Biomedical subjects

A Ullrich

Publications and source records attributed to A Ullrich.

At least 343 records · Page 19Linked to original sources

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

Primary structure and mRNA localization of protein F1, a growth-related protein kinase C substrate associated with synaptic plasticity.

Protein F1 is a neuron-specific, synaptic-enriched, membrane-bound substrate of protein kinase C (PKC) whose phosphorylation is related to synaptic plasticity in the adult. The sequence of 26 N-terminal amino acids was determined from purified rat protein F1. A 78-mer synthetic oligonucleotide designed from the partial N-terminal sequence enabled identification of protein F1 cDNA clones in a rat brain library. F1 protein is a 226 amino acid protein encoded by a 1.5 kb brain-specific, developmentally-regulated mRNA. Transcripts for protein F1 can be detected at birth, and their level declines after maturation. A full-length cDNA clone was transcribed and translated in vitro. Translation products could be immunoprecipitated with anti-F1 antibodies. In situ hybridization analysis revealed protein F1 transcripts in hippocampal pyramidal cells, but not in granule cells. In cerebellum, granule cells contained protein F1 mRNA, while Purkinje cells did not. Co-localization of protein F1 with protein kinase C-II [PKC-II (beta)], rather than PKC-I (gamma) suggests that PKC-II may phosphorylate protein F1.

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

Characterization of an endogenous substrate of the insulin receptor in cultured cells.

Using antiphosphotyrosine antibodies, we have characterized the tyrosine phosphorylation of an endogenous substrate of the insulin receptor in Fao hepatoma cells and in Chinese hamster ovary cells transfected with a eukaryotic expression vector containing the human insulin receptor cDNA. In Fao cells, besides the beta-subunit of the insulin receptor, a protein with a molecular mass between 170 and 210 kDa designated pp185, undergoes tyrosine phosphorylation immediately after insulin stimulation reaching a maximum level within 30 s. After 4 h of continuous insulin stimulation, the labeling of pp185 decreased to less than half of its original intensity, whereas the insulin receptor was unchanged. After 24 h of insulin stimulation, the phosphotyrosine-containing insulin receptor decreased by 75% owing to down-regulation, whereas the pp185 was completely undetectable. By several biochemical and physiological criteria, the pp185 is distinct from the insulin receptor. The pp185 and the beta-subunit of the insulin receptor were strongly labeled with [32P]orthophosphate, but in contrast to the insulin receptor, the pp185 was not labeled by cross-linking with 125I-insulin or surface 125I iodination. Unlike the insulin receptor, the pp185 was extracted from Fao cells without detergent, and tryptic phosphopeptide mapping of the pp185 and the insulin receptor yielded distinct patterns. Thus, the pp185 is not located at the external face of the plasma membrane and does not bind insulin. Treatment of Fao cells with the phorbol ester, phorbol 12-myristate 13-acetate, stimulated the phosphorylation of two proteins with molecular weights of 170 and 210 kDa which were immunoprecipitated with the anti-phosphotyrosine antibody. Subsequent insulin stimulation increased the phosphorylation of the 210 kDa protein, but the pp185 was not detected. Increasing the concentration of the human insulin receptor in the Chinese hamster ovary cells by transfection with a plasmid containing the human insulin receptor cDNA caused a higher level of tyrosine phosphorylation of the beta-subunit and the pp185. These data support the notion that the insulin signal may be transmitted to a cellular substrate (pp185) which may initiate insulin action at intracellular sites.

Animals↗

PubMed1987

Down-regulation of protein kinase C is due to an increased rate of degradation.

The phorbol-ester-induced loss of protein kinase C that has been documented in many cell types appears to be a critical event in the generation of a cellular refractory state. We have investigated here the synthesis and degradation of the protein kinase C polypeptide in order to determine why its steady-state amounts are depleted in response to phorbol esters. These results indicate that depletion is due to an increased rate of degradation, with no change either in mRNA amounts or in rates of polypeptide synthesis.

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

Human insulin receptors mutated at the ATP-binding site lack protein tyrosine kinase activity and fail to mediate postreceptor effects of insulin.

Transfected Chinese hamster ovary cell lines were developed that expressed equivalent numbers of either normal human receptor or receptor that had alanine substituted for Lys-1018 in the ATP-binding domain of the beta subunit. The mutated receptor was processed into subunits and bound insulin but lacked protein tyrosine kinase activity. Five effects of insulin were assayed: deoxyglucose uptake, S6 kinase activity, endogenous protein-tyrosine phosphorylation, glycogen synthesis, and thymidine uptake. In each case, cells bearing normal human receptors were 10-100-fold more sensitive to insulin than the parental cells. Cells with the mutant receptor behaved like the parental cells with respect to S6 kinase activation, endogenous substrate phosphorylation, glycogen synthesis, and thymidine uptake, but their deoxyglucose uptake was significantly depressed and relatively insensitive to insulin. The analyses led to the following conclusions: substitution of alanine for lysine at amino acid 1018 inactivates the kinase activity of the receptor; a kinase-negative receptor can be properly processed and bind insulin; insulin-dependent deoxyglucose uptake, S6 kinase activation, endogenous substrate phosphorylation, glycogen synthesis, and thymidine incorporation into DNA are mediated by the normal but not by the kinase-deficient human receptor.

Adenosine Triphosphate↗

PubMed1987

Synthesis of messenger RNAs for transforming growth factors alpha and beta and the epidermal growth factor receptor by human tumors.

Human tumors and tumor cell lines were analyzed for the presence of mRNA coding for transforming growth factors alpha and beta (TGF-alpha and -beta) and the epidermal growth factor receptor. TGF-alpha mRNA was not detectable in hematopoietic tumor cell lines but was found in a variety of solid tumor cells, particularly carcinomas. Many of the tumors that contained TGF-alpha mRNA also expressed high levels of epidermal growth factor receptor mRNA. The concentration of TGF-alpha in the media of several tumor cell lines did not necessarily correlate with TGF-alpha mRNA levels, as a substantial fraction of TGF-alpha can remain cell associated. The levels of TGF-beta mRNA in tumor cell lines and tumor specimens were variable, but higher in tumors than in the adjacent normal tissues.

Cell Line↗

PubMed1987

Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.

The HER-2/neu oncogene is a member of the erbB-like oncogene family, and is related to, but distinct from, the epidermal growth factor receptor. This gene has been shown to be amplified in human breast cancer cell lines. In the current study, alterations of the gene in 189 primary human breast cancers were investigated. HER-2/neu was found to be amplified from 2- to greater than 20-fold in 30% of the tumors. Correlation of gene amplification with several disease parameters was evaluated. Amplification of the HER-2/neu gene was a significant predictor of both overall survival and time to relapse in patients with breast cancer. It retained its significance even when adjustments were made for other known prognostic factors. Moreover, HER-2/neu amplification had greater prognostic value than most currently used prognostic factors, including hormonal-receptor status, in lymph node-positive disease. These data indicate that this gene may play a role in the biologic behavior and/or pathogenesis of human breast cancer.

Axilla↗

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

Structure and nucleotide sequence of a Drosophila melanogaster protein kinase C gene.

Genomic and cDNA clones encoding a Drosophila melanogaster protein kinase C (PKC) homologue were identified using a bovine PKC cDNA probe. The cDNA clones contain a single open reading frame that encodes a 639 amino acid, 75-kd protein having extensive homology with bovine, human and rat PKC and homology with the kinase domains of other serine, threonine and tyrosine kinases. The Drosophila PKC gene is localized to region 53E of chromosome 2. The gene spans approximately 20 kb and contains at least 14 exons. Messenger RNA for PKC could not be detected in 0-3 h Drosophila embryos. Adult flies contain three PKC transcripts of 4.3, 4.0 and 2.4 kb.

Amino Acid Sequence↗

PubMed1987

Protein kinase C.

The combined application of protein structural techniques, immunochemistry, and molecular biology has permitted an analysis of the differential expression of the genes for the three related protein kinases, C-alpha, -beta, and gamma. The evidence now suggests that the type of protein kinase C in a cell may govern the nature and mechanisms of functional responses.

Amino Acid Sequence↗

PubMed1987

Short-latency somatosensory evoked potentials in brain death.

Simultaneous recording of somatosensory evoked potentials to median nerve stimulation above the upper and lower neck in brain-dead patients revealed that all cervical responses were preserved in 10%, whereas a marked reduction in amplitude or even loss of N 13b at the level of the C2 spinous process was observed in 90%. Of the patients, 55% revealed an additional loss of N 13a, recorded at the level of the C7 spinous process; in 15% all cortical and spinal evoked potentials were missing, but Erb's point waves were still normal. These results suggest two different origins of the main negative waves (N 13a and N 13b), recorded above the upper and lower cervical spinal cord. N 13a (C7) is supposed to arise in the dorsal horn at the C6/7 level, N 13b (C2) in the cervicomedullary junction.

Adolescent↗

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

Tissue-specific and developmentally regulated transcription of the insulin-like growth factor 2 gene.

Transcription of the rat and human IGF-2 gene loci is unusually complex. The pattern of expression of these genes varies both between tissues and within a given tissue during different stages of development. Alternative splicing or possibly transcriptional initiation events generate variant IGF-2 mRNAs that contain different 5'-untranslated leader sequences. These leader exon sequences are shared with non-IGF-2 mRNAs. Certain noncoding IGF-2 gene sequence elements are transcribed extensively and are found in multiple copies elsewhere in the human genome. Furthermore, IGF-2 mRNA levels are particularly high in a variety of human malignancies.

Age Factors↗