Epidermal Growth Factor Receptor Agonists Research Articles

Adrenergic and V1-ergic Agonists/Antagonists Affecting Recovery from Brain Trauma in the Lund Project Act on Astrocytes

The Lund project (1992) recommended treatment with clonidine (α2-adrenergic agonist) and metoprolol (β1-adrenergic antagonist) to improve recovery after brain trauma, and discouraged use of the V1 agonist vasopressin (ADH). Brain effects of these drugs and the ability of a post-traumatic elevation of extracellular K+ concentrations ([K+]o) to activate mechanism(s) leading to secondary cytotoxic (cellular) edema were then virtually unknown. Now, it is established that interactions occur between effects on astrocytes by high [K+]o and vasopressin or α2- and β1-adrenergic agonists and antagonists, and that the effects modify edema and thus intracranial pressure. In mouse astrocytes in primary cultures, reliably expressing characteristics of their in vivo counterparts, high [K+]o and each of the transmitters agonists activate a signal mechanism, transactivation, in which Ca2+ entry through depolarization-mediated channel opening or stimulation of Gq- or Gi/o protein-coupled receptors via PKC-, Ca2+- and metalloproteinase-mediated signaling leads to release of an epidermal growth factor (EGF) receptor agonist. Minor, but important, differences exist between individual pathways. The agonist released by dexmedetomidine decreases neuronal vulnerability to oxidative damage by a paracrine effect, and in all cases the released EGF receptor agonist has autocrine effects. These include mitogen-activated protein (MAP) kinase-mediated phosphorylation of astrocytic extracellular-regulated kinase (ERK), and with high [K+]o also the cotransporter NKCC1, accumulating Na+ and K+ together with 2 Cl- and water, causing edema. This effect, exerted specifically on astrocytes, is enhanced by β1-adrenergic or vasopressinergic V1 signaling, explaining the beneficial effect of β1-adrenergic antagonists and why vasopressin should be omitted in edema treatment after brain trauma. Keywords: Brain edema, α2-adrenergic agonist, β1-adrenergic antagonist, NKCCI, EGF receptor, ERK, AKT, vasopressin, potassium, astrocytes

The von Hippel-Lindau Tumor Suppressor Protein Promotes c-Cbl-Independent Poly-Ubiquitylation and Degradation of the Activated EGFR

Somatic mutations or reduced expression of the von Hippel-Lindau (VHL) tumor suppressor occurs in the majority of the clear cell renal cell carcinoma (ccRCC) and is a causal factor for the pathogenesis of ccRCC. pVHL was reported to suppress the oncogenic activity of Epidermal Growth Factor Receptor (EGFR) by reducing the expression of the EGFR agonist TGF-α and by reducing the translation efficiency of EGFR itself. Furthermore, it was reported that pVHL down-regulates activated EGFR by promoting efficient lysosomal degradation of the receptor. These modes of negative regulation of EGFR by pVHL were dependent on Hypoxia Inducible Factor (HIF). In this study, we report that HIF was not the only factor stabilizing the activated EGFR in VHL-deficient ccRCC cells. Down-regulation of endogenous HIF in these cells had little effect on the turnover rates of the activated EGFR. Furthermore, neither pretreatment with lysomomal inhibitors pretreatment nor down-regulation of c-Cbl, a major E3 ubiquitin ligase that targets the activated EGFR for lysosomal degradation, significantly increased the stabilities of EGFR in VHL-expressing ccRCC cells. In contrast, pretreatment with proteasomal inhibitors extended EGFR lifetime and led to similar EGFR half-lives in VHL-expressing and VHL-deficient ccRCC cells. Down-regulation of c-Cbl in VHL-deficient ccRCC cells revealed that the c-Cbl and pVHL collaborated to down-regulate the activated EGFR. Finally, we found that pVHL promoted the poly-ubiquitylation of the activated EGFR, and this function was c-Cbl-independent. Thus these results indicate that pVHL limits EGFR signaling by promoting c-Cbl-independent poly-ubiquitylation of the activated receptor, which likely results in its degradation by proteasome.

Radiotherapy

Radiotherapy

Epidermal growth factor receptor kinase activity is required for gap junction closure and for part of the decrease in ovarian follicle cGMP in response to LH

The meiotic cell cycle in mouse oocytes is arrested in prophase, and then restarted when LH acts on the surrounding granulosa cells. The granulosa cells keep meiosis arrested by providing a source of cGMP that diffuses into the oocyte through gap junctions, and LH restarts the cell cycle by closing the junctions and by decreasing granulosa cell cGMP, thus lowering oocyte cGMP. Epidermal growth factor receptor (EGFR) activation is an essential step in triggering LH-induced meiotic resumption, but its relationship to the cGMP decrease in the follicle is incompletely understood, and its possible function in causing gap junction closure has not been investigated. Here, we use EGFR agonists (epiregulin and amphiregulin) and an EGFR kinase inhibitor (AG1478) to study the function of the EGFR in the signaling pathways leading to the release of oocytes from prophase arrest. Our results indicate that the EGFR kinase contributes to LH-induced meiotic resumption in two different ways. First, it is required for gap junction closure. Second, it is required for an essential component of the decrease in follicle cGMP. Our data show that the EGFR kinase-dependent component of the cGMP decrease is required for LH-induced meiotic resumption, but they also indicate that an as yet unidentified pathway accounts for a large part of the cGMP decrease.

Serotonin increases ERK1/2 phosphorylation in astrocytes by stimulation of 5-HT2B and 5-HT2C receptors

We have previously shown that fluoxetine causes ERK1/2 phosphorylation in cultured mouse astrocytes mediated exclusively by stimulation of 5-HT2B receptors (Li et al., 2008b). This raises the question whether this is also the case for serotonin (5-HT) itself. In the present study serotonin was found to induce ERK1/2 phosphorylation by stimulation of 5-HT2B receptors with high affinity (EC50: 20–30pM), and by stimulation of 5-HT2C receptor with low affinity (EC50: 1μM or higher). ERK1/2 phosphorylation induced by stimulation of either 5-HT2B or 5-HT2C receptors was mediated by epidermal growth factor (EGF) receptor transactivation (Peng et al., this issue), shown by the inhibitory effect of AG1478, an inhibitor of the EGF receptor tyrosine kinase, and GM6001, an inhibitor of Zn-dependent metalloproteinases, and thus of 5-HT2B receptor-mediated EGF receptor agonist release. It is discussed that the high potency of the 5-HT2B-mediated effect is consistent with literature data for binding affinity of serotonin to cloned human 5-HT2B receptors and with observations of low extracellular concentrations of serotonin in brain, which would allow a demonstrated moderate and modality-dependent increase in specific brain areas to activate 5-HT2B receptors. In contrast the relevance of the observed 5-HT2C receptors on astrocytes is questioned.

Stimulation by vasopressin of ERK phosphorylation and vector-driven water flux in astrocytes is transactivation-dependent

Vasopressin acts on astrocytic G q protein- and phospholipase C-coupled V 1 receptors. In mesangial cells, which also express the V 1 receptor, it stimulates cell growth by activating mitogen-activated protein kinase (MAP kinase) secondary to transactivation of the epidermal growth factor (EGF) receptor. Transactivation is an intracellular/extracellular process, in which activation of a G q or a G i/o protein-coupled receptor leads to metalloproteinase-catalyzed shedding of an EGF receptor agonist, which stimulates EGF receptors on the same cell and/or its neighbor(s). The goal of the present study was to investigate if vasopressin signaling is mediated by transactivation also in astrocytes and whether such a transactivation is required for its ability to facilitate vector-driven water fluxes. Vasopressin concentrations between 10 − 12 and 10 − 6 M were found to lead to phosphorylation (activation) of extracellular regulated kinase 1 and 2 (ERK 1/2). Phosphorylation of ERK 1/2 could be completely inhibited by either AG1478, an inhibitor of the EGF receptor-activated tyrosine kinase, or GM6001, an inhibitor of Zn 2+-activated metalloproteinases, indicating the involvement of transactivation. Exposure to a hypotonic medium caused an immediate (within one min) increase in cell water volume (demonstrated by decrease of fluorescence quenching of calcein), part of which was dependent upon the presence of vasopressin, added at a concentration of 1 × 10 − 8 M. This vasopressin-dependent component persisted throughout the duration of the experiment (22 min). The effect of vasopressin was abolished in the presence of AG1478, indicating its dependence upon transactivation, and by U0126 an inhibitor of the MAP kinase/ERK kinase (MEK), and thus of ERK 1/2 phosphorylation.

Transcription-Dependent Epidermal Growth Factor Receptor Activation by Hepatocyte Growth Factor

The mechanisms and biological implications of coordinated receptor tyrosine kinase coactivation remain poorly appreciated. Epidermal growth factor receptor (EGFR) and c-Met are frequently coexpressed in cancers, including those associated with hepatocyte growth factor (HGF) overexpression, such as malignant astrocytoma. In a previous analysis of the HGF-induced transcriptome, we found that two EGFR agonists, transforming growth factor-alpha and heparin-binding epidermal growth factor-like growth factor (HB-EGF), are prominently up-regulated by HGF in human glioma cells. We now report that stimulating human glioblastoma cells with recombinant HGF induces biologically relevant EGFR activation. EGFR phosphorylation at Tyr(845) and Tyr(1068) increased 6 to 24 h after cell stimulation with HGF and temporally coincided with the induction of transforming growth factor-alpha (~5-fold) and HB-EGF (~23-fold) expression. Tyr(845) and Tyr(1068) phosphorylation, in response to HGF, was inhibited by cycloheximide and actinomycin D, consistent with a requirement for DNA transcription and RNA translation. Specifically, blocking HB-EGF binding to EGFR with the antagonist CRM197 inhibited HGF-induced EGFR phosphorylation by 60% to 80% and inhibited HGF-induced S-G(2)-M transition. CRM197 also inhibited HGF-induced anchorage-dependent cell proliferation but had no effect on HGF-mediated cytoprotection. These findings establish that EGFR can be activated with functional consequences by HGF as a result of EGFR ligand expression. This transcription-dependent cross-talk between the HGF receptor c-Met and EGFR expands our understanding of receptor tyrosine kinase signaling networks and may have considerable consequences for oncogenic mechanisms and cancer therapeutics.

Chemokines and squamous cancer of the head and neck: targets for therapeutic intervention?

The biological properties of squamous carcinoma cells are intimately regulated by a multitude of cytokines and growth factors; the most well studied of these include epidermal growth factor receptor agonists and members of the transforming growth factor-β family. The recent explosion of research in the field of chemokine function as a mediator of tumor progression has led to the possibility that these small, immunomodulatory proteins also play key roles in squamous carcinogenesis and may, therefore, be potential targets for novel therapeutic approaches.

601 POSTER Head and neck squamous carcinoma cell lines exhibit an intact EGFR signaling pathway and variable response to EGFR agonist and antagonist

The in vivo results with our previously reported irreversible labeled inhibitor [11C]-ML03 suggested that more chemically stable inhibitors, labeled with a longer-lived radioisotope, could be better candidates for molecular imaging of epidermal growth factor receptor (EGFR) positive tumors. On the basis of this hypothesis we synthesized three new irreversible tyrosine kinase (TK) inhibitors with various chemical reactivities. The three new inhibitors were successfully labeled on the anilino moiety with [124I], starting with the 6-amino-4-[(3-tributylstannylphenyl)amino]-quinazoline (9) precursor. The cell-free results, obtained with these new irreversible inhibitors, indicated that compounds 5 (α-chloro-acetamide derivative) and 6 (4-dimethylamino-but-2-enoic amide derivative) possessed high potencies toward the EGFR with an irreversible inhibition effect. Compound 4 (α-methoxy-acetamide derivative) was found to be less potent, with only a partially irreversible effect. The high potency of compounds 5 and 6 toward the EGFR establishes their potential as PET agents for molecular imaging of EGFR positive tumors. Their prospect as PET biomarkers is further being investigated.

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

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

Mammary ductal morphogenesis requires paracrine activation of stromal EGFR via ADAM17-dependent shedding of epithelial amphiregulin

Epithelial-mesenchymal crosstalk is essential for tissue morphogenesis, but incompletely understood. Postnatal mammary gland development requires epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AREG), which generally must be cleaved from its transmembrane form in order to function. As the transmembrane metalloproteinase ADAM17 can process AREG in culture and Adam17(-/-) mice tend to phenocopy Egfr(-/-) mice, we examined the role of each of these molecules in mammary development. Tissue recombination and transplantation studies revealed that EGFR phosphorylation and ductal development occur only when ADAM17 and AREG are expressed on mammary epithelial cells, whereas EGFR is required stromally, and that local AREG administration can rescue Adam17(-/-) transplants. Several EGFR agonists also stimulated Adam17(-/-) mammary organoid growth in culture, but only AREG was expressed abundantly in the developing ductal system in vivo. Thus, ADAM17 plays a crucial role in mammary morphogenesis by releasing AREG from mammary epithelial cells, thereby eliciting paracrine activation of stromal EGFR and reciprocal responses that regulate mammary epithelial development.

Antagonism of Rat β-Cell Voltage-dependent K+ Currents by Exendin 4 Requires Dual Activation of the cAMP/Protein Kinase A and Phosphatidylinositol 3-Kinase Signaling Pathways

Antagonism of voltage-dependent K+ (Kv) currents in pancreatic beta-cells may contribute to the ability of glucagon-like peptide-1 (GLP-1) to stimulate insulin secretion. The mechanism and signaling pathway regulating these currents in rat beta-cells were investigated using the GLP-1 receptor agonist exendin 4. Inhibition of Kv currents resulted from a 20-mV leftward shift in the voltage dependence of steady-state inactivation. Blocking cAMP or protein kinase A (PKA) signaling (Rp-cAMP and H-89, respectively) prevented the inhibition of currents by exendin 4. However, direct activation of this pathway alone by intracellular dialysis of cAMP or the PKA catalytic subunit (cPKA) could not inhibit currents, implicating a role for alternative signaling pathways. A number of phosphorylation sites associated with phosphatidylinositol 3 (PI3)-kinase activation were up-regulated in GLP-1-treated MIN6 insulinoma cells, and the PI3 kinase inhibitor wortmannin could prevent antagonism of beta-cell currents by exendin 4. Antagonists of Src family kinases (PP1) and the epidermal growth factor (EGF) receptor (AG1478) also prevented current inhibition by exendin 4, demonstrating a role for Src kinase-mediated trans-activation of the EGF tyrosine kinase receptor. Accordingly, the EGF receptor agonist betacellulin could replicate the effects of exendin 4 in the presence of elevated intracellular cAMP. Downstream, the PKCzeta pseudosubstrate inhibitor could prevent current inhibition by exendin 4. Therefore, antagonism of beta-cell Kv currents by GLP-1 receptor activation requires both cAMP/PKA and PI3 kinase/PKCzeta signaling via trans-activation of the EGF receptor. This represents a novel dual pathway for the control of Kv currents by G protein-coupled receptors.

Selective abrogation of the proinvasive activity of the trefoil peptides pS2 and spasmolytic polypeptide by disruption of the EGF receptor signaling pathways in kidney and colonic cancer cells.

Trefoil peptides (TFFs) are now considered as scatter factors, proinvasive and angiogenic agents acting through cyclooxygenase-2 (COX-2)- and thromboxane A2 receptor (TXA2-R)-dependent signaling pathways. As expression and activation levels of the epidermal growth factor receptor (EGFR) predict the metastatic potential of human colorectal cancers, the purpose of this study was to establish whether the EGF receptor tyrosine kinase (EGFR-TK) contributes to cellular invasion induced by TFFs in kidney and colonic cancer cells. Both the dominant negative form of the EGFR (HER-CD533) and the EGFR-TK inhibitor ZD1839 (Iressa) abrogated cellular invasion induced by pS2, spasmolytic polypeptide (SP) and the src oncogene, but not by ITF and the TXA2-R. Similarly, EGFR-TK inhibition by ZD1839 reversed the invasive phenotype promoted by the constitutively activated form of the EGFR (EGFRvIII) and the EGFR agonists transforming growth factor alpha (TGFalpha), amphiregulin and EGF. We also provide evidence that TFFs, EGFRvIII, and TGFalpha trigger common proinvasive pathways using the PI3'-kinase and Rho/Rho- kinase cascades. These findings identify the EGFR-TK as a key signaling element for pS2- and SP-mediated cellular invasion. It is concluded that although pS2, SP and ITF belong to the same family of inflammation- and cancer-associated regulatory peptides, they do not control identical signaling networks.

α-Adrenergic stimulation of ERK phosphorylation in astrocytes is α 2-specific and may be mediated by transactivation

The highly specific α 2-adrenergic agonist, dexmedetomidine, has hypnotic-sedative, anesthetic-sparing and analgesic effects, and it protects neurons against ischemia. The α 1-adrenergic agonist, phenylephrine, does not share dexmedetomidine’s pharmacological properties, although both dexmedetomidine and phenylephrine increase free cytosolic Ca 2+ ([Ca 2+] i) in astrocytes, and most of dexmedetomidine’s actions in the brain are exerted on postjunctional receptors. α 2-Adrenergic receptors are abundant on astrocytes. Dexmedetomidine-mediated ‘down-streamn’ signal transduction was therefore investigated in primary cultures of mouse astrocytes and contrasted with that of phenylephrine. The cultures were incubated with dexmedetomidine concentrations known to be pharmacologically active and to act specifically on α 2-adrenergic receptors (25–100 nM). ERK 1/2 phosphorylation was measured using specific antibodies. Peak increases of ERK 1/2 phosphorylation occurred at 50 nM dexmedetomidine, with less effect at higher concentrations. Phenylephrine caused ERK phosphorylation only at a concentration high enough to exert non subtype-specific effects (10 μM), and this effect was counteracted by the α 2-adrenergic antagonist atipamezole. The phosphorylation of ERK was reduced by tyrphostin AG1478, an inhibitor of phosphorylation of the epidermal growth factor receptor (EGFR), and by heparin, which neutralizes heparin-binding epithelial growth factor (HB-EGF), suggesting the involvement of a transactivation process, in which α 2-adrenergic stimulation leads to proteolytic shedding of HB-EGF (and perhaps other EGFR agonists) from transmembrane-spanning precursors.

Urea signalling to immediate-early gene transcription in renal medullary cells requires transactivation of the epidermal growth factor receptor.

Signalling by physiological levels of urea (e.g. 200 mM) in cells of the mammalian renal medulla is reminiscent of activation of a receptor tyrosine kinase. The epidermal growth factor (EGF) receptor may be transactivated by a variety of G-protein-coupled receptors, primarily through metalloproteinase-dependent cleavage of a membrane-anchored EGF precursor. In the murine inner medullary collecting duct (mIMCD3) cell line, urea (200 mM) induced prompt (1-5 min) tyrosine phosphorylation of the EGF receptor. Pharmacological inhibition of EGF receptor kinase activity with AG1478 or PD153035 blocked urea-inducible transcription and expression of the immediate-early gene, Egr-1. AG1478 blocked, either fully or partially, other hallmarks of urea signalling including Elk-1 activation and extracellular signal-regulated kinase phosphorylation. EGF receptor kinase inhibition also blocked the cytoprotective effect of urea observed in the context of hypertonicity-inducible apoptosis. EGF receptor transactivation was likely to be attributable to metalloproteinase-dependent ectodomain shedding of an EGF receptor agonist because both specific and non-specific inhibitors of metalloproteinases blocked the urea effect. Heparin-binding EGF (HB-EGF), in particular, was implicated because the diphtheria toxin analogue and highly specific antagonist of HB-EGF, CRM197, also blocked urea-inducible transcription. In aggregate, these data indicate that signalling in response to urea in renal medullary cells requires EGF receptor transactivation, probably through autocrine action of HB-EGF.

Glucagon-like peptide 1 induces pancreatic beta-cell proliferation via transactivation of the epidermal growth factor receptor.

We previously provided evidence that glucagon-like peptide 1 (GLP-1) induces pancreatic beta-cell growth nonadditively with glucose in a phosphatidylinositol (PI) 3-kinase- and protein kinase C zeta-dependent manner. However, the exact mechanism by which the GLP-1 receptor (GLP-1R), a member of the G protein-coupled receptor (GPCR) superfamily, activates the PI 3-kinase signaling pathway to promote beta-cell growth remains unknown. We hypothesized that the GLP-1R could activate PI 3-kinase and promote beta-cell proliferation through transactivation of the epidermal growth factor (EGF) receptor (EGFR), an event possibly linked to GPCRs via activation of c-Src and the production of putative endogenous EGF-like ligands. Both the c-Src inhibitor PP1 and the EGFR-specific inhibitor AG1478 blocked GLP-1-induced [(3)H]thymidine incorporation in INS(832/13) cells as well as in isolated rat islets, while only AG1478 inhibited the proliferative action of betacellulin (BTC), an EGFR agonist. Both compounds also suppressed GLP-1-induced PI 3-kinase activation. A time-dependent increase in tyrosine phosphorylation of the EGFR in response to GLP-1 was observed in INS(832/13) cells. This transactivation of the EGFR was sensitive to both the pharmacological agents PP1 and AG1478. The action of GLP-1 and BTC on INS cell proliferation was found to be not additive. Overexpression of a dominant-negative EGFR in INS cells with a retroviral expression vector curtailed GLP-1-induced beta-cell proliferation. GLP-1 treatment of INS cells caused a decrease in cell surface-associated BTC, as shown by FACS analysis. Also, the metalloproteinase inhibitor GM6001 and an anti-BTC neutralizing antibody suppressed the GLP-1 proliferative effect. Finally, coculturing the prostatic cancer cell line LNCaP that lacks GLP-1 responsiveness with INS cells increased LNCaP cell proliferation in the presence of GLP-1, thus revealing that INS cells secrete a growth factor in response to GLP-1. GM6001 and an anti-BTC neutralizing antibody suppressed increased LNCaP cell proliferation in the presence of GLP-1 in the coculture experiments. The results are consistent with a model in which GLP-1 increases PI 3-kinase activity and enhances beta-cell proliferation via transactivation of the EGFR that would require the proteolytic processing of membrane-anchored BTC or other EGF-like ligands.

Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-kappaB.

The glial glutamate transporter GLT-1 may be the predominant Na(+)-dependent glutamate transporter in forebrain. Expression of GLT-1 correlates with astrocyte maturation in vivo and increases during synaptogenesis. In astrocyte cultures, GLT-1 expression parallels differentiation induced by cAMP analogs or by coculturing with neurons. Molecule(s) secreted by neuronal cultures contribute to this induction of GLT-1, but little is known about the signaling pathways mediating this regulation. In the present study, we determined whether growth factors previously implicated in astrocyte differentiation regulate GLT-1 expression. Of the six growth factors tested, two [epidermal growth factor (EGF) and transforming growth factor-alpha] induced expression of GLT-1 protein in cultured astrocytes. Induction of GLT-1 protein was accompanied by an increase in mRNA and in the V(max) for Na(+)-dependent glutamate transport activity. The effects of dibutyryl-cAMP and EGF were additive but were independently blocked by inhibitors of protein kinase A or protein tyrosine kinases, respectively. The induction of GLT-1 in both EGF- and dibutyryl-cAMP-treated astrocytes was blocked by inhibitors targeting phosphatidylinositol 3-kinase (PI3K) or the nuclear transcription factor-kappaB. Furthermore, transient transfection of astrocyte cultures with a constitutively active PI3K construct was sufficient to induce expression of GLT-1. These data suggest that independent but converging pathways mediate expression of GLT-1. Although an EGF receptor-specific antagonist did not block the effects of neuron-conditioned medium, the induction of GLT-1 by neuron-conditioned medium was completely abolished by inhibition of PI3K or nuclear factor-kappaB. EGF also increased expression of GLT-1 in spinal cord organotypic cultures. Together, these data suggest that activation of specific signaling pathways with EGF-like molecules may provide a novel approach for limiting excitotoxic brain injury.

Identification of the Minimal Requirements for Binding to the Human Epidermal Growth Factor (EGF) Receptor Using Chimeras of Human EGF and an EGF Repeat of Drosophila Notch

Many proteins contain so-called epidermal growth factor (EGF)-like domains that share the characteristic spacing of cysteines and glycines with members of the EGF family. They are, however, functionally unrelated, despite the fact that the three-dimensional structure of these EGF-like domains, also, is often very similar to that of the EGF receptor agonists. In the present study, we linked an EGF-like repeat from the Drosophila Notch protein to the N- and C-terminal linear tail sequences of human EGF (hEGF), and we showed that this chimera (E1N6E) is unable to bind or activate the hEGF receptor. This recombinant protein was then used as a basic construct for identifying the minimal requirements for high affinity EGF receptor binding and activation. We selectively reintroduced a limited number of important hEGF-derived residues, and by using this unique approach, we were able to make hEGF/Notch chimeras that, compared with wild type hEGF, showed nearly 100% binding affinity and mitogenic activity on HER-14 cells expressing the hEGF receptor.

A Single Amino Acid Exchange, Arg-45 to Ala, Generates an Epidermal Growth Factor (EGF) Mutant with High Affinity for the Chicken EGF Receptor

The finding that human epidermal growth factor (hEGF) and human transforming growth factor (hTGF) alpha bind with similar affinity to the human EGF receptor but differ in their affinity for the chicken EGF receptor was used as a model system to study ligand-receptor interaction of EGF receptor agonists. We previously constructed domain-exchange mutants of hEGF and hTGF alpha and found that the region COOH-terminal of the sixth cysteine residue in hTGF alpha is important for high affinity binding to the chicken EGF receptor (Kramer, R. H., Lenferink, A. E. G., Lammerts van Bueren-Koornneef, I., van der Meer, A., van de Poll, M. L. M., and van Zoelen, E. J. J. (1994) J. Biol. Chem. 269, 8708-8711). To analyze this domain in more detail, we now constructed four additional chimeras in which either the region between the sixth cysteine residue and the highly conserved Leu-47 was exchanged or the region COOH-terminal of Leu-47. A mutant in which the latter region in hEGF was replaced by hTGF alpha (designated E6ET) showed intermediate binding affinity, whereas replacement of the former region in hEGF by hTGF alpha was sufficient to generate a mutant (designated E6TE) with a similar high affinity for the chicken EGF receptor as wild type hTGF alpha. Furthermore, a deletion mutant of hEGF lacking three COOH-terminal amino acids, EGF50, showed intermediate binding affinity for the chicken EGF receptor similar to E6ET, but upon additional deletions (EGF49 and EGF48), this initial gain in affinity was lost. A systematic analysis of the region between the sixth cysteine residue and Leu-47 showed that the low affinity of hEGF for the chicken EGF receptor is mainly due to the presence of Arg-45. Replacement of the positively charged Arg-45 by Ala, the corresponding amino acid in hTGF alpha, was sufficient to generate a mutant growth factor with high affinity for the chicken EGF receptor. This indicates that in hEGF Arg-45 may play an important role in receptor binding. A model is proposed in which positively charged amino acids close to or within the receptor recognition site of hEGF prohibit high affinity binding to the chicken EGF receptor due to electrostatic repulsion of positively charged amino acids in the putative ligand binding domain of the chicken EGF receptor.