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J Schlessinger

Publications and source records attributed to J Schlessinger.

At least 271 records · Page 15Linked to original sources
PubMed1989

EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling.

Binding of EGF to cells expressing human EGF receptor stimulated rapid tyrosine phosphorylation of phospholipase C-II (PLC-II), as revealed by immunoblotting analysis with phosphotyrosine-specific antibodies. Tyrosine phosphorylation of PLC-II was stimulated by low physiological concentrations of EGF (1 nM), was quantitative, and was already maximal after a 30 sec incubation with 50 nM EGF at 37 degrees C. Interestingly, antibodies specific for PLC-II were able to coimmunoprecipitate the EGF receptor and antibodies against EGF receptor also coimmunoprecipitated PLC-II. According to this analysis, approximately 1% of EGF receptor molecules were associated with PLC-II molecules. The protein tyrosine kinase inhibitor tyrphostin RG50864, which blocks EGF-dependent cell proliferation, blocked EGF-induced tyrosine phosphorylation of PLC-II, its association with EGF receptor, and EGF-induced Ca2+ release. Hence, EGF-induced tyrosine phosphorylation of PLC-II may be a regulatory event linking the tyrosine kinase activity of EGF receptor to the PIP2 hydrolysis signaling pathway.

Antineoplastic Agents↗
PubMed1989

All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor.

Activation of the epidermal growth factor (EGF) receptor kinase leads to autophosphorylation and to the phosphorylation of various cellular substrates. The three known autophosphorylation sites of EGF receptor are located at the carboxyl-terminal tail where they probably act to compete with and thus modulate substrate phosphorylation. Mutational analysis and microsequencing techniques have been used to localize and identify new autophosphorylation site(s) of the EGF receptor. We have compared the phosphopeptide maps of human EGF receptor, and two deletion mutants lacking 63 and 126 amino acids from the carboxyl-terminal tail with the phosphopeptide maps of HER/neu and a chimeric EGF receptor containing the carboxyl-terminal tail of HER2/neu. HER2/neu is highly homologous to the EGF receptor, and it probably functions as a growth factor receptor for as yet unidentified growth factor. On the basis of this analysis, we have concluded that all autophosphorylation sites of EGF receptor and HER2/neu are located in their carboxyl-terminal tails. Utilizing the EGF receptors with carboxyl-terminal deletions, we were also able to identify tyr1086 as an additional autophosphorylation site of EGF receptor. Direct microsequencing of a phosphorylated tryptic peptide from the human EGF receptor confirmed this assignment.

Amino Acid Sequence↗
PubMed1989

Metabolic effects induced by epidermal growth factor (EGF) in cells expressing EGF receptor mutants.

The epidermal growth factor (EGF) receptor tyrosine kinase activity is required for both the earliest EGF-stimulated post-binding events (enhancement of inositol phosphate formation and Ca2+ influx, activation of Na+/H+ exchange), and the ultimate EGF-induced mitogenic response. To assess the role of EGF receptor kinase in EGF-induced metabolic effects (2-deoxyglucose and 2-aminoisobutyric acid uptake), we used NIH3T3 cells (clone 2.2), which do not possess endogenous EGF receptors and which were transfected with cDNA constructs encoding either wild type or kinase-deficient human EGF receptor (HER). In addition, we tested the importance of three HER autophosphorylation sites (Tyr-1068, Tyr-1148, and Tyr-1173) in transduction of EGF-stimulated 2-deoxyglucose uptake. Taking our data together, we conclude the following: (i) HER tyrosine kinase activity is required to elicit EGF stimulation of both 2-deoxyglucose and 2-aminoisobutyric acid uptake; (ii) mutations on individual HER autophosphorylation sites, Tyr-1068, Tyr-1148, and Tyr-1173 do not impair EGF-stimulated 2-deoxyglucose uptake.

Aminoisobutyric Acids↗
PubMed1989

HER2 cytoplasmic domain generates normal mitogenic and transforming signals in a chimeric receptor.

We have investigated the biological function of an unidentified human growth factor, the ligand of the putative HER2 receptor, by characterizing the signalling properties of its receptor. HER2 (or c-erbB-2), the human homolog of the rat neu proto-oncogene, encodes a transmembrane glycoprotein of the tyrosine kinase family that appears to play an important role in human breast carcinoma. Since a potential ligand for HER2 has not yet been identified, it has been difficult to analyze the biochemical properties and biological function of this cell surface protein. For this reason, we replaced the HER2 extracellular domain with the closely related ligand binding domain sequences of the epidermal growth factor (EGF) receptor, and examined the ligand-induced biological signalling potential of this chimeric HER1-2 protein. This HER1-2 receptor is targetted to the cell surface of transfected NIH 3T3 cells, forms high and low affinity binding sites, and generates normal mitogenic and cell transforming signals upon interaction with EGF or TGF alpha. The constitutive activation of wild-type HER2 in transfected NIH 3T3 cells suggests the possibility that these cells synthesize the as yet unidentified HER2 ligand and activate HER2 by an autocrine mechanism.

Animals↗
PubMed1989

Functional analysis of the ligand binding site of EGF-receptor utilizing chimeric chicken/human receptor molecules.

The epidermal growth factor (EGF)-receptor is composed of an extracellular ligand-binding region connected by a single transmembrane region to the cytoplasmic kinase domain. In spite of its importance for understanding signal transduction, the ligand-binding domain of the EGF-receptor is not yet defined. We describe the identification of a major ligand-binding domain of the EGF-receptor by utilizing chimeras between the human EGF-receptor and the chicken EGF-receptor. This approach is based on the fact that murine EGF binds to the chicken EGF-receptor with 100-fold lower affinity as compared to the human EGF-receptor. Hence, the substitution of various domains of the chicken EGF-receptor by domains of the human EGF-receptor may restore the higher binding affinity towards EGF, characteristic of the human receptor. We show that chimeric chicken/human EGF-receptor, which contains domain III of the extracellular region of the human receptor, behaves like the human EGF-receptor with respect to EGF binding affinity and biological responsiveness. However, a chimeric chicken/human EGF-receptor containing domains I and II of the human receptor behaves like the chicken rather than the human EGF-receptor. Moreover, two different monoclonal antibodies which compete for the binding of EGF to EGF-receptor recognize specifically domain III of the human EGF-receptor. It is concluded that domain III which is flanked by the two cysteine-rich domains is a major ligand-binding domain of the EGF-receptor.

Animals↗
PubMed1989

Differential signalling potential of insulin- and IGF-1-receptor cytoplasmic domains.

The human receptors for insulin-like growth factor 1 (IGF-1) and insulin, and two chimeric receptors consisting of ligand-binding, extracellular insulin receptor and intracellular IGF-1 receptor structures, have been expressed in NIH-3T3 fibroblasts. All four receptor types were synthesized, processed and transported to the cell surface to form high-affinity binding sites. All normal and chimeric receptors had an active tyrosine kinase which was regulated by homologous or heterologous ligands respectively. In addition, cell surface receptors were internalized efficiently and subjected to accelerated degradation in the presence of ligand. While all four types of receptor stimulated glucose transport with similar efficiency, they displayed significant differences in their mitogenic signalling potentials. Receptors with an IGF-1 receptor cytoplasmic domain were 10 times more active in stimulating DNA synthesis than the insulin receptor. In NIH-3T3 cells overexpressing wild-type and chimeric receptors, maximal growth responses obtained with IGF-1 or insulin alone were equivalent to those obtained with 10% fetal calf serum. We conclude that in the cell system employed the receptors for IGF-1 and insulin mediate short-term responses similarly, but display distinct characteristics in their long-term mitogenic signalling potentials.

Animals↗
PubMed1989

Cytoplasmic domains determine signal specificity, cellular routing characteristics and influence ligand binding of epidermal growth factor and insulin receptors.

The cell surface receptors for insulin and epidermal growth factor (EGF) both employ a tyrosine-specific protein kinase activity to fulfil their distinct biological roles. To identify the structural domains responsible for various receptor activities, we have generated chimeric receptor polypeptides consisting of major EGF and insulin receptor structural domains and examined their biochemical properties and cellular signalling activities. The EGF-insulin receptor hybrids are properly synthesized and transported to the cell surface, where they form binding competent structures that are defined by the origin of their extracellular domains. While their ligand binding affinities are altered, we find that these chimeric receptors are fully functional in transmitting signals across the plasma membrane and into the cell. Thus, EGF receptor and insulin receptor cytoplasmic domain signalling capabilities are independent of their new heterotetrameric or monomeric environments respectively. Furthermore, the cytoplasmic domains carry the structural determinants that define kinase specificity, mitogenic and transforming potential, and receptor routing.

Animals↗
PubMed1989

Intermolecular transphosphorylation between insulin receptors and EGF-insulin receptor chimerae.

The insulin receptor, a glycoprotein consisting of two extracellular alpha- and two transmembrane beta-subunits, is thought to mediate hormone action by means of its tyrosine-specific protein kinase activity. To explore the mechanism of insulin receptor phosphorylation we have used NIH3T3 cells transfected with two receptor constructs: one encoding a chimeric receptor composed of the extracellular domain of the human EGF receptor and the cytosolic domain of the human insulin receptor beta-subunit, and a second construct encoding a kinase-defiecient human insulin receptor. Stimulation of these cells with EGF induced tyrosine autophosphorylation of the EGF-insulin receptor chimera (150 kd) and tyrosine phosphorylation of the beta-subunit of the kinase-deficient insulin receptor (95 kd). The phosphopeptides of the autophosphorylated cytoplasmic domain of the EGF-insulin receptor chimera were comparable to those of the transphosphorylated beta-subunit of the kinase-deficient insulin receptor and of the wild-type human insulin receptor. When immunoaffinity purified EGF-insulin receptor hybrids and kinase-deficient insulin receptors were used in a cell lysate phosphorylation assay, it was found that addition of EGF produced 32P-labeling of both receptor species. We conclude that EGF acting directly through the EGF-insulin receptor chimera causes transphosphorylation of the kinase-deficient insulin receptor. These data support the notion that autophosphorylation of the insulin receptor may proceed by an intermolecular mechanism.

Animals↗
PubMed1989

Phospholipase C-gamma, a substrate for PDGF receptor kinase, is not phosphorylated on tyrosine during the mitogenic response to CSF-1.

Quiescent mouse NIH3T3 cells expressing a transduced human c-fms gene encoding the receptor for colony stimulating factor-1 (CSF-1) were stimulated with mitogenic concentrations of platelet-derived growth factor (PDGF) or CSF-1. Immunoprecipitated phospholipase C-gamma (PLC-gamma) was phosphorylated on tyrosine and calcium was mobilized following treatment of intact cells with PDGF. In contrast, only trace amounts of phosphotyrosine were incorporated into PLC-gamma and no intracellular calcium signal was detected after CSF-1 stimulation. Similarly, CSF-1 treatment did not stimulate phosphorylation of PLC-gamma on tyrosine in a CSF-1-dependent. SV40-immortalized mouse macrophage cell line that expresses high levels of the CSF-1 receptor. In fibroblasts, antiserum to PLC-gamma co-precipitated a fraction of the tyrosine phosphorylated form of the PDGF receptor (PDGF-R) after ligand stimulation, implying that phosphorylated PDGF-R and PLC-gamma were associated in a stable complex. Pre-treatment of cells with orthovanadate also led to tyrosine phosphorylation of PLC-gamma which was significantly enhanced by PDGF, but not by CSF-1. Thus, although the PDGF and CSF-1 receptors are structurally related and appear to be derived from a single ancestor gene, only PDGF-induced mitogenesis in fibroblasts correlated with tyrosine phosphorylation of PLC-gamma.

Animals↗
PubMed1989

Inhibition of human tumor growth in nude mice by a conjugate of doxorubicin with monoclonal antibodies to epidermal growth factor receptor.

Monoclonal antibodies that recognize the extracellular domain of the epidermal growth factor receptor (mAb108) were conjugated with doxorubicin through a dextran bridge. Several antibody-drug conjugates, containing different amounts of doxorubicin, retained binding capacity to human epidermoid carcinoma (KB) cells overexpressing epidermal growth factor receptors. Slight decrease in drug cytotoxicity was seen in in vitro tests, as determined either by inhibition of thymidine incorporation into cells or by reduction in number and size of KB-cell colonies. Yet, when tested in vivo against KB tumor xenografted into nude mice, the anti-epidermal growth factor-receptor drug conjugates with high drug-substitution levels were significantly more effective than free doxorubicin, antibody alone, mixture of dextran-doxorubicin and antibody, or drug conjugated with irrelevant antibody. When the labile covalent bonds linking antibody to dextran bridge were stabilized by reduction, the therapeutic efficacy of the conjugate was markedly decreased. These results show that antibodies against the extracellular domain of the epidermal growth factor can deliver doxorubicin specifically and efficiently to tumor sites that express high receptor levels exerting a specific antitumor effect.

Animals↗
PubMed1989

Receptor for acidic fibroblast growth factor is related to the tyrosine kinase encoded by the fms-like gene (FLG).

We have previously isolated a human gene from an endothelial cell cDNA library encoding a putative tyrosine kinase; we have designated this gene the fms-like gene (FLG). To analyze the gene product(s) of FLG, we have generated rabbit polyclonal antibodies directed against a synthetic peptide from FLG and used it to immunoprecipitate biosynthetically labeled FLG protein from a variety of human cell lines. These antibodies specifically recognized glycoprotein(s) of 100, 120, and 135 kDa with protein cores of 90 and 110 kDa. Acidic fibroblast growth factor (aFGF) stimulated tyrosine kinase activity of FLG in vitro and in living cells, suggesting that FLG encodes the membrane receptor for aFGF. Further supporting evidence came from cross-linking experiments on intact cells with the covalent cross-linking agent disuccinimidyl suberate and 125I-labeled aFGF as a specific probe. The cross-linked 125I-labeled aFGF-aFGF receptor complex was specifically immunoprecipitated with FLG antipeptide antibodies. It appears, therefore, that the receptor(s) for aFGF is related to the FLG gene product.

Animals↗
PubMed1989

Evidence that autophosphorylation of solubilized receptors for epidermal growth factor is mediated by intermolecular cross-phosphorylation.

Structurally distinguishable mutants of human epidermal growth factor receptor (EGFR) were used to investigate the mechanism of EGFR autophosphorylation. Mutant receptors generated by site-directed mutagenesis were expressed in transfected NIH 3T3 cells lacking endogenous receptors. After coincubation of cell lysates in the presence or absence of EGF, receptor immunoprecipitates were incubated with [gamma-32P]ATP. A kinase-negative mutant EGFR (K721A), in which Lys-721 in the ATp binding site was replaced by an alanine residue, was shown to be phosphorylated in an EGF-dependent manner by an enzymatically active EGFR deletion mutant lacking two autophosphorylation sites. A mutant EGFR lacking the EGF-binding domain as well as the phosphorylation sites also phosphorylated the kinase-negative mutant. In both cases the kinase-negative mutant K721A was phosphorylated on sites virtually identical to the sites that are autophosphorylated by wild-type recombinant or native human EGFRs. With four different site-specific anti-EGFR antibodies, it was shown that deletion mutants devoid of epitopes recognized by the antibodies were coimmunoprecipitated together with wild-type or mutant receptors recognized by the antibodies. This indicates that EGFR oligomers were preserved during immunoprecipitation. On the basis of these results, we propose that autophosphorylation of solubilized EGFR is mediated by intermolecular cross-phosphorylation, probably facilitated by receptor oligomerization.

Animals↗
PubMed1989

Characterization of internalization and endosome formation of epidermal growth factor in transfected NIH-3T3 cells by computerized image-intensified three-dimensional fluorescence microscopy.

Computerized image-intensified fluorescence microscopy has been used to quantify routing and subcellular concentrations of rhodaminated EGF (Rh-EGF) during its receptor-mediated endocytosis in two transfected NIH-3T3 cell lines expressing 2 X 10(5) and 1.5 X 10(6) receptors per cell, respectively. A series of images were digitized by focusing at different depths through the volume of a single cell. The digitized pictures were corrected for fluorescence photobleaching, and removal of out-of-focus fluorescence contributions by deconvolution using the point spread function of the microscope optics (Agard, D. A., and J. W. Sedat. 1980. Proc. Soc. Photo-Opt. Instr. Eng. 264:110-117) allowed automatic computer analysis of the time dependence of endosomal vesicle size and fluorescence intensity in a live cell and also enabled the study of isolated vesicles. An increase in the amount of fluorescence bound to the cell surface, either by increasing the number of receptors expressed per cell or the concentration of Rh-EGF in the incubation drop, yielded an increase in the total fluorescence of internalized vesicles without an increase in their number and area. The linear relation between fluorescence intensity and area for vesicles at different times indicates that EGF concentration is conserved. This is compatible with fusion of small vesicles to form larger ones. However, as endocytosis proceeds, a twofold increase in the slope of the fluorescence vs. area plots is observed for larger vesicles, suggesting that active sorting causes the EGF to be concentrated. Alternatively, this factor could be produced by cumulative fluorescence contributions from stacked membranes. Since coated pits are internalized independent of their occupancy with EGF receptor, we propose that endocytosis does not involve a mechanism specifically recognizing occupied receptor but is rather triggered by a global intracellular event.

Animals↗
PubMed1989

Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells.

The response of malignant and nonmalignant human breast cell lines to the growth inhibitory effects of monoclonal antibodies against the epidermal growth factor (EGF) receptor was studied. A series of human breast cell lines, which express EGF receptor, were used: MDA-468, MDA-231, and Hs578T human breast cancer cells and the transformed human mammary epithelial cell lines 184A1N4 and 184A1N4-T that have been benzo[a]pyrene immortalized and further transformed with SV40T, respectively. Four antibodies of two different classes were tested: 225 immunoglobulin G (IgG), 108.4 IgG, 96 immunoglobulin M (IgM), and 42 IgM. All four antibodies inhibited the anchorage-dependent and -independent, EGF-stimulated growth of 184A1N4 and 184A1N4-T cells, respectively, and this growth inhibition could be reversed by the addition of increasing concentrations of EGF. In contrast, the antibodies inhibited the anchorage-dependent and -independent growth of MDA-468 cells in the absence of exogenous EGF suggesting that the antibodies were acting to block access of an endogenously produced ligand to the EGF receptor. In the presence of antibody and increasing concentrations of EGF, MDA-468 cell growth was first stimulated then inhibited as the EGF concentration increased, thus, uncovering the growth stimulatory potential of low concentrations of EGF in these cells. Data is presented that indicates MDA-468 cells secrete a transforming growth factor with autocrine growth stimulatory capabilities. The growth of MDA-231 and Hs578T cells, which contain activated ras oncogenes, was not inhibited by the antibodies and the growth of these cell lines was not stimulated by EGF. Of the cell lines studied only MDA-468 cells appear to possess an autocrine growth stimulatory capacity.

Antibodies, Monoclonal↗
PubMed1989

Ligand-independent tyrosine phosphorylation of EGF receptor and the erbB-2/neu proto-oncogene product is induced by hyperosmotic shock.

Treatment of intact cells with media containing high concentrations of ionic and non-ionic solutes induced increased tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and the protein product of the erbB-2/neu proto-oncogene. This self phosphorylation occurred in the absence of EGF or other growth factors. High concentrations of solutes did not activate phosphorylation of either isolated EGF receptor or EGF receptor solubilized by non-ionic detergents. No evidence for receptor dimerization was found in response to hyperosmotic shock. Since receptor dimers have been implicated in the EGF-induced activation of EGF receptor, hyperosmotic shock may activate EGF receptor by a different mechanism.

Cell Line↗
PubMed1988

Efficacy of antibodies to epidermal growth factor receptor against KB carcinoma in vitro and in nude mice.

Iodine-125-labeled monoclonal antibody 108.4 (108.4 mAb), raised against the extracellular domain of the epidermal growth factor (EGF) receptor, was shown to visualize sc xenografts of human oral epidermoid carcinoma (KB) cells in nude mice. In vitro, although EGF caused an increase in the number of KB cell colonies (150% at a concentration of 160 mM), the anti-EGF receptor antibodies reduced clone formation. At a concentration at which EGF caused a 50% increase in colony number, the addition of a 100-fold molar excess of 108.4 mAb resulted in a decrease in the number of cell colonies to 20% of the original value. Therefore, the effect of antibody on the KB tumor was studied in vivo in three different modes of tumor transplantation. Antitumor activity was demonstrated first by retardation (versus controls) of the growth of tumor cells as sc xenografts (P greater than .017), then by prolongation of the life span of animals with the ip form of the tumor (P less than .001), and finally on an experimental lung metastasis by a reduction in the number and size of tumors (P less than .05). When the anti-EGF receptor antibodies were added together with cisplatin, the antitumor effect was greatly enhanced, suggesting that the toxic activity of these agents is synergistic (P less than .007). The antitumor effect persisted when animals were treated with the F(ab)'2 fragment of the antibody, although it was less efficient. The Fab fragment of the antibody, whose ability to bind to the cell-associated receptor was completely conserved, did not affect the growth of the tumor. The activity manifested by the F(ab)'2 fragment of the anti-EGF receptor antibodies suggested that the antitumor effect was not due to immune mechanisms requiring the Fc portion of the antibody.

Animals↗