Epidermal growth factor receptor occupancy inhibits vaccinia virus infection

Vaccinia virus encodes VGF, an early protein of relative molecular mass 19,000 (19K) which, from amino-acid residues 45 to 85, is homologous in 19 residues to epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha). The conserved sequence includes a region of high homology (6 out of 10 amino acids) from residues 71 to 80, corresponding to the third disulphide loop of both EGF and TGF-alpha. This region has recently been shown to contain a binding region of TGF-alpha for the EGF receptor, and this raises the question of whether vaccinia virus utilizes the EGF receptor in order to bind to and infect cells. We now show that occupancy of the EGF receptor inhibits vaccinia virus infection. Inhibition is observed in a dose-dependent fashion by pre-treatment with either EGF or synthetic decapeptide antagonists of EGF's mitogenic activity which correspond to the sequence of the third disulphide loop of VGF or TGF-alpha. The relative ability of the peptides to inhibit vaccinia virus infection parallels their binding affinity to the EGF receptor.     

Autophosphorylation sites on the epidermal growth factor receptor

The epidermal growth factor (EGF) receptor is a tyrosine-specific protein kinase with autophosphorylating activity. A 300 amino acid-long region of the receptor's cytoplasmic domain matches (35-90% homology) sequences of transforming proteins from the src family and includes a putative nucleotide binding site. Several of the src transforming proteins have tyrosine kinase activity, but v-erb-B, which appears to be a truncated EGF receptor, is virtually identical to the receptor over this region and yet lacks detectable kinase activity. To locate possible acceptor sites in the v-erb-B protein, we have mapped these sites in the human EGF receptor. We report here that three tyrosine sites near the C-terminus are phosphorylated in vitro. In intact cells, we find that EGF stimulates phosphorylation of several sites, the tyrosine 14 residues from the C-terminus being modified the most extensively. The equivalent site is absent in the v-erb-B protein of avian erythroblastosis virus (AEV) and may influence tyrosine kinase activity.     

Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences

Each of six peptides derived from the human epidermal growth factor (EGF) receptor very closely matches a part of the deduced sequence of the v-erb-B transforming protein of avian erythroblastosis virus (AEV). In all, the peptides contain 83 amino acid residues, 74 of which are shared with v-erb-B. The AEV progenitor may have acquired the cellular gene sequences of a truncated EGF receptor (or closely related protein) lacking the external EGF-binding domain but retaining the transmembrane domain and a domain involved in stimulating cell proliferation. Transformation of cells by AEV may result, in part, from the inappropriate acquisition of a truncated EGF receptor from the c-erb-B gene.     

Similarity between the vaccinia virus 19K early protein and epidermal growth factor

An analysis of the 1,217-amino acid residue sequence of the precursor of mouse epidermal growth factor (mEGF) revealed regions of considerable similarity with bovine factor X, a blood coagulation factor. Similarities of mEGF itself with factor X, pancreatic secretory trypsin inhibitor and, most strikingly, transforming growth factor I (TGF-I) have been observed. On the basis of the comparisons described here, it seems that the presumptive 140-residue 19K early protein (relative molecular mass (Mr) 19,000) of vaccinia virus from residues 40-91 shows an overall identity of 36% (19/53 residues) with both mEGF and urogastrone (human epidermal growth factor, hEGF); a single deletion is assumed for vaccinia virus 19K protein which allows the six Cys residues (positions 45-80) to be aligned with those of mEGF or hEGF. This protein is encoded in the 10.3-kilobase (kb) inverted terminal repeat. Because it is an early protein with an EGF-like central portion, the 19K vaccinia virus protein may have an autocrine function and may be required for DNA synthesis.     

ATP-stimulated interaction between epidermal growth factor receptor and supercoiled DNA

The receptor for epidermal growth factor (EGF) has been identified as a transmembrane glycoprotein that has tyrosine-specific kinase activity. The kinase activity of the receptor is enhanced in the presence of EGF (or related peptides), and the phosphorylation of a number of substrates, as well as autophosphorylation of the receptor, has been reported. Analogous findings have been described for the insulin receptor and the receptor for platelet-derived growth factor (PDGF). Thus, a number of hormone receptors and several viral transforming proteins appear to share the highly unusual property of tyrosine-specific kinase activity. Nevertheless, the specific relationship between tyrosine kinase activity and the control of cell growth and replication is unknown. It is known that after the initial binding of EGF to the plasma membrane, the hormone together with its receptor is rapidly internalized in endocytic vesicles and the hormone is eventually degraded in lysosomes. It is possible that the function of EGF is simply to stimulate internalization of its receptor, and that as a result of its altered location the receptor is able to phosphorylate a cytoplasmic component or even interact directly with a nuclear component. We now report that the purified receptor for EGF is able to interact with and nick supercoiled double-stranded DNA in an ATP-stimulated manner.     

Retinoids specifically enhance the number of epidermal growth factor receptors

Retinoids elicit many biological and biochemical responses from cells in vitro. One widely used criterion for the responsiveness of cells to retinoids is inhibition of growth; retinoids reduce the saturation density and/or growth rate of many normal and tumorigenic cell lines. Propagation of eukaryotic cells has been demonstrated to be dependent on the presence of macromolecular growth factors such as epidermal growth factor (EGF), which can stimulate proliferation of epithelial and fibroblastic cell lines. We now describe the effect of retinoids on the binding of EGF to its receptor. Retinoic acid enhances binding of 125I-labelled EGF to various fibroblastic and epidermal cell lines. It has no marked effect on the affinity of this growth factor for its receptor, but increases the number of EGF receptor sites. Retinoic acid has little effect on the binding of concanavalin A (Con A) and insulin, indicating the specific nature of the action of retinoids on cell-surface glycoproteins. Treatment of cells with the phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) and retinoic acid shows poor antagonism between these compounds on EGF binding. It has been previously shown that retinoids induce or stimulate differentiation of embryonal carcinoma cells. EGF binding can be used as a marker to monitor differentiation of these cells.     

A chimaeric receptor allows insulin to stimulate tyrosine kinase activity of epidermal growth factor receptor

The cell surface receptors for insulin and epidermal growth factor (EGF) appear to share a common evolutionary origin, as suggested by structural similarity of cysteine-rich regions in their extracellular domains and a highly conserved tyrosine-specific protein kinase domain. Only minor similarity is found outside this catalytic domain, as expected for receptors that have different ligand specificities and generate different biological signals. The EGF receptor is a single polypeptide chain but the insulin receptor consists of distinct alpha and beta subunits that function as an alpha 2 beta 2 heterotetrameric receptor complex. Provoked by this major structural difference in two receptors that carry out parallel functions, we have designed a chimaeric receptor molecule comprising the extracellular portion of the insulin receptor joined to the transmembrane and intracellular domains of the EGF receptor to investigate whether one ligand will activate the tyrosine kinase domain of the receptor for the other ligand. We show here that the EGF receptor kinase domain of the chimaeric protein, expressed transiently in simian cells, is activated by insulin binding. This strongly suggests that insulin and EGF receptors employ closely related or identical mechanisms for signal transduction across the plasma membrane.     

The neu oncogene encodes an epidermal growth factor receptor-related protein

The neu oncogene is repeatedly activated in neuro- and glioblastomas derived by transplacental mutagenesis of the BDIX strain of rat with ethylnitrosourea. Foci induced by the DNAs from such tumours on NIH 3T3 cells contain the neu oncogene and an associated phosphoprotein of relative molecular mass 185,000 (p185). Previous work has shown that the neu gene is related to, but distinct from, the gene encoding the EGF receptor (c-erb-B). Here we describe a neu complementary DNA clone isolated from a cell line transformed by this oncogene; the clone has biological activity in a focus-forming assay. The nucleotide sequence of this clone predicts a 1,260-amino-acid transmembrane protein product similar in overall structure to the EGF receptor. We found that 50% of the predicted amino acids of neu and the EGF receptor are identical; greater than 80% of the amino acids in the tyrosine kinase domain are identical. Our results suggest strongly that the neu gene encodes the receptor for an as yet unidentified growth factor.     

Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane

The receptor for epidermal growth factor (EGF) is a 170,000-180,000 molecular weight single-chain glycoprotein of 1,186 amino acids. Its sequence suggests that it has an external EGF-binding domain, formed by the NH2-terminal 621 amino acids, linked to a cytoplasmic region by a single membrane-spanning segment. In the cytoplasmic portion, starting 50 residues from the membrane, there is a 250-residue stretch similar to the catalytic domain of the src gene family of retroviral tyrosine protein kinases, and, indeed, a tyrosine-specific protein kinase activity intrinsic to the receptor is stimulated when EGF is bound. Increased tyrosine phosphorylation of cellular proteins, detected in A431 cells following EGF binding, may be important in the mitogenic signal pathway. Tumour promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), counteract this increase, as well as causing loss of a high affinity class of EGF binding sites. The major receptor for TPA has been identified as the serine/threonine-specific Ca2+/phospholipid-dependent diacylglycerol-activated protein kinase, protein kinase C. By substituting for diacylglycerol, TPA stimulates protein kinase C. Protein kinase C phosphorylates purified EGF receptor at specific sites, and this reduces EGF-stimulated tyrosine protein kinase activity. TPA treatment of A431 cells increases serine and threonine phosphorylation of the EGF receptor at the same sites, which suggests that the reduction of EGF receptor kinase activity in TPA-treated cells is a consequence of the receptor's phosphorylation by the kinase. We have attempted to identify these phosphorylation sites and show here that protein kinase C phosphorylates threonine 654 in the human EGF receptor. This threonine is in a very basic sequence nine residues from the cytoplasmic face of the plasma membrane in the region before the protein kinase domain; it is thus in a position to modulate signalling between this internal domain and the external EGF-binding domain.     

Epidermal growth factor-dependent phosphorylation of lipocortin

Lipocortin-like proteins are a family of steroid-induced inhibitors of phospholipase activity with potential anti-inflammatory activity. Related proteins have been detected in a variety of tissues and species. The best characterized form is a protein of relative molecular mass (Mr) approximately 40,000 (40K), which is phosphorylated in vivo by protein tyrosine kinases and by protein serine-threonine kinases. It has been proposed that the phospholipase inhibitory activity of lipocortin can be regulated by its phosphorylation. In the A431 cell line, a protein of approximately 35K is phosphorylated by the protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Here we report that human lipocortin is phosphorylated near its amino terminus by the EGF receptor/kinase. By peptide mapping and immunological analyses, we show that lipocortin and the endogenous 35K substrate for the EGF receptor/kinase from A431 cells are the same protein.     

The solution structure of human epidermal growth factor

The epidermal growth factors (EGFs) are powerful mitogens for a wide variety of cells in culture; human EGF (hEGF), known as urogastrone, also inhibits gastric acid secretion in vivo. The transforming growth factors (TGF-alpha) are related to the EGF family both in sequence and activity and EGF-like sequences are often observed in a wide range of functionally unrelated proteins. Attempts to examine the structure of EGF by diffraction methods have not yet succeeded because of difficulties with crystallization. We report here a three-dimensional structure of a biologically active derivative (residues 1-48) of the 53-residue human EGF. An analysis of high resolution 1H nuclear magnetic resonance (NMR) spectra was used together with a combination of distance geometry, restrained energy minimization and restrained molecular dynamics methods. The three-dimensional structure provides a basis for understanding the properties of EGFs and for predicting the structures of homologous sequences in other proteins.     

Desensitisation of cultured glial cells to epidermal growth factor by receptor down-regulation.

Epidermal growth factor (EGF), which can be purified from the mouse submaxillary gland or from pregnant human urine, is a potent multiplication-stimulating factor for several types of cultured cells, including human fibroblasts and glial cells. The molecule binds with high affinity and saturation kinetics to a cell-surface receptor, is subsequently internalised and finally degraded. The binding event is accompanied by a reduction in the number of EGF receptors. This phenomenon--'receptor down-regulation'--has been demonstrated with several hormones and may be a general principle for the modulation of binding groups on the outer cell surface. Further, it has been proposed that receptor loss acts to regulate the cellular response to the binding ligand. The present study provides direct experimental support for this hypothesis. It demonstrates that down-regulation of EGF receptors on glial cells causes desensitisation of the mitogenic response of these cells to subsequent stimulation with EGF.     

正常人口腔粘膜中表皮生长因子及其受体的表达

目的：研究人口腔粘膜中表皮生长因子（EGF）及其受体（EGF－R）的分布状态,方法：采用免疫组织化学方法,结果：EGF主要分布于口腔上皮底层细胞中,并随细胞向上分化而逐渐减少,其免疫染色出现在细胞浆中,EGF－R也主要在基底层细胞表达,除存在于细胞浆外,在细胞核中亦见表达,结论：表皮生长因子及其受体对口腔上皮细胞生长,分化的调节有重要作用,但EGF－R在细胞核中的表达,其作用尚需深入研究。     

Two growth factor signalling pathways in fibroblasts distinguished by pertussis toxin

The primary action of a family of mitogens including bombesin, bradykinin, vasopressin and alpha-thrombin is to activate the hydrolysis of polyphosphoinositides. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by phospholipase C is mediated through coupling of surface receptors to a GTP-binding protein (Gp protein) which, in some cells, is inactivated by the toxin of Bordetella pertussis. It is not known whether this signalling pathway is involved in initiating DNA replication, whereas it has been firmly established that reinitiation of DNA synthesis can be triggered without activation of PtdIns(4,5)P2 hydrolysis by, for example, EGF (epidermal growth factor), FGF (fibroblast growth factor) and insulin/IGF-I (insulin-like growth factor-I), members of a class of mitogens known to activate receptor tyrosine kinases. Taking advantage of the fact that Chinese hamster lung fibroblasts respond to either class of mitogens and that their Gp protein appears to be sensitive to pertussis toxin, we have now analysed the toxin's effect on reinitiation of DNA synthesis and find that it inhibits up to 95% of thrombin-induced mitogenicity without affecting EGF- or FGF-induced DNA synthesis and proliferation. These findings strongly suggest that activation of PtdIns(4,5)P2-phospholipase C has a determinant function in growth control, and confirm the existence of alternative growth factor-signalling pathways independent of polyphosphoinositide breakdown.     

Transformation by murine and feline sarcoma viruses specifically blocks binding of epidermal growth factor to cells.

Normal cells in culture have membrane receptors for epidermal growth factor (EGF); EGF stimulates cells to divide by binding to these receptors. Cells transformed by murine and feline sarcoma viruses rapidly lose the ability to bind EGF, whereas cells transformed by the DNA tumour viruses, polyoma and SV40, or infected with non-transforming RNA tumour viruses have normal levels of functional EGF receptors. The results suggest that a product of the sarcoma virus genome specifically changes cell EGF receptors; the sarcoma gene product may, then, be functionally related to EGF.     

Local aggregation of hormone-receptor complexes is required for activation by epidermal growth factor.

An analogue of epidermal growth factor (EGF) which is virtually devoid of biological activity retains receptor binding activity but cannot form cell surface clusters or patches. Bivalent anti-EGF antibodies restore both bioactivity and patch formation. The sensitivity of fibroblasts to native EGF can also be enhanced greatly by these antibodies, especially in hormone-resistant cell lines.     

Nucleotide sequence of epidermal growth factor cDNA predicts a 128,000-molecular weight protein precursor

Epidermal growth factor (EGF) has a profound effect on the differentiation of specific cells in vivo, and has been shown to be a potent mitogenic factor for a variety of cultured cells, of both ectodermal and mesodermal origin (see ref. 1 for review). This 53-amino acid polypeptide of known sequence contains six cysteine residues, which are thought to form three intrachain disulphide bonds. Urogastrone, a polypeptide bearing anti-gastric secretory activity isolated from human urine, which is presumably synthesized in submandibular and Brunner's glands, shares extensive sequence homology (70%) with EGF and may represent the human EGF equivalent. Here we present the sequence of a mouse EGF cDNA clone, which suggests that EGF is synthesized as a large protein precursor of 1,168 amino acids. Our data indicate that the discrepancy between EGF levels in male and female mouse submaxillary glands (MSGs) is due to different EGF mRNA levels in these tissues, and suggest that precursor EGF processing may differ from that described previously for other polypeptide hormones.     

Production and auto-induction of transforming growth factor-alpha in human keratinocytes

Transforming growth factor-alpha (TGF-alpha) is a polypeptide which is structurally related to epidermal growth factor (EGF) and binds to the EGF receptor. TGF-alpha synthesis occurs in a variety of neoplastic cells and during early fetal development but has not been reported in normal cells of the adult organisms. TGF-alpha has therefore been regarded as an embryonic growth factor which is inappropriately expressed during neoplasia. Here we report that primary cultures of normal human keratinocytes synthesize TGF-alpha. Furthermore, we show that addition of EGF or TGF-alpha to these cultures induces TGF-alpha gene expression, suggesting that a mechanism of auto-induction exists. Analysis of normal skin biopsies using in situ hybridization and immunohistochemistry demonstrates the in vivo presence of TGF-alpha messenger RNA and protein in the stratified epidermis.     

Abelson murine leukaemia virus transformation involves loss of epidermal growth factor-binding sites

Malignant transformation by mammalian RNA sarcoma viruses has previously been shown to involve a reduction in receptor sites for a well characterized 6,000-molecular weight (MW) growth-promoting substance, designated epidermal growth factor (EGF). Although Abelson murine leukaemia virus (AbLV) resembles sarcoma viruses in its ability to transform embryo fibroblasts in cell culture, AbLV induces a rapid B-cell lymphoid leukaemia rather than fibrosarcomas in vivo. The major translational product of AbLV is a highly phosphorylated polyprotein of MW 120,000 which exhibits an associated tyrosine-specific protein kinase activity and probable transforming function. We show here that AbLV transformation resembles transformation by RNA sarcoma viruses with respect to the abolition of EGF-binding sites. EGF binding is restored to control levels following loss of polyprotein expression in morphological revertants of AbLV-transformed clones and remains uninfluenced in cell lines infected with transformation-defective (td) AbLV mutants encoding polyproteins deficient in protein kinase activity. These findings indicate that AbLV transformation involves a polyprotein-associated, tyrosine-specific protein kinase activity which mediates its effect through a mechanism resulting directly or indirectly in the abolition of EGF-binding sites.