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Biomedical subjects

A Ullrich

Publications and source records attributed to A Ullrich.

At least 325 records · Page 18Linked to original sources
PubMed1988

Biological activities of EGF-receptor mutants with individually altered autophosphorylation sites.

In vitro site-directed mutagenesis was used to replace individually the three known autophosphorylation sites of the epidermal growth factor (EGF)-receptor (i.e. Tyr1173, Tyr1148 and Tyr1068) by phenylalanine, a residue which cannot serve as a phosphate acceptor site. In another mutant, Tyr1173 was substituted by a serine residue. The cDNA constructs encoding either mutant or wild-type EGF-receptors were transfected into NIH-3T3 cells devoid of endogenous EGF-receptors. The mutant receptors were expressed on the cell surface and displayed typical high- and low-affinity binding sites for [125I]EGF. Phorbol ester (PMA) modulated the binding affinity of wild-type and mutant receptors in a similar manner. Mutant EGF-receptors exhibited EGF-dependent tyrosine kinase activity leading to self-phosphorylation and phosphorylation of exogenous substrates both in vitro and in living cells. The internalization and degradation of EGF-receptors were not affected by the mutations. Cells expressing mutant EGF-receptors became mitogenically responsive to EGF, indicating that none of the vital functions of the EGF-receptor were critically impaired by the loss of individual autophosphorylation sites. Maximal mitogenic stimulation correlated with the number of wild-type or mutant receptors per cell, highly expressing cells showing higher maximal stimulation. However, the dose-response curves of cells expressing mutant receptors were slightly shifted to lower concentrations of EGF, rendering the cells mitogenically responsive to lower doses of EGF than cells expressing normal EGF-receptor at similar expression levels. Basal [3H]thymidine incorporation in the presence of 0.5% calf serum was consistently higher for cells expressing mutant receptors, while the response to stimulation with 10% calf serum was not affected.

Animals↗
PubMed1988

Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites.

Epidermal growth factor (EGF)-receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH-3T3 cells lacking endogenous EGF-receptors. Kinetic parameters of the kinase of wild-type and mutant receptors were compared. Both wild-type and mutant EGF-receptors had a Km(ATP) 1-3 microM for the autophosphorylation reaction, and a Km(ATP) of 3-7 microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF-receptor of A431 cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr1173 of the EGF-receptor (KGSTAENAEYLRV) was phosphorylated by wild-type receptor with a Km of 110-130 microM, and the peptide inhibited autophosphorylation with a Ki of 150 microM. Mutant EGF-receptors phosphorylated the peptide substrate with a Km of 70-100 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates angiotensin II and a peptide derived from c-src. The Km of angiotensin II phosphorylation was reduced from 1100 microM for wild-type receptor to 890 microM for mutant receptor and for c-src peptide from 1010 microM to 770 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr1173 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650-750 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1988

Truncation of the human EGF receptor leads to differential transforming potentials in primary avian fibroblasts and erythroblasts.

The transforming capacity of the normal and mutant human EGF receptor (EGFR) was investigated in primary chicken cells. In fibroblasts, both N- and C-terminal truncations resulted in a weak, additive oncogenic activity. However, not even double truncations caused a v-erbB-like phenotype. Upon EGF-binding, on the other hand, both normal and C-terminally truncated EGFRs resembled v-erbB in their fibroblast transforming potential. In erythroblasts, N-terminal truncation was sufficient to induce constitutive self-renewal, which was enhanced by deletion of 32 C-terminal amino acids but abolished by a larger truncation of 202 amino acids. In contrast to the normal EGFR, the receptor lacking 32 C-terminal amino acids resembled v-erbB in conferring erythropoietin independence for spontaneous differentiation to the transformed erythroblasts. Our results indicate that the C-terminal domain of the EGFR is non-essential in fibroblast transformation, but seems to be crucial for both self renewal induction and specificity of receptor function in erythroblasts.

Animals↗
PubMed1988

Chromosomal mapping of the gene for the type II insulin-like growth factor receptor/cation-independent mannose 6-phosphate receptor in man and mouse.

The gene for insulin-like growth factor II (IGF-II) receptor (IGF2R) that has recently been found, by DNA sequencing, to be identical to the cation-independent mannose 6-phosphate receptor (CIM6PR) has been mapped in the human and murine species. Cloned cDNAs for human and rat IGF-II receptors were used to probe Southern blots of somatic cell hybrid DNA and for in situ chromosomal hybridization. The genes are located in a region of other conserved syntenic genes on the long arm of human chromosome 6, region 6q25----q27, and mouse chromosome 17, region A-C. The CIM6PR/IGF2R locus in man is asyntenic with the genes encoding IGF-II (IGF2), the IGF-I receptor (IGF1R), and the cation-dependent mannose 6-phosphate receptor (CDM6PR).

Animals↗
PubMed1988

Amplified expression of the HER2/ERBB2 oncogene induces resistance to tumor necrosis factor alpha in NIH 3T3 cells.

Functional characterization of oncogene products that induce cellular transformation has progressed rapidly in recent years. However, less is known about the mechanism(s) by which the transformed cells may escape destruction by host immune defenses and form tumors. A recently described oncogene that has an important association with aggressive human breast carcinoma is "HER2," for human epidermal growth factor receptor 2. The oncogene has also been called NGL and human c-erbB-2 (ERBB2). In this paper we show that amplification of HER2 oncogene expression can induce resistance of NIH 3T3 cells to the cytotoxic effects of recombinant tumor necrosis factor alpha (rTNF-alpha) or macrophages. Resistance is accompanied by an increased dissociation constant for rTNF-alpha binding to high-affinity receptors on the HER2-transformed NIH 3T3 cells. The resistance phenotype is independent of transformation since NIH 3T3 cells transformed by the activated human homologue of the Harvey-ras oncogene (HRAS) retain high-affinity binding sites for rTNF-alpha as well as sensitivity to its cytotoxic effects. These results suggest that HER2 may potentiate tumorigenesis by inducing tumor cell resistance to host defense mechanisms.

Animals↗
PubMed1988

Ligand-induced stimulation of epidermal growth factor receptor mutants with altered transmembrane regions.

The epidermal growth factor (EGF) receptor is a transmembrane glycoprotein composed of a large extracellular ligand-binding region connected to the cytoplasmic kinase domain by a single transmembrane (TM) region. To explore the role of the TM region in the process of receptor activation, we have generated EGF-receptor mutants with altered TM regions by utilizing in vitro site-directed mutagenesis. The TM regions of two mutant receptors were either extended (designated i626-3) or shortened (designated d625.3) by three hydrophobic amino acid residues. In the other two mutant receptors, hydrophobic amino acids were substituted by charged residues--i.e., Val-627 was replaced by glutamic acid (designated V627E) or Leu-642 was replaced by an arginine residue (designated L642R). NIH 3T3 cells lacking endogenous EGF receptors were transfected with constructs encoding either wild-type or mutant receptors and shown to express the receptor molecules using 125I-labeled EGF binding and immunoprecipitation experiments. The mutant receptors were expressed on the cell surface as polypeptides of Mr 170,000 exhibiting typical high- and low-affinity binding sites for 125I-labeled EGF. Similar to its effect on wild-type receptors, phorbol 12-myristate 13-acetate abolished the mutant-receptor high-affinity binding sites for EGF. Moreover, EGF was able to stimulate the kinase activities of wild-type and mutant receptors both in vitro and in living cells. The mutant receptors were also able to undergo EGF-induced receptor dimerization as revealed by cross-linking experiments with a bifunctional covalent cross-linking agent. These results are compatible with an intermolecular allosteric oligomerization model for receptor activation rather than with a model based on an intramolecular mechanism for receptor activation.

Animals↗
PubMed1988

Ligand activation of overexpressed epidermal growth factor receptors transforms NIH 3T3 mouse fibroblasts.

The cell surface receptor for the mitogenic peptide epidermal growth factor (EGF) is involved in control of normal cell growth and may play a role in the genesis of human neoplasia such as squamous carcinoma and glioblastoma. Soft-agar growth and focus-formation experiments with NIH 3T3 mouse fibroblasts transfected with an expression plasmid demonstrated the ligand-dependent transforming potential of the human EGF receptor without structural alterations. Activation of overexpressed normal receptor alone appears to be sufficient for transformation of NIH 3T3 cells in vitro.

Animals↗
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↗
PubMed1988

Endocytotic uptake, processing, and retroendocytosis of human biosynthetic proinsulin by rat fibroblasts transfected with the human insulin receptor gene.

The cellular itinerary and processing of insulin and proinsulin were studied to elucidate possible mechanisms for the observed in vivo differences in the biologic half-lives of these two hormones. A rat fibroblast cell line transfected with a normal human insulin receptor gene was used. Due to gene amplification, the cells express large numbers of receptors and are ideal for studying a ligand, such as proinsulin, that has a low affinity for the insulin receptor. Competitive binding at 4 degrees C showed that the concentration of unlabeled insulin and proinsulin that is needed to displace 50% of tracer insulin or proinsulin was 0.85-0.95 nM and 140-150 nM, respectively. Binding to surface receptors and internalization occur at rates that are four to five times faster in cells incubated with insulin compared with proinsulin. Chloroquine led to an increase in cell-associated radioactivity of approximately 1.4-fold in cells incubated with insulin or proinsulin, but inhibited the appearance of degraded insulin by 54% and degraded proinsulin by only 10%. To study the fate of internalized ligand, cells were incubated with insulin and proinsulin until steady state binding occurred. Surface bound ligand was removed by an acid wash and the remaining cell-associated radioactivity represented internalized ligand. Cells were then reincubated in 37 degrees C buffer and the cell-associated radioactivity and radioactivity released into the medium were analyzed by TCA precipitation, Sephadex G-50, and HPLC. The results demonstrated that proinsulin more readily bypasses the intracellular degradative machinery and is therefore released intact from the cell via the retroendocytotic pathway. These results may help to explain the prolonged metabolic clearance rate and biologic responsiveness of proinsulin in vivo.

Animals↗
PubMed1988

Growth hormone stimulates insulin gene expression in cultured human fetal pancreatic islets.

The total body mass of the human fetus increases about 100-fold from 10-20 weeks of gestation, and peak serum GH concentrations occur at 20 weeks. Since insulin has an essential growth-promoting influence in the fetus, these experiments were designed to determine whether GH can function as a growth factor with insulin-releasing activity by stimulating insulin gene expression during embryogenesis. Pancreatic islets were isolated from human fetuses (n = 36) of 18-22 weeks gestational age. Insulin gene expression was quantified by measuring insulin mRNA by blot hybridization analysis using a [32P] cDNA probe encoding human insulin. We found that by 48 h of culture insulin gene expression was stimulated by 1.25 micrograms recombinant human GH/mL medium to 216% of the control value (n = 2). Insulin secretory capacity was expressed as a fractional stimulation ratio (FSR = F2/F1) of insulin release rates during two successive 1-h static incubations. After 48 h of culture 1.25 micrograms/mL GH stimulated the FSR value to 273% of the control value (n = 5). We conclude that recombinant human GH significantly enhances the steady state level of insulin mRNA concurrent with an increase in insulin secretory capacity, hence providing evidence for a regulatory function of GH on insulin gene expression during fetal development.

Gene Expression Regulation↗
PubMed1988

Defects in human insulin receptor gene expression.

The insulin receptor plays a central role in mediating the biological actions of insulin. We have used Epstein-Barr virus-transformed lymphocytes (EBV-lymphocytes) to investigate the receptor defects in patients with genetic forms of insulin resistance. Within the normal population, we found a close correlation between the number of insulin receptors on the surface of EBV-lymphocytes and the cellular content of insulin receptor mRNA. In addition, we have used the cloned human insulin receptor cDNA to investigate the nature of the mutations causing the reduction in the number of insulin receptors in EBV-lymphocytes from three insulin resistant patients. One patient with leprechaunism has a marked reduction in the level of receptor mRNA, which probably accounts for the extremely slow rate of receptor biosynthesis measured in this patient's cells. The remaining two patients with type A extreme insulin resistance are sisters, the products of a consanguineous marriage, who have normal levels of insulin receptor mRNA. We have previously shown that the insulin receptor precursor is synthesized at a normal rate in these patients' cells, thus suggesting a defect in the posttranslational processing of the receptor or in its translocation to the plasma membrane.

Gene Expression Regulation↗
PubMed1988

Restriction-fragment-length polymorphism in insulin-receptor gene and insulin resistance in NIDDM.

Restriction-enzyme analysis of genomic DNA from 52 White and Hispanic nondiabetic subjects and 51 subjects with non-insulin-dependent diabetes (NIDDM) was carried out with insulin-receptor cDNA probes. A polymorphic 5.8-kilobase SstI fragment was found in 12 (23.5%) of 51 NIDDM subjects but only in 4 (7.7%) of 52 nondiabetic control subjects. This association is significant by chi 2-analysis (P less than .05). Furthermore, the nondiabetic subjects with the polymorphism were found to have hyperinsulinemia and/or nondiagnostic glucose tolerance. The polymorphism is a genetic marker for a phenotype that is neither necessary nor, by itself, sufficient for NIDDM. Nevertheless, it may indicate that insulin resistance functionally related to an insulin-receptor gene polymorphism is the proximal cause of NIDDM in at least one subset of the population.

Aged↗
PubMed1988

Abrogation of the requirements for added growth factors in 3T3 cells constitutively expressing the p53 and IGF-1 genes.

Two constructs (co-transfected with selectable markers) were used to establish cell lines from BALB/c3T3 cells: a p53 mini-gene under the control of an LTR promoter and a synthetic IGF-1 coding sequence driven by the SV40 early promoter. BALB/c3T3 cells do not grow in plasma or in 1% serum or in soft agar and in defined media require both platelet-derived growth factor (PDGF) and insulin (or IGF-1). Cells carrying only the LTR/p53 mini-gene grew well in plasma (but not in 1% serum), in soft agar and in serum-free medium containing only insulin. Cells carrying only the SV40/IGF-1 gene grew (but not too vigorously) in soft agar and grew well in serum-free medium containing PDGF but no insulin. Cells carrying both constructs grew very well in plasma, in soft agar and in serum-free medium without PDGF nor insulin. The results indicate that the requirements for growth of BALB/c3T3 cells can be reduced to two genes: IGF-1 and a gene replacing PDGF (p53 in our case). An unexpected, but interesting observation was that cells carrying the LTR/p53 gene grew slower in 10% serum than in plasma.

Animals↗