Biosynthesis of the epidermal growth factor receptor in human squamous cell carcinoma lines: secretion of the truncated receptor is not common to epidermal growth factor receptor-hyperproducing cells

The biosynthesis of the EGF receptor was examined in the epidermoid carcinoma cell line A431 and five novel cell lines from human squamous cell carcinomas possessing high numbers of EGF receptors. Newly synthesized EGF receptors were visualized by labeling with [35S]methionine and immunoprecipitation with a monoclonal anti-EGF receptor antibody. In addition, the processing of the EGF receptor and its intracellular transport was analyzed by distinguishing cell surface receptors from intracellular receptors and by treating cells with inhibitors such as tunicamycin, monensin and brefeldin A. These analyses revealed that in all the tumor cell lines the EGF receptor is synthesized as a glycosylated protein of Mr 160,000 which is converted to the receptor of Mr 170,000 through posttranslational glycosylation. The receptors of Mr 160,000 and 170,000 appeared to possess high mannose type oligosaccharide chains because endoglycosidase H treatment reduced their molecular weights by approximately 30,000. A431 was the only tumor cell line studied that secreted the truncated EGF receptor of Mr 110,000. In A431 cells, the truncated EGF receptor was generated from a protein of Mr 60,000 through tunicamycin- and monensin-sensitive glycosylation. A431 cells treated with monensin secreted the truncated receptor as a Mr 95,000 form.     

Aspects of the metabolism of the epidermal growth factor receptor in A431 human epidermoid carcinoma cells.

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.     

Glycosylation of the epidermal growth factor receptor in A-431 cells. The contribution of carbohydrate to receptor function

A-431 cells were treated with inhibitors of either N-linked glycosylation (tunicamycin or glucosamine) or of N-linked oligosaccharide processing (swainsonine or monensin) to examine the glycosylation of epidermal growth factor (EGF) receptors and to determine the effect of glycosylation modification on receptor function. The receptor was found to be an Mr = 130,000 polypeptide to which a relatively large amount of carbohydrate is added co-translationally in the form of N-linked oligosaccharides. Processing of these oligosaccharides accounts for the 10,000-dalton difference in electrophoretic migration between the Mr = 160,000 precursor and Mr = 170,000 mature forms of the receptor. No evidence was found for O-linked oligosaccharides on the receptor. Mr = 160,000 receptors resulting from swainsonine or monensin treatment were present on the cell surface and retained full function, as judged by 125I-EGF binding to intact cells and detergent-solubilized extracts and by in vitro phosphorylation in the absence or presence of EGF. On the other hand, when cells were treated with tunicamycin or glucosamine, ligand binding was reduced by more than 50% in either intact cells or solubilized cell extracts. The Mr = 130,000 receptors synthesized in the presence of these inhibitors were not found on the cell surface. In addition, no Mr = 130,000 phosphoprotein was detected in the in vitro phosphorylation of tunicamycin or glucosamine-treated cells. It appears, therefore, that although terminal processing of N-linked oligosaccharides is not necessary for proper translocation or function of the EGF receptor, the addition of N-linked oligosaccharides is required.     

Effects of epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate on metabolism of the epidermal growth factor receptor in normal human fibroblasts.

The biosynthesis, phosphorylation, and degradation of the epidermal growth factor (EGF) receptor were examined in normal human fibroblasts. The receptor was initially synthesized as an Mr = 160,000 immature form which matured to an Mr = 170,000 form in a monensin-sensitive manner. Tunicamycin treatment led to the accumulation of an Mr = 130,000 protein. The receptor was phosphorylated on serine and threonine residues in normally growing and quiescent cells, and treatment with EGF or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a two- to threefold increase in receptor-bound phosphate. EGF increased the amount of phosphoserine and phosphothreonine and caused the appearance of a minor amount of phosphotyrosine. TPA increased the levels of phosphoserine and phosphothreonine exclusively. Prior treatment with TPA inhibited the EGF-dependent appearance of phosphotyrosine in the receptor. Analysis of tryptic phosphopeptides revealed that six of the seven major peptides were common to the receptor from cells treated with EGF or TPA. EGF strongly stimulated [3H]thymidine incorporation in confluent cells, increased final saturation density three to fourfold, and increased whole-cell levels of phosphotyrosine about threefold. Treatment of cells with TPA before addition of EGF inhibited all three of these EGF-dependent responses. EGF also decreased the receptor half-life from 15 h to 1 h, but this was not inhibited by TPA. TPA alone had no detectable effect on the receptor half-life.     

Change in metabolic turnover is an alternate mechanism increasing cell surface epidermal growth factor receptor levels in tumor cells

The biosynthesis and metabolic turnover of the epidermal growth factor (EGF) receptor was examined in a human pancreatic carcinoma cell line, UCVA-1. This cell line has been shown to possess a much higher level of EGF receptors than is expected solely from receptor gene/mRNA dosage. Analysis of the biosynthesis using metabolic labeling, immunological quantitation, and inhibitor treatment revealed that the naked EGF receptor in UCVA-1 cells is a protein of Mr 130,000 that is matured consecutively as a Mr 160,000 and 170,000 glycoprotein through post-translational glycosylation. Analysis of the metabolic turnover using pulse-chase labeling and inhibitor treatment revealed that the rate of EGF receptor synthesis in UCVA-1 cells was similar to that in two squamous cell carcinoma cell lines, NA and Ca9-22, which also have high numbers of EGF receptors, but because of gene amplification. In contrast, the rate of receptor degradation in UCVA-1 cells was significantly slower than in the other two cell lines. These results suggest that the retarded metabolic turnover may constitute a unique mechanism for elevating cell surface EGF receptor levels in some tumor cells independent of gene amplification.     

Epidermal growth factor-induced truncation of the epidermal growth factor receptor

NIH-3T3 cells expressing the human epidermal growth factor (EGF) receptor were used in experiments to determine the fate of the EGF receptor in cells continuously exposed to EGF. EGF receptor was immunoprecipitated from cells labeled for 12 h with [35S] methionine in the absence or presence of 10 nM EGF. As expected, a single Mr = 170,000 polypeptide representing the mature EGF receptor was immune-precipitated from control cells. Surprisingly, immune precipitates from EGF-treated cells contained a prominent Mr = 125,000 receptor species, in addition to the Mr = 170,000 mature receptor. The Mr = 125,000 species was shown to be derived from the Mr = 170,000 form by pulse-chase experiments, in which the Mr = 170,000 receptor chased into the Mr = 125,000 form when EGF was included during the chase and by partial proteolysis. Both proteins became extensively phosphorylated on tyrosine residues in immune precipitate kinase assays. Treatment of immune precipitates with endoglycosidase F changed the apparent molecular weight of the Mr = 170,000 receptor to Mr = 130,000 and of the Mr = 125,000 form to Mr = 105,000, indicating that the appearance of the Mr = 125,000 protein was probably due to proteolysis. Antibody against the carboxyl terminus of the mature EGF receptor recognized the Mr = 125,000 protein, whereas antibody against the amino terminus did not. Incubation of cells with leupeptin prior to and during EGF addition inhibited processing to the Mr = 125,000 species. Methylamine and low temperature also inhibited the EGF-induced processing to the Mr = 125,000 form. These data suggest a possible role for proteolysis of the EGF receptor in receptor function.     

Synthesis of epidermal growth factor receptor in human A431 cells. Glycosylation-dependent acquisition of ligand binding activity occurs post-translationally in the endoplasmic reticulum

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.     

The mouse leukocyte adhesion proteins Mac-1 and LFA-1: studies on mRNA translation and protein glycosylation with emphasis on Mac-1

Translation in vitro of mRNA and immunoprecipitation with specific rabbit antisera showed that the unglycosylated precursor polypeptides of the mouse Mac-1 and lymphocyte function associated antigen (LFA-1) alpha subunits are 130,000 Mr and 140,000 Mr, respectively. Furthermore, polysomes purified by using anti-Mac-1 IgG yielded a similar major product of translation in vitro of Mr = 130,000. The Mac-1 and LFA-1 alpha subunit translation products are immunologically noncross-reactive, showing that differences between these related proteins are not due to post-translational processing. Mac-1 and LFA-1 alpha subunits could only be in vitro translated from mRNA from cell lines the surfaces of which express the corresponding Mac-1 and LFA-1 alpha-beta complexes, showing tissue-specific expression is regulated at the mRNA level. The glycosylation of Mac-1 was examined by both translation in vitro in the presence of dog pancreas microsomes and by biosynthesis in vivo and treatment with tunicamycin, endoglycosidase H, and the deglycosylating agent trifluoromethane sulfonic acid. High mannose oligosaccharides are added to the Mac-1 alpha and beta polypeptide backbones of Mr = 130,000 and 72,000, respectively, to yield precursors of Mr = 164,000 and 91,000, respectively. The alpha and beta subunit precursors are then processed with partial conversion of high mannose to complex type carbohydrate to yield the mature subunits of Mr = 170,000 and 95,000, respectively.     

The oligosaccharide moieties of the epidermal growth factor receptor in A-431 cells. Presence of complex-type N-linked chains that contain terminal N-acetylgalactosamine residues

The receptor for epidermal growth factor (EGF) in the human epidermoid carcinoma cell line A-431 is a glycoprotein of apparent molecular weight = 170,000. During biosynthesis, the receptor is first detected as a precursor of apparent Mr = 160,000. In this report we describe our studies on the structures of the oligosaccharide moieties of the mature receptor and its precursor. A-431 cells were grown in medium containing radioactive sugars and the radiolabeled receptors were purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled glycopeptides were prepared from the purified receptor by proteolysis, and their structures were examined by a variety of techniques. The mature EGF receptor contains both complex-type and high mannose-type Asn-linked oligosaccharides in the approximate ratio of 2 to 1, while the precursor contains only high mannose-type chains. A number of experimental results demonstrate that the mature receptor does not contain oligosaccharides in O-linkage through N-acetylgalactosamine to either serine or threonine. The high mannose-type oligosaccharides in both precursor and mature receptor can be cleaved by endo-beta-N-acetylglucosaminidase H and occur in the mature receptor as Man9GlcNAc2 (6%), Man8GlcNAc2 (49%), Man7GlcNAc2 (25%), and Man6GlcNAc2 (20%), whereas, in the receptor precursor the high mannose chains occur primarily as Man8GlcNAc2 (70%). The complex-type oligosaccharides in the mature receptor are predominantly tri- or tetraantennary species and are unusual in several respects. (i) Many of the chains do not contain sialic acid, while the remaining chains contain 1-2 sialic acid residues. (ii) Half of the [3H] mannose-derived radioactivity was recovered as [3H] fucose and the remaining half as [3H] mannose, indicating that there may be an average of 3 fucose residues/chain. (iii) About one-third of the [3H] glucosamine-derived radioactivity in these glycopeptides was recovered as N-acetylgalactosamine and these residues are all alpha-linked and occur at the nonreducing termini. These data demonstrate that the complex-type Asn-linked oligosaccharides in the EGF receptor from A-431 cells contain sugar residues related to human blood type A. In light of other recent studies, these results suggest that in A-431 cells blood group determinants in surface glycoproteins are contained in Asn-linked but not O-linked oligosaccharides.     

Biosynthesis and maturation of lactase-phlorizin hydrolase in the human small intestinal epithelial cells.

The biosynthesis and maturation of the human intestinal lactase-phlorizin hydrolase (LPH; EC 3.2.1.23-3.2.1.62) has been studied in cultured intestinal biopsies and mucosal explants. Short time pulse labelling revealed on high mannose intermediate of Mr 215,000 which was converted upon endo-beta-N-acetylglucosaminidase H (endo-H) digestion to a polypeptide of Mr 200,000. The brush border form of LPH was revealed after longer pulse periods and has Mr 160,000. It possesses mainly complex oligosaccharide chains and, owing to its partial endo-H sensitivity, at least one chain of the high mannose type. Leupeptin partially inhibited the appearance of the Mr-160,000 polypeptide. Monensin treatment of biopsies resulted in the modification of the Mr-160,000 species to the Mr-140,000 molecule, which was endo-H sensitive. Pulse-chase analysis indicated a slow post-translational processing of the high mannose precursor (Mr 215,000) to yield the mature brush-border form (Mr 160,000) of LPH. Our results further indicate that LPH is synthesized as a single polypeptide precursor which is intracellularly cleaved to yield the mature brush border of LPH. The data presented suggest that this cleavage occurs during the translocation of the molecule across the Golgi complex.     

Proteolytic cleavage of epidermal growth factor receptor. A Ca2+-dependent, sulfhydryl-sensitive proteolytic system in A431 cells

The Mr = 160,000 epidermal growth factor (EGF) receptor in A431 cells is partially cleaved during membrane isolation to a Mr = 145,000 polypeptide containing both EGF binding and phosphate acceptor sites. We show that the proteolytic degradation of the EGF receptor depends upon the presence of Ca2+ in the medium used to scrape the cells from the substratum. Only the high molecular weight form of the receptor is detected in membranes prepared in the absence of Ca2+. Ca2+-dependent proteolysis occurs rapidly (t1/2 approximately 5 min) following cell scraping. Proteolysis results in a decrease in EGF-dependent phosphorylation of the receptor while retaining EGF binding capacity. In addition, membranes containing the uncleaved form of the receptor reveal a substantial increase in EGF-dependent phosphorylation of proteins with Mr approximately 80, 89, and 185 X 10(3). In the presence of Ca2+, addition of iodoacetic acid to the scraping medium strongly inhibits receptor fragmentation, whereas other inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and pepstatin) have no effect. The results implicate a role for a Ca2+-dependent, SH-sensitive protease in EGF receptor degradation. Prevention of proteolysis yields membrane preparations with highly active EGF-dependent kinase system.     

Epidermal Growth Factor Induces Internalization But Not Degradation of the Epidermal Growth Factor Receptor in a Human Breast Cancer Cell Line

Abstract

Metabolism of the epidermal growth factor (EGF) receptor was studied in the MDA-MB-231 human breast cancer cell line. As in normal fibroblasts the EGF receptor from MDA-MB-231 cells was synthesized from a M_r =160,000 precursor and tunicamycin treatment of cells resulted in accumulation of a M_r =130,000 polypeptide. Unlike normal fibroblasts in which a M_r =170,000 mature form of the EGF receptor was found, MDA-MB-231 cells contained a M_r =172,000 mature form. Addition of EGF to MDA-MB-231 cells led to rapid internalization of EGF receptors, however, internalization did not affect receptor half-life and receptors did not recycle to the cell surface. EGF receptors could be visualized by immunofluorescence and remained sequestered in intracellular membranous structures following internalization. EGF was degraded slowly by MDA-MB-231 cells relative to degradation of EGF by normal cells. A high endogenous level of in vivo phosphorylation of threonine 654 of the EGF receptor was found in MDA-MB-231 cells and treatment of cells with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further stimulated phosphorylation of this residue. EGF induced receptor internalization resulted in dephosphorylation of threonine 654. The significance of these unusual properties of EGF receptor metabolism in MDA-MB-231 cells is discussed.     

Biogenesis of the somatogenic receptor in rat liver

Certain structural characteristics, in particular the type of oligosaccharide chains associated with the rat liver somatogenic (GH) receptors, were studied in different isolated organelles involved in receptor biosynthesis, maturation, and binding, with the use of ligand-affinity cross-linking, incubation with various oligosaccharide chain-cleaving enzymes, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In an endoplasmic reticulum-enriched fraction, a somatogenic receptor with Mr 33,000, after correction for bound ligand (assuming a 1:1 binding ratio of ligand to receptor) was found to contain N-linked high mannose oligosaccharide chain(s). In an intermediate density fraction, enriched in cis-Golgi, a major receptor of Mr 43,000 was found to contain N-linked complex type of oligosaccharide chains. In a low density membrane fraction, containing trans-Golgi complex membranes and endocytic vesicles, three receptors of Mr 95,000, 55,000, and 43,000 were found. These three receptors contain N-linked complex-type oligosaccharide chains. Neuraminidase treatment resulted in a decrease of the Mr 95,000 and 43,000 receptors to Mr 81,000 and 39,000, respectively. Two specific somatogenic receptors of Mr 95,000 and 43,000 containing N-linked complex type of oligosaccharides were found in an isolated plasma membrane-enriched fraction. When isolated hepatocytes were analyzed, the Mr 95,000 receptor was found to be the major labeled species. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (first dimension nonreducing and the second dimension reducing conditions), showed that the Mr 43,000 receptor is contained within the Mr 95,000 receptor. The data suggest that the Mr 33,000 receptor found in endoplasmic reticulum constitutes a precursor to the Mr 43,000 receptor and that the Mr 43,000 receptor is complexed with an unknown subunit during transport through the Golgi complex to form an Mr 95,000 receptor present on the cell surface.     

Biosynthesis of the human sucrase-isomaltase complex. Differential O-glycosylation of the sucrase subunit correlates with its position within the enzyme complex

The biosynthesis and maturation of human sucrase-isomaltase (SI, EC 3.2.1.48-10), was studied in cultured small intestinal biopsy specimens and mucosa explants. Pulse-chase experiments with [35S]methionine revealed one high mannose intermediate of Mr = 210,000 (pro-SIh) which was processed at a slow rate to an endo H-resistant, mature form of Mr = 245,000 (pro-SIc). The fully core-glycosylated form (Mr = 212,000) was detected only when 1-deoxynojirimycin was added to the culture medium, thus indicating that the core sugars undergo rapid processing by rough endoplasmic reticulum membrane-bound glycosidases. The data presented showed that trypsin specifically and instantaneously (within 1 min) cleaves pro-SIc to two subunits Ic (Mr = 145,000) and Sc (Mr = 130,000). Elastase and chymotrypsin are not effective. Enzymic and chemical deglycosylations of SI with endo-beta-N-acetylglucosaminidase F/glycopeptidase F and trifluoromethanesulfonic acid (TFMS) as well as probing for the binding capacity of SI to Helix pomatia lectin demonstrated that pro-SIc, Ic, and Sc are N- and O-glycosylated. Furthermore, the results were indicative of a posttranslational O-glycosylation of pro-SI, since (i) the earliest detectable precursor form, pro-SIh, did not bind to H. pomatia lectin and (ii) its deglycosylation products with both endo-beta-N-acetylglucosamidase H and TFMS were identical. Both the Sc and Ic subunits contain eight N-linked glycan units, at least one of which is of the high mannose type and found on Sc. Finally, Sc, but not Ic, was shown to display at least four populations varying in their content of O-linked glycans. The heterogeneous O-glycosylation pattern of Sc could be correlated with the distal position of this subunit (and its O-glycosylation sites) within the pro-SI molecule, thus affecting the extent of O-linked oligosaccharide processing and their subsequent presentation on the mature molecule.     

Characterization of a specific alpha-mannosidase involved in oligosaccharide processing in Saccharomyces cerevisiae

Fractionation of a crude extract from Saccharomyces cerevisiae X-2180 on Sepharose 6B in the presence of 0.5% Triton X-100 resolves two enzyme fractions containing alpha-mannosidase activity. Fraction I which is excluded from the gel contains alpha-mannosidase activity toward both p-nitrophenyl-alpha-D-mannopyranoside and Man9GlcNAc oligosaccharide as substrates, whereas Fraction II which is included in the gel contains only oligosaccharide alpha-mannosidase activity. The latter enzyme is very specific and removes a single mannose residue from Man9GlcNAc, whereas the alpha-mannosidase activity of Fraction I removes several mannose residues from Man9GlcNAc oligosaccharide. High resolution 1H NMR analysis of the Man8GlcNAc formed from Man9GlcNAc in the presence of the alpha-mannosidase of Fraction II showed only a single isomer with the following structure: (see formula; see text) This specific enzyme is most probably involved in processing of oligosaccharide during biosynthesis of mannoproteins. The mannose analog of 1-deoxynojirimycin (50-500 microM), dideoxy-1,5-imino-D-mannitol, inhibits the oligosaccharide alpha-mannosidase activities of Fractions I and II to about the same extent, but has no effect on the nonspecific alpha-mannosidase which acts on p-nitrophenyl-alpha-D-mannopyranoside.     

Epidermal growth factor receptor. Characterization of a monoclonal antibody specific for the receptor of A431 cells

A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells was obtained after fusion of immunized BALB/c mouse spleen cells with NS-1 myeloma cells. Specific binding of the antibody to the plasma membrane of A431 cells was demonstrated by indirect immunofluorescence and electron microscopy. The antibody did not react with human KB cells, normal rat kidney cells, or Swiss 3T3 cells. The antibody is an IgG3K; it specifically immunoprecipitated a Mr approximately 170,000 protein from radiolabeled A431 cell extracts. This protein is phosphorylated in a EGF-dependent manner in intact A431 cells and in Triton X-100-solubilized plasma membranes. The specificity of the interaction of the antibody with the Mr = 170,000 protein was confirmed by electrophoretic transfer of A431 cell proteins to nitrocellulose followed by incubation with the antibody and 125I-protein A. When 125I-EGF was covalently cross-linked to its receptor, the 125I-EGF-receptor complex was specifically precipitated by the antibody. The monoclonal antibody did not inhibit the binding of 125I-EGF to its receptor in intact A431 cells and also failed to stimulate the phosphorylation of the Triton X-100-solubilized EGF receptor. The results indicate that the antibody and EGF bind to different sites on the EGF receptor. The antibody will be useful for isolating the EGF receptor in an unactivated form.     

1-deoxynojirimycin impairs oligosaccharide processing of alpha 1-proteinase inhibitor and inhibits its secretion in primary cultures of rat hepatocytes

1-Deoxynojirimycin was found to inhibit oligosaccharide processing of rat alpha 1-proteinase inhibitor. In normal hepatocytes alpha 1-proteinase inhibitor was present in the cells as a 49,000 Mr high mannose type glycoprotein with oligosaccharide side chains having the composition Man9GlcNAc and Man8GlcNAc with the former in a higher proportion. Hepatocytes treated with 5 mM 1-deoxynojirimycin accumulated alpha 1-proteinase inhibitor as a 51,000 Mr glycoprotein with carbohydrate side chains of the high mannose type, containing glucose as measured by their sensitivity against alpha-glucosidase, the largest species being Glc3Man9GlcNAc. Conversion to complex oligosaccharides was inhibited by the drug. In addition, increasing concentrations of 1-deoxynojirimycin inhibited glycosylation resulting in the formation of some alpha 1-proteinase inhibitor with two instead of three oligosaccharide side chains. 5 mM 1-deoxynojirimycin inhibited the secretion of alpha 1-proteinase inhibitor by about 50%, whereas secretion of albumin was unaffected. The oligosaccharides of alpha 1-proteinase inhibitor secreted from 1-deoxynojirimycin-treated cells were characterized by their susceptibility to endoglucosaminidase H, incorporation of [3H]galactose, and [3H]fucose and concanavalin A-Sepharose chromatography. It was found that 1-deoxynojirimycin did not completely block oligosaccharide processing, resulting in the formation of alpha 1-proteinase inhibitor molecules carrying one or two complex type oligosaccharides. Only these alpha 1-proteinase inhibitor molecules processed to the complex type in one or two of their oligosaccharide chains were nearly exclusively secreted. This finding demonstrates the importance of oligosaccharide processing for the secretion of alpha 1-proteinase inhibitor.     

Regulation of epidermal growth factor receptor number and phosphorylation by fasting in rat liver

The binding of 125I-epidermal growth factor (EGF) to microsomal membrane preparations from the livers of rats fasted for 72 h or fed control or high carbohydrate diets was examined to determine whether alterations in nutrient intake could affect the EGF receptor system. Fasted rats had 40-50% less membrane binding than did control or carbohydrate-fed rats. Scatchard analysis of the binding data indicated that the decrease in EGF binding in fasted rats was due to a decrease in receptor number with no change in receptor affinity. Cross-linking of 125I-EGF to EGF receptors with disuccinimidyl suberate revealed specific binding of a Mr 170,000 protein, which was diminished by approximately 75% in fasting, and a Mr = 150,000 protein, which accounted for 40-50% of the total labeling in the control and carbohydrate-fed rats and which was relatively unchanged by fasting. The sum of the labeling of the 2 bands was reduced by approximately 40% in fasting and is consistent with the reduction in EGF binding detected by Scatchard analysis. EGF stimulated a 1.5-3-fold increase in 32P incorporation into one major protein of 170 kDa in all 3 groups. Basal and EGF-stimulated autophosphorylation of 170 kDa, when normalized for protein, was 75% lower in membranes from fasted animals, compared to those from control or carbohydrate-fed rats. The comparable reduction of 125I-EGF binding to, and 32P incorporation into, the 170-kDa EGF receptor protein suggested that kinase activity/receptor was unaffected by fasting. Moreover, EGF receptor kinase activity in the 3 groups was comparable for an exogenous substrate, as judged by equal basal and EGF-stimulated phosphorylation of Val5-angiotensin II, when normalized for total EGF-binding capacity. These results suggest that fasting regulates EGF receptor kinase activity primarily by regulation of the number of hepatic EGF receptors. The possibility exists that some in vivo effects of fasting may be mediated by a reduction in EGF receptor levels.     

Retention of glucose on oligosaccharide chains linked to the LH/hCG receptor prevents cell surface expression

In the first step of asparagine-linked oligosaccharide chain maturation, terminal glucose residues are removed from the high mannose oligosaccharide core by glucosidases I and II. The role that glucose residues play in trafficking the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor from the endoplasmic reticulum to the cell surface was investigated. Glucosidases I and II were inhibited by incubating 293 T cells transiently transfected with LH/hCG receptor cDNA with 5 mM 1-deoxynojirimycin (DNJ). DNJ treatment resulted in a marked reduction in cell surface [(125)I]hCG binding. Similar results were obtained from glucosidase I-deficient Lec 23 Chinese hamster ovarian (CHO) cells and wild-type CHO cells that were transiently transfected with LH/hCG receptor cDNA. Immunoprecipitation followed by Western blotting of transfected 293 T cells incubated in the presence or absence of 5 mM DNJ revealed that there is substantially less receptor in DNJ-treated cells than in control cells. These results show that the removal of glucose residues is necessary for trafficking the LH/hCG receptor to the cell surface.