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Biomedical subjects

J Schlessinger

Publications and source records attributed to J Schlessinger.

At least 343 records · Page 19Linked to original sources
PubMed1985

The Drosophila EGF receptor gene homolog: conservation of both hormone binding and kinase domains.

Chicken v-erB probe was used to isolate a unique clone of Drosophila melanogaster DNA. It maps by in situ hybridization to position 57F on chromosome 2. A complete nucleotide sequence of the coding region has been obtained. The putative Drosophila EGF receptor protein is similar in overall organization to the human homolog. It shows three distinct domains: an extracellular putative EGF binding domain, a hydrophobic transmembrane region, and a cytoplasmic kinase domain. The overall amino acid homology is 41% in the extracellular domain and 55% in the kinase domain. Two cysteine-rich regions, a hallmark of the human ligand-binding domain, have also been conserved. Fusion of the coding sequences of the kinase and extracellular domains generating the receptor gene must have occurred over 800 million years ago.

Amino Acid Sequence↗
PubMed1985

Antibodies against a synthetic peptide as a probe for the kinase activity of the avian EGF receptor and v-erbB protein.

The transforming protein v-erbB of avian erythroblastosis virus (AEV) displays extensive sequence homology with the presumptive protein-tyrosine kinase domain of the human EGF receptor and with the src protein-tyrosine kinase family of oncogenes. However, no kinase activity has previously been demonstrated for the v-erbB protein. Here antibodies generated against a synthetic peptide from the C terminus of human EGF receptor are shown to immunoprecipitate the EGF receptor from human and avian cells, as well as the v-erbB proteins from AEV-transformed cells that become phosphorylated on tyrosine residues upon the addition of gamma-32P-ATP. The immunoprecipitates are also able to phosphorylate exogenous tyrosine-containing substrates. Hence, it is likely that both avian EGF receptor and v-erbB proteins are protein tyrosine-specific protein kinases. Since the kinase activity of v-erbB protein cannot be regulated by EGF, it is proposed that the tyrosine protein kinase function of v-erbB may be constitutively activated.

Alpharetrovirus↗
PubMed1985

Amplification and overexpression of the EGF receptor gene in primary human glioblastomas.

The expression of epidermal growth factor (EGF) receptor in brain tumours of glial origin was studied at the protein, mRNA and genomic levels. Four out of 10 glioblastomas that overexpress EGF receptor also have gene amplification. The amplified genes appear to be rearranged, generating an aberrant mRNA in at least one of these tumours. Such receptor defects may be relevant to tumorigenesis of human glioblastomas.

Brain Neoplasms↗
PubMed1984

Monoclonal antibodies associated with sodium channel block nerve impulse and stain nodes of Ranvier.

Monoclonal antibodies were generated against native eel electroplax sodium channels in their natural membrane. These antibodies block nerve conduction in rat central (optic) and peripheral (sciatic) nerve. The antibody binding to eel electroplax membrane fragments and to rat brain synaptosomes can be modulated by neurotoxins. Thus it implies that active sites of the sodium channels are immunogenic in their natural membrane. Unlike the antibodies described in the past, our antibodies recognize antigenic determinants which are associated with the physiological activity of the channel and have been conserved through evolution.

Animals↗
PubMed1984

Production of EGF-containing polypeptides in Xenopus oocytes microinjected with submaxillary gland mRNA.

The biosynthesis of epidermal growth factor (EGF), a 6045 dalton mitogen produced in the mouse submaxillary gland under androgen regulation, was studied using Xenopus oocytes. Microinjection of total, unfractionated gland mRNA together with [35S]cysteine resulted in the production of a secretory polypeptide of approximately 9000 daltons, specifically immunoprecipitable with anti-EGF antibodies. A minor amount of a similarly immunoreactive 9000 dalton secretory polypeptide was produced from the sucrose gradient 9S fraction of gland mRNA. Other, more intensely labeled polypeptides, a cytoplasmic 125 000 dalton and a secretory 110 000 dalton protein were immunoprecipitated from oocytes injected with the greater than 25S mRNA fraction. The biosynthesis of both can hardly be detected in oocytes injected with unfractionated mRNA. All three polypeptides are produced under androgen regulation and share common immunoreactive properties. Northern blot analysis using a 76 nucleotide synthetic EGF cDNA probe revealed hybridization with a single 28S mRNA species. This, and the apparent interrelation between the three polypeptides, suggest that a gland-specific processing protein, encoded by a 9S mRNA, is required to produce the 9000 dalton pro-EGF from the nascent translation product of EGF mRNA.

Animals↗
PubMed1984

Antibodies to two defined regions of the transforming protein pp60src interact specifically with the epidermal growth factor receptor kinase system.

Antibodies generated against two synthetic peptides corresponding to two defined regions on the transforming protein of Rous sarcoma virus, pp60src, interact specifically with the epidermal growth factor (EGF)-receptor kinase. An antibody directed against a synthetic peptide corresponding to the major phosphorylation site of pp60src interacts specifically with EGF receptor and immunoprecipitates a functional EGF-receptor kinase. The second antibody, which binds close to a region on the src molecule that is required for its kinase activity, also binds to EGF-receptor kinase and prevents the autophosphorylation of the receptor molecules. Neither antibody binds to intact cells, but they do recognize various forms of the solubilized receptor. It is concluded that at least two cytoplasmic domains of the EGF receptor are antigenically and presumably also structurally related to specific domains on pp60src.

Amino Acid Sequence↗
PubMed1984

Biologically active synthetic fragments of epidermal growth factor: localization of a major receptor-binding region.

A primary receptor-binding region of mouse epidermal growth factor (EGF) was identified by comparing the relative affinities of selected synthetic fragments with overlapping sequences in the EGF receptor-binding assay, using human foreskin fibroblasts. Only synthetic peptides containing the amino acid residues 20-31 in the mouse EGF sequence showed the ability to compete with 125I-labeled EGF in binding to EGF receptors. The affinities of the cyclic EGF fragment [Ala20]EGF-(14-31) and the linear [(S-acetamidomethyl)-Cys20,31]-EGF-(20-31) were approximately 1/10(4) of the affinity of EGF. Despite their reduced receptor affinities, these two peptides exhibited the in vitro biological activities of native EGF, while fragments from other regions of the EGF molecule were devoid of these biological properties. The peptides induced DNA synthesis in human foreskin fibroblasts as measured by [3H]thymidine incorporation into DNA. They also induced EGF receptor clustering and activated the EGF-sensitive kinase, enhancing the autophosphorylation of EGF receptors in a dose-related manner. Moreover, a major antigenic determinant of EGF for rabbit anti-EGF antibodies was identified within this same localized region of the EGF molecule by competition experiments utilizing the synthetic EGF fragments. The predominant EGF antigenic determinant(s) was also found within the fragment [(S-acetamidomethyl)Cys20,31]-EGF-(20-31). The accessibility of the residues in positions 20-31 for antibody recognition is consistent with the conclusion that these residues constitute or contain a major receptor-binding region for EGF.

Binding, Competitive↗
PubMed1984

Interaction between major histocompatibility complex antigens and epidermal growth factor receptors on human cells.

It has been suggested that products of the major histocompatibility complex, the MHC, of vertebrates function in many processes of recognition and ligand binding at the cell surface. Here we show that binding of polyclonal and monoclonal antibodies against human MHC antigens, HLA, reduced the binding of epidermal growth factor (EGF) to its membrane receptors on A-431 tumor cells and on normal human fibroblasts. Binding of EGF at 37 degrees C similarly inhibited the binding of Fab fragments and intact Ig anti-HLA to human cells. The inhibitory effect of anti-HLA antibodies was rapid and dependent upon temperature and antibody concentration and valence. Fluorescence microscopy qualitatively confirmed the binding data and showed that MHC antigens and EGF-receptors do not co-cluster in the membrane.

Animals↗
PubMed1984

Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells.

Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h.

Animals↗
PubMed1984

Incorporation of fluorescent gangliosides into human fibroblasts: mobility, fate, and interaction with fibronectin.

Rhodamine- and fluorescein-labeled gangliosides were used as probes to investigate the distribution, dynamics, and fate of plasma membrane-bound gangliosides on cultured human fibroblasts. When sparse cultures of fibroblasts were incubated with the fluorescent ganglioside derivatives, their surfaces became highly fluorescent. The fluorescent gangliosides were taken up by the cells in a time- and temperature-dependent manner and were not removed from the cell surface by trypsin or serum. Thus, the gangliosides appeared to be stably incorporated into the lipid bilayer of the plasma membrane. Fluorescent photobleaching recovery measurements showed that the inserted gangliosides were free to diffuse in the plane of the membrane with a high diffusion coefficient of approximately 10(-8) cm2/s. When the ganglioside-treated cells were washed and incubated in fresh medium, the surface gangliosides became internalized with time, and localized in the perinuclear region of the fibroblasts. In dense cultures of fibroblasts, a large fraction of the fluorescent gangliosides were organized in a fibrillar network and were immobile on the time scale of fluorescent photobleaching recovery measurements. Using antifibronectin antibodies and indirect immunofluorescence, these gangliosides were found to co-distribute with fibrillar fibronectin. Thus, exogenous gangliosides appear to be stably inserted into the lipid bilayer of the plasma membrane and to diffuse freely in its plane as well as form a less mobile state with the fibrillar networks of fibronectin associated with the cells.

Antibodies↗
PubMed1984

Expression of epidermal growth factor receptors in human brain tumors.

The expression of receptors for epidermal growth factor (EGF-R) was determined in 29 samples of brain tumors from 22 patients. Primary gliogenous tumors, of various degrees of cancer, five meningiomas, and two neuroblastomas were examined. Tissue samples were frozen in liquid nitrogen immediately after the operation and stored at -70 degrees until use. Cerebral tissue samples from 11 patients who died from diseases not related to the central nervous system served as controls. Immunoprecipitation of functional EGF-R-kinase complexes revealed high levels of EGF-R in all of the brain tumors of nonneuronal origin that were examined. The level of EGF-R varied between tumors from different patients and also between specimens prelevated from different areas of the same tumor. In contrast, the levels of EGF-R from control specimens were invariably low. The biochemical properties of EGF-R in brain tumor specimens were found to be indistinguishable from those of the well-characterized EGF-R from the A-431 cell line, derived from human epidermoid carcinomas. Human brain EGF-R displays a molecular weight of 170,000 by polyacrylamide-sodium dodecyl sulfate gel electrophoresis. It is phosphorylated mainly in tyrosine residues and shows a 2-dimensional phosphopeptide map similar to that obtained with the phosphorylated EGF-R from membranes of A-431 cells. Our observations suggest that induction of EGF-R expression may accompany the malignant transformation of human brain cells of nonneuronal origin.

Adult↗
PubMed1983

Biological role of epidermal growth factor-receptor clustering. Investigation with monoclonal anti-receptor antibodies.

Monoclonal anti-epidermal growth factor (EGF) receptor antibodies were used as a diagnostic tool for the investigation of the role of the receptor molecule in the transduction of the biological effects mediated by EGF. The specificity of the antibodies was established by immunoprecipitation of the receptor from biosynthetically labeled cells. The previously described (Schreiber, A. B., Lax, I., Yarden, Y., Eshhar, Z., and Schlessinger, J. (1981) Proc. Natl. Acad. Sci U. S. A. 78, 7535-7539) 2F2-IgM antibody binds to or close to the binding site of the growth factor on the receptor molecule; it induces receptor clustering and internalization and triggers early and delayed effects of EGF. A monovalent Fab fragment of the 2G2-IgM antibody stimulates the EGF-receptor sensitive protein kinase, but does not induce receptor clustering and DNA synthesis. When cell-bound 2G2-Fab fragments are cross-linked with anti-mouse Ig antibodies, both receptor clustering and mitogenic activity are restored. A second monoclonal antibody against EGF-receptor denoted TL5-IgG does not interfere with the binding of EGF to the receptor, fails to induce receptor clustering, and does not possess any intrinsic bioactivity. The cross-linking of cell-bound TL5-IgG with anti-mouse Ig antibodies leads to the stimulation of DNA synthesis. It is concluded that the EGF-receptor, when properly triggered, contains all the biochemical attributes necessary for the initiation of biological effects. Receptor clustering, even when mediated via domains distinct from the hormone binding site on the receptor molecule, appears as a necessary and sufficient signal for the induction of DNA synthesis.

Animals↗
PubMed1983

The dynamic interrelationships of actin and vinculin in cultured cells.

The dynamic state of cytoskeletal proteins actin and vinculin was studied in living cells using microinjection of fluorescently-labeled proteins combined with fluorescence photobleaching recovery (FPR). It is shown that both proteins maintain a dynamic equilibrium between their diffusible pools in the cytoplasms and their "organized" cytoskeletal fraction. These interrelationships could be simulated in model systems consisting of isolated substrate attached membranes. It was demonstrated that fluorophore bound vinculin was incorporated into the exposed focal contacts and that this binding was largely actin independent. These results are in line with the hypothesis that local contacts induce binding of vinculin to the endofacial surface of the membranes and that this region serves as a nucleation center for the assembly of actin bundles.

Actins↗
PubMed1983

Appearance of functional EGF receptor kinase during rodent embryogenesis.

Mouse and rat embryonic tissues at various stages of development were examined for epidermal growth factor (EGF) receptor kinase activity. The phosphorylated EGF receptor from embryonic tissues appeared as a band of mol. wt. 170 000 daltons on SDS gels. It was clearly demonstrable in the developing mouse fetus from 10 days of gestation onwards. The distribution of the EGF receptor kinase was studied in various tissues of 13 day mouse fetuses. The activity was apparent in the skin, developing skeletal muscles and various internal organs but was notably absent in the liver and brain. The amnion was found to be one of the richest sources of activity while the yolk sac was negative, and the placenta was weakly positive. In 16 day rat fetuses the distribution was quite similar to that of the 13 day mouse fetus. The liver acquired EGF receptor kinase activity by 18 days of gestation and had high activity in neonates. Phosphoamino acid analysis revealed that phosphotyrosine was the major labelled amino acid residue in the embryonic tissues. Thus, the EGF receptor of fetal tissues as studied by immune precipitation and phosphorylation appears to be a similar entity to that found in adult mammalian tissues. This functional EGF receptor kinase activity could first be detected at the time of onset of organogenesis.

Age Factors↗
PubMed1983

Monoclonal antibody reactive with the human epidermal-growth-factor receptor recognizes the blood-group-A antigen.

The hybridoma antibody TL5, which precipitates the EGF receptor from the human epidermoid carcinoma cell line A431, has been shown to recognize the blood-group-A carbohydrate structure. This conclusion has been reached from studies of (a) the binding of the antibody to glycoproteins and haemagglutination of erythrocytes with known blood-group-antigen activities and (b) the inhibition of binding of the antibody to a radiolabelled blood-group-A-active glycoprotein by structurally defined oligosaccharides.

ABO Blood-Group System↗
PubMed1983

Monoclonal antibodies against epidermal growth factor receptor induce prolactin synthesis in cultured rat pituitary cells (GH3).

The addition of epidermal growth factor (EGF) to cultured rat pituitary cells (GH3) leads to increased synthesis of prolactin and to partial inhibition of cell proliferation. Monoclonal antibodies generated against EGF receptor from human epidermoid carcinoma (A-431) cells were used to characterize the EGF receptor kinase system of GH3 cells and to investigate the role of the hormone-receptor complex in the expression of the prolactin gene in these cells. The EGF receptor of GH3 cells is a 170,000-dalton protein associated with a protein kinase. It is similar but not identical to the EGF receptor identified in other tissues. The immunoprecipitated membrane receptor is phosphorylated on both serine and tyrosine residues. The monoclonal antibody denoted 2G2-IgM binds to EGF receptor on GH3 cells. Like EGF, the monoclonal antibody induced the synthesis of prolactin and morphological changes in these cells. Hence, EGF receptor in GH3 cells, when properly triggered, contains all of the biological attributes necessary for the induction of EGF-induced gene expression and morphological changes in GH3 cells.

Animals↗