EGF receptor

EVOLUTIONARY HOMOLOGS

Table of contents

Additional mechanisms for EGFR activation

PC12 cells respond to a variety of external stimuli, such as growth factors, neurotransmitters, and membrane depolarization, by activating the Ras/mitogen-activated protein kinase pathway. Both depolarization-induced calcium influx and treatment with bradykinin stimulate tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Depolarization and bradykinin triggered signals involve EGFR function upstream of SHC and MAP kinase. Bradykinin-stimulated EGFR transactivation is critically dependent on the presence of extracellular calcium; when triggered by ionophore treatment, calcium influx is already sufficient to induce EGFR tyrosine phosphorylation. Taken together, these results establish calcium-dependent EGFR transactivation as a signaling mechanism mediating activation of the Ras/mitogen-activated protein kinase pathway in neuronal cell types (Zwick, 1997).

The epidermal growth factor receptor (EGFR) and the neu oncoprotein become rapidly tyrosine-phosphorylated upon stimulation of Rat-1 cells with the G-protein coupled receptor (GPCR) agonists endothelin-1, lysophosphatic acid and thrombin, suggesting that there is an intracellular mechanism for transactivation. Specific inhibition of EGFR function by either the selective tyrphostin AG1478 or a dominant-negative EGFR mutant suppresses MAP kinase activation and strongly inhibit induction of fos gene expression and DNA synthesis. These results demonstrate a role for RTKs as downstream mediators in GPCR mitogenic signalling and suggest a ligand-independent mechanism of RTK activation through intracellular signal crosstalk (Daub, 1996).

The epidermal growth factor receptor (EGFR) tyrosine kinase recently was identified as providing a link to mitogen-activated protein kinase (MAPK) in response to G protein-coupled receptor (GPCR) agonists in Rat-1 fibroblasts. This cross-talk pathway is also established in other cell types such as HaCaT keratinocytes, primary mouse astrocytes and COS-7 cells. Transient expression of either Gq- or Gi-coupled receptors in COS-7 cells allows GPCR agonist-induced EGFR transactivation, and lysophosphatidic acid (LPA)-generated signals involve the docking protein Gab1. The increase in SHC tyrosine phosphorylation and MAPK stimulation through both Gq- and Gi-coupled receptors is reduced strongly upon selective inhibition of EGFR function. Inhibition of phosphoinositide 3-kinase does not affect GPCR-induced stimulation of EGFR tyrosine phosphorylation, but inhibits MAPK stimulation, upon treatment with both GPCR agonists and low doses of EGF. The Src tyrosine kinase inhibitor PP1 strongly interfers with LPA- and EGF-induced tyrosine phosphorylation and MAPK activation downstream of EGFR. These results demonstrate an essential role for EGFR function in signaling through both Gq- and Gi-coupled receptors and provide novel insights into signal transmission downstream of EGFR for efficient activation of the Ras/MAPK pathway (Daub, 1997).

Lysophosphatidic acid (LPA) utilizes a G-protein-coupled receptor to activate the small GTP-binding protein Rho and to induce rapid remodeling of the actin cytoskeleton. The signal transduction from LPA receptors to Rho activation has been studied. Analysis of the G-protein-coupling pattern of LPA receptors (by labeling activated G-proteins with [alpha-32P]GTP azidoanilide) reveals interaction with proteins of the Gq, Gi, and G12 subfamilies. In COS-7 cells, expression of GTPase-deficient mutants of Galpha12 and Galpha13 trigger Rho activation as measured by increased Rho-GTP levels. In Swiss 3T3 cells, incubation with LPA or microinjection of constitutively active mutants of Galpha12 and Galpha13 induce formation of actin stress fibers and assembly of focal adhesions in a Rho-dependent manner. Interestingly, the LPA-dependent cytoskeletal reorganization is suppressed by microinjected antibodies directed against Galpha13, whereas Galpha12-specific antibodies show no inhibition. The tyrosine kinase inhibitor tyrphostin A 25 and the epidermal growth factor (EGF) receptor-specific tyrphostin AG 1478 completely block actin stress fiber formation caused by LPA or activated Galpha13, but not the effects of activated Galpha12. Expression of the dominant negative EGF receptor mutant EGFR-CD533 also markedly prevents the LPA- and Galpha13-induced actin polymerization. Coexpression of EGFR-CD533 and activated Galpha13 in COS-7 cells result in decreased Rho-GTP levels, when compared with expression of activated Galpha13 alone. These data indicate that in Swiss 3T3 cells, G13 (but not G12) is involved in the LPA-induced activation of Rho. These results suggest an involvement of the EGF receptor in this pathway (Gohla, 1998).

When growth hormone binds to its receptor, which belongs to the cytokine receptor superfamily, activates the Janus kinase Jak2 (see Drosophila Hopscotch). Jak2 provides tyrosine-kinase activity and initiates an activation of several key intracellular proteins (for example, mitogen-activated protein (MAP) kinases) that eventually execute the biological actions induced by growth hormone, including the expression of particular genes. In contrast to receptors that themselves have tyrosine kinase activity, the signaling pathways leading to MAP kinase activation triggered by growth hormone are poorly understood, but appear to be mediated by the proteins Grb2 and Shc (see Drosophila Shc). Growth hormone stimulates tyrosine phosphorylation of the receptor for epidermal growth factor (Egf-r) and its association with Grb2; at the same time, Egf-r stimulates MAP kinase activity in liver, an important target tissue of growth hormone. Expression of Egf-r and its mutants reveals that growth-hormone-induced activation of MAP kinase and expression of the transcription factor c-fos requires phosphorylation of tyrosines on Egf-r, but not its own intrinsic tyrosine-kinase activity. The tyrosine at residue 1,068 of the Egf-r is proposed to be one of the principal phosphorylation sites and a Grb2-binding site stimulated by growth hormone via Jak2. These results indicate that the role of Egf-r in signaling by growth hormone is to be phosphorylated by Jak2, thereby providing docking sites for Grb2 and activating MAP kinases and gene expression, independent of the intrinsic tyrosine kinase activity of Egf-r. This may represent a novel cross-talk pathway between the cytokine receptor superfamily and growth factor receptor (Yamauchi, 1997).

Tenascin-C (TN-C) (see Drosophila Tenascin major) is induced in pulmonary vascular disease, where it colocalizes with proliferating smooth muscle cells (SMCs) and epidermal growth factor (Egf). Cultured SMCs require TN-C for Egf-dependent growth on type I collagen. In this study, the regulation and function of TN-C was explored in SMCs. A matix metalloproteinase (MMP) inhibitor (GM6001) suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-C expression in a beta3 integrin- dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of Egf. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001, restores the Egf growth response and "rescues" cells from apoptosis. The mechanism by which TN-C facilitates Egf-dependent survival and growth was then investigated. TN-C interactions with alphavbeta3 integrins modify SMC shape, and Egf-dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of Egf-rs, tyrosine-phosphorylated proteins, and increased activation of Egf-rs after addition of Egf. Cross-linking SMC beta3 integrins replicates the effect of TN-C on Egf-r clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating beta3 integrin ligands in type I collagen. In turn, alphavbeta3 interactions with TN-C alter SMC shape and increase Egf-r clustering and Egf-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces apoptosis (Jones, 1997).

Adhesion of human primary skin fibroblasts and ECV304 endothelial cells to immobilized matrix proteins (beta1 or alphav integrin antibodies) stimulates tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. This tyrosine phosphorylation is transiently induced, reaching maximal levels 30 min after adhesion, and it occurs in the absence of receptor ligands. Similar results are observed with EGF receptor-transfected NIH-3T3 cells. Use of a kinase-negative EGF receptor mutant demonstrates that the integrin-stimulated tyrosine phosphorylation is due to activation of the receptor's intrinsic kinase activity. Integrin-mediated EGF receptor activation leads to Erk-1/MAP kinase induction, as shown by treatment with the specific inhibitor tyrphostin AG1478 and by expression of a dominant-negative EGF receptor mutant. EGF receptor and Erk-1/MAP kinase activation by integrins does not lead per se to cell proliferation, but is important for entry into S phase in response to EGF or serum. EGF receptor activation is also required for extracellular matrix-mediated cell survival. Adhesion-dependent MAP kinase activation and survival are regulated through EGF receptor activation in cells expressing this molecule above a threshold level of 5000 receptors per cell. These results demonstrate that integrin-dependent EGF receptor activation is a novel signaling mechanism involved in cell survival and proliferation in response to the extracellular matrix. The molecular mechanisms underlying EGF receptor activation by integrins remain to be defined. Integrin-dependent activation of the EGF receptor and of other tyrosine kinases such as p125Fak occurs through distinct mechanisms. In fact, cytochalasin D, which disrupts the actin cytoskeleton, strongly inhibits p125Fak tyrosine phosphorylation, but does not affect EGF receptor tyrosine phosphorylation in response to integrins. This indicates that the organization of actin cytoskeleton is not a primary event in integrin-dependent EGF receptor activation, while it is required to bring together signaling proteins leading to p125Fak tyrosine phosphorylation. These data also indicate that p125Fak tyrosine phosphorylation is not required in integrin-mediated EGF receptor activation. Moreover, integrins and EGF receptors can associate on the membrane, forming a molecular complex, as shown by co-immunoprecipitation experiments, while association of integrins with p125Fak cannot be detected by this technique. Preliminary experiments also show that PtdIns-3 kinase is not involved in integrin-induced EGF receptor tyrosine phosphorylation, as detected by the use of specific inhibitors (Moro, 1998).

Activated epidermal growth factor receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between signaling and endocytosis is not well understood. EGF stimulation of NR6 cells induces a specific, rapid and transient activation of Rab5a. EGF also enhances translocation of the Rab5 effector, early endosomal autoantigen 1 (EEA1), from cytosol to membrane. The activation of endocytosis, fluid phase and receptor mediated, by EGF is enhanced by Rab5a expression, but not by Rab5b, Rab5c, or Rab5a truncated at the NH2 and/or COOH terminus. Dominant negative Rab5a (Rab5:N34) blocks EGF-stimulated receptor-mediated and fluid-phase endocytosis. EGF activation of Rab5a function is dependent on tyrosine residues in the COOH-terminal domain of the EGF receptor (EGFR). Removal of the entire COOH terminus by truncation (c'973 and c'991) abrogates ligand-induced Rab5a activation of endocytosis. A 'kinase-dead' EGFR fails to stimulate Rab5a function. However, another EGF receptor mutant (c'1000), with the kinase domain intact and a single autophosphorylation site, effectively signals Rab5 activation. These results indicate that EGFR and Rab5a are linked via a cascade that results in the activation of Rab5a and that appears essential for internalization. The results point to an interdependent relationship between receptor activation, signal generation and endocytosis (Barbieri, 2000).

Autoinhibition of EGFR

The orphan receptor tyrosine kinase ErbB2 (also known as HER2 or Neu) transforms cells when overexpressed, and it is an important therapeutic target in human cancer. Structural studies have suggested that the oncogenic (and ligand-independent) signalling properties of ErbB2 result from the absence of a key intramolecular 'tether' in the extracellular region that autoinhibits other human ErbB receptors, including the epidermal growth factor (EGF) receptor. Although ErbB2 is unique among the four human ErbB receptors, this study shows that it is the closest structural relative of the single EGF receptor family member in Drosophila melanogaster (dEGFR). Genetic and biochemical data show that dEGFR is tightly regulated by growth factor ligands, yet a crystal structure, presented in this study, shows that it, too, lacks the intramolecular tether seen in human EGFR, ErbB3 and ErbB4. Instead, a distinct set of autoinhibitory interdomain interactions hold unliganded dEGFR in an inactive state. All of these interactions are maintained (and even extended) in ErbB2, arguing against the suggestion that ErbB2 lacks autoinhibition. It is therefore suggested that normal and pathogenic ErbB2 signalling may be regulated by ligands in the same way as dEGFR. These findings have important implications for ErbB2 regulation in human cancer, and for developing therapeutic approaches that target novel aspects of this orphan receptor (Alvarado, 2009).

Recycling, endocytosis and downregulation of EGFR

Calcium-modulating cyclophilin ligand (CAML) is a ubiquitous protein that has been implicated in signaling from the cell surface receptor TACI in lymphocytes, although its role and mechanism of action are unknown. To study its function in the mouse, the CAML gene was disrupted; it was found to be required for early embryonic development, but not for cellular viability. CAML-deficient cells have severely impaired proliferative responses to the epidermal growth factor (EGF). Although EGF-induced activation of signaling intermediates and internalization of the EGF receptor (EGFR) are normal in the absence of CAML, the recycling of internalized receptors to the plasma membrane is defective, leading to its reduced surface accumulation. CAML normally associates directly with the kinase domain of the EGFR in a ligand-dependent manner. These data implicate CAML in EGFR signaling and suggest that it may play a role in receptor recycling during long-term proliferative responses to EGF (Tran, 2003).

Upon activation, the EGFR is rapidly internalized and later enters a complex sorting process. c-Cbl binds directly to and ubiquitinates internalized receptors, which are subsequently delivered to the lysosome for degradation. Alternatively, a significant fraction of endocytosed receptors escapes c-Cbl-mediated ubiquitination and becomes dephosphorylated before being diverted toward the recycling route. However, the factors that identify and sort this cargo into the recycling endosome are currently unclear. The direct physical binding of CAML to the EGFR in a ligand-dependent fashion, especially to the more rapidly migrating species most likely corresponding to the nonubiquitinated and tyrosine-hypophosphorylated receptor, is consistent with its proposed function in receptor recycling. The finding that CAML directly interacts with the core kinase domain of the receptor may also be consistent with its potential role in recycling, since intrinsic receptor kinase activities have been implicated in regulating EGFR trafficking, including recycling, but not lysosomal targeting. Receptor mutants lacking this region are efficiently degraded in the lysosome in a constitutive fashion and yet fail to undergo other aspects of normal routing. CAML may by preferentially binding to nonubiquitinated receptors to aid in their return to the cell surface.These data suggest that CAML may regulate EGFR recycling not by deubiquitinating the receptor but more likely by influencing trafficking steps downstream of the ubiquitination machinery. The majority of accumulated EGFR in CAML-negative cells does not appear to be multiply monoubiquitinated in response to ligand. The hypothesis is favored, therefore, that the accumulated EGFR in CAML-negative cells represents a population of molecules unable to complete the recycling process in the absence of CAML (Tran, 2003).

Plasma membrane receptors can be endocytosed through clathrin-dependent and clathrin-independent pathways. The epidermal growth factor receptor (EGFR), when stimulated with low doses of EGF, is internalized almost exclusively through the clathrin pathway, and it is not ubiquitinated. At higher concentrations of ligand, however, a substantial fraction of the receptor is endocytosed through a clathrin-independent, lipid raft-dependent route, as the receptor becomes ubiquitinated. A ubiquitination-impaired EGFR mutant is internalizes through the clathrin pathway, whereas an EGFR/ubiquitin chimera, that can signal solely through its ubiquitin (Ub) moiety, is internalizes exclusively by the non-clathrin pathway. Non-clathrin internalization of ubiquitinated EGFR depends on its interaction with proteins harboring the Ub-interacting motif, as shown through the ablation of three Ub-interacting motif-containing proteins: eps15, eps15R, and epsin. Thus, eps15s and epsin perform an important function in coupling ubiquitinated cargo to clathrin-independent internalization (Sigismund, 2005).

Down-regulation of mitogenic signaling in mammalian cells relies in part on endosomal trafficking of activated receptors into lysosomes, where the receptors are degraded. These events are mediated by ubiquitination of the endosomal cargo and its consequent sorting into multivesicular bodies that form at the surfaces of late endosomes. Tumor susceptibility gene 101 (tsg101) recently was found to be centrally involved in this process. TSG101 (see Drosophila Tumor suppressor protein 101) interacts with HRS, an early endosomal protein, and disruption of this interaction impedes endosomal trafficking and endocytosis-mediated degradation of mitogenic receptors. TSG101/HRS interaction occurs between a ubiquitin-binding domain of TSG101 and two distinct proline-rich regions of HRS, and is modulated by a C-terminal TSG101 sequence that resembles a motif targeted in HRS. Mutational perturbation of TSG101/HRS interaction prevents delivery of epidermal growth factor receptor (EGFR) to late endosomes, resulted in the cellular accumulation of ubiquitinated EGFR in early endosomes, and inhibited ligand-induced down-regulation of EGFR. These results reveal the TSG101 interaction with HRS as a crucial step in endocytic down-regulation of mitogenic signaling and suggest a role for this interaction in linking the functions of early and late endosomes (Lu, 2003).

The ubiquitin ligase Cbl mediates ubiquitination of activated receptor tyrosine kinases (RTKs) and interacts with endocytic scaffold complexes, including CIN85/endophilins, to facilitate RTK endocytosis and degradation. Several mechanisms regulate the functions of Cbl to ensure the fine-tuning of RTK signalling and cellular homeostasis. One regulatory mechanism involves the binding of Cbl to Sprouty2, which sequesters Cbl away from activated epidermal growth factor receptors (EGFRs). Sprouty2 associates with CIN85 and acts at the interface between Cbl and CIN85 to inhibit EGFR downregulation. The CIN85 SH3 domains A and C bind specifically to proline-arginine motifs present in Sprouty2. Intact association between Sprouty2, Cbl and CIN85 is required for inhibition of EGFR endocytosis as well as EGF-induced differentiation of PC12 cells. Moreover, Sprouty4, which lacks CIN85-binding sites, does not inhibit EGFR downregulation, providing a molecular explanation for functional differences between Sprouty isoforms. Sprouty2 therefore acts as an inducible inhibitor of EGFR downregulation by targeting both the Cbl and CIN85 pathways (Haglund, 2005).

Knockdown of growth factor receptor binding protein 2 (Grb2) by RNA interference strongly inhibits clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR). To gain insights into the function of Grb2 in EGFR endocytosis, cell lines were generated in which endogenous Grb2 was replaced by yellow fluorescent protein (YFP)-tagged Grb2 expressed at the physiological level. In these cells, Grb2-YFP fully reversed the inhibitory effect of Grb2 knockdown on EGFR endocytosis and, moreover, trafficked together with EGFR during endocytosis. Overexpression of Grb2-binding protein c-Cbl does not restore endocytosis in Grb2-depleted cells. However, EGFR endocytosis is rescued in Grb2-depleted cells by chimeric proteins consisting of the Src homology (SH) 2 domain of Grb2 fused to c-Cbl. The 'knockdown and rescue' analysis revealed that the expression of Cbl-Grb2/SH2 fusions containing RING finger domain of Cbl restores normal ubiquitylation and internalization of the EGFR in the absence of Grb2, consistent with the important role of the RING domain in EGFR endocytosis. In contrast, the carboxy-terminal domain of Cbl, when attached to Grb2 SH2 domain, had 4 times smaller endocytosis-rescue effect compared with the RING-containing chimeras. Together, the data suggest that the interaction of Cbl carboxy terminus with CIN85 has a minor and a redundant role in EGFR internalization. It is concluded that Grb2-mediated recruitment of the functional RING domain of Cbl to the EGFR is essential and sufficient to support receptor endocytosis (Huang, 2005).

Ligand-induced activation of the epidermal growth factor receptor (EGFR) initiates multiple signal-transduction pathways as well as trafficking events that relocalize the receptors from the cell surface to intracellular endocytic compartments. Although there is growing awareness that endocytic transport can play a direct role in signal specification, relatively little is known about the molecular mechanisms underlying this link. This study shows that human Sprouty 2 (hSpry2), a protein that has been implicated in the negative regulation of receptor tyrosine kinase (RTK) signaling, interferes with the trafficking of activated EGFR specifically at the step of progression from early to late endosomes. This effect is mediated by the binding of hSpry2 to the endocytic regulatory protein, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), and leads to a block in intracellular signal propagation. These observations suggest that EGFR signaling is controlled by a novel mechanism involving trafficking-dependent alterations in receptor compartmentalization (Kim, 2007).

Clathrin-mediated endocytosis (CME) is the major pathway of epidermal growth factor receptor (EGFR) internalization. It is commonly believed that CME mediates long-term attenuation of EGFR signaling by targeting the receptor for degradation. However, the EGFR can also be internalized through one or more clathrin-independent pathway, and it remains unclear why distinct mechanisms of internalization have evolved. This study reports that EGFRs internalized via CME are not targeted for degradation, but instead are recycled to the cell surface. By contrast, clathrin-independent internalization preferentially commits the receptor to degradation. This finding has profound implications for signaling, as by skewing EGFR fate toward recycling rather than degradation, CME prolongs the duration of signaling. The data show that CME determines the longevity of some EGFR-activated signaling pathways and that EGF-dependent biological responses, such as DNA synthesis, absolutely require CME. Thus, CME of the EGFR unexpectedly has a greater impact on receptor signaling than on receptor degradation (Sigismund, 2008).

EGFR mutation and interspecies substitution

Epidermal growth factor receptor (EGFR) is a key regulator of keratinocyte biology. However, the physiological role of EGFR in vivo has not been well established. To analyze the role of EGFR in skin, transgenic mice were generated expressing an EGFR dominant negative mutant in the basal layer of epidermis and outer root sheath of hair follicles. Mice expressing the mutant receptor display short and waved pelage hair and curly whiskers during the first weeks of age, but subsequently pelage and vibrissa hairs become progressively sparser and atrophic. Eventually, most mice present severe alopecia. Histological examination of the skin of transgenic mice shows striking alterations in the development of hair follicles: they fail to enter into catagen stage. These alterations eventually lead to necrosis and disappearance of the follicles, accompanied by strong infiltration of the skin with inflammatory elements. The interfollicular epidermis of these mice shows marked hyperplasia, expression of hyperproliferation-associated keratin K6 and increased 5-bromo-2-deoxyuridine incorporation. EGFR function is inhibited in transgenic skin keratinocytes, since in vivo and in vitro autophosphorylation of EGFR is almost completely abolished on EGF stimulation. These results implicate EGFR in the control of hair cycle progression, and provide new information about its role in epidermal growth and differentiation (Murillas, 1995).

The wa-2 mutation was mapped previously to the vicinity of the EGF/TGF-alpha receptor (EGFR) gene. EGFR mRNA and protein are expressed in wa-2 liver and skin at levels that are comparable to those in the corresponding normal tissues, and the ability of wa-2 EGFR to bind ligand is unaltered. However, ligand-dependent autophosphorylation of wa-2 EGFR is diminished 5- to 10-fold in vitro, and the ability of wa-2 EGFR to phosphorylate an exogenous substrate is reduced by > 90% compared with that of the control receptor. EGF-induced tyrosine phosphorylation, including that of EGFR itself, is also diminished in skin, particularly at lower dose of exogenous EGF. There is a single-nucleotide transversion resulting in the substitution of a glycine for a conserved valine residue near the amino terminus of the tyrosine kinase domain. The importance of this mutation was confirmed by showing that its introduction into an otherwise normal EGFR markedly reduces the receptor's tyrosine kinase activity in transfected Chinese hamster ovary cells. In situ hybridization analysis demonstrated expression of EGFR predominantly in the outer root sheath of active hair follicles in neonatal mice. This pattern of EGFR expression is consistent with the effect of the wa-2 mutation on hair structure, and reveals a critical role for signaling by this ligand/receptor system in skin (Luetteke, 1994).

Although the tyrosine kinase activity of wa-2 EGF receptors is significantly impaired, NIH 3T3 cells lacking endogenous EGF receptors but overexpressing recombinant wa-2 EGF receptor cDNA are mitogenically responsive to EGF. While young and adult wa-2 mice are healthy and fertile, 35% of wa-2 mice born of homozygous wa-2 mothers die of malnutrition because of impaired maternal lactation (Fowler, 1995).

Mice lacking the epidermal growth factor receptor (EGFR) exhibit strain-dependent phenotypes ranging from placental to postnatal skin, lung and brain defects. After birth, all mutant mice develop a progressive neurodegeneration in the frontal cortex, olfactory bulb and thalamus, characterized by massive apoptosis and upregulation of c-fos. These defects occur in a strain-independent manner, since neither rescue of the placental phenotype by aggregation of diploid 129/Sv EGFR mutant and tetraploid wild-type embryos, nor promotion of lung maturation by transplacental dexamethasone administration alters the course of neurodegeneration. VEGF is not induced during the degenerative process, excluding hypoxia and ischemia as causes of cell death. A migratory disorder is detected in the hippocampus with nests of ectopic neurons, which are also apoptotic. Cerebral cortices from Egf-r mutants contain lower numbers of GFAP positive astrocytes, which display reduced proliferation in vitro. Since Egf-r is expressed in the affected cell-types, these results define a specific function for Egf-r in the proliferation and/or differentiation of astrocytes and in the survival of postmitotic neurons (Sibilia, 1998).

Signaling by the epidermal growth factor (Egf) receptor (Egfr) has been studied intensively, but for most cell types the analysis is complicated by the fact that Egfr not only homodimerizes but can also form heterodimers with other Egfr family members. Heterodimerization is a particular problem in the study of Egfr mutants, where the true phenotype of the mutants is confounded by the contribution of the heterodimer partner to signal transduction. The murine hemopoietic cell line BaF/3, which does not express Egfr family members, was used to express either wild-type (WT) Egfr, three kinase-defective Egfr mutants (V741G, Y740F, and K721R), or a C-terminally truncated Egfr (CT957) and their responses to Egf were measured. Under the appropriate conditions Egf can stimulate cell proliferation of BaF/3 cells expressing WT or CT957 Egfrs but not that of cells expressing the kinase-defective mutants. However, Egf promotes the survival of BaF/3 cells expressing either of the kinase-defective receptors (V741G and Y740F), indicating that these receptors can still transmit a survival signal. Analysis of the early signaling events by the WT, V741G, and Y740F mutant Egf receptors indicates that Egf stimulates comparable levels of Shc phosphorylation, Shc-GRB-2 association, and activation of Ras, B-Raf, and Erk-1. Blocking the mitogen-activated protein kinase (MAPK) signaling pathway with the specific inhibitor PD98059 abrogates completely the Egf-dependent survival of cells expressing the kinase-defective Egfr mutants but has no effect on the Egf-dependent proliferation mediated by WT and CT957 Egfrs. Similarly, the Src family kinase inhibitor PP1 abrogates Egf-dependent survival without affecting proliferation. However blocking phosphatidylinositol-3-kinase or JAK-2 kinase with specific inhibitors does arrest growth factor-dependent cell proliferation. Thus, Egfr-mediated mitogenic signaling in BaF/3 cells requires an intact Egfr tyrosine kinase activity and appears to depend on the activation of both the JAK-2 and PI-3 kinase pathways. Activation of the Src family of kinases or of the Ras/MAPK pathway can, however, be initiated by a kinase-impaired Egfr and is linked to survival (Walker, 1998).

Pancreatic acini and islets are believed to differentiate from common ductal precursors through a process requiring various growth factors. Epidermal growth factor receptor is expressed throughout the developing pancreas. The pancreatic phenotype of Egfr deficient mice, which generally die from epithelial immaturity within the first postnatal week, has been analyzed. The pancreata appear macroscopically normal. The most striking feature of the Egfr minus islets is that instead of forming circular clusters, the islet cells are mainly located in streak-like structures directly associated with pancreatic ducts. Based on BrdU-labelling, proliferation of the neonatal Egfr minus beta-cells is significantly reduced and the difference persists even at 7-11 days of age. Analysis of embryonic pancreata reveals impaired branching morphogenesis and delayed islet cell differentiation in the mutant mice. Islet development was analyzed further in organ cultures of E12.5 pancreata. The proportion of insulin-positive cells is significantly lower in the mutant explants, indicating delayed differentiation of the beta cells. Branching of the epithelium into ducts is also impaired. Matrix metalloproteinase (MMP-2 and MMP-9) activity is reduced 20% in mutant late-gestation pancreata, as measured by gelatinase assays. Furthermore, the levels of secreted plasminogen activator inhibitor-1 (PAI-1) are markedly higher, while no apparent differences are seen in the levels of active uPA and tPa between mutant and wild-type pancreata. These findings suggest that the perturbation of EGF-R-mediated signaling can lead to a generalized proliferation defect of the pancreatic epithelia associated with a delay in beta cell development and disturbed migration of the developing islet cells as they differentiate from their precursors. Upregulated PAI-1 production and decreased gelatinolytic activity correlates to this migration defect. An intact EGF-R pathway appears to be a prerequisite for normal pancreatic development (Miettinen, 2000).

The physiological role of any of the epidermal growth factor (EGF) receptor tyrosine kinases has yet to be determined in zebrafish. A zebrafish homolog of EGFR (egfr) was isolated that shows a 63% amino acid overall identity to human EGFR but with 90% amino acid identity in the kinase domain. Whole mount in situ hybridization shows ubiquitous distribution of egfr transcripts during gastrulation, somitogenesis and later stages. When expressed in Chinese hamster ovary cells, zebrafish Egfr is a functional receptor that responds to EGF by receptor tyrosine phosphorylation and activation of MAP kinase. The function of zebrafish Egfr in vivo was determined by inhibiting its activity using EGFR kinase inhibitors and antisense morpholinos (MO); these treatments, respectively, inhibit Egfr kinase activity and translation of egfr messenger RNA into protein. The zebrafish is a particularly excellent model for studying cardiovascular development because zebrafish are transparent allowing direct visualization of the heart and circulation in the blood vessels. Inhibition of zebrafish Egfr activity in vivo impedes blood flow via the outflow tract into the aorta and impedes circulation in the axial and intersegmental vessels by 80 h post-fertilization. Analysis of the heart shows that the heart chambers and pericardial sacs are dilated and the outflow tracts are narrowed. Together these results suggest that zebrafish Egfr has a cardiovascular function in the developing zebrafish that is required for normal circulation (Goishi, 2003).

Mice lacking the epidermal growth factor receptor (EGFR) develop epithelial defects and a neurodegenerative disease and die within the first month of birth. By employing a conditional knock-in approach using the human EGFR cDNA, mice humanized for EGFR (hEGFR^KI/KI) were generated. Homozygous hEGFR^KI/KI mice are viable and live up to six months. However, these mice are growth retarded and show skin and hair defects similar to Egfr^-/- mutants. Interestingly, the neurodegeneration is fully rescued in hEGFR^KI/KI mice, however, they develop a severe heart hypertrophy with semilunar valve abnormalities. Moreover, hEGFR^KI/KI mice display accelerated chondrocyte and osteoblast differentiation, a phenotype that is also present in Egfr^-/- mice and has not been previously described. The severity of the phenotypes correlates with the expression levels of the hEGFR^KI allele, which is not efficiently expressed in epithelial and bone cells, but is expressed at similar and even higher levels as the endogenous Egfr in brain and heart. These results demonstrate that mice humanized for EGFR display tissue-specific hypomorphic phenotypes and describe a novel function for EGFR in bone development (Sibilia, 2003).

EGFR, cell migation, axon development and axon guidance

Epidermal growth factor receptors (EGFRs) have been implicated in the control of migration in the telencephalon, but the mechanism underlying their contribution is unclear. Expression of a threshold level of EGFRs confers chemotactic competence in stem cells, neurons and astrocytes in cortical explants. This level of receptor expression is normally achieved by a subpopulation of cells during mid-embryonic development. Cells that express high levels of EGFR are located in migration pathways, including the tangential pathway to the olfactory bulb via the rostral migratory stream (RMS), the lateral cortical stream (LCS) leading to ventrolateral cortex and the radial pathway from proliferative zones to cortical plate. The targets of these pathways express the ligands HB-EGF and/or TGFalpha. To test the idea that EGFRs mediate chemotactic migration in these pathways, the size of the population of cells expressing threshold levels of EGFRs was increased in vivo by viral transduction. The results suggest that EGFRs mediate migration radially to the cortical plate and ventrolaterally in the LCS, but not tangentially in the RMS. Within the bulb, however, EGFRs also mediate radial migration. These findings suggest that developmental changes in EGFR expression, together with changes in ligand expression regulate the migration of specific populations of cells in the telencephalon by a chemoattractive mechanism (Caric, 2001).

Neural progenitor proliferation, differentiation and migration are continually active in the rostral migratory stream of the adult brain. The receptor tyrosine kinase ErbB4 is expressed prominently by the neuroblasts present in the subventricular zone and the rostral migratory stream. The neuregulins (NRG1-NRG3), which have been identified as ErbB4 ligands, are detected either in the stream or in adjacent regions. Mice deficient in ErbB4 expressed under the control of either the nestin or the hGFAP promoter have altered neuroblast chain organization and migration and deficits in the placement and differentiation of olfactory interneurons. These findings suggest that ErbB4 activation helps to regulate the organization of neural chains that form the rostral migratory stream and influences the differentiation of olfactory interneuronal precursors (Anton, 2004).

Most cortical interneurons arise from the subcortical telencephalon, but the molecules that control their migration remain largely unidentified. Different isoforms of Neuregulin-1 are expressed in the developing cortex and in the route that migrating interneurons follow toward the cortex, whereas a population of the migrating interneurons express ErbB4, a receptor for Neuregulin-1. The different isoforms of Neuregulin-1 act as short- and long-range attractants for migrating interneurons, and perturbing ErbB4 function in vitro decreases the number of interneurons that tangentially migrate to the cortex. In vivo, loss of Neuregulin-1/ErbB4 signaling causes an alteration in the tangential migration of cortical interneurons and a reduction in the number of GABAergic interneurons in the postnatal cortex. These observations provide evidence that Neuregulin-1 and its ErbB4 receptor directly control neuronal migration in the nervous system (Flames, 2004).

In postnatal developing optic nerves, astrocytes organize their processes in a cribriform (appearing like a sieve; perforated with many small openings) network to group axons into bundles. In neonatal rat optic nerves in vivo, the active form of EGFR tyrosine kinase is abundantly present when the organization of astrocytes and axons is most actively occurring. Blocking activity of EGFR tyrosine kinase during the development of rat optic nerves in vivo inhibits astrocytes from extending fine processes to surround axons. In vitro, postnatal optic nerve astrocytes, stimulated by EGF, organize into cribriform structures which look remarkably like the in vivo structure of astrocytes in the optic nerve. In addition, when astrocytes are co-cultured with neonatal rat retinal explants in the presence of EGF, astrocytes that are adjacent to the retinal explants, re-organize to an astrocyte-free zone into which neurites grow out from the retinal tissue. It is hypothesized that in the developing optic nerve, EGFR activity directs the formation of a histoarchitectural structure of astrocytes that surrounds axons and provides a permissive environment for axon development (Liu, 2004).

Receptor tyrosine kinases of the EGFR family exert their various effects on cellular function through the formation of different dimeric receptor complexes. To investigate the functional impact of EGFR-HER2 heterodimers on migration of glial tumour cells, different HER2 constructs, including a constitutively active (HER2VE) and a dominant-negative (HER2VEKA) receptor, were stably transfected in the EGFR-overexpressing human glioma cell line LN18. Interference of EGFR activation through HER2VEKA inhibited cellular migration, whereas EGFR activation through HER2VE increased migration. These results were corroborated by inhibition of EGFR-HER2 signalling with tyrosine kinase inhibitors, because only the blocking of both receptors in HER2VE-cells with the bi-specific inhibitor AEE788 downregulated migration to levels comparable with those in HER2VEKA cells. The non-migratory phenotype was mediated through upregulation of N-cadherin and its recruitment to the cell membrane in HER2VEKA cells; downregulation of N-cadherin by RNAi restored migration in HER2VEKA cells and N-cadherin was also downregulated in migrating HER2VE-cells. Downregulation of N-cadherin levels in the plasma membrane was accompanied by a direct interaction of the EGFR-HER2 and N-cadherin-beta-catenin complexes, leading to tyrosine phosphorylation of beta-catenin. These results indicate that HER2 affects glial-cell migration by modulating EGFR-HER2 signal transduction, and that this effect is mediated by N-cadherin (Rappl, 2008).

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