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Biomedical subjects

A Ullrich

Publications and source records attributed to A Ullrich.

At least 271 records · Page 15Linked to original sources
PubMed1990

Receptor-mediated internalization of insulin requires a 12-amino acid sequence in the juxtamembrane region of the insulin receptor beta-subunit.

The juxtamembrane region of the insulin receptor (IR) beta-subunit contains an unphosphorylated tyrosyl residue (Tyr960) that is essential for insulin-stimulated tyrosyl phosphorylation of some endogenous substrates and certain biological responses (White, M.F., Livingston, J.N., Backer, J.M., Lauris, V., Dull, T.J., Ullrich, A., and Kahn, C.R. (1988) Cell 54, 641-649). Tyrosyl residues in the juxtamembrane region of some plasma membrane receptors have been shown to be required for their internalization. In addition, a juxtamembrane tyrosine in the context of the sequence NPXY [corrected] is required for the coated pit-mediated internalization of the low density lipoprotein receptor. To examine the role of the juxtamembrane region of the insulin receptor during receptor-mediated endocytosis, we have studied the internalization of insulin by Chinese hamster ovary (CHO) cells expressing two mutant receptors: IRF960, in which Tyr960 has been substituted with phenylalanine, and IR delta 960, in which 12 amino acids (Ala954-Asp965), including the putative consensus sequence NPXY [corrected], were deleted. Although the in vivo autophosphorylation of IRF960 and IR delta 960 was similar to wild type, neither mutant could phosphorylate the endogenous substrate pp185. CHO/IRF960 cells internalized insulin normally whereas the intracellular accumulation of insulin by CHO/IR delta 960 cells was 20-30% of wild-type. However, insulin internalization in the CHO/IR delta 960 cells was consistently more rapid than that occurring in CHO cells expressing kinase-deficient receptors (CHO/IRA1018). The degradation of insulin was equally impaired in CHO/IR delta 960 and CHO/IRA1018 cells. These data show that the juxtamembrane region of the insulin receptor contains residues essential for insulin-stimulated internalization and suggest that the sequence NPXY [corrected] may play a general role in directing the internalization of cell surface receptors.

Amino Acid Sequence↗
PubMed1990

Receptors for insulin and insulin-like growth factor-I can form hybrid dimers. Characterisation of hybrid receptors in transfected cells.

We have demonstrated the formation of hybrid insulin/insulin-like growth factor-I(IGF-I) receptors in transfected rodent fibroblasts, which overexpress human receptors, by examining reactivity with species- and receptor-specific monoclonal antibodies. In NIH 3T3 and Rat 1 fibroblasts, endogenous IGF-I receptors were unreactive with anti-(human insulin receptor)monoclonal antibodies (47-9, 25-49, 83-14, 83-7, 18-44). However, in transfected cells expressing high levels of insulin receptors, 60-80% of high-affinity IGF-I receptors reacted with these antibodies, as assessed either by inhibition of ligand binding in intact cells or by precipitation of solubilized receptors. Conversely, endogenous insulin receptors in NIH 3T3 cells were unreactive with anti-(IGF-I receptor) antibodies alpha IR-3 and 16-13. However, approx. 50% of high-affinity insulin receptors reacted with these antibodies in cells expressing high levels of human IGF-I receptors. The hybrid receptors in transfected cells bound insulin or IGF-I with high affinity. However, responses to these ligands were asymmetrical, in that binding of IGF-I inhibited subsequent binding of insulin, but prior binding of insulin did not affect the affinity for IGF-I. The existence of hybrid receptors in normal tissues could have important implications for metabolic regulation by insulin and IGF-I.

Animals↗
PubMed1990

Overexpression of protein kinase C alpha-subtype in Swiss/3T3 fibroblasts causes loss of both high and low affinity receptor numbers for epidermal growth factor.

The full length cDNA coding for the alpha form of protein kinase C (PKC) was introduced into Swiss/3T3 cells using a retroviral expression system. This has enabled the generation of a series of cell lines stably expressing high levels of PKC alpha enzyme, as well as the appropriate control cell lines, carrying an integrated vector but lacking PKC alpha cDNA insert. PKC alpha-overexpressing cell lines did not display a transformed morphology nor were they capable of growth in soft agar. However, these cells exhibited enhanced growth rates especially under low serum conditions. Using Scatchard plot analysis of 125I-epidermal growth factor (EGF) binding to its receptor on the surface of PKC alpha-overproducing cells revealed reduction in EGF receptor numbers when compared to control cells, without change in affinities of remaining receptors. These data indicated that reduced receptor numbers for EGF were among the phenotypic changes occurring in these cells underlying their diminished dependence on external factors for growth. Furthermore, we provide evidence that reduced EGF receptor numbers found on the cell surface of PKC alpha-overproducing cells resulted from the decreased biosynthesis of EGF receptor molecules, which correlated also with lower levels of mRNA transcripts coding for the EGF receptor found in these cells. Hence, our studies imply that PKC alpha is involved in a cellular mechanism regulating the expression of EGF receptor molecules in Swiss/3T3 cells. Thus, deregulation of PKC alpha, i.e. by increasing its expression levels in specific cells may affect, in turn the expression of cell surface receptors including the EGF receptor. Similar molecular mechanisms may be involved in initial stages of neoplasia and tumor promotion.

Animals↗
PubMed1990

Characterization of an insulin receptor mutant lacking the subunit processing site.

An insulin receptor mutant was constructed utilizing site-directed mutagenesis to delete the Arg-Lys-Arg-Arg basic amino acid cleavage site (positions 720-723) from the cDNA encoding the human insulin proreceptor. This mutant was transfected into Chinese hamster ovary cells. Immunoprecipitation of metabolically labeled cells revealed a 205-kDa proreceptor which bound to wheat germ agglutinin. Processed 130-kDa alpha and 95-kDa beta subunits were also observed and contained approximately 20% as much protein as the proreceptor on a molar basis. Trypsin digestion of intact metabolically labeled cells decreased the proreceptor band by 80%. Pulse-chase studies revealed a half-life of 28 h for the proreceptor. When cells were photolabeled with 125I-B2(2-nitro-4-azidophenylacetyl)-des-PheB1 (NAPA)-insulin, the proreceptor incorporated 10% as much label as the 130-kDa alpha subunit in spite of a 5-fold molar excess. Incubation of NAPA-labeled cells at 37 degrees C for 20 min resulted in 60% of the labeled subunits, but little labeled proreceptor, becoming resistant to trypsin degradation. Immunoprecipitation of NAPA-insulin-stimulated cells with anti-phosphotyrosine antibodies revealed that 62% of the processed labeled receptors, but very little proreceptor, contained phosphotyrosine. Thus, this mutant receptor is synthesized, glycosylated, and expressed on the cell surface as uncleaved proreceptor, although some processing to alpha and beta subunits still occurs. It exhibits a markedly decreased affinity for insulin, and when insulin is bound to, demonstrates defective internalization, down-regulation, and autophosphorylation. These data suggest that cleavage of the mutant proreceptor into subunits is required not only for the development of high affinity binding sites, but also for normal transduction of the signal which activates the beta subunit tyrosine kinase.

Affinity Labels↗
PubMed1990

Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body.

We compared the internalization and intracellular sorting of epidermal growth factor receptor (EGF-R) and point mutant kinase-negative EGF-R separately expressed in NIH 3T3 cells lacking endogenous receptor. Both EGF-Rs internalized rapidly, but kinase-negative receptor was surface down-regulated only with monensin or at 20 degrees C. Furthermore, EGF internalized by mutant receptor alone was, in significant proportion, returned to the cell surface undegraded. Hence unlike wild-type receptor, kinase-negative EGF-R recycles. By electron microscopy the early pathways of endocytosis for the two receptors were identical; however, after 10-20 min the pathways diverged at the multivesicular body (MVB). Wild-type EGF-R, destined for degradation, localized to internal vesicles, while kinase-negative EGF-R, destined for recycling, localized to surface membranes of the MVBs and moved to small tubulovesicles. We conclude that sorting of internalized receptor for degradation or recycling can occur through spatial segregation within the MVB, and sorting of EGF-R is controlled by tyrosine kinase activity.

Animals↗
PubMed1990

Effect of phospholipase C-gamma overexpression on PDGF-induced second messengers and mitogenesis.

Platelet-derived growth factor (PDGF) stimulates phospholipase C (PLC) activity and the phosphorylation of the gamma isozyme of PLC (PLC-gamma) in vitro and in living cells. The role of PLC-gamma in the phosphoinositide signaling pathway was addressed by examining the effect of overexpression of PLC-gamma on cellular responses to PDGF. Overexpression of PLC-gamma correlated with PDGF-induced tyrosine phosphorylation of PLC-gamma and with PDGF-induced breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2). However, neither bradykinin- nor lysophosphatidic acid-induced phosphoinositide metabolism was enhanced in the transfected cells, suggesting that the G protein-coupled phosphoinositide responses to these ligands are mediated by other PLC isozymes. The enhanced PDGF-induced generation of inositol trisphosphate (IP3) did not enhance intracellular calcium signaling or influence PDGF-induced DNA synthesis. Thus, enzymes other than PLC-gamma may limit PDGF-induced calcium signaling and DNA synthesis. Alternatively, PDGF-induced calcium signaling and DNA synthesis may use biochemical pathways other than phosphoinositide metabolism for signal transduction.

Animals↗
PubMed1990

Expression of the p185 encoded by HER2 oncogene in normal and transformed human tissues.

The human homolog of the rat neu oncogene, HER2 (also termed c-erbB2) has been demonstrated in amplified form in human breast tumors with poor prognosis. Although amplification of the gene correlates with expression of a 185-kDa transmembrane glycoprotein, no extensive information is available regarding the extent of tissue and tumor specificity of this gene product. We have addressed this issue by immunohistochemically evaluating the expression of p185 HER2 in normal tissue and various tumors using monoclonal antibodies (MAbs) to distinct epitopes of its extracellular domain. No detectable levels of p185 HER2 were found in fetal tissues analyzed, with the exception of renal tubules in 2 out of 3 specimens tested and in intestinal epithelium. In adult tissues, detectable levels of this glycoprotein were found in a restricted number of cell types, the expression being heterogeneous among individuals and cell histotypes. Among the neoplasms assayed p185 HER2 was expressed in 46% of primary breast cancers, in 28% of ovarian tumors and in 30% of colon rectum malignancies. No male breast adenocarcinomas were p185-positive. A large number of other tumors tested revealed only a low incidence of expression of the p185. In metastatic breast tumors p185 HER2 was demonstrated homogeneously among multiple autologous lesions and almost invariably (80%) the expression of p185 in the primary lesion correlated with that of the deriving metastases. Our findings indicate that the expression of the p185 HER2 represents a tumor marker of clinical relevance in breast cancer. Whether this holds true for other malignancies remains to be explored.

Antibodies, Monoclonal↗
PubMed1990

Characterization of murine monoclonal antibodies reactive to either the human epidermal growth factor receptor or HER2/neu gene product.

High levels of expression of either the epidermal growth factor receptor or the receptor-like HER2/neu gene product p185HER2 have been observed in a variety of human malignancies. Because of the association of this high level expression with certain human tumors, we have generated a panel of monoclonal antibodies specific for either the epidermal growth factor receptor or p185HER2 to study their structure, function, and antigenic domains in the normal and neoplastic states. We used the epidermoid carcinoma line A431 to generate five monoclonal antibodies which immunoprecipitate the epidermal growth factor receptor. These monoclonal antibodies bind to the extracellular domain of the epidermal growth factor receptor and demonstrate variable effects on epidermal growth factor binding. We used a stably transfected NIH 3T3 cell line expressing the HER2/neu gene to produce and characterize 10 monoclonal antibodies which immunoprecipitate p185HER2. These monoclonal antibodies bind to the extracellular domain of p185HER2 and do not cross-react with the epidermal growth factor receptor. The characteristics and potential applications of these monoclonal antibodies will be discussed.

Antibodies, Monoclonal↗
PubMed1990

Functionally distinct insulin receptors generated by tissue-specific alternative splicing.

Cloning of the insulin receptor cDNA has earlier revealed the existence of two alternative forms of the receptor differing by the presence or absence of 12 amino acids near the C-terminus of the receptor alpha-subunit. This insert has been shown by others to be encoded by a discrete exon, and alternative splicing of this exon leads to tissue-specific expression of two receptor isoforms. We have studied the functional significance of the receptor isoforms and have confirmed that they are generated by alternative splicing. When cDNAs encoding the two forms of the insulin receptors are expressed in Rat 1 cells, the receptor lacking the insert (HIR-A) has a significantly higher affinity for insulin than the receptor with the insert (HIR-B). This difference in affinity is maintained when insulin binding activity is assayed in solution using detergent solubilized, partially purified receptors. These data, combined with the tissue specificity of HIR-A and HIR-B expression, suggest that alternative splicing may result in the modulation of insulin metabolism or responsiveness by different tissues.

Amino Acid Sequence↗
PubMed1990

The tyrosine phosphorylated carboxyterminus of the EGF receptor is a binding site for GAP and PLC-gamma.

Phospholipase C-gamma (PLC-gamma) and GTPase activating protein (GAP) are substrates of EGF, PDGF and other growth factor receptors. Since either PLC-gamma or GAP also bind to the activated receptors it was suggested that their SH2 domains are mediating this association. We attempted to delineate the specific region of the EGF receptor that is responsible for the binding, utilizing EGF receptor mutants, PLC-gamma, and a bacterially expressed TRP E fusion protein containing the SH2 domains of GAP. As previously shown, tyrosine autophosphorylation of the wild-type receptor wsa crucial in mediating the association and in agreement, a kinase negative EGF receptor could bind PLC-gamma or TRP E GAP SH2, but only when cross tyrosine phosphorylated by an active EGF receptor kinase. The importance of autophosphorylation for association was confirmed by demonstrating that a carboxy-terminal deletion of the EGFR missing four autophosphorylation sites bound these proteins poorly. To study the role of EGF receptor autophosphorylation further, a 203 amino acid EGF receptor fragment was generated with cyanogen bromide that contained all known tyrosine autophosphorylation sites. This fragment bound both TRP E GAP SH2 and PLC-gamma but only when tyrosine phosphorylated. This data localizes a major binding site for SH2 domain containing proteins to the carboxy-terminus of the EGF receptor and points to the importance of tyrosine phosphorylation in mediating this association.

Animals↗
PubMed1990

Localization of the gene for amiloride binding protein on chromosome 7 and RFLP analysis in cystic fibrosis families.

The apical sodium channel is essential for sodium reabsorption by the kidney. Its activity is blocked by the diuretic amiloride. Using a human cDNA coding for the amiloride binding protein (ABP), the corresponding structural gene was mapped to human chromosome 7q34-q36 by in situ hybridization. This region flanks the region implicated in cystic fibrosis (7q32). Because an alteration of the amiloride sensitive sodium channel function has been suggested in cystic fibrosis, a possible link between the ABP gene and this disease was analyzed by restriction fragments length polymorphism (RFLP) analyses. From this study, it appears that the gene coding for ABP is not directly modified by mutations causing cystic fibrosis.

Amine Oxidase (Copper-Containing)↗
PubMed1990

Stress experienced by physicians and nurses in the cancer ward.

This study examined occupational stress amongst medical staff on cancer wards. The sample consisted of 91 nurses and 57 physicians from 13 institutions in Bavaria, F.R.G. Strong associations emerged between specific, situational stressors and reported psychosomatic complaints. In particular, interpersonal difficulties, whether on or off the job, related to physical distress amongst nurses. For doctors, dissatifaction with the job and working conditions related to general malaise. Certain characteristics of the carer (sex, profession, age) and of the institutional environment (e.g. presence of trainees, size of institution) were also linked with stress and complaint levels.

Adult↗
PubMed1990

Human kidney amiloride-binding protein: cDNA structure and functional expression.

Phenamil, an analog of amiloride, is a potent blocker of the epithelial Na+ channel. It has been used to purify the porcine kidney amiloride-binding protein. Synthetic oligonucleotides derived from partial sequences have been used to screen a human kidney cDNA library and to isolate the cDNA encoding the human amiloride-binding protein. The primary structure was deduced from the DNA sequence analysis. The protein is 713 residues long, with a 19-amino acid signal peptide. The mRNA was expressed in 293-S and NIH 3T3 cells, yielding a glycoprotein (i) that binds amiloride and amiloride analogs with affinities similar to the amiloride receptor associated with the apical Na+ channel in pig kidney membranes and (ii) that is immunoprecipitated with monoclonal antibodies raised against pig kidney amiloride-binding protein.

Amiloride↗
PubMed1990

High-affinity epidermal growth factor binding is specifically reduced by a monoclonal antibody, and appears necessary for early responses.

We have tested the effects of an mAb directed against the protein core of the extracellular domain of the human EGF receptor (mAb108), on the binding of EGF, and on the early responses of cells to EGF presentation. We used NIH 3T3 cells devoid of murine EGF receptor, transfected with a cDNA encoding the full-length human EGF receptor gene, and fully responsive to EGF. The binding to saturation of mAb108 to the surface of these cells at 4 degrees C and at other temperatures specifically reduced high-affinity binding of EGF, but did not change the dissociation constant or the estimated number of binding sites for low-affinity binding of EGF. The kinetics of EGF binding to the transfected cells were measured to determine the effects of the mAb on the initial rate of EGF binding at 37 degrees C. Interestingly, high-affinity EGF receptor bound EGF with an intrinsic on-rate constant 40-fold higher (9.8 x 10(6) M-1.s-1) than did low-affinity receptor (2.5 x 10(5) M-1.s-1), whereas the off-rate constants, measured at 4 degrees C were similar. Cells treated with the mAb or with phorbol myristate acetate displayed single on-rate constants similar to that for the low-affinity receptors. At low doses of EGF ranging from 0.4 to 1.2 nM, pretreatment of cells with mAb108 inhibited by 50-100% all of the early responses tested, including stimulation of tyrosine-specific phosphorylation of the EGF receptor, turnover of phosphatidyl inositol, elevation of cytoplasmic pH, and release of Ca2+ from intracellular stores. At saturating doses of EGF (20 nM) the inhibition of these early responses by prebinding of mAb108 was overcome. On the basis of these results, we propose that the high-affinity EGF receptors are necessary for EGF receptor signal transduction.

Animals↗
PubMed1990

Separate endocytic pathways of kinase-defective and -active EGF receptor mutants expressed in same cells.

Ligand binding to the membrane receptor for EGF induces its clustering and internalization. Both receptor and ligand are then degraded by lysosomal enzymes. A kinase defective point mutant (K721A) of EGF receptor undergoes internalization similarly to the wild-type receptor. However, while internalized EGF molecules bound to either the wild-type or mutant receptors are degraded, the K721A mutant receptor molecules recycle to the cell surface for reutilization. To investigate the mechanism of receptor trafficking, we have established transfected NIH-3T3 cells coexpressing the kinase-negative mutant (K721A) together with a mutant EGF receptor (CD63) with active kinase. CD63 was chosen because it behaves like wild-type EGF receptor with respect to biological responsiveness and cellular routing but afforded immunological distinction between kinase active and inactive mutants. Although expressed in the same cells, the two receptor mutants followed their separate endocytic itineraries. Like wild-type receptor, the CD63 mutant was downregulated and degraded in response to EFG while the kinase-negative mutant K721A returned to the cell surface for reutilization. Intracellular trafficking of EGF receptor must be determined by a sorting mechanism that specifically recognizes EGF receptor molecules according to their intrinsic kinase activity.

Animals↗
PubMed1990

Domain deletion in the extracellular portion of the EGF-receptor reduces ligand binding and impairs cell surface expression.

Cultured NIH-3T3 cells were transfected with cDNA constructs encoding human epidermal growth factor-receptor (EGF-R)* and two deletion mutants in the extracellular portion of the receptor molecule. One mutant is devoid of 124 amino-terminal amino acids, and the other lacks 76 residues. Mutant receptors were not delivered to the cell surface unless the transfected cells contained also endogenous EGF-Rs, suggesting that receptor interaction complements the mutation and allows surface display of mutant receptors. Immunoprecipitation experiments revealed an association between mutant and endogenous EGF-Rs when both proteins were expressed in the same cell. Hence, receptor-oligomers may exist in the plane of the membrane even in the absence of ligand binding, and oligomerization may play a role in normal trafficking of EGF-Rs to the cell surface. Mutant receptors retained partial ligand binding activity as 125I-labeled EGF was covalently cross-linked to both mutant receptors, and EGF stimulated, albeit weakly, their protein tyrosine kinase activity. Both mutant EGF-Rs bind EGF with a 10-fold lower affinity than that of the solubilized wild type EGF-R. These results provide further evidence that the region flanked by the two cysteine-rich domains plays a crucial role in defining ligand-binding specificity of EGF-R.

Allosteric Regulation↗
PubMed1990

Retrovirus-mediated transfer of an adenovirus gene encoding an integral membrane protein is sufficient to down regulate the receptor for epidermal growth factor.

We have used retrovirus-mediated gene transfer to introduce sequences encoding a 10,400-molecular-weight (10.4K) adenovirus protein previously shown to down regulate the receptor for epidermal growth factor (EGF) into two murine cell lines that possess human EGF receptors (EGF-Rs). Assays for receptor expression showed that acute infection resulted in rapid, constitutive down regulation of the EGF-R via a pathway that appears to be endosome mediated. This represents the first demonstration that 10.4K expression in the absence of other virus-encoded proteins is sufficient to elicit this response. The usefulness of this approach for the study of 10.4K-mediated signal transduction in cells with a nontransformed phenotype is discussed.

Adenoviridae↗