Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor

Epidermal growth factor (EGF) receptor from A-431 cells was purified by affinity chromatography with monoclonal anti-receptor antibodies. The purified radiolabeled receptor was incubated with EGF and then analyzed by gel electrophoresis under nondenaturing conditions. In these gels, the EGF receptor migrates in two forms: a fast-migrating (low) form and an EGF-induced slow-migrating (high) form. On the basis of the various control and calibration experiments described, it is concluded that the low form represents the monomeric 170-kilodalton EGF receptor and the high form represents an EGF receptor dimer. The binding of EGF causes a rapid, temperature-sensitive dimerization of the EGF receptor. Receptor dimerization is fully reversible and involves saturable, noncovalent interactions that are stable at neutral pH and in nonionic detergents. Both the monomeric and dimeric forms of the receptor bind EGF and undergo self-phosphorylation. The dimeric form of the receptor may possess higher ligand binding affinity, and it seems to be phosphorylated earlier than the monomeric form following the addition of EGF and [gamma-32P]ATP. On the basis of these results, it is concluded that receptor oligomerization is an intrinsic property of the occupied EGF receptor and that it may play a role in the activation of the kinase function and the subsequent transmembrane signaling process.     

Epidermal growth factor receptor kinase translocation and activation in vivo

The rat liver epidermal growth factor (EGF) receptor was assessed for EGF-dependent autophosphorylation as well as phosphorylation of a defined exogenous substrate in purified plasmalemma and Golgiendosome fractions isolated from rat liver homogenates. While EGF-dependent kinase activity was readily detected in plasmalemma the corresponding activity in Golgi-endosome fractions required detergent. Consequent to the systemic injection of EGF in vivo, the majority (approximately 60%) of receptor as evaluated by 125I-EGF binding was rapidly lost (T 1/2 approximately 8 min) from the plasmalemma and correspondingly accumulated in the Golgi-endosome fraction in a dose-dependent manner. Electron microscope radioautography of 125I-EGF uptake into Golgi-endosome fractions identified internalization into lipoprotein-filled vesicles of heterogenous size and shape but not into stacked saccules of the Golgi apparatus. Evaluation of receptor kinase activity in plasmalemma fractions isolated at various times after EGF injection in vivo showed more rapid loss of EGF-dependent autophosphorylation activity (T 1/2 approximately 10 s) than of receptor content (T 1/2 approximately 8 min). In contrast to the EGF receptor kinase of the plasmalemma fraction, kinase activity accumulating in endosomes was activated, i.e. maximally stimulated, in the absence of EGF or Triton X-100 in vitro. Furthermore, following the peak time of accumulation of EGF receptor kinase in endosomes (5-15 min) EGF-dependent autophosphorylation activity and EGF receptor content were lost more slowly (T 1/2 approximately 27 and 87 min for the loss of autophosphorylation activity and receptor content, respectively). The rapidity of translocation of activated EGF receptor into endosomes (30 s) and the dose response to low levels (1 microgram) of EGF injected are consistent with a physiological role for internalized EGF receptor kinase activity.     

Epidermal growth factor receptor transactivation mediates tumor necrosis factor-induced hepatocyte replication

Tumor necrosis factor (TNF) has multiple biological effects such as participating in inflammation, apoptosis, and cell proliferation, but the mechanisms of its effects on epithelial cell proliferation have not been examined in detail. At the early stages of liver regeneration, TNF functions as a priming agent for hepatocyte replication and increases the sensitivity of hepatocytes to growth factors such as transforming growth factor alpha (TGFalpha); however, the mechanisms by which TNF interacts with growth factors and enhances hepatocyte replication are not known. Using the AML-12 hepatocyte cell line, we show that TNF stimulates proliferation of these cells through transactivation of the epidermal growth factor receptor (EGFR). The transactivation mechanism involves the release of TGFalpha into the medium through activation of the metalloproteinase TNFalpha-converting enzyme (also known as ADAM 17). Binding of the ligand to EGFR initiates a mitogenic cascade through extracellular signal-regulated kinases 1 and 2 and the partial involvement of protein kinase B. TNF-induced release of TGFalpha and activation of EGFR signaling were inhibited by TNFalpha protease inhibitor-1, an agent that interferes with TNFalpha-converting enzyme activity. We suggest that TNF-induced transactivation of EGFR may provide an early signal for the entry of hepatocytes into the cell cycle and may integrate proliferative and survival pathways at the start of liver regeneration.     

Epidermal growth factor receptor threonine and serine residues phosphorylated in vivo

The epidermal growth factor (EGF) receptor is regulated by EGF-stimulated autophosphorylation and by phorbol ester-stimulated, protein kinase C (Ca2+/phospholipid-dependent enzyme) mediated phosphorylation at identified sites. The EGF receptor contains additional phosphorylation sites including a prominent phosphothreonine and several phosphoserines which account for the majority of phosphate covalently bound to the receptor in vivo. We have identified three of these sites in EGF receptor purified from 32P-labeled A431 cells. The major phosphothreonine was identified as threonine 669 in the EGF receptor sequence. Phosphoserine residues were identified as serines 671 and 1046/1047 of the EGF receptor. Two other phosphoserine residues were localized to tryptic peptides containing multiple serine residues located carboxyl-terminal to the conserved protein kinase domain. The amino acid sequences surrounding the three identified phosphorylation sites are highly conserved in the EGF receptor and the protein products of the v-erb B and neu oncogenes. Analysis of predicted secondary structure of the EGF receptor reveals that all of the phosphorylation sites are located near beta turns. In A431 cells phosphorylation of the serine residues was dependent upon serum. In mouse B82 L cells transfected with a wild type human EGF receptor. EGF increased the 32P content in all tryptic phosphopeptides. A mutant EGF receptor lacking protein tyrosine kinase activity was phosphorylated only at threonine 669. Regulated phosphorylation of the EGF receptor at these threonine and serine residues may influence aspects of receptor function.     

Epidermal growth factor receptor expression related to differentiation capacity in normal and transformed keratinocytes

Epidermal growth factor (EGF) and Ca2+ have been indicated to play a major role in skin development. We have used normal keratinocytes, SV40-transformed keratinocytes (SVK14) and various squamous carcinoma cell (SCC) lines as in vitro model system to study the effect of the extracellular Ca2+ concentration of EGF-receptor expression in relation to the capability of cells to differentiate. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-12B2 greater than SCC-4, as judged from Ca2+-ionophore-induced cornified envelope formation. Under normal Ca2+ conditions, all cell lines (except for SCC-15) exhibited two classes of EGF-binding sites. The number of low-affinity binding sites increased considerably as cells were less able to differentiate, while the apparent dissociation constant (kd) was similar in all cell lines. In contrast, the properties of high-affinity EGF binding varied in the various cell lines without a clear relationship to the degree of differentiation capacity. Lowering the extracellular Ca2+ concentration to 0.06 mM resulted in a decrease of Ca2+ ionophore-induced cornified envelope formation, demonstrating the decreased ability to differentiate under these conditions. The decreased ability to differentiate was accompanied by a marked increase in the number of EGF-binding sites, but without a change of the kd. Furthermore, no high-affinity EGF-binding sites were detectable under these conditions. Finally, addition of Ca2+ to low Ca2+-cultured cells caused a rapid decrease of EGF binding in all cell lines, most prominently in normal keratinocytes and SCC-12F2 cells. The data presented demonstrate: The combination of normal keratinocytes, SVK14 and the various SCC lines provides an attractive model system to study differentiation in vitro; EGF-receptor expression is related to the state of differentiation, both phenomena being sensitive to the external Ca2+ concentration; and EGF-receptor expression is related to the capability of cells to differentiate.     

Epidermal growth factor receptor tyrosine kinase mediates Ras activation by gonadotropin-releasing hormone

Gonadotropin releasing hormone (GnRH) contributes to the maintenance of gonadotrope function by increasing extracellular signal-regulated kinase (ERK) activity subsequent to binding to its cognate G-protein-coupled receptor. As the GnRH receptor exclusively interacts with G(q/11) proteins and as receptor expression is regulated in a beta-arrestin-independent fashion, it represents a good model to systematically dissect underlying signaling pathways. In alphaT3-1 gonadotropes endogenously expressing the GnRH receptor, GnRH challenge resulted in a rapid increase in ERK activity which was attenuated by the epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitor AG1478. In COS-7 cells transiently expressing the human GnRH receptor, agonist-induced ERK activation was independent of free Gbetagamma subunits but could be mimicked by short-term phorbol ester treatment. Most notably, G(q/11)-induced ERK activation was sensitive to N17-Ras and to expression of the C-terminal Src kinase but also to other dominant negative mutants of signaling components localized upstream of Ras, like Shc and the EGFR. GnRH as well as phorbol esters led to Ras activation in COS-7 and alphaT3-1 cells, which was dependent on Src and EGFR tyrosine kinases, indicating that both tyrosine kinases act downstream of protein kinase C (PKC) and upstream of Ras. However, Src did not contribute to Shc tyrosine phosphorylation. GnRH or phorbol ester challenge resulted in PKC-dependent EGFR autophosphorylation. Furthermore, a 5-min phorbol ester treatment was sufficient to trigger tyrosine phosphorylation of the platelet-derived growth factor-beta receptor in L cells. Thus, in several cell systems PKC is able to stimulate Ras via activation of receptor tyrosine kinases.     

Epidermal growth factor receptor down-regulation induced by UVA in human keratinocytes does not require the receptor kinase activity

Activation of the epidermal growth factor (EGF) receptor by EGF, its ligand, results in receptor internalization and down-regulation, which requires receptor kinase activity, phosphorylation, and ubiquitination. In contrast, we have found here in human HaCaT keratinocytes that exposure to UVA induces EGF receptor internalization and down-regulation without receptor phosphorylation and ubiquitination. The presence of the receptor kinase activity inhibitor AG1478 increased UVA-induced receptor down-regulation, whereas it inhibited EGF-induced receptor down-regulation. These observations demonstrate that, in contrast to EGF, receptor kinase activity is not required for receptor down-regulation by UVA. Concurrent with receptor down-regulation, caspases were activated by UVA exposure. The presence of caspase inhibitors blocked receptor down-regulation in a pattern similar to poly(ADP)-ribose polymerase cleavage. Much more receptor down-regulation was observed after UVA exposure in apoptotic detached cells in which caspase is activated completely. These results indicate that UVA-induced receptor down-regulation is dependent on caspase activation. Similar to UVA, both UVB and UVC induced receptor down-regulation, in which receptor kinase activity is not required, whereas caspase activation is involved. Inhibition of EGF receptor down-regulation increased receptor activation and activation of its downstream survival signaling ERK and AKT after UVA exposure. Preventing the activation of each of these pathways enhanced apoptosis induced by UVA. These findings suggest that EGF receptor down-regulation by UVA may play an important role in the execution of the cell suicide program by attenuating its anti-apoptotic function and thereby preventing cell transformation and tumorigenesis in vivo.     

Epidermal growth factor receptor transactivation mediates substance P-induced mitogenic responses in U-373 MG cells

Ligand-induced activation of G protein-coupled receptors is emerging as an important pathway leading to the activation of certain receptors with intrinsic tyrosine kinase activity, such as the epidermal growth factor receptor (EGFR). Substance P (SP) exerts many effects via activation of its G protein-coupled receptor (neurokinin-1, NK-1). SP participates in acute inflammation and activates key proteins involved in mitogenic pathways, such mitogen-activated protein kinases (MAPKs), stimulating DNA synthesis. We tested the hypothesis that SP-induced MAPK activation and DNA synthesis require activation of the EGFR. In U-373 MG cells, which express functional NK-1, SP induced tyrosine phosphorylation of several proteins including EGFR. SP induced formation of an activated EGFR complex containing the adapter proteins SHC and Grb2, but not c-Src. SP activated the MAPK pathway as shown by increased Erk2 kinase activity. SP induced Erk2 activation, and DNA synthesis was inhibited in cells transfected with a dominant negative EGFR plasmid lacking kinase activity, as well as in cells treated with a specific EGFR inhibitor. In addition, pertussis toxin, an inhibitor of Galpha(iota) protein subunits, prevented SP-induced EGFR transactivation and subsequent DNA synthesis. Our results implicate EGFR as an essential regulator in SP/NK-1-induced activation of the MAPK pathway and cell proliferation in U-373 MG cells, and these events are mediated by a pertussis toxin-sensitive Galpha protein. We suggest that this mechanism by which SP controls cell proliferation is an important pathway in tissue restoration and healing.     

Epidermal growth factor induction of front-rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2

Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front-rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front-rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front-rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front-rear polarity and forward movement.     

N-arginine dibasic convertase is a specific receptor for heparin-binding EGF-like growth factor that mediates cell migration.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a mitogen and chemotactic factor, binds to two receptor tyrosine kinases, erbB1 and erbB4. Now we demonstrate that HB-EGF also binds to a novel 140 kDa receptor on MDA-MB 453 cells. Purification of this receptor showed it to be identical to N-arginine dibasic convertase (NRDc), a metalloendopeptidase of the M16 family. Binding to cell surface NRDc and NRDc in solution was highly specific for HB-EGF among EGF family members. When overexpressed in cells, NRDc enhanced their migration in response to HB-EGF but not to EGF. Conversely, inhibition of endogenous NRDc expression in cells by antisense morpholino oligonucleotides inhibited HB-EGF-induced cell migration. Anti-erbB1 neutralizing antibodies completely abrogated the ability of NRDc to enhance HB-EGF-dependent migration, demonstrating that this NRDc activity was dependent on erbB1 signaling. Although NRDc is a metalloproteinase, enzymatic activity was not required for HB-EGF binding or enhancement of cell migration; neither did NRDc cleave HB-EGF. Together, these results suggest that NRDc is a novel specific receptor for HB-EGF that modulates HB-EGF-induced cell migration via erbB1.     

EGFR基因在非小细胞肺癌、乳腺癌中突变的研究

表皮生长因子受体(EGFR)基因酪氨酸激酶域体细胞突变与非小细胞肺癌(NSCLC)患者对酪氨酸激酶抑制剂吉非替尼敏感性密切相关。文章分析和检测本院75例非小细胞肺癌、10例乳腺癌患者石蜡包埋标本EGFR基因突变状况。采用PCR技术进行EGFR基因19和21外显子突变分析。结果显示:75例NSCLC患者中有13例(13/75,17.33%)酪氨酸激酶域存在体细胞突变。其中7例(7/75,9.33%)为19外显子缺失突变,6例(6/75,8%)为21外显子替代突变(2573T>G,L858R)。病理分型显示,腺癌突变率高于其他几种类型NSCLC。乳腺癌患者均为免疫组化HER-2阳性女性,EGFR基因的19、21外显子中未见突变发生。中国非小细胞肺癌患者总突变率高于高加索人种,女性患者较男性患者突变率高,提示肺腺癌的患者突变率高可能在吉非替尼的治疗中获益。     

Role of epidermal growth factor receptor in basal and stimulated colonic epithelial cell migration in vitro.

Colonic mucosal wounds are repaired, in part, by epithelial migration. Signaling mechanisms regulating this migration are poorly characterized. This study aimed to examine the role that the epidermal growth factor (EGF) receptor (EGF-R) and its ligands, EGF and transforming growth factor-alpha (TGF-alpha), play in migration in wounded in vitro models of colonic epithelium. Migration was assessed over 24 h in circular wounds made in confluent monolayers of LIM1215 human colon cancer cells. EGF and TGF-alpha stimulated migration twofold from 4 h after wounding. Basal migration and the motogenic effects of short chain fatty acids and hepatocyte growth factor were mediated through enhanced binding of TGF-alpha to EGF-R, while trefoil peptide-mediated motogenesis required EGF-R activation independently of TGF-alpha binding. Activation of protein kinase C (PKC) stimulated migration, an effect more potent than, and independent of, EGF-R activation. However, neither inhibition of PKC by Ro 31-8220 nor depletion of PKC by pretreatement with phorbol myristate acetate attenuated EGF-R-mediated motogenesis. In conclusion, EGF-R activation via TGF-alpha binding, or intracellularly, mediates basal LIM1215 migration and the effects of several motogens, with the exception of PKC activators. Since EGF-R and PKC have physiological activators in vivo, they may control colonic mucosal repair processes following injury.     

Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo.

Connective tissue growth factor (CTGF) is a novel cysteine-rich, secreted protein. Recently, we found that inhibition of the endogenous expression of CTGF by its antisense oligonucleotide and antisense RNA suppresses the proliferation and migration of vascular endothelial cells. In the present study, the following observations demonstrated the angiogenic function of CTGF in vitro and in vivo: (i) purified recombinant CTGF (rCTGF) promoted the adhesion, proliferation and migration of vascular endothelial cells in a dose-dependent manner under serum-free conditions, and these effects were inhibited by anti-CTGF antibodies; (ii) rCTGF markedly induced the tube formation of vascular endothelial cells, and this effect was stronger than that of basic fibroblast growth factor or vascular endothelial growth factor; (iii) application of rCTGF to the chicken chorioallantoic membrane resulted in a gross angiogenic response, and this effect was also inhibited by anti-CTGF antibodies. (iv) rCTGF injected with collagen gel into the backs of mice induced strong angiogenesis in vivo. These findings indicate that CTGF is a novel, potent angiogenesis factor which functions in multi-stages in this process.     

Integrin alpha5/beta1 mediates fibronectin-dependent epithelial cell proliferation through epidermal growth factor receptor activation

Human integrin alpha5 was transfected into the integrin alpha5/beta1-negative intestinal epithelial cell line Caco-2 to study EGF receptor (EGFR) and integrin alpha5/beta1 signaling interactions involved in epithelial cell proliferation. On uncoated or fibronectin-coated plastic, the integrin alpha5 and control (vector only) transfectants grew at similar rates. In the presence of the EGFR antagonistic mAb 225, the integrin alpha5 transfectants and controls were significantly growth inhibited on plastic. However, when cultured on fibronectin, the integrin alpha5 transfectants were not growth inhibited by mAb 225. The reversal of mAb 225-mediated growth inhibition on fibronectin for the integrin alpha5 transfectants correlated with activation of the EGFR, activation of MAPK, and expression of proliferating cell nuclear antigen. EGFR kinase activity was necessary for both MAPK activation and integrin alpha5/beta1-mediated cell proliferation. Although EGFR activation occurred when either the integrin alpha5-transfected or control cells were cultured on fibronectin, coprecipitation of the EGFR with SHC could be demonstrated only in the integrin alpha5-transfected cells. These results suggest that integrin alpha5/beta1 mediates fibronectin-induced epithelial cell proliferation through activation of the EGFR.     

Epidermal growth factor receptor expression in human gliomas

Summary The expression of epidermal growth factor receptor (EGFR) was determined in cryosections of 42 human gliomas using biotinylated epidermal growth factor (B-EGF) and two monoclonal antibodies (mAb) against EGFR. All gliomas were found to express EGFR when examined with B-EGF, whereas 33 expressed EGFR when examined with the two mAbs. The highly malignant gliomas (glioblastomas and anaplastic astrocytomas) had a more heterogeneous staining pattern and a larger proportion of tumour cells staining strongly with B-EGF than did the low-grade gliomas (astrocytomas, oligodendrogliomas, mixed gliomas, and ependymomas). This indicates that high-grade gliomas contain more tumour cells rich in EGFR than do the low-grade gliomas. Reactive astrocytes, ependymal cells, and many types of nerve cells (cerebral cortical pyramidal cells, pyramidal and granular hippocampal cells, Purkinje cells, cerebellar granular cells and neurons in the molecular layer of the cerebellum) expressed EGFR, whereas small neurons and normal glial cells were not found to express EGFR.     

表皮生长因子受体及其变异性质

EGFR主要由三个结构区域组成,即EGF结合区域、跨膜区域和依赖EGF蛋白激酶区域。EGFR被激活后,不仅能使许多底物蛋白磷酸化,自身也发生磷酸化,这些磷酸化作用在信息传导中有重要作用。EGFR与EGF结合后,其复合体以胞饮方式内容形成受体小体,除部分被降解外,大部分在高尔基GERL区加工后到达细胞核调节基因表达。本文通过对受体激活模型和用体外遗传变异方法对变异EGFR的研究阐述了EGFR的结构与功能的关系。     

Leukaemic cell growth and aggregation in vitro

Experiments were performed with two lymphoblastic mouse leukaemia, L1210 VA II cell population growing in vitro for 90 and 400 days. These cells were cultured for 9 days in Eagle's solution without exchanging the medium, and each day a sample was taken for determination of its density, viability of cells and cell diameter. In the cells of the 400 day old population the presence and number of aggregates was checked. Lipid, nucleotide, nucleic acid, DNA and protein contents were determined in these cells. The present experiment in vitro was arranged to observe an aggregation of leukaemia cells during culture without change of the medium. The density and viability of cells changes of their metabolism in this system may stimulate the situation in vivo after dissemination of cells to a new place.