clinically correlates with histone H4-Y72 phosphorylation, H4-K20 monomethylation, and the Ki-67 proliferation marker. These findings uncover a mechanism by which EGFR transduces signal to chromatin to regulate DNA synthesis and repair (Chou, 2014).

Interaction of EGFR with actin and Src

Epidermal growth factor receptor (EGFR) binds directly to Filamentous-actin by EGFRs actin binding domain (ABD). This ABD is located in a 12 amino acid long sequence (amino acid 984-996), situated between the tyrosine kinase domain and the main autophosphorylation sites. Activation of the EGFR by its ligand EGF elicits alterations in F-actin content and cytoskeleton organization, which results in an altered cell morphology. NIH-3T3 fibroblasts expressing EGFRs lacking the ABD were analyzed for their EGF-induced capacity to invade a bone marrow stromal cell (BMSC) monolayer. The fibroblasts display a reduction in the percentage of cytoskeleton-associated EGFRs. EGF-induced tyrosine kinase activity is unaffected by the mutation. Cells expressing the mutant EGFRs hardly invade a BMSC monolayer upon EGF stimulation, in contrast to cells expressing wild-type EGFRs. Using the same cells, no difference is observed in PDGF-induced invasion: the ligand is as potent in both cell types as EGF is in wild-type cells. Inhibition of both the phosphatidyl inositol-3-kinase (PI-3-K) and lipoxygenase pathways in wild-type cells mimic the effect of the ABD deletion. These results point to an important role for the ABD of the EGFR in EGF-induced tissue invasion (van der Heyden, 1997a).

In the epidermal growth factor (EGF)-receptor signal transduction cascade, the non-receptor tyrosine kinase c-Src (see Drosophila Src oncogene 1) becomes activated upon EGF stimulation. c-Src associates with the cytoskeleton and co-isolates with actin filaments upon EGF treatment of NIH-3T3 cells transfected with the EGF receptor. Immunofluorescence studies show colocalization of F-actin and endogenous c-Src predominantly around endosomes and not on stress fibers and cell-cell contacts. Stimulation of EGF receptor-transfected NIH-3T3 cells with EGF induces an activation and translocation of c-Src to the cytoskeleton. These processes depend on the presence of the actin binding domain of the EGF-receptor, since in cells expressing EGF-receptors that lack this domain, EGF fails to induce an activation and a translocation to the cytoskeleton of c-Src. These data suggest a role for the actin binding domain of the EGF-receptor in the translocation of c-Src (Van der Heyden, 1997b).

Receptor-mediated endocytosis allows the specific removal of cell surface receptors and their cargo from the plasma membrane and targets them to endosomes, where they are sorted for downregulation or recycling. This process is initiated by recruitment of the receptor into a clathrin-coated pit at the plasma membrane, a structure formed by assembly of clathrin and adaptors into a protein lattice on the membrane's cytosolic face. Polymerization of clathrin into a hexagonal array provides a scaffold for organizing the adaptors, which recognize sequence motifs in the cytoplasmic domains of internalized receptors. A novel aspect of ligand-induced endocytosis of the epidermal growth factor receptor (EGFR) is described. Receptor signaling, upon ligand binding, stimulates modification and recruitment of clathrin. A partial explanation for the difference between constitutively endocytosed receptors and those whose endocytosis is ligand induced is that the adaptor recognition signal is constitutively accessible in the former, but cryptic in the latter, until ligand binding has occurred. For example, ligand binding to EGFR causes receptor tyrosine kinase activation and autophosphorylation. The implication of downstream signaling and effects on clathrin in several examples of ligand-induced endocytosis suggests a possible relationship between these processes, particularly in the case of the dramatic clathrin recruitment following receptor tyrosine kinase activation (Wilde, 1999).

Epidermal growth factor (EGF) binding to its receptor causes rapid phosphorylation of the clathrin heavy chain at tyrosine 1477, which lies in a domain controlling clathrin assembly. EGF-mediated clathrin phosphorylation is followed by clathrin redistribution to the cell periphery and is the product of downstream activation of SRC kinase by EGF receptor signaling. In cells lacking SRC kinase, or cells treated with a specific SRC family kinase inhibitor, EGF stimulation of clathrin phosphorylation and redistribution does not occur, and EGF endocytosis is delayed. These observations demonstrate a role for SRC kinase in modification and recruitment of clathrin during ligand-induced EGFR endocytosis and thereby define a novel effector mechanism for regulation of endocytosis by receptor signaling (Wilde, 1999).

Tyrosine 1477 has been identified as the site of pp60c-src-mediated clathrin heavy chain (CHC) phosphorylation during ligand-induced endocytosis of EGFR. Tyrosine 1477 is conserved in the three mammalian CHC sequences that have been determined and in the CHC of D. melanogaster, D. discoidum, and S. cerevisiae. Structural analysis of the light chain-binding region of CHC confirms that tyrosine 1477 is solvent exposed and located near the predicted clathrin light chain-binding site. The clathrin light chain subunits negatively regulate spontaneous clathrin assembly, so that cellular clathrin assembly is adaptor dependent. It is possible that tyrosine phosphorylation of residue 1477 causes an increase in clathrin assembly by directly affecting CHC interactions or that it affects assembly indirectly by negating the inhibitory effects that light chains have on assembly. Alternatively, tyrosine phosphorylation of CHC could recruit a protein that might enhance assembly or enhance transport of clathrin to the cell periphery. Whether clathrin recruitment directly influences EGF uptake by increasing the local concentration of clathrin and promoting coated pit formation or whether the regulatory mechanism is more complex, possibly relating to changing the intracellular dynamics of clathrin, will be the focus of future studies. It should be pointed out that the phosphorylation and recruitment of clathrin represent only a subset of the molecular requirements for EGF endocytosis. In mouse fibroblasts, CHC phosphorylation is necessary and sufficient for clathrin recruitment in some cell lines (SV40 transformed) but not sufficient in others (3T3-like), implicating additional factors. In the case of a pathway as important as regulated endocytosis, it would not be surprising if, in different tissues, different SRC family kinases could mediate clathrin phosphorylation (Wilde, 1999).

There is a requirement for the nonreceptor tyrosine kinase, cellular Src (c-Src), in epidermal growth factor (EGF)-induced mitogenesis, and a synergistic interaction between c-Src and EGF receptor (EGFR) in tumorigenesis. Although endocytic internalization of EGFR may be thought to attenuate EGF-stimulated signaling, recent evidence suggests that signaling through Ras can be amplified by repeated encounters of endosome-localized receptor. Shc.Grb2.Sos complexes with the plasma membrane, where Ras resides almost exclusively. Based on these reports, EGFR trafficking behavior was examined in a set of single and double c-Src/EGFR C3H10T1/2 overexpressors to determine if c-Src affects basal receptor half-life, ligand-induced internalization, and/or recycling. Overexpression of c-Src causes no change in EGFR half-life but does produce an increase in the internalization rate constant of EGF.EGFR complexes, when the endocytic apparatus is not stoichiometrically saturated; this effect of c-Src on EGFR endocytosis is negligible at high receptor occupancy in cells overexpressing the receptor. In neither case are EGFR recycling rate constants affected by c-Src. These data indicate a functional role for c-Src in receptor internalization, which in turn could alter some aspects of EGFR signaling related to mitogenesis and tumorigenesis (Ware, 1997).

Table of contents

EGF receptor : Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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