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Query: UNIPROT:P04626 (erbB-2)
 5,251 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The expression and activities of epidermal growth factor (EGF) receptor and a highly related protein (Mr approximately 190,000 protein; p190) were characterized from a human glioma cell line, KE. p190 was specifically immunoprecipitated with a monoclonal antibody with specificity against the EGF receptor (Mr approximately 170,000; p170). Furthermore, both proteins were shown to possess tyrosine-protein kinase activities, although p170 required the presence of EGF to undergo autophosphorylation, whereas p190 appeared to be constitutively activated. Partial and total proteolytic polypeptide analyses of the two proteins suggested that their phosphopeptides were nearly identical and were phosphorylated on similar amino acid residues. However, a number of alterations were observed between [35S]methionine-labeled polypeptides of p170 and p190. This was also supported by the finding that the size of the protein cores of p170 and p190 was different. This observation is in agreement with Northern blot analysis in which KE cells expressed a novel EGF receptor RNA of 10.5 kilobases in addition to the previously reported 10-kilobase RNA. Southern blot analysis of the EGF receptor gene also revealed some amplification, approximately 4- to 5-fold; however, no significant rearrangements were noted in the KE cell DNA. These results suggest that p190 represents an endogeneous structurally and functionally altered EGF receptor.
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PMID:Expression of an altered epidermal growth factor receptor by human glioblastoma cells. 341

It was previously demonstrated that the epidermal growth factor (EGF) receptor in human A431 cells undergoes a slow post-translational modification by which it acquires EGF binding capacity (Slieker, L.J., and Lane, M.D. (1985) J. Biol. Chem. 260, 687-690). In this report, the role of glycosylation in the acquisition of ligand binding activity and in the intracellular translocation of the receptor precursor is characterized. Human A431 cells were incubated with [35S]methionine, and 35S-labeled EGF receptors were purified either by immunoprecipitation (total receptor) or by adsorption to an EGF affinity matrix (high affinity, or active receptor). The half-time for receptor activation is approximately 30 min and precedes its acquisition of resistance to endo-beta-N-acetylglucosaminidase H (t 1/2 = 75 min), a medial Golgi event. Activation is blocked by tunicamycin and is markedly slowed (t 1/2 = 120 min) by 1-deoxynojirimycin, an inhibitor of glucosidase I. In the latter case, the oligosaccharide chains are not further processed to complex forms. Treatment of the active high mannose receptor with endo-beta-N-acetylglucosaminidase H generates a fully active aglycoreceptor polypeptide, indicating that core oligosaccharide addition is a prerequisite for activation but that oligosaccharide chains are not intrinsically required for EGF binding. Subcellular fractionation studies showed that the EGF receptor is activated in the endoplasmic reticulum and that translocation from that organelle is extremely slow (t 1/2 = 75 min). Since the latter translocation rate approximates that for the acquisition of the resistance to endoglycosidase H, transit from the endoplasmic reticulum appears to be rate-limiting for the maturation of the receptor. Both tunicamycin and 1-deoxynojirimycin inhibit exit from the endoplasmic reticulum in parallel with their effects on the acquisition of binding activity. Immunoprecipitation of 35S-labeled EGF receptor with antiphosphotyrosine antibody in the presence of ATP suggested that the autophosphorylation activity of the receptor is also acquired post-translationally. The possible correlation of this to EGF binding activity is discussed.
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PMID:Synthesis of epidermal growth factor receptor in human A431 cells. Glycosylation-dependent acquisition of ligand binding activity occurs post-translationally in the endoplasmic reticulum. 349 Apr 80

Stimulation of epidermal growth factor (EGF) receptor autophosphorylation by EGF and phosphorylation of a Mr 52,000 protein endogenous to the membrane extracts were decreased 6-12-fold in liver membrane extracts from mice homozygous for either the ob/ob or db/db mutation when compared to controls. Liver membranes from the mutant mice bound 4-5-fold less 125I-EGF/unit of protein than did their normal littermates, but exhibited normal EGF binding affinity. Similar decreases in EGF binding were noted in liver membranes from homozygous fa/fa Zucker rats, another obese, hyperinsulinemic animal model, when compared to values from control animals. We also immunoprecipitated hepatic EGF receptors from mice injected with [35S]methionine, and found that livers from db/db mice contained approximately 35% of the labeled EGF receptors found in control animals. Both ob/ob and db/db mice had serum immunoreactive EGF levels similar to or lower than those found in unaffected littermates, suggesting that ligand-mediated down-regulation of receptors was not the cause of the decreased EGF binding. In one mutant, db/db, the decreased binding was associated with a 6-fold decrease in the levels of liver EGF receptor mRNA transcripts; in the ob/ob mice, at most a 2-fold decrease in the level of liver EGF receptor transcripts was observed. EGF binding to cultured peritoneal fibroblasts derived from db/db mice was normal, suggesting that the abnormality in the mutant mice might result from altered environmental or tissue-specific factors rather than an abnormal receptor gene. This was supported by Southern blot analysis of DNA from these animals, which showed identical restriction fragment patterns for the EGF receptor gene in both control and mutant animals. These data indicate that three distinct strains of obese hyperglycemic rodents have decreased levels of hepatic EGF receptors, and suggest that this decrease may result from altered environmental stimuli or tissue-specific factors rather than a primary defect in the EGF receptor gene.
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PMID:Decreased levels of hepatic epidermal growth factor receptors in obese hyperglycemic rodents. 362 63

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.
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PMID:Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis. 608 49

The biosynthesis and posttranslational metabolism of the epidermal growth factor (EGF) receptor were examined in the A431 human epidermoid carcinoma cell line. Polyclonal antibody against the receptor specifically immunoprecipitated two [35S]methionine-labeled proteins of Mr = 160,000 and 170,000. Pulse chase experiments showed the Mr = 160,000 protein to be a precursor of the Mr = 170,000 protein. Preincubation with tunicamycin resulted in immunoprecipitation of a single band of Mr = 130,000, whereas monensin inhibited maturation to the Mr = 170,000 form. Digestion of the Mr = 160,000 and 170,000 proteins with endoglycosidase H resulted in the appearance of Mr = 130,000 and 165,000 proteins, respectively. Prolonged pulse-chase experiments indicated that the half-life of the receptor is ca. 20 h in the absence of EGF and 5 h in the presence of EGF. Approximately three- to five-fold more phosphate is incorporated into the mature receptor upon addition of EGF, due primarily to increases in levels of phosphotyrosine and phosphoserine. Phosphate was also present on the Mr = 160,000 protein and the Mr = 130,000 protein found in the presence of tunicamycin.
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PMID:Aspects of the metabolism of the epidermal growth factor receptor in A431 human epidermoid carcinoma cells. 632 85

Using a monoclonal antibody to the human epidermal growth factor (EGF) receptor (EGF-R1), we have followed the metabolism of the receptor and the pathway of its internalization in KB cells after the addition of EGF. Measurement of surface binding of 125I-labeled EGF showed that about 80% of EGF binding activity disappeared from the plasma membrane after a 10-min exposure to EGF at 37 degrees C. Immunoprecipitation of the receptor from [35S]methionine-labeled cell extracts with EGF-R1 showed that EGF caused the receptor to be degraded with a half-life of 40 min. Immunofluorescence using EGF-R1 showed an EGF-dependent redistribution of the EGF receptor. In cells not exposed to EGF, almost all of the receptor was diffusely distributed on the cell surface. After EGF addition, the receptor was rapidly internalized, first appearing in small punctate organelles characteristic of receptosomes and then in larger perinuclear lysosome-like structures. By 120 min almost all of the immunoreactive EGF receptor had disappeared from the cells. Immunocytochemistry at the electron microscopic level confirmed these light microscopic findings. The diffusely distributed receptor on the cell surface first clustered into clathrin-coated pits in the presence of EGF, next was internalized into receptosomes, appeared transiently in transreticular Golgi elements, and finally was seen in lysosomes. This EGF-dependent down-regulation and degradation of the EGF receptor in KB cells provides a striking example of ligand-dependent clustering and internalization of a receptor, followed by degradation in lysosomes of both ligand and receptor.
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PMID:Down-regulation of the epidermal growth factor receptor in KB cells is due to receptor internalization and subsequent degradation in lysosomes. 632 24

Conflicting results concerning the ability of the epidermal growth factor (EGF) receptor to associate with and/or activate phosphatidylinositol (PtdIns) 3-kinase have been published. Despite the ability of EGF to stimulate the production of PtdIns 3-kinase products and to cause the appearance of PtdIns 3-kinase activity in antiphosphotyrosine immunoprecipitates in several cell lines, we did not detect EGF-stimulated PtdIns 3-kinase activity in anti-EGF receptor immunoprecipitates. This result is consistent with the lack of a phosphorylated Tyr-X-X-Met motif, the p85 Src homology 2 (SH2) domain recognition sequence, in this receptor sequence. The EGF receptor homolog, ErbB2 protein, also lacks this motif. However, the ErbB3 protein has seven repeats of the Tyr-X-X-Met motif in the carboxy-terminal unique domain. Here we show that in A431 cells, which express both the EGF receptor and ErbB3, PtdIns 3-kinase coprecipitates with the ErbB3 protein (p180erbB3) in response to EGF. p180erbB3 is also shown to be tyrosine phosphorylated in response to EGF. In contrast, a different mechanism for the activation of PtdIns 3-kinase in response to EGF occurs in certain cells (PC12 and A549 cells). Thus, we show for the first time that ErbB3 can mediate EGF responses in cells expressing both ErbB3 and the EGF receptor.
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PMID:ErbB3 is involved in activation of phosphatidylinositol 3-kinase by epidermal growth factor. 751 47

The Shc gene encodes three overlapping proteins which all contain a carboxy-terminal SH2 domain. Shc proteins are ubiquitously expressed and are downstream targets and effectors of activated tyrosine kinases (TK). We investigated tyrosine-phosphorylation of Shc proteins in normal and transformed cells. In tumor cells with known TK gene alterations Shc proteins were constitutively phosphorylated and complexed with the activated TK. No constitutive Shc phosphorylation was found in primary cell cultures and normal tissues. In 14 of 27 tumor cell lines with no reported TK alterations, Shc proteins were constitutively phosphorylated and formed stable complexes with novel tyrosine-phosphorylated polypeptides. Ten distinct Shc-associated phosphoproteins were identified with molecular weights ranging from 30 to 200 kDa. In a subset of carcinoma cell lines, phosphorylated Shc proteins complexed with a p175 phosphoprotein that was identified as the constitutively activated EGFR. In one glioblastoma cell line, a Shc-associated p190 was identified as the activated PDGFR. In 13 of 14 acute leukemia samples phosphorylated Shc proteins were constitutively complexed with a p140 phosphoprotein. Some of the Shc-associated phosphoproteins (EGFR, PDGFR, erbB-2, Met, bcr-abl, H4-ret) bound both the Shc- and Grb2-SH2 domains in vitro; others (p175; p70-p80) only the Shc-SH2 domain and yet others (p140) only the Grb2-SH3 domains. These results indicate that Shc proteins are common substrates of constitutively activated TKs and that the analysis of Shc phosphorylation allow the identification of tumors with constitutive TK activation.
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PMID:Constitutive phosphorylation of Shc proteins in human tumors. 767 49

The effect of herbimycin A on the biosynthesis of epidermal growth factor (EGF) receptor was examined in human epidermoid carcinoma A431 cells. Cells were pulse-labelled with [35S]methionine, and EGF receptor biosynthesis was quantified by immunoprecipitation using a monoclonal anti-(EGF receptor) antibody. In the presence of herbimycin A, an immature 160 kDa EGF receptor precursor accumulated in 1 h and disappeared completely in 4 h. Pulse-labelled 160 kDa receptor precursor in the absence of herbimycin A, however, was converted normally into a 170 kDa one by chase with herbimycin A. Herbimycin A affected neither the synthesis of the secreted form of EGF receptor devoid of cytoplasmic domain, nor that of the transferrin receptor in A431 cells. The herbimycin A-induced degradation of 160 kDa EGF receptor precursor was not inhibited by an inhibitor of lysosomal enzymes, NH4Cl. Endoglycosidase H digestion of the 160 kDa precursor converted it into the deglycosylated 130 kDa precursor peptide. These results suggested that herbimycin A selectively acted on the EGF receptor precursor during the synthesis of the 160 kDa form, probably on the cytoplasmic domain, to form an aberrant molecule which was subjected to rapid degradation in the endoplasmic reticulum.
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PMID:Accelerated degradation of 160 kDa epidermal growth factor (EGF) receptor precursor by the tyrosine kinase inhibitor herbimycin A in the endoplasmic reticulum of A431 human epidermoid carcinoma cells. 803 92

To identify serum-inducible genes in the insulin-producing cell line beta TC-1, a library subtraction screening procedure was performed on serum-deprived (G0) and serum-restimulated (G1) insulin-producing beta TC-1 cells. A cDNA containing a motif with strong homology to Src homology 2 (SH2) domains was found using this procedure and called Shb. The Shb cDNA contains two methionine codons in its N-terminus and thus may code for two proteins of 67 and 56 kDa, each with one SH2 domain in its C-terminus. No other structural similarity to proteins with catalytic activity could be detected, suggesting that Shb is a so called adaptor. Shb contains the proline-rich sequence PPPGPGR between the two proposed initiator methionines which resembles a sequence for binding to Src homology 3 (SH3) domains. A second proline-rich sequence was detected after the second methionine codon. The Shb cDNA hybridized to a similar or identical mRNA of 3.1 kb expressed in mouse brain, liver, kidney, heart, NIH3T3 fibroblasts and beta TC-1 cells. Western blot analysis of the same tissues using an antiserum directed against a synthetic peptide corresponding to a part of the SH2 domain of Shb, revealed reactivity with two proteins of 56 and 67 kDa. In addition, a third reactive component of 40 kDa was detected in most tissues. Transfection and transient expression of the Shb cDNA in COS-1 cells yielded increased expression of the 67, 56 and 40 kDa proteins. Transfection and stable expression of the Shb cDNA in pig aortic endothelial cells showed increased expression primarily of the 67 kDa protein. A fusion protein consisting of the SH2 domain of Shb linked to glutathione S-transferase showed increased binding to glycoproteins of cells stimulated with platelet-derived growth factor (PDGF-BB). Furthermore, the autophosphorylated PDGF beta-receptor but not the autophosphorylated epidermal growth factor (EGF) receptor bound specifically to immobilized fusion protein. It is concluded that Shb is a novel SH2-containing protein with proline-rich domains and therefore probably involved in the signal-transduction of some ligand-activated tyrosine kinase receptors.
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PMID:Shb is a ubiquitously expressed Src homology 2 protein. 830 79


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