Biomedical subjects

J Schlessinger

Publications and source records attributed to J Schlessinger.

At least 307 records · Page 17Linked to original sources

PubMed1988

Localization of a major receptor-binding domain for epidermal growth factor by affinity labeling.

Epidermal growth factor (EGF) receptor was affinity labeled with 125I-labeled EGF, using bifunctional covalent cross-linking agents. The affinity-labeled receptor was isolated and cleaved with CNBr to yield a single-labeled fragment, which was unequivocally identified by site-specific antibodies and other methods to encompass residues 294 to 543 of the EGF receptor. On the basis of amino acid sequence conservation, the extracellular portion of EGF receptor can be divided into four domains. The labeled CNBr fragment contains the entire sequence which is flanked by the two cysteine-rich domains of extracellular portion of the EGF receptor denoted as domain III. On the basis of these and other results, we propose that domain III contributes most of the interactions that define ligand-binding specificity of the EGF receptor.

Affinity Labels↗

PubMed1988

Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.

The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors.

Amino Acid Sequence↗

PubMed1988

Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor.

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.

Animals↗

PubMed1987

Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells.

The epidermal growth factor receptor (EGFR) gene is frequently amplified and/or overexpressed in human malignancies. To investigate the biological effects of its overexpression, we constructed a eukaryotic vector containing human EGFR cDNA. Introduction of this construct led to reconstitution of functional EGF receptors in NR6 mutant cells, which are normally devoid of this receptor. Transfection of NIH 3T3 resulted in no significant alterations in growth properties. However, EGF addition led to the formation of densely growing transformed foci in liquid culture and colonies in semisolid medium. NIH 3T3-EGFR clonal lines, which expressed the EGF at 500- to 1000-fold levels over control NIH 3T3 cells, demonstrated a marked increase in DNA synthesis in response to EGF. Thus EGF receptor overexpression appears to amplify normal EGF signal transduction. Finally, high levels of EGFR expression, which conferred a transformed phenotype to NIH 3T3 cells in the presence of ligand, were demonstrated in representative human tumor cell lines that contained amplified copies of the EGFR gene.

Animals↗

PubMed1987

On the tertiary structure of the extracellular domains of the epidermal growth factor and insulin receptors.

Alignment of the sequences, the identification of conserved residue patterns and secondary structure predictions indicate that the extra-cellular regions of the human and Drosophila epidermal growth factor (EGF), c-erb-B2 and human insulin receptors each contain two large, homologous domains (L) which are probably comprised of at least four short alpha-helices followed by turns of conserved length and beta-strands. In the human and Drosophila EGF and c-erb-B2 receptors these homologous domains are each followed by a series of smaller cystine-rich domains (S) to give a gene-duplicated structure of L1S11S12S13L2S21S22S23. In the human insulin receptor, the second series of cystine domains is replaced by a different sequence. These duplicated structures are probably organised as a pseudo-symmetrical dimer. There are two 'hyper-variable' regions, one at the end of the large domains and one in the cystine-rich sequences, which are candidates for hormone or growth-factor binding.

Amino Acid Sequence↗

PubMed1987

Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing.

Cultured NIH 3T3 cells devoid of endogenous EGF receptors were transfected with cDNA constructs encoding either the human EGF receptor or an EGF receptor mutant in which Lys721, a key residue in the ATP binding site, was replaced with an alanine residue. The mutant receptor was properly processed, and it displayed both high- and low-affinity surface binding sites. Unlike the wild-type receptor, the mutant receptor did not possess intrinsic protein-tyrosine kinase activity. The initial rate of EGF internalization was similar for wild-type and mutant EGF receptors. Surprisingly, the mutant receptors were not down regulated, but appeared to recycle in transfected cells. These data suggest that degradation of normal EGF receptors after endocytosis is due to the kinase activity endogenous to this receptor. A single amino acid substitution rendered a "down-regulated" receptor into a receptor that can recycle from cytoplasmic compartment back to the cell surface.

Adenosine Triphosphate↗

PubMed1987

Identification of the bombesin receptor on murine and human cells by cross-linking experiments.

The bombesin receptor present on the surface of murine and human cells was identified using 125I-labeled gastrin-releasing peptide as a probe, the cross-linking agent disuccinimidyl suberate, and sodium dodecyl sulfate gels. A clone of NIH-3T3 cells which possesses approximately 80,000 bombesin receptors/cell with a single binding constant of approximately 1.9 X 10(-9) M was used in these studies. In addition, we used Swiss 3T3 cells and a human glioma cell line which possesses approximately 100,000 and approximately 55,000 bombesin receptors/cell, respectively. Under conditions found optimal for binding, it is demonstrated that 125I-labeled gastrin-releasing peptide can be cross-linked specifically to a glycoprotein of apparent molecular mass of 65,000 daltons on the surface of the NIH-3T3 cells. Similar results were obtained when the cross-linked product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions. Moreover, the cross-linking reaction is specific and saturable and the 65,000-dalton polypeptide is not observed when the cross-linking experiments were performed with a NIH-3T3 cell line which is devoid of bombesin receptors. Interestingly, glycoproteins with apparent molecular weights of 75,000 were labeled specifically by 125I-labeled gastrin-releasing peptide when similar experiments were performed with Swiss 3T3 cells and with human glioma cell line GM-340. These different molecular weights may indicate differential glycosylation as treatment with the enzyme N-glycanase reduced the apparent molecular weight of the cross-linked polypeptide to 45,000. On the basis of these results it is concluded that the cross-linked polypeptides represent the bombesin receptor or the ligand-binding subunit of a putative larger bombesin receptor expressed on the surface of these cells.

Animals↗

PubMed1987

A chimeric, ligand-binding v-erbB/EGF receptor retains transforming potential.

Comparison of amino acid sequences from human epidermal growth factor (EGF) receptor and avian erythroblastosis virus erbB oncogene product suggests that v-erbB represents a truncated avian EGF receptor gene product. Although both proteins are transmembrane tyrosine kinases, the v-erbB protein lacks most of the extracellular ligand-binding domain and a 32-amino acid cytoplasmic sequence present in the human EGF receptor. To test the validity of the proposed origin of v-erbB and to investigate the functional significance of the deleted extracellular sequences, a chimeric gene encoding the extracellular and the transmembrane domain of the human EGF receptor joined to sequences coding for the cytoplasmic domain of the avian erbB oncogene product was constructed. When expressed in Rat1 fibroblasts, this reconstituted gene product (HER-erbB) was transported to the cell surface and bound EGF. Its autophosphorylation activity was stimulated by interaction with the ligand. Expression of the HER-erbB chimera led to anchorage-independent cell growth in soft agar and EGF-induced focus formation in Rat1 monolayers. Thus, it appears that v-erbB protein sequences in the chimeric receptor retain their transforming activity under the influence of the human extracellular EGF-binding domain.

Animals↗

PubMed1987

Self-phosphorylation of epidermal growth factor receptor: evidence for a model of intermolecular allosteric activation.

The membrane receptor for epidermal growth factor (EGF) is a 170,000-dalton glycoprotein composed of an extracellular EGF-binding domain and a cytoplasmic kinase domain connected by a stretch of 23 amino acids traversing the plasma membrane. The binding of EGF to the extracellular domain activates the cytoplasmic kinase function even in highly purified preparations of EGF receptor, suggesting that the activation occurs exclusively within the EGF receptor moiety. Conceivably, kinase activation may require the transfer of a conformational change through the single transmembrane region from the ligand binding domain to the cytoplasmic kinase region. Alternatively, ligand-induced receptor-receptor interactions may activate the kinase and thus bypass this requirement. Both mechanisms were contrasted by employing independent experimental approaches. The following lines of evidence support an intermolecular mechanism for the activation of the detergent-solubilized receptor: the EGF-induced receptor self-phosphorylation has a parabolic dependence on the concentration of EGF receptor, cross-linking of EGF receptors by antibodies or lectins stimulates receptor self-phosphorylation, immobilization of EGF receptor on various solid matrices prevents EGF from activating the kinase function, and cross-linking of EGF receptors increases their affinity toward EGF. On the basis of these results, an allosteric aggregation model is formulated for the activation of the cytoplasmic kinase function of the receptor by EGF. This model may be relevant to the mechanism by which the mitogenic signal of EGF is transferred across the membrane.

Adenosine Triphosphate↗

PubMed1987

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.

Allosteric Regulation↗

PubMed1987

An angiogenic growth factor is expressed in human glioma cells.

Progression to increased malignancy frequently occurs in human brain tumors of glial origin and usually involves neovascularization--a massive proliferation of endothelial cells into the tumor tissue. We have shown previously that subversion of a normal growth factor-related pathway is frequently associated with human gliomas. Here we show that human glioma cell lines express the gene encoding the angiogenic peptide endothelial cell growth factor (ECGF) or acidic fibroblast growth factor (a-FGF) and that an ECGF-like polypeptide is produced by these cells. The glioma-derived growth factor was partially purified from cell extracts by heparin-Sepharose affinity chromatography where it eluted at 1.5 M sodium chloride. On reversed-phase h.p.l.c., growth factor activity for endothelial cells was eluted at the same concentration of acetonitrile as found for bovine brain-ECGF, also a potent mitogen for endothelial cells. Moreover, human glioma cells possess specific cell surface receptors for ECGF and are mitogenically stimulated by exogenous addition of this growth factor. Glioma derived-ECGF may therefore have a dual influence: first, by autocrine growth-stimulation of human gliomas and, second, by paracrine-stimulation of endothelial cell proliferation which results in neovascularization of the tumor tissue.

Cell Line↗

PubMed1987

An insertional mutant of epidermal growth factor receptor allows dissection of diverse receptor functions.

Cultured NIH-3T3 cells devoid of endogenous EGF-receptors were transfected with cDNA constructs encoding normal human EGF-receptor and with a construct encoding an insertional mutant of the EGF-receptor containing four additional amino acids in the kinase domain after residue 708. Unlike the wild-type receptor expressed in these cells which exhibits EGF-stimulatable protein tyrosine kinase activity, the mutant receptor lacks protein tyrosine kinase activity both in vitro and in vivo. Despite this deficiency the mutant receptor is properly processed, it binds EGF and it exhibits both high and low affinity binding sites. Moreover, it undergoes efficient EGF-mediated endocytosis. However, EGF fails to stimulate DNA synthesis and is unable to stimulate the phosphorylation of S6 ribosomal protein in cells expressing this receptor mutant. Hence, it is proposed that the protein tyrosine kinase activity of EGF-receptor is essential for the initiation of S6 phosphorylation and for DNA synthesis induced by EGF. However, EGF-receptor processing, the expression of high and low affinity surface receptors and receptor internalization, require neither kinase activity nor receptor autophosphorylation. Interestingly, phorbol ester (TPA) fails to abolish the high affinity state and is also unable to stimulate the phosphorylation of this receptor mutant. This result is consistent with the notion that kinase-C phosphorylation of EGF-receptor is essential for the loss of high affinity EGF-receptors caused by TPA.

Animals↗

PubMed1987

Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand.

Structural features of v-kit, the oncogene of HZ4 feline sarcoma virus, suggested that this gene arose by transduction and truncation of cellular sequences. Complementary DNA cloning of the human proto-oncogene coding for a receptor tyrosine kinase confirmed this possibility: c-kit encodes a transmembrane glycoprotein that is structurally related to the receptor for macrophage growth factor (CSF-1) and the receptor for platelet-derived growth factor. The c-kit gene is widely expressed as a single, 5-kb transcript, and it is localized to human chromosome 4 and to mouse chromosome 5. A c-kit peptide antibody permitted the identification of a 145,000 dalton c-kit gene product that is inserted in the cellular plasma membrane and is capable of self-phosphorylation on tyrosine residues in both human glioblastoma cells and transfected mouse fibroblasts. Our results suggest that p145c-kit functions as a cell surface receptor for an as yet unidentified ligand. Furthermore, carboxy- and amino-terminal truncations that occurred during the viral transduction process are likely to have generated the transformation potential of v-kit.

Amino Acid Sequence↗

PubMed1987

Onset of endogenous synthesis of epidermal growth factor in neonatal mice.

We have analyzed mouse fetuses and neonates for the presence of epidermal growth factor (EGF)-specific mRNA. No detectable EGF-specific mRNA was found in fetuses, fetal membranes, or placentae from Day 9 of gestation through birth or in the early neonatal period. While the kidneys begin to produce EGF specific transcripts by two weeks postpartum, the salivary glands begin to produce detectable levels of EGF mRNA only after weaning and even then at levels far below the adult amount. Reports of EGF and EGF-related material in rodent fetuses failed to determine whether this material was of maternal or fetal origin. We now conclude that authentic EGF in these embryos is probably of maternal origin. We have performed experiments designed to determine whether EGF can be transported into the fetus. A small percentage of 125I-EGF administered to pregnant females either systemically or directly into the uterine arteries reached the fetus itself. The uterus and the placenta attained a high level of labeling, whereas the amniotic fluid and yolk sac were virtually devoid of the tracer. In the neonatal period, milk may be the physiologically relevant source of EGF. We have found that 125I-EGF ingested by neonates was absorbed into the circulation, reached many internal organs, and was eventually excreted in the urine. Previously demonstrated EGF receptors in mouse embryonic cell types may be activated by either alpha type transforming growth factor or maternal EGF transported via the placenta.

Animals↗

PubMed1987

Increased expression of the putative growth factor receptor p185HER2 causes transformation and tumorigenesis of NIH 3T3 cells.

The HER2 gene encodes a cell-surface glycoprotein with extensive homology to the epidermal growth factor receptor. Recently it was found to be amplified in about 30% of primary human breast malignancies. In experiments designed to assess the role of the HER2 gene in oncogenesis, we found that overexpression of unaltered HER2 coding sequences in NIH 3T3 cells resulted in cellular transformation and tumorigenesis.

Animals↗

PubMed1987

High-affinity interleukin 2 binding by an oncogenic hybrid interleukin 2-epidermal growth factor receptor molecule.

Both interleukin 2 (IL-2) and epidermal growth factor (EGF) receptors exist in two forms that differ with respect to affinity for their ligand. Only the high-affinity receptors appear to be responsible for the proliferation signal delivered upon binding of the growth factor. Fibroblasts transfected with IL-2 receptor cDNA generate only low-affinity receptors for IL-2, but fusion of membranes from these fibroblasts with T-cell membranes converts some receptors to high affinity, indicating the involvement of a T cell-specific factor in the generation of high-affinity receptors. We have constructed a chimeric cDNA molecule containing the extracellular IL-2-binding domain of the IL-2 receptor cDNA and the transmembrane and intracellular tyrosine kinase domains of the EGF receptor cDNA. When transfected into fibroblasts, this IL-2-EGF receptor cDNA generated high-affinity receptors for IL-2. Moreover, fibroblasts transfected with the chimeric molecule were morphologically transformed and produced rapidly growing tumors in nude mice.

Animals↗

PubMed1987

A mutant epidermal growth factor receptor with defective protein tyrosine kinase is unable to stimulate proto-oncogene expression and DNA synthesis.

Cultured NIH-3T3 cells devoid of endogenous epidermal growth factor (EGF) receptors were transfected with cDNA expression constructs encoding either normal human EGF receptor or a receptor mutated in vitro at Lys-721, a residue that is thought to function as part of the ATP-binding site of the kinase domain. Unlike the wild-type EGF-receptor expressed in these cells, which exhibited EGF-dependent protein tyrosine kinase activity, the mutant receptor lacked protein tyrosine kinase activity and was unable to undergo autophosphorylation and to phosphorylate exogenous substrates. Despite this deficiency, the mutant receptor was normally expressed on the cell surface, and it exhibited both high- and low-affinity binding sites. The addition of EGF to cells expressing wild-type receptors caused the stimulation of various responses, including enhanced expression of proto-oncogenes c-fos and c-myc, morphological changes, and stimulation of DNA synthesis. However, in cells expressing mutant receptors, EGF was unable to stimulate these responses, suggesting that the tyrosine kinase activity is essential for EGF receptor signal transduction.

Adenosine Triphosphate↗

PubMed1987

Human HER2 (neu) promoter: evidence for multiple mechanisms for transcriptional initiation.

We localized the 5' region of the human gene HER2 in a cloned fragment of genomic DNA. This clone contained exons 1 to 4 of HER2, spanning the coding sequence for the first 191 amino acids. The promoter region of HER2 was identified upstream to exon 1 by nuclease S1 mapping and by a functional assay in which the promoter region drives the expression of a chloramphenicol acetyltransferase gene. The HER2 promoter is different from the promoter of the epidermal growth factor receptor gene (HER1), and the GC boxes which are typical of the promoter of the epidermal growth factor receptor gene are absent from the HER2 promoter. One major and two minor RNA start sites located at nucleotides 178, 244, and 257 upstream to the initiator ATG were identified. The first one is 21 and 70 base pairs downstream from typical TATAA and CAAT boxes, respectively. This indicates that transcription of HER2/neu can be regulated by a mechanism involving a TATA box, as well as by other unidentified regulatory elements.

Base Sequence↗