Prediction of the three-dimensional structures of the nerve growth factor and epidermal growth factor binding proteins (kallikreins) and an hypothetical structure of the high molecular weight complex of epidermal growth factor with its binding protein.

We have predicted the three-dimensional structures of the serine protease subunits (gamma-NGF, alpha-NGF, and EGF-BP) of the high molecular weight complexes of nerve growth factor (7S NGF) and epidermal growth factor (HMW-EGF) from the mouse submandibular gland (from the X-ray crystal structures of two related glandular kallikreins). The conformations of three of the six loops surrounding the active site are relatively well defined in the models of gamma-NGF and EGF-BP, but three other loops are likely to have flexible conformations. Although the amino acid sequence of alpha-NGF is closely related to those of gamma-NGF and EGF-BP, it is catalytically inactive. Model-building studies on alpha-NGF suggested that mutations (in alpha-NGF) just prior to the active site serine (195) and an unusual N-terminal sequence are consistent with alpha-NGF having a zymogen-like conformation (similar to that in chymotrypsinogen). An hypothetical model of the quaternary structure of HMW-EGF has been constructed using this model of EGF-BP and the NMR structure of murine EGF. The C-terminal arm of EGF was modeled into the active site of EGF-BP based on data indicating that the C-terminal arginine of EGF occupies the S1 subsite of EGF-BP. Data suggesting one of the surface loops of EGF-BP is buried in the HMW-EGF complex and symmetry constraints were important in deriving a schematic model. A molecular docking program was used to fit EGF to EGF-BP.     

Molecular species of epidermal growth factor carrying immunosuppressive activity

The suppression of antibody formation to sheep red cells in mice by partially purified fractions of mouse submaxillary gland was shown to be caused by epidermal growth factor (EGF). Purification of EGF by the method of Savage and Cohen resolved three components referred to as EGF a, EGF b, and EGF c. All three induced premature eye opening in neonatal mice, but only EGF a (identified as EGF 1-53) had full immunosuppressive activity. EGF c was shown by micropeptide mapping of chymotryptic and thermolytic digests and amino-terminal analysis to differ from EGF a only by the presence of beta-aspartyl instead of an asparaginyl residue. EGF b differed from EGF a in that it lacked the N-terminal asparagine. EGF shortened enzymatically at its carboxy terminal by two or five amino acids did not have any immunosuppressive activity. These findings suggest that, in contrast to some other biological effects of EGF, intact amino and carboxy terminals are required for the expression of immunosuppressive activity.     

Regulation of the epidermal growth factor receptor by phosphorylation

The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self-phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37 degrees C and exhibits a very low Km for ATP (0.2 microM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self-phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self-phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function.     

Epidermal growth factor induces changes of interaction between epidermal growth factor receptor and actin in intact cells

The epidermal growth factor receptor (EGFR) is a cyto-skeleton-binding protein. Although purified EGFR can interact with acting in vitro and normally at least 10% of EGFR exist in the insoluble cytoskeleton fraction of A431 cells, interaction of cytosolic EGFR with actin can only be visualized by fluorescence resonance energy transfer when epidermal growth factor presents in the cell medium. Results indicate that the correct orientation between EGFR and actin is important in the signal transduction process.     

Secretion of human epidermal growth factor by Corynebacterium glutamicum

AIMS: To examine the secretion of human epidermal growth factor (hEGF) by Corynebacterium glutamicum. METHODS AND RESULTS: We recently showed that a novel protein-secretion system in C. glutamicum could produce Streptomyces mobaraensis transglutaminase. In the present study, the industrially important protein hEGF was secreted into the culture medium in a fully active form by C. glutamicum and accumulated at a rate of up to 156 mg l(-1) day(-1). CONCLUSIONS: These results demonstrated that the hEGF protein could be secreted in an active form by C. glutamicum. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data confirmed that the pharmaceutically important human protein hEGF could be efficiently secreted in an active form by the C. glutamicum protein-expression system. Moreover, we demonstrated that this bacterium has potential as a host for the industrial-scale production of human proteins.     

Hepatocyte growth factor induces epithelial cell motility through transactivation of the epidermal growth factor receptor

Hepatocyte growth factor (HGF) is a potent inducer of motility in epithelial cells. Since we have previously found that activation of the epidermal growth factor receptor (EGFR) is an absolute prerequisite for induction of motility of corneal epithelial cells after wounding, we investigated whether induction of motility in response to HGF is also dependent on activation of the EGFR. We now report that HGF induces transactivation of the EGFR in an immortalized line of corneal epithelial cells, in human skin keratinocytes, and in Madin-Darby canine kidney cells. EGFR activation is unconditionally required for induction of motility in corneal epithelial cells, and for induction of a fully motile phenotype in Madin-Darby canine kidney cells. Activation of the EGFR occurs through amphiregulin and heparin-binding epidermal growth factor-like growth factor. Early after HGF stimulation, blocking EGFR activation does not inhibit extracellular-signal regulated kinase 1/2 (ERK1/2) activation by HGF, but the converse is seen after approximately 1 h, indicating the existence of EGFR-dependent and -independent routes of ERK1/2 activation. In summary, HGF induces transactivation of the EGFR in epithelial cells, and this is a prerequisite for induction of full motility.     

重组人表皮生长因子的稳定性

考察了不同条件下重组人表皮生长因子（rhEGF）的稳定性。结果表明,不同的保存条件（物理状态、温度、pH、浓度等）对rhEGF的稳定性有显影响,使用适当的添加剂（保护剂、抗氧剂、抑菌等）可以提高rhEGF的稳定性。上述结果为rhEGF的制剂学研究提供了依据。     

Role of epidermal growth factor-stimulated protein kinase in control of proliferation of A431 cells

Epidermal growth factor (EGF), which stimulates tyrosine-specific protein kinase activity both in vivo and in vitro, inhibits proliferation of A431 human epidermoid carcinoma cells. After mutagenesis clonal cell lines that were resistant to the growth inhibitory effects of EGF were selected. All six variants examined contained decreased EGF-stimulated protein kinase. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in variant cell lines with up to tenfold reductions in both activities. This result suggests that both EGF binding and kinase activities reside in the same or closely coupled molecules. The effect of EGF on growth of two resistant variants was examined in detail. Clone 29 contains approximately 50% and clone 4 contains approximately 20% of the EGF-stimulated protein kinase activity of the parental A431 cell line. In serum-supplemented medium, EGF stimulated proliferation of clone 29 but did not affect growth of clone 4. In a 1:1 mixture of DME and F-12 medium without serum, EGF caused both clone 29 and clone 4 to grow as well as in 10% serum. These variants, which were selected for resistance to the growth inhibitory effects of EGF, thus exhibit a strong mitogenic response to EGF. This result suggests that resistance to the growth inhibitory effect of EGF may involve both a decrease in EGF-stimulated protein kinase and an alteration in the response pathway.     

Structure of domain III of the blood-stage malaria vaccine candidate, Plasmodium falciparum apical membrane antigen 1 (AMA1)

Apical membrane antigen 1 of the malarial parasite Plasmodium falciparum (Pf AMA1) is a merozoite antigen that is considered a strong candidate for inclusion in a malaria vaccine. Antibodies reacting with disulphide bond-dependent epitopes in AMA1 block invasion of host erythrocytes by P.falciparum merozoites, and we show here that epitopes involving sites of mutations in domain III are targets of inhibitory human antibodies. The solution structure of AMA1 domain III, a 14kDa protein, has been determined using NMR spectroscopy on uniformly 15N and 13C/15N-labelled samples. The structure has a well-defined disulphide-stabilised core region separated by a disordered loop, and both the N and C-terminal regions of the molecule are unstructured. Within the disulphide-stabilised core, residues 443-447 form a turn of helix and residues 495-498 and 503-506 an anti-parallel beta-sheet with a distorted type I beta-turn centred on residues 500-501, producing a beta-hairpin-type structure. The structured region of the molecule includes all three disulphide bonds. The previously unassigned connectivities for two of these bonds could not be established with certainty from the NMR data and structure calculations, but were determined to be C490-C507 and C492-C509 from an antigenic analysis of mutated forms of this domain expressed using phage display. Naturally occurring mutations in domain III that are located far apart in the primary sequence tend to cluster in the region of the disulphide core in the three-dimensional structure of the molecule. The structure shows that nearly all the polymorphic sites have a high level of solvent accessibility, consistent with their location in epitopes recognised by protective antibodies. Even though domain III in solution contains significant regions of disorder in the structure, the disulphide-stabilised core that is structured is clearly an important element of the antigenic surface of AMA1 recognised by protective antibodies.     

Proteolytic domains of the epidermal growth factor receptor of human placenta

Microsomal membranes from human placenta, which bind 5–20 pmol of ¹²⁵I-epidermal growth factor (EGF) per mg protein, have been affinity-labeled with ¹²⁵I-EGF either spontaneously or with dimethylsuberimidate. Coomassie blue staining patterns on SDS polyacrylamide gels are minimally altered, and the EGF-receptor complex appears as a specifically labeled band of 180,000 daltons which is not removed by urea, neutral buffers, or chaotropic salts but is partially extracted by mild detergents. Limited proteolysis by alpha chymotrypsin and several other serine proteases yields labeled fragments of 170,000, 130,000, 85,000, and 48,000 daltons. More facile cleavage by papain or bromelain rapidly degrades the hormone-receptor complex to smaller labeled fragments of about 35,000 and 25,000 daltons. These fragments retain the binding site for EGF, are capable of binding EGF, and remain associated with the membrane. Alpha chymotryptic digestion of receptor solubilized by detergents yields the same fragments obtained with intact vesicles, suggesting that the fragments may represent intrinsic proteolytic domains of the receptor.     

表皮生长因子受体与肺脏发育的关系

表皮生长因子受体(Epidermal growth factor receptor,EGFR)是一种跨膜蛋白受体,是ErbB家族成员之一,具有酪氨酸激酶活性。EGFR与相应的配体结合引起EGFR形成同源或异源二聚体启动胞内信号转导,激活下游多种信号转导途径,产生生物学效应,RAS/RAF/MEK/ERK通路与细胞增殖、分化和凋亡有关;PI3K/PDK1/AKT通路与细胞的迁移和粘附有关。EGFR能促进肺泡II型上皮细胞的成熟和肺表面活性物质的合成、分泌。EGFR对哺乳动物肺脏的作用呈现时空效应及剂量依赖效应,EGFR的下调表达则会引起肺脏发育不成熟;而EGFR过度表达促进肺肿瘤细胞的增殖、侵袭和转移。文章综述了EGFR及其调节信号通路的研究进展,以及EGFR与动物肺脏发育不成熟和肺癌之间的关系。     

Nerve growth factor-induced reduction in epidermal growth factor responsiveness and epidermal growth factor receptors in PC12 cells: an aspect of cell differentiation.

The rat pheochromocytoma clone PC12 responds to nerve growth factor through the expression of a number of differentiated neuronal properties. One of the most rapid changes is a large, transient increase in the activity of ornithine decarboxylase. These cells also show an increase in ornithine decarboxylase activity in response to the mitogen, epidermal growth factor, but do not respond morphologically as they do to nerve growth factor. Specific, high-affinity epidermal growth factor receptors are present on the cells. When the cells are differentiated with nerve growth factor, the response to epidermal growth factor is markedly diminished and there is a marked reduction in the binding of epidermal growth factor to the cells.     

Renal origin of rat urinary epidermal growth factor

The origin of rat urinary epidermal growth factor (EGF) has been investigated. Unilateral nephrectomy decreased the concentration, total output of EGF and EGF/creatinine ratio by approximately 50%, while the output of creatinine was unchanged. Removal of the submandibular glands and duodenal Brunner's glands, organs known to produce EGF, had no influence on the output of EGF in urine. Renal clearance of EGF exceeded that of creatinine, and after bilateral nephrectomy or bilateral ligation of the ureters, the concentration of creatinine in serum increased, while the concentration of EGF was below the detection limit of the assay. Renal production of EGF was confirmed by immunohistochemistry demonstrating EGF immunoreactivity in the afferent arteriole of the juxtaglomerular apparatus. EGF in the submandibular glands and in urine was found to differ with chromatofocusing and reverse-phase HPLC. At isoelectric focusing the pI of submandibular EGF was 4.8 and 5.4 while that of urinary EGF was 5.3 and 6.4.In conclusion, this study demonstrates that urinary EGF mainly originates from the kidneys and is localized to the renal juxtaglomerular apparatus.     

Mass production of human epidermal growth factor using fed-batch cultures of recombinant Escherichia coli

Fed-batch cultures of recombinant E. coli HB101 harboring expression plasmid pTRLBT1 or pTREBT1, with acetate concentration monitoring, are investigated to obtain high cell density and large amounts of human epidermal growth factor (hEGF). The expression plasmid pTRlBT1 contains a synthetic hEGF gene attached downstream of the N-terminal fragment of the trp L gene preceded by the trp promoter. The expression plasmid pTREBT1 contains the same coding sequence attached downstream of the N-terminal fragment of the trp E gene preceded by the trp promoter, trp L gene, and attenuator region. E. coli harboring pTREBT1 produces 0.56 mg/L hEGE and immediately degrades it. On the other hand E. coli harboring pTRLBT1 produces 6.8 mg/L hEGF and does not decompose it. Prominent inclusion bodies are observed in E. coli cells harboring pTRLBT1 using an election microscope. To Cultivate E. coli harboring pTRLBT1, a fed-batch culture system, divided into a cell growth step and an hEGF production step, is carried out. The cells grow smoothly without acetate-induced inhibition. Cell concentration and hEGF quantity reach the high values of 21 g/L and 60 mg/L, respectively.     

Combined effects of somatomedin-like growth factors with fibroblast growth factor or epidermal growth factor in DNA synthesis in rabbit chondrocytes

Summary The somatomedin-like growth factors cartilage-derived factor (CDF) and multiplication-stimulating activity (MSA) stimulate DNA synthesis and proliferation of rabbit costal chondrocytes under serum-free conditions. Previously, we suggeted that CDF and MSA act on chondrocytes in an early G₁ phase to stimulate DNA synthesis. CDF and MSA have synergistic effects with epidermal growth factor (EGF) or fibroblast growth factor (FGF) in stimulating DNA synthesis of the cells. The mode of combined action of CDF or MSA with EGF or FGF in chondrocytes was studied by sequential treatments with these agents. EGF or FGF had synergistic effects with CDF or MSA in stimulating DNA synthesis, even when added 10 h after the latter. Synergism was also observed in cells pretreated with CDF or MSA; That is, the cultures were treated for 5 h with CDF or MSA and then washed, and treated with FGF or EGF. However, when CDF or MSA was added more than 5 h after EGF or FGF, no synergism of effects was observed. These findings suggest that the cultured chondrocytes become activated to interact with FGF or EGF for commitment to DNA synthesis when they are exposed to somatomedin-like growth factors at an early stage in the G₁ phase. Thus chondrocytes are under a different mechanism of growth control from fibroblastic cells.Abbreviations CDF cartilage-derived factor - MSA multiplication-stimulating activity - EGF epidermal growth factor - FGF fibroblast growth factor     

Density-induced down regulation of epidermal growth factor receptors

Summary Previous studies have shown that cell density can regulate the binding of several growth factors. To determine whether cell density exerts a uniform effect on the expression of epidermal growth factor (EGF) receptors, seven cell lines were examined in detail. For each cell line, EGF binding was found to decrease as cell density increases. Scatchard analysis of the binding data reveals that decreases in EGF binding are due to reductions in the number of cell surface EGF receptors. The only apparent exception is the effect of cell density on the binding of EGF to A-431 cells. For these cells, increases in cell density lead to two effects: decreases in the number of high affinity EGF receptors and increases in the total number of EGF receptors. In addition to the effects of cell density on EGF receptors, it was determined that increases in cell density can coordinately down-regulate receptors for as many as four different growth factors. Overall, the findings described in this report for EGF and those previously described for transforming growth factor type-β (TGF-β) and fibroblast growth factor (FGF) demonstrate the existence of a common mechanism for down-regulating growth factor receptors. This work was supported by grants from the Nebraska Department of Health (89-51), the National Cancer Institute (Laboratory Research Center Support Grant, CA36727), and the American Cancer Society (Core Grant ACS SIG-16). EDITOR'S STATEMENT The paper by Rizzino et al. demonstrates that receptor number decreases as a function of cell density. This may represent a mechanism by which cell proliferation is reduced as cell density increases.     

人表皮生长因子的研究进展

人表皮生长因子是激活表皮生长因子受体的生长因子家族的典型成员,由人体的多个组织器官合成与分泌,通过结合受体激活一系列信号途径,调控细胞的增殖、分化和迁移等。近年来,有关人表皮生长因子的研究已扩展到其在人类生理和病理作用的领域,尤其在组织再生和伤口愈合方面成为研究热点。文中综述了人表皮生长因子的研究进展,简要描述了其基因和蛋白的结构与特点、作用机制与生物学效应,重点介绍该生长因子在胃肠溃疡愈合、皮肤伤口修复和肿瘤病理过程中的作用与影响,从而为相关研究提供辅助信息。