The immunofluorescent detection of phosphorylated receptors of the epidermal growth factor during their internalization in A-431 cells]

Phosphorylated receptors of the epidermal growth factor (EGF) were localized in the human epidermoid carcinoma cells using immunofluorescent staining with antibody to phosphotyrosine. The application of EGF at 4 degrees C was seen to induce a characteristic fluorescence of the cell margins, whereas no cell staining occurs in the absence of EGF. After a 1 hour incubation of cells at 37 degrees C, within which the internalized EGF receptor complexes are accumulated in the juxtanuclear compartment near the para-Golgi region, the staining with antiphosphotyrosine antibody reveals the receptors in this region. It is concluded that the internalized EGF-receptor complexes may remain in the phosphorylated state.     

Direct visualization of the phosphorylated epidermal growth factor receptor during its internalization in A-431 cells

Epidermal growth factor (EGF) rapidly stimulates receptor autophosphorylation in A-431 cells. After 1 min the phosphorylated receptor can be identified at the plasma membrane using an anti- phosphotyrosine antibody. With further incubation at 37 degrees C, approximately 50% of the phosphorylated EGF receptor was internalized (t1/2 = 5 min) and associated with the tubulovesicular system and later with multivesicular bodies, but not the nucleus. During this period, there was no change in the extent or sites of phosphorylation. At all times the phosphotyrosine remained on the cytoplasmic side of the membrane, opposite to the EGF ligand identified by anti-EGF antibody. These data indicate that (a) the tyrosine-phosphorylated EGF receptor is internalized in its activated form providing a mechanism for translocation of the receptor kinase to substrates in the cell interior; (b) the internalized receptor remains intact for at least 60 min, does not associate with the nucleus, and does not generate any tyrosine-phosphorylated fragments; and (c) tyrosine phosphorylation alone is not the signal for receptor internalization.     

Recycling of epidermal growth factor-receptor complexes in A431 cells: identification of dual pathways

The intracellular sorting of EGF-receptor complexes (EGF-RC) has been studied in human epidermoid carcinoma A431 cells. Recycling of EGF was found to occur rapidly after internalization at 37 degrees C. The initial rate of EGF recycling was reduced at 18 degrees C. A significant pool of internalized EGF was incapable of recycling at 18 degrees C but began to recycle when cells were warmed to 37 degrees C. The relative rate of EGF outflow at 37 degrees C from cells exposed to an 18 degrees C temperature block was slower (t1/2 approximately 20 min) than the rate from cells not exposed to a temperature block (t1/2 approximately 5-7 min). These data suggest that there might be both short- and long-time cycles of EGF recycling in A431 cells. Examination of the intracellular EGF-RC dissociation and dynamics of short- and long-time recycling indicated that EGF recycled as EGF-RC. Moreover, EGF receptors that were covalently labeled with a photoactivatable derivative of 125I-EGF recycled via the long-time pathway at a rate similar to that of 125I-EGF. Since EGF-RC degradation was also blocked at 18 degrees C, we propose that sorting to the lysosomal and long-time recycling pathway may occur after a highly temperature-sensitive step, presumably in the late endosomes.     

Internalization of functional epidermal growth factor:receptor/kinase complexes in A-431 cells

We have isolated and partially purified an intracellular vesicle fraction from A-431 cells that contains both epidermal growth factor (EGF) and enzymatically active EGF:receptor/kinase. Exposure of intact A-431 cells to EGF leads to an accumulation of both EGF and kinase activity in this vesicle fraction. The accumulation is time- and temperature-dependent and is blocked by inhibitors of energy production. The EGF receptor in internalized vesicles is capable of autophosphorylation and, in the presence of Ca2+, of phosphorylation of the previously isolated 35-kDa protein (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). The demonstration of an EGF-induced increase in kinase activity of an internalized vesicle fraction lends credence to the hypothesis that EGF-induced endocytosis of the receptor is of physiological significance in the response of cells to this ligand. In addition, these results are consistent with the suggestion that the phosphorylation of the 35-kDa protein is associated with internalization of the EGF:receptor/kinase complex.     

Purification of denatured epidermal growth factor-receptor from A431 human epidermoid carcinoma cells

A rapid and simple method was developed for isolating denatured epidermal growth factor (EGF)-receptor suitable for use in preparation of polyclonal antisera. Membranes from A431 cells (which possess unusually high numbers of EGF-receptors) were phosphorylated in vitro with [gamma-32P]ATP and run on preparative sodium dodecyl sulfate (SDS)-polyacrylamide gels. The Mr 170,000 major phosphorylated region was excised from the gels, eluted, and protein chromatographed on SDS-hydroxylapatite. Fractions containing the Mr 170,000 tyrosine-phosphorylated protein were pooled, concentrated, and rerun on preparative SDS gels. The protein eluted from these gels was judged to be highly purified, based on peptide mapping and on comparison of proteins immunoprecipitated by monoclonal antibody against the EGF-receptor with proteins precipitated by polyclonal antibody prepared against the Mr 170,000 protein described here. The polyclonal antiserum recognized native and denatured EGF-receptor from human, rat, and mouse cells and should prove useful in studying EGF-receptor synthesis and function.     

Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431

We have prepared a conjugate of epidermal growth factor (EGF) and ferritin that retains substantial binding affinity for cell receptors and is biologically active. Glutaraldehyde-activated EGF was covalently linked to ferritin to produce a conjugate that contained EGF and ferritin in a 1:1 molar ratio. The conjugate was separated from free ferritin by affinity chromatography using antibodies to EGF. Monolayers of human epithelioid carcinoma cells (A-431) were incubated with EGF:ferritin at 4 degrees C and processed for transmission electron microscopy. Under these conditions, approximately 6 X 10(5) molecules of EGF:ferritin bound to the plasma membrane of each cell. In the presence of excess native EGF, the number of bound ferritin particles was reduced by 99%, indicating that EGF:ferritin binds specifically to cellular EGF receptors. At 37 degrees C, cell-bound EGF:ferritin rapidly redistributed in the plane of the plasma membrane to form small groups that were subsequently internalized into pinocytic vesicles. By 2.5 min at 37 degrees C, 32% of the cell-bound EGF:ferritin was localized in vesicles. After 2.5 min, there was a decrease in the proportion of conjugate in vesicles with a concomitant accumulation of EGF:ferritin in multivesicular bodies. By 30 min, 84% of the conjugate was located in structures morphologically identified as multivesicular bodies or lysosomes. These results are consistent with other morphological and biochemical studies utilizing 125I-EGF and fluorescein-conjugated EGF.     

Activation of the calcium channels of A-431 cells by epidermal growth factor

The cell-attached version of patch-clamp technique was used to search for calcium permeable channels in human carcinoma A-431 cells. With 100 mM CaCl2 in pipette, the inward currents were recorded having the mean unitary conductance of 2.8 pS and the reversal potential (obtained by linear extrapolation) equal to +25.5 mV. Application of the epidermal growth factor (EGF) into the bath extracellular solution produced a transient increase in probability for these channels to be open. The effect developed with a delay of about 20 seconds to last thereafter for 36 seconds (mean values). We propose that these channels mediate EGF-induced increase in concentration of cytosolic free calcium.     

Cytoskeletal changes in A-431 cells under the action of epidermal growth factor]

By the use of rhodamine-phalloidin, the distribution of actin in A-431 cells during the action of epidermal growth factor (EGF) has been studied. Changes in the pattern of staining are observed in 30-60 s after addition of the EGF. Microvilli and wrinkles are created on the cell surface. Following a 5-10 min action of EGF, rhodamine-phalloidin stained intensely ruffles and cell borders. After 60 min, the ruffling of cell surface disappeared, and actin was seen concentrating on the cell borders only. Electron microscopy of the EGF-treated A-431 cells lysed by Triton X-100 also revealed some vigorous fibrillar bunches on the cell edges.     

Presence of mannose phosphate on the epidermal growth factor receptor in A-431 cells

In this report, we demonstrate a novel post-translational modification of the epidermal growth factor (EGF) receptor. This modification involves the presence of phosphate, previously thought to exist only on amino acid residues in the EGF receptor, on oligosaccharides of the receptor. We have utilized several independent approaches to determine that mannose phosphate is present on the EGF receptor in A-431 cells. Following metabolic labeling with 32P, immunoisolation of the EGF receptor, and digestion with Pronase radioactivity was determined to be present on high mannose type oligosaccharides by concanavalin A chromatography. Also, after acid hydrolysis of in vivo 32P-labeled EGF receptor, radioactivity was detected that co-migrated with mannose 6-phosphate on two-dimensional thin layer electrophoresis. This radiolabeled material co-eluted with a mannose 6-phosphate standard from a high pressure liquid chromatography anion exchange column. Last, an acid hydrolysate of [3H]mannose-labeled EGF receptor contained two radiolabeled fractions, as analyzed by thin layer electrophoresis, and the radioactivity in one of these fractions was substantially reduced by alkaline phosphatase treatment prior to electrophoresis. These experiments indicate that the mature EGF receptor in A-431 cells contains mannose phosphate. This is a novel modification for membrane receptors and has only been reported previously for lysosomal enzymes and a few secreted proteins.     

Characterization of the metabolic turnover of epidermal growth factor receptor protein in A-431 cells

The metabolism of the receptor for epidermal growth factor (EGF) in A-431 cells has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. The rate of EGF receptor degradation (t1/2 = 20 hr) was faster than the rate of degradation of total cell protein (t1/2 = 52 hr). When EGF was added at the beginning of the chase, the half-life of prelabeled receptor decreased to 8.9 hr. This decrease was specific, as the level of total cellular protein and another plasma membrane protein, the transferrin receptor, were relatively unaffected by EGF. The carbohydrate portion of the receptor is degraded, in the presence or absence of EGF, at approximately the same rate as the protein moiety. The amount of EGF receptor protein in A-431 cells has been quantitated by radiolabeling total cellular protein and quantitating the immunoprecipitable receptor. The EGF receptor constitutes approximately 0.15% of the total cell protein in A-431 cells. These cells, therefore, have approximately 30 times more EGF receptor protein than fibroblasts. The EGF receptor constitutes an even higher proportion of 3H-glucosamine- or 3H-mannose-labeled macromolecules in A-431 cells, 1.5% or 5.2%, respectively. The EGF receptor from A-431 cells can easily be identified by submitting carbohydrate-labeled, solubilized cells to electrophoresis as described by Laemmli (1970).     

Ultrastructural changes in the plasma membrane of A-431 cells exposed to epidermal growth factor

The plasma membrane ultrastructural changes after the action of epidermal growth factor were studied in A-431 cells using freeze-fracture methods. The incubation with EGF (100 ng/ml, 0 degree C, 60 min) led to a decrease in density of intramembrane particles on the P surface of ventral cell membrane, while the number of coated pits increased there. The revealed effects of EGF may be related to direct consequences of EGF-receptor complex formation, because all the temperature dependent steps of its processing were blocked. The data obtained testify to an active involvement of the membrane ventral surface in the formation of cell response towards growth factors.     

Rapid stimulation of pinocytosis in human carcinoma cells A-431 by epidermal growth factor

Horseradish peroxidase (HRP) uptake was used to measure fluid-phase pinocytosis in monolayers of human epithelioid carcinoma cells (A-431). Histochemistry confirmed that cell-associated HRP was restricted to intracellular vesicles. Biochemical methods showed that HRP uptake in control cultures was directly proportional to the duration of exposure. The addition of low concentrations of epidermal growth factor (EGF) to the incubation media produced a 10-fold increase in the initial rate of pinocytosis. The EGF effect was rapid (within 30 s) but transient; the rate of pinocytosis returned to control levels within 15 min. Metabolic inhibitors reduced the EGF-stimulated rate of pinocytosis by greater than 90%. A conjugate of EGF and ferritin (F:EGF) was used to simultaneously compare the intracellular locations of EGF and HRP. Much of F:EGF was internalized in approximately 100-nm vesicles, while most of the HRP was located in much larger vesicles (range 0.1--1.2 micrometer) which also contained F:EGF. The tumor-promoter 12-0-tetradecanoyl-phorbol-13-acetate, which shares several biological activities with EGF, was also effective in stimulating an increase in the rate of pinocytosis.     

Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A-431 cells

Epidermal growth factor (EGF) stimulates the incorporation of 32Pi and [3H]inositol into phosphatidylinositol (5-10-fold) in A-431 cells. EGF also stimulates the incorporation of 32Pi into phosphatidic acid (up to 10-fold). These effects are attributed to an acceleration of the turnover of phosphatidylinositol as a consequence of the binding of EGF to its membrane receptor. The extent of the phosphatidylinositol response to EGF parallels the extent of hormone binding. The phosphatidylinositol response to EGF appears to be dependent on an influx of calcium since (a) external calcium is required for the enhancement of phosphatidylinositol turnover, (2) the accumulation of 45Ca by A-431 cells is stimulated by EGF, (3) blockage of calcium influx with LaCl3 inhibits stimulation of phosphatidylinositol turnover, and (4) calcium influx via ionophore A23187 is sufficient to stimulate phosphatidylinositol turnover. Since the binding, internalization, and degradation of 125I-labeled EGF in A-431 cells are unaffected by the omission of calcium from the medium, external calcium and phosphatidylinositol turnover are not necessary for the internalization and degradation of the EGF-receptor complex.     

Rapid rounding of human epidermoid carcinoma cells A-431 induced by epidermal growth factor

Epidermal growth factor (EGF) induces rapid rounding of A-431 human epidermoid carcinoma cells in Ca(++)-free medium. Cell rounding is not induced by a variety of other polypeptide hormones, antiserum to cell membranes, local anesthetics, colchicine, cytochalasin B, or cyclic nucleotides. However, trypsin, like EGF, induces rounding of A- 431 cells in the absence of Ca(++). Both trypsin- and EGF-induced rounding are temperature dependent, appear to be energy dependent, and are inhibited by cytochalasins, suggesting that the active participation of microfilaments in cell rounding. However, a medium transfer experiment suggests that EGF-induced rounding is not attributable to secretion of a protease, and a number of serine protease inhibitors have no effect on the EGF-induced rounding process. Cell rounding is not attributable to the slight stimulation by EGF of the release of Ca(++) that is observed in the Ca(++)-free medium, as stimulation of such release by the ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 neither induces cell rounding nor blocks EGF-induced rounding. Cells that have rounded up after treatment with EGF or trypsin spread out upon addition of Ca(++) to the medium, even in the continuing presence of EGF or typsin. Like the cell-rounding process, the cell-spreading process is temperature dependent, appears to be energy dependent, and is inhibited by cytochalasin B. Thus, EGF does not destroy the ability of the cell to spread; rather, in the presence of the EGF (or trypsin), cell spreading and the maintenance of the flattened state become dependent on external Ca(++). Because untreated cells remain flattened in the absence of Ca(++), the data suggest that EGF may disrupt Ca(++)-independent mechanisms of adhesion normally present in A-431 cells.     

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.     

Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells

Human epithelioid carcinoma A-431 cells are known to express unusually large numbers of receptors for the polypeptide hormone epidermal growth factor. The current studies demonstrate that this cell line also expresses 5- to 10-fold more low density lipoprotein (LDL) receptors per cell than either human fibroblasts or Chinese hamster ovary (CHO) cells. As visualized with an LDL-ferritin conjugate, the LDL receptors in A-431 cells appeared in clusters that were distributed uniformly over the cell surface, occurring over flat regions of the membrane as well as over the abundant surface extensions. Only 4% of the LDL receptors were located in coated pits. The LDL receptors in A-431 cells showed the same affinity and specificity as the LDL receptors in human fibroblasts and other cell types. In addition, they were subject to feedback regulation by sterols in the same manner as the LDL receptors in other cells. However, in contrast to other cell types in which the receptor-bound LDL is internalized with high efficiency, in the A-431 cells only a small fraction of the receptor-bound LDL entered the cell. In CHO cells approximately 66% of the LDL receptors were located over coated regions of membrane, and the efficiency of LDL internalization was correspondingly 10-fold higher than in A-431 cells. These findings support the concept that the rate of LDL internalization is proportional to the number of LDL receptors in coated pits and that the inefficiency of internalization in the A-431 cells is caused by a limitation in the ability of these cells to incorporate their LDL receptors into coated pits.     

Changes in the cell surface and cytoskeleton of A-431 cells under the action of epidermal growth factor

Certain changes in human carcinoma A-431 are found by scanning electron microscopy. The early cell response to growth factor (after 10 minutes) involves a disappearance of microvilli, an appearance of ruffles and rounded cells, along with a decrease in cell attachment to the substrate. The cell surface changes correlate with the state of cytoskeleton elements: the material stained with iron hematoxylin is accumulated in ruffle formation sites. Retractional fibrils filled with the cytoskeleton material result from a decrease in the cell area.     

Regulation of casein kinase II activity by epidermal growth factor in human A-431 carcinoma cells

In order to characterize more fully the mechanism by which casein kinase II is regulated in mammalian cells, the effect of epidermal growth factor (EGF) on the activity of the kinase in human A-431 carcinoma cells was examined. Treatment of cells with EGF prior to lysis consistently resulted in a transient 4-fold increase in the activity of cytosolic casein kinase II. Activity rose sharply between 20 and 30 min, peaked at approximately 50 min, and returned to basal levels by approximately 120 min. Similar results were obtained using the casein kinase II specific peptide substrate, Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu, or DNA topoisomerase II (which is specifically modified by the kinase in vivo and serves as a high affinity substrate in vitro) as the phosphate acceptor in assays. Identification of casein kinase II as the stimulated activity was confirmed by partial proteolytic mapping and phosphoamino acid analysis of modified topoisomerase II, by inhibition at nanomolar levels of heparin or micromolar levels of nonradioactive GTP, and by the ability to employ radioactive GTP as a direct phosphate donor. The EGF stimulation of casein kinase II was dependent on the availability of intracellular (but not extracellular) calcium. In addition, hormonal action was modulated by calcium/phospholipid-dependent protein kinase (protein kinase C). Casein kinase II stimulation did not require an increase in the concentration of the kinase, protein synthesis, the continual presence of a small effector molecule, or a direct interaction with the EGF receptor/tyrosine kinase. In contrast, hormonal activation of the kinase was dependent on the phosphorylation of casein kinase II or a terminal stimulatory factor.     

EGF-dependent association of 20S-proteasome and alpha-RNP particles with the epidermal growth factor receptor in A-431 cells

Here we demonstrate that the epidermal growth factor (EGF) induces association of prosomes (20S-proteasomes) with its receptor in A-431 cells. Additionally, ligand-dependent association of ribonucleoprotein particles (alpha-RNP), containing small ALU-like RNA, with the EGF receptor was demonstrated. A suggestion has been put forward on the involvement of prosomes and alpha-RNP in the EGF signal transmission to different stages of gene expression.     

Epidermal growth factor potentiates cyclic AMP accumulation in A-431 cells.

Epidermal growth factor (EGF) treatment of A-431 cells potentiates up to 5-fold the intracellular cyclic AMP (cAMP) accumulation induced by isoproterenol, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine (IBMX). EGF potentiates cAMP accumulation in several epithelial cell lines which overexpress the EGF receptor including A-431 cells, HSC-1 cells, and MDA-468 cells, and in the A-431-29S clone which expresses a normal complement of EGF receptors. Although EGF potentiates cAMP accumulation, EGF by itself does not measurably alter the basal level of cAMP. EGF rapidly enhances cAMP accumulation (within 1 to 3 min) in A-431 cells treated with these cAMP-elevating agents. EGF potentiation of cAMP accumulation does not reflect enhancement of beta-adrenergic receptor activation and is not a consequence of intracellular cAMP elevation or the concomitant activation of cAMP-dependent protein kinase. Since EGF potentiates accumulation of both intracellular and extracellular cAMP in isoproterenol-treated A-431 cells, EGF does not potentiate intracellular cAMP accumulation by inhibition of cAMP export. EGF potentiation of cAMP accumulation is pertussis toxin-insensitive and does not result from EGF inhibition of cAMP degradation in A-431 cells. These results demonstrate that EGF transmembrane signaling includes an interaction with a component of the adenylate cyclase system and that this interaction stimulates cAMP synthesis resulting in enhancement of cAMP accumulation.