共查询到20条相似文献,搜索用时 31 毫秒
1.
H S Shiah T Y Chen C M Chang J T Chow H J Kung J Hwang 《The Journal of biological chemistry》1992,267(33):24034-24040
Epidermal growth factor (EGF) was fused to the carboxyl end of a modified pseudomonas exotoxin A that has its toxin binding domain deleted. This chimeric toxin designated as PE(delta Ia)-EGF kills A431 cells through the EGF receptor-mediated pathway. In this study, we used a random mutagenesis approach to make point mutations on EGF, followed by replacing the wild type EGF in PE(delta Ia)-EGF with these EGF mutants. We have constructed 14 different PE(delta Ia)-EGFmutants, and examined their EGF receptor binding activity as well as their cytotoxicity to A431 cells. Our results showed that individual mutations of Val19 to Glu and Val34 to Asp in the EGF domain of PE(delta Ia)-EGFmutants resulted in an increase in the binding affinity to EGF receptor and cytotoxicity to A431 cells. On the other hand, individual mutations of His16 to Asp and Gly18 to Ala in the EGF domain of PE(delta Ia)-EGFmutants lead to a decrease in the binding affinity to EGF receptor and cytotoxicity to A431 cells. In addition, mutations of any of the cysteine residues of EGF in PE(delta Ia)-EGFmutants resulted in the loss of their binding activity to EGF receptor and a corresponding loss of their cytotoxicity. This study indicates that the cytotoxicity of PE(delta Ia)-EGFmutant to EGF receptor-bearing cells may be used as an indicator to screen mutations of EGF important in EGF-receptor interactions. 相似文献
2.
The rotational diffusion of epidermal growth factor (EGF) bound to its specific receptor on the surface of human carcinoma A431 cells was studied by means of time-resolved phosphorescence anisotropy measurements. The rotational mobility was measured on the total population of EGF receptors by using a saturating concentration of EGF conjugated with a phosphorescent label, erythrosin, or on the subpopulation of high-affinity EGF receptors by using a low concentration of labeled EGF. At 4 degrees C, the rotational correlation times for both the high-affinity and total (mostly low affinity) receptor populations were in the range of 60-100 microns. Elevation of the temperature to 37 degrees C resulted in a lengthening of the rotational correlation time of the total receptor population to 200-300 microns, confirming a previous study of receptor microaggregation. The high-affinity EGF receptors were completely immobilized at 37 degrees C (rotational correlation time greater than 500 microns). The data are consistent with a model involving association of the cytoskeleton with the high-affinity receptors at 37 degrees C, but not at 4 degrees C. 相似文献
3.
S Iwashita K Mitsui Y Shoji-Kasai M Senshu-Miyaike 《The Journal of biological chemistry》1990,265(18):10702-10708
While a cAMP-dependent protein kinase (protein kinase A) has been suggested to phosphorylate epidermal growth factor (EGF) receptor in vitro, both intrinsic and EGF- or potent phorbol tumor promoter-induced phosphorylation of EGF receptor were found to be depressed in human epidermoid carcinoma A431 cells by prior incubation of the cells with various protein kinase A activators (e.g. cholera toxin, forskolin, cAMP analogues, or a combination of prostaglandin E1 and 3-isobutyl-1-methylxanthine). Protein kinase A activators did not change significantly either the number of EGF receptors or their affinity for EGF. The tryptic phosphopeptide map of EGF receptors from cells treated with cholera toxin alone or cholera toxin followed by EGF revealed unique peptides whose serine phosphorylation was preferentially depressed. However, the catalytic subunit of protein kinase A phosphorylated no threonine and little serine in the EGF receptors in the plasma membranes of isolated A431 cells in vitro, while serine residues in an unidentified 170-kDa membrane protein(s) other than EGF receptor were heavily phosphorylated. Pretreatment of the cells with forskolin blocked 1,2-diacylglycerol induction by EGF; growth inhibition by nanomolar levels of EGF could be partially restored by the presence of forskolin. These results indicate that an increase in intracellular cAMP modulates the EGF receptor signal transduction system by reducing EGF-induced production of diacylglycerol without direct phosphorylation of EGF receptors by protein kinase A in A431 cells. 相似文献
4.
A direct radioimmunoassay for human epidermal growth factor receptor using 32P-autophosphorylated receptor 总被引:3,自引:0,他引:3
A simple and reproducible radioimmunoassay of the epidermal growth factor (EGF) receptor which uses 32P-labeled EGF receptor and anti-receptor monoclonal antibodies is reported. In vitro phosphorylation of A431 cell membranes with [gamma-32P]ATP in the presence of 20% dimethyl sulfoxide (which stimulates autophosphorylation of the EGF receptor) and 10 microM Na3VO4 (a potent inhibitor of phosphotyrosyl protein phosphatase) provides radiolabeled EGF receptor for radioimmunoassay without further purification. The most selective phosphorylation of the EGF receptor is achieved at ATP concentrations of 0.1-0.2 microM, which corresponds to the reported Km value for the autophosphorylation reaction of the EGF receptor (W. Weber, P.J. Bertics, and G.N. Gill, 1984, J. Biol. Chem. 259, 14631-14939). The incorporation of 32P into EGF receptors increases in proportion to the increase of ATP concentration up to 6 mol of labeled phosphate at 2.0 microM ATP. The label is entirely on tyrosine residues. The cell membranes can be stored at -70 degrees C for 3 months without loss of immunoreactivity and autophosphorylating activity. Standard curves for the radioimmunoassay were constructed employing either A431 cell membranes or whole cell homogenates containing a known amount of EGF receptor. The assay can detect 7 X 10(10) EGF receptor molecules or 20 ng of the receptor protein, and can quantitatively distinguish the difference in EGF receptor numbers between A431 cells and 29E2 and KB cells with 10-fold and 15-fold fewer receptors than A431 cells, respectively. 29E2 cells and KB cells express twofold more immunoreactive EGF receptors than EGF-binding sites. In contrast, A431 cells possess the same number of immunoreactive sites and receptor sites for EGF binding. To assess total EGF receptor expression, it is necessary to use a method which detects EGF receptors regardless of their intrinsic kinase activity, or capacity to bind EGF. This radioimmunoassay detects immunoreactive receptor molecules, even those which do not bind EGF. 相似文献
5.
Human esophageal carcinoma cells have fewer, but higher affinity epidermal growth factor receptors 总被引:2,自引:0,他引:2
Squamous cell carcinomas have recently been shown to contain increased numbers of epidermal growth factor (EGF) receptors. Since EGF has an important role in epithelial growth and differentiation, it is possible that modulation of its receptor may have an important role in neoplasia. In an attempt to further explore the relationship of EGF receptor expression to malignant transformation, we examined 14 squamous cell carcinoma cell lines of the esophagus for the number and affinity of EGF receptors. Seven cell lines were newly isolated by this laboratory and recently characterized. The seven additional cell lines were obtained from Japan (4 cell lines) and South Africa (3 cell lines). Surprisingly, we found that esophageal carcinomas contained lowered quantities of surface EGF receptors (2- to 100-fold) and that the affinity of the EGF receptor was increased (6- to 100-fold) when compared to normal esophageal epithelial cells. Moreover, the biologic response of esophageal carcinoma cells to EGF differed markedly from that of other squamous cell tumor cells exhibiting elevated numbers of receptors, such as A431 and SCC-15. Human esophageal carcinoma cells were maximally stimulated by the addition of 5 ng/ml of EGF, similar to normal esophageal keratinocytes, but in contrast to normal cells were not inhibited by the higher concentrations tested (up to 40 ng/ml). On the other hand, addition of any EGF to the medium (beyond that normally present in serum) was found to dramatically inhibit the growth of A431 and SCC-15 cells. Our findings indicate that squamous cell neoplasia is not dependent upon increased numbers of cell surface EGF receptors, that EGF receptor number may have a determinant role in EGF cell toxicity, and that the stimulatory response of cells to EGF may reflect a complex function of EGF receptor number, affinity, and occupancy. 相似文献
6.
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. 相似文献
7.
We have constructed hybrid proteins in which the toxic A chains of ricin or diptheria toxin have been linked to either asialofetuin, fetuin, or epidermal growth factor (EGF). Both ASF-RTA and ASF-DTA are potent toxins on cultured rat hepatocytes, cells that display the asialoglycoprotein receptor. Toxicity of these two compounds is restricted to hepatocytes and can be blocked by asialoglycoproteins but not the native glycoproteins or asialoagalactoglycoprotein derivatives, indicating that the toxicity of the conjugates is mediated by the hepatic asialoglycoprotein receptor. The EGF-RTA conjugate is an extremely potent toxin on cells that can bind the hormone, but is only poorly effective on cells that are unable to bind EGF. The EGF-DTA conjugate, in contrast, is unable to kill 3T3 cells and is at least two orders of magnitude less effective than EGF-RTA on A431 cells, a cell line with 1-2 X 10(6) EGF receptors per cell. However, when EGF-RTA and EGF-DTA were tested on primary liver hepatocyte cultures, which were susceptible to both ASF-RTA and ASF-DTA, both EGF conjugates were potent toxins. Sensitivity of the hepatocyte cultures to ricin toxicity increases slightly during a 52-hr culture period. In contrast, sensitivity to EGF-RTA and ASF-RTA decline dramatically during this period. Receptors for both ligands remain plentiful on the cell surface during this time. 相似文献
8.
Killing of cultured hepatocytes by conjugates of asialofetuin and EGF linked to the A chains of ricin or diphtheria toxin 总被引:2,自引:0,他引:2
A disulfide-linked conjugate between asialofetuin (ASF) and the toxic A chain (RTA) of ricin is as potent a toxin for cultured rat hepatocytes as our previously described conjugate between ASF and fragment A of diphtheria toxin (DTA). An RTA conjugate of epidermal growth factor (EGF) was a potent toxin for 3T3 cells. In contrast, EGF-DTA was essentially nontoxic for 3T3 cells. We have now examined the toxicity of EGF-RTA and EGF-DTA on cultured hepatocytes. The EGF-DTA conjugate, nontoxic to 3T3 cells, is also a potent toxin for hepatocytes. We also observed a decrease with time of culture in the sensitivity of hepatocytes to the ASF and EGF conjugates. This decrease is not a result of a decrease in EGF or asialoglycoprotein receptors. 相似文献
9.
When human epidermoid carcinoma A431 cells labeled with 32Pi to steady state specific activity were treated either with epidermal growth factor (EGF) or with active phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate, labeling of 160 kDa EGF receptors isolated by immunoprecipitation with monoclonal anti-EGF receptor IgG was increased 2- to 3-fold. These treatments produced no significant increase in 32Pi labeling of acid-precipitable material present in detergent extracts of the cells. Phosphoamino acid analysis of radiolabeled EGF receptors isolated from these cells revealed several differences: the relative abundance of phosphotyrosine in EGF receptors was increased in cells treated with EGF, but decreased in cells treated with TPA; the overall relative abundance of phosphothreonine in EGF receptors was decreased in cells treated with EGF, but remained constant within the limits of experimental detection in cells treated with TPA. Two-dimensional mapping of the radiolabeled phosphopeptides produced from EGF receptors isolated by immunoprecipitation and treated with trypsin revealed 9 independent labeled regions, 2 of which contained phosphothreonine and were present only in EGF- or TPA-treated cells. These two phosphopeptide regions were more highly labeled in cells treated with TPA than with EGF. 相似文献
10.
Association of ganglioside-protein conjugates into cell and Sendai virus. Requirement for the HN subunit in viral fusion 总被引:7,自引:0,他引:7
We have prepared several electron and light microscopic labels of epidermal growth factor (EGF) to analyse the morphologic features of its binding and internalization by cultured cells. These include a ferritin conjugate of EGF, a covalent conjugate of EGF and horseradish peroxidase (EGF-HRP), a colloidal gold marker system using EGF-HRP as a primary antigen, and a covalent complex of EGF with rhodamine-labelled lactalbumin. All of the light and electron microscopic labels showed similar patterns of binding. EGF initially bound to diffusely distributed cell surface receptors at 4 degrees C. The EGF-receptor complexes clustered into clathrin-coated pits on the cell surface only when the temperature was raised to 37 degrees C. In KB and Swiss 3T3 cells, this was followed by rapid internationalization into receptosomes, compartmentalization into the Golgi system, clustering in the clathrin-coated regions of the Golgi, and finally delivery into lysosomes from the Golgi. This general pathway was seen in Swiss 3T3 cells which have a low number of EGF receptors, KB cells which have a moderate number of receptors and A431 cells that have a high number of receptors. However, the ruffling activity induced in A431 cells by EGF produced some internalization through macropinosomes, making the pathway of entry more difficult to evaluate. Double label experiments showed that EGF is internalized together with alpha 2-macroglobulin and adenovirus particles. These data clarify the route of entry of EGF in different cell types using multiple labels, and shows that it enters cells through the same coated pit entry pathway as most other ligands previously examined. 相似文献
11.
J A Fernandez-Pol 《The Journal of biological chemistry》1985,260(8):5003-5011
A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells, denoted 2D1-IgM, was generated after fusion of immunized BALB/c mouse spleen cells with SP2/0-Ag14 myeloma cells. Specific binding of 2D1-IgM to the A431 cell-surface receptor for EGF was demonstrated by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Scatchard analysis of 125I-EGF binding to A431 cells demonstrated that 2D1-IgM treatment did not change the number of EGF receptors, but caused an increase in the affinity of EGF receptors from a population of low affinity to a uniform population of high affinity. Like EGF, 2D1-IgM induced phosphorylation of EGF receptors and EGF receptor clustering. As in the case of EGF, a biphasic growth response with stimulation of DNA synthesis at low and inhibition at high concentrations of 2D1-IgM was evident in A431 cells. The intrinsic "EGF-like" bioactivity of 2D1-IgM was enhanced by the presence of EGF. These results suggest that the binding of 2D1-IgM to the EGF receptor at a different site from that to which EGF binds can initiate an effective EGF-like biological response; and the EGF-like biological effects of 2D1-IgM may be mediated by a population of high affinity EGF receptors which may be involved in the control of cellular growth. 相似文献
12.
Direct visualization and quantitative analysis of epidermal growth factor-induced receptor clustering 总被引:3,自引:0,他引:3
N van Belzen P J Rijken W J Hage S W de Laat A J Verkleij J Boonstra 《Journal of cellular physiology》1988,134(3):413-420
Several observations have indicated that clustering of growth factor receptors plays an important role in the action of growth factors. In this investigation, we have used the label fracture method to study the effects of epidermal growth factor (EGF) on the lateral distribution of its receptors in A431 epidermoid carcinoma cells. This method allows a direct visualization of immunogold-labeled plasma membrane receptors on ultrastructural level and in addition permits an quantitative analysis of their lateral distribution. EGF receptors were immunogold-labeled according to standard procedures with the monoclonal anti-EGF receptor antibody 2E9 (IgG1), which binds to the EGF receptor in a 1:1 ratio. In the absence of EGF, EGF receptors located on the surface of A431 cells were found to be clustered, as deduced from Poisson variance analysis (p less than 0.001). Following treatment of A431 cells with EGF, receptor clustering increased rapidly, reaching the maximum within 10 min. Maximal clustering was maintained for 1 h, after which the lateral distribution of receptors returned to the control situation within another hour. 相似文献
13.
Epidermal growth factor receptors in the human glioblastoma cell line SF268 differ from those in epidermoid carcinoma cell line A431 总被引:1,自引:0,他引:1
M Westphal G R Harsh M L Rosenblum R G Hammonds 《Biochemical and biophysical research communications》1985,132(1):284-289
Two established human tumor cell lines, epidermoid carcinoma line A431 and glioblastoma line SF268, were studied to compare the interaction of each with epidermal growth factor (EGF). SF268 cells bound [125I] EGF with 35-40 fold higher affinity than did the A431 cells. The EGF binding sites of both lines were photoaffinity labeled using 2,4-NAPS-[125I] EGF, a photoreactive derivative of EGF. Extracts of photolysed cells analyzed by SDS-PAGE showed a difference between the two cell lines in the high molecular weight component corresponding to the EGF receptor. EGF in a dose range from 0.3-200 nM had no effect on thymidine incorporation by SF268 cells, whereas thymidine incorporation by A431 cells was markedly inhibited by EGF. 相似文献
14.
Relation of epidermal growth factor receptor concentration to growth of human epidermoid carcinoma A431 cells 总被引:19,自引:0,他引:19
T Kawamoto J Mendelsohn A Le G H Sato C S Lazar G N Gill 《The Journal of biological chemistry》1984,259(12):7761-7766
The relation between the concentration of epidermal growth factor (EGF) receptor/kinase and effects of EGF on cell proliferation has been studied using variant A431 cells and antagonist anti-EGF receptor monoclonal antibodies. Clonal A431 cell variants selected for escape from the EGF-mediated growth inhibition of parental A431 cells all have reduced concentrations of EGF receptor/kinase; Harvey sarcoma virus-transformed A431 cells, which have escaped from EGF-mediated growth inhibition, also have reduced EGF receptors. Three clonal variants which have reacquired EGF-mediated growth inhibition have 2- to 4-fold more EGF receptor than their respective parent variant. A biphasic response with stimulation at low and inhibition at high concentrations of EGF was especially evident in revertants of clone 29. Three separate antagonist monoclonal anti-EGF receptor antibodies block the growth inhibitory effects of EGF and uncover EGF-mediated growth stimulation. These studies indicate that in A431 cell variants a continuum of ligand-activated EGF receptors determines proliferative responses from low concentrations of active receptors under basal conditions to intermediate concentrations causing growth stimulation to high concentrations, causing inhibition of cell proliferation. 相似文献
15.
J A Berkers P M van Bergen en Henegouwen A J Verkleij J Boonstra 《Biochimica et biophysica acta》1990,1052(3):453-460
Epidermoid carcinoma A431 cells exhibit two classes of epidermal growth factor (EGF) receptors as deduced from Scatchard analysis. Steady-state binding of EGF to isolated A431 membranes indicated, however, the presence of only one class of EGF binding sites. The apparent dissociation constant (Kd) of these sites was approx. 0.45 nM which is similar to that of the high-affinity receptor of intact A431 cells. These results suggest that the vesicle receptor population consists only of high-affinity receptors. However, further studies indicated that the binding sites were similar to the low-affinity class, since binding of EGF could be blocked entirely by 2E9, a monoclonal anti-EGF receptor antibody which is able to inhibit specifically EGF binding to low-affinity receptors in A431 cells. The difference in affinity of the receptors in membrane vesicles as compared to intact cells may be explained by differences in biophysical parameters such as diffusion-limited EGF binding and receptor distribution. Based upon these considerations, it is concluded that membrane vesicles of A431 cells contain one class of EGF receptors which are apparently identical to the low-affinity receptors of intact cells. 相似文献
16.
Properties of a monoclonal antibody to epidermal growth factor receptor with implications for the mechanism of action of EGF. 总被引:10,自引:3,他引:7
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A monoclonal antibody, EGR/ G49 , raised against the receptor for epidermal growth factor (EGF) present in A431 cells inhibits EGF binding by decreasing the affinity of the major population of low affinity receptors while leaving the minor high affinity population relatively unperturbed. The antibody, which binds to a carbohydrate determinant at a site distinct from the EGF binding site, induces clustering and internalisation of the receptor without stimulating the EGF receptor-kinase or affecting its ability to undergo stimulation by EGF. It is toxic to A431 cells and induces morphological changes similar to those seen when these cells are challenged with EGF in the concentration range 1-10 nM. These results suggest that high and low affinity EGF receptors can be distinguished and that they may serve different functions. 相似文献
17.
Bachran C Sutherland M Heisler I Hebestreit P Melzig MF Fuchs H 《Experimental biology and medicine (Maywood, N.J.)》2006,231(4):412-420
Saponins are a group of plant glycosides consisting of a steroid or triterpenoid aglycone to which one or more sugar chains are attached. They exhibit cell membrane-permeabilizing properties and, thus, have been investigated for their therapeutic potential. Recently, at a non-permeabilizing concentration saponinum album from Gypsophila paniculata L. has been described to enhance the cytotoxicity of a chimeric toxin in a cell culture model. To elucidate whether this enhancing effect is also mediated by other saponins, we analyzed the ability of seven different saponins to enhance the cytotoxicity of a targeted chimeric toxin. The chimeric toxin is composed of saporin, a plant ribosome-inactivating toxin, a cleavable adapter, and human epidermal growth factor (EGF). Cytotoxicity on EGF receptor (EGFR)-bearing cells was analyzed both alone and after combined application of saponin and chimeric toxin. Only two of the tested saponins, quillajasaponin and saponinum album, enhanced cytotoxicity by more than 1,000-fold, whereas the enhancement factors of the other saponins were only approximately 10-fold. In contrast to saponinum album, quillajasaponin enhanced the cytotoxicity both on control cells lacking EGFR and on target cells, indicating that, in this case, the enhancement is not target cell receptor specific. This is also the case for some of the saponins with low enhancement factors. Saponinum album resulted in a more than 13,600-fold receptor-specific enhancement, decreasing the 50% inhibitory concentration (IC(50)) from 2.4 nM to 0.18 pM, which renders it the best option to promote saporin-3-based drug uptake while retaining specificity for the EGFR. 相似文献
18.
P J Parker S Young W J Gullick E L Mayes P Bennett M D Waterfield 《The Journal of biological chemistry》1984,259(15):9906-9912
A431 cells have been used as an immunogen for generating monoclonal antibodies against the epidermal growth factor (EGF) receptor. Two immunoglobulin M and eight immunoglobulin G3 anti-EGF receptor antibodies were cloned. All ten antibodies immunoprecipitated biosynthetically labeled mature A431 cell EGF receptor and were able to recognize the receptor in Western blotting. However, none of the antibodies immunoprecipitated precursor polypeptides of the A431 cell EGF receptor, neither did they recognize EGF receptors from human foreskin fibroblasts, human placenta, nor a human-mouse hybrid cell expressing EGF receptor. The antibodies were found to bind to glycolipids from A431 cells and it was shown that the determinant involved was the blood group A antigen. It appears that this determinant is present on both the EGF receptor and glycolipids of A431 cells but is not expressed on EGF receptors from other human cells tested. One of the monoclonal antibodies raised was used for immunoaffinity purification of the EGF receptor. The procedure took advantage of the carbohydrate nature of the antigenic determinant by employing sugar-specific elution. The mild conditions permitted the purification of A431 cell EGF receptor (70-80% pure) that possessed an intrinsic EGF-stimulated tyrosine kinase activity with a specific activity of about 20 nmol/min/mg. 相似文献
19.
Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431 总被引:38,自引:30,他引:38
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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. 相似文献
20.
P M van Bergen en Henegouwen J C den Hartigh P Romeyn A J Verkleij J Boonstra 《Experimental cell research》1992,199(1):90-97
In this paper we describe our investigations on the association of receptors for the epidermal growth factor (EGF) with the cytoskeleton of A431 cells. In order to determine which filamentous system the EGF receptors are associated to, the cytoskeletal fraction to which these receptors bind was isolated. Second, the possible colocalization of EGF receptors with different cytoskeletal elements was examined in A431 cells. By selective extractions of the A431 cytoskeletons, it is shown that more than 90% of the cytoskeleton-associated EGF receptors are removed from the cytoskeletons together with the actin filamentous system. During several cycles of poly- and depolymerization of actin isolated from A431 cells, the EGF receptor precipitates together with the actin containing filaments, indicating that EGF receptors are able to bind in vitro to actin filaments. With immunofluorescence studies we show that EGF receptors especially colocalize with actin filaments. These results demonstrate that the EGF receptor is associated specifically with actin filaments in A431 cells. 相似文献