High affinity epidermal growth factor receptors on the surface of A431 cells have restricted lateral diffusion

Rhodamine-labelled epidermal growth factor (Rh-EGF) was shown to bind to A431 cells grown at low density both to a small number of high affinity receptors (KD = 2.8 X 10(-10) M; fraction of total binding sites approximately 0.12) and also to a large number of low affinity receptors (KD = 4 X 10(-9) M; fraction of total binding sites approximately 0.88). Measurements of the lateral diffusion of EGF receptors on the cell surface were made using Rh-EGF and the technique of fluorescence photobleaching recovery. The high affinity receptors (labelled with 1.6 X 10(-10) M Rh-EGF, 5% of EGF binding sites occupied) did not show lateral mobility over the temperature range 3 degrees-37 degrees C. The low affinity receptors (labelled with 2.4 X 10(-7) M Rh-EGF, 90% of EGF sites occupied) showed at least 75% fluorescence recovery after photobleaching, and lateral diffusion coefficients of approximately 2 X 10(-10) cm2/s. These results show that the two populations of EGF receptors defined by binding studies differ in their freedom to diffuse laterally. The observation that the high affinity receptors are immobile indicates that lateral diffusion of receptors, at least over a distance of a few hundred nanometres or more, may not be required for the action of low concentrations of EGF.     

Average density and size of microclusters of epidermal growth factor receptors on A431 cells.

For some hormone receptors, the early events of signal transduction depend on their molecular arrangement and interactions at the cell surface. An understanding of the mechanism of signal transduction in general needs a careful analysis of the receptor distribution. Here, we present the first quantitative measurement of epidermal growth factor receptor distribution on A431 cells obtained by scanning fluorescence correlation spectroscopy. Prior to epidermal growth factor binding, the A431 cell membrane presents an average surface density of 7.7-8.4 microclusters/microns 2, each containing an average of 130 receptors.     

Actin-binding proteins involved in the capping of epidermal growth factor receptors in A431 cells.

A capping process of epidermal growth factor receptors (EGF-Rs) was used for the study of the relation between the receptors and the actin-binding proteins (spectrin, vinculin, annexin I) that may be involved in EGF-R-cytoskeleton interaction. In intact, adherent A431 cells, EGF-Rs were diffusively distributed on the cell surface. Spectrin, vinculin, and annexin I were located beneath the plasma membrane. An abundance of EGF-Rs as well as submembrane proteins was observed in regions of membrane ruffles and cell-cell contacts. Annexin I was localized also in cytoplasm being attached to filamentous structures surrounding the nucleus and extending to the cell periphery. Under polyvalent ligand treatment, EGF-Rs of adherent cells were aggregated on one side of the cell. Spectrin, vinculin, and annexin I dislocated together with EGF-Rs and were concentrated under plasma membrane at regions where cap formation took place. In suspended A431 cells only spectrin was located under the plasma membrane whereas annexin I and vinculin were diffusively distributed through the cells. During cap formation only spectrin was colocalized with EGF-Rs. The results confirmed the major role of spectrin as a receptor-microfilament linking protein.     

Nonrandom distribution of epidermal growth factor receptors on the plasma membrane of human A431 cells

The localization of epidermal growth factor (EGF) receptors over the plasma membranes of human epidermoid carcinoma A431 cells was analyzed at the electron microscopic level using surface replica techniques and conventional thin sections, in combination with immunocytochemistry. Immunolabeling was performed using two distinct monoclonal antibodies directed against the extracellular portion of the receptor, followed by protein A-colloidal gold conjugates. Unexpectedly, with the first monoclonal antibody used, the distribution of the receptors in both unfixed and glutaraldehyde-fixed cells was clearly regionalized, showing a preferential localization of the immunolabeling at the cell periphery as well as over the areas rich in microvilli and in coated and uncoated pits. A similar pattern of distribution was observed also with the other monoclonal antibody, but only when the cells were fixed with glutaraldehyde before immunolabeling. Treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate modifies this distribution, inducing a more disperse pattern. Our observations suggest that a minor group of EGF receptors, which may represent the high-affinity receptors, presents a regional distribution, similar to that described for typical recycling receptors.     

Recycling of epidermal growth factor in A431 cells

The fate of epidermal growth factor (EGF) after internalization by A431 cells was studied. First, cells containing 125I-EGF-receptor complexes in endosomes were obtained. Subsequent incubation of the cells at 37 degrees C resulted in the recycling of 125I-EGF from endosomes to the cell surface in the receptor-bound state and the gradual release of recycled ligand into the medium. The excess of unlabeled EGF blocked both rebinding and re-internalization of recycled 125I-EGF to produce enhanced accumulation of ligand in the medium. The rate of recycling was shown to be much higher than that of EGF degradation.     

Biosynthesis of the epidermal growth factor receptor in A431 cells.

A monoclonal antibody R1 against the human epidermal growth factor receptor has been used to study biosynthesis in the carcinoma cell line A431. Two glycoproteins of apparent mol. wts. 95 000 and 160 000 were immunoprecipitated from cells labelled for short times with [35S]methionine or [3H]mannose. Pulse-chase studies show the 160 000 mol. wt. glycoprotein to be a precursor of the 175 000 mol. wt. receptor, but do not establish a precursor role for the 95 000 mol. wt. glycoprotein. Limited proteolysis, peptide mapping, endoglycosidase digestion and the use of monensin and tunicamycin show that the 95 000 mol. wt. glycoprotein is structurally related to the 160 000 mol. wt. glycoprotein and that both glycoproteins have approximately 22 000 - 28 000 mol. wt. of oligosaccharide side chains. Monensin blocks conversion of the 160 000 to the 175 000 mol. wt. mature receptor, a process which involves complexing several of its N-linked oligosaccharide chains. Pulse-chase studies showed that an immunoprecipitable polypeptide of 115 000 mol. wt., or 95 000 mol. wt., in the presence of monensin, was secreted into the medium at late chase times. The possible mechanisms for the origins of all the receptor-related polypeptides are discussed.     

Electric field-induced redistribution and postfield relaxation of epidermal growth factor receptors on A431 cells

The lateral mobility of the epidermal growth factor (EGF) receptor in the plane of the plasma membrane of cultured A431 cells was investigated using direct and indirect fluorescent probes to measure the generation and relaxation of electric field-induced receptor asymmetry. A steady electric field of 15 V/cm for 30 min at 23 degrees C induced a redistribution of the unoccupied EGF receptor such that there was approximately a three-fold higher concentration of receptors at the cathode-facing pole. After termination of the field, the unoccupied receptors back diffused at 37 degrees C with a rate corresponding to a diffusion coefficient of 2.6-3.5 X 10(-10) cm2/s. No diffusion was detected at 4 degrees C. Formation of the hormone-receptor complex is known to induce receptor clustering and internalization. By inhibiting internalization with metabolic poisons, we were able to study the cell surface mobility of clusters of the hormone-receptor complex. The same degree of asymmetry was induced when the occupied receptor was exposed to an electric field and the rate of back diffusion of clusters of the hormone-receptor complex corresponded to a diffusion coefficient of 0.68-0.95 X 10(-10) cm2/s. Although the unoccupied receptor is somewhat more mobile than the hormone-receptor complex, it was still far less mobile than one would predict for an unconstrained protein imbedded in a phospholipid bilayer.     

Participation of membrane skeleton proteins in aggregation of epidermal growth factor receptors in A431 cells.

Polyclonal antibody against alpha-spectrin of chicken erythrocytes was prepared. This antibody as well as anti-vinculin and anti-annexin I and II, were used for localization of the antigens in A431 cells during translocation of epidermal growth factor receptors (EGF-Rs) on cell surface. During aggregation of EGF-Rs only spectrin and actin aggregates colocalized with the "capped" receptors in adherent as well as in suspended cells. Physiological implication of spectrin involvement in EGF-Rs redistribution in A431 cells is discussed.     

Compartmentalization dynamics of the epidermal growth factor in A431 cells

Dynamics of compartmentalization of epidermal growth factor (EGF) in human carcinoma A431 cells during the first hour after initiation of endocytosis was examined by methods of the organelle fractionation on a 20% Percoll gradient and of the microfluorimetric visualization of endocytosis of rhodamine-labeled EGF (EGF-R). EGF was revealed in small vesicles localized in the peripheral region of cytoplasm in a few minutes after endocytosis initiation. During centrifugation in Percoll these vesicles (endosomes), with an average density of 1.038 g/ml, were seen co-sedimented with Golgi membranes. By one hour after initiation of endocytosis, EGF-R was accumulated in perinuclear zone, in a trans-Golgi region, as numerous big luminous centres that were apparently MB-endosomes and had the same density in Percoll as did small peripheral endosomes. Such centres appeared in several cells already within 5-10 minutes. In A431 cells EGF did not reach lysosomes within 60 minutes, because no accumulation of 125I-EGF was shown in lysosome corresponding regions of Percoll gradient (average density 1.070 g/ml).     

Epidermal growth factor induces tyrosine phosphorylation of epidermal growth factor receptors not occupied with ligand in intact A431 cells.

The mechanism of epidermal growth factor receptor (EGF-R) autophosphorylation in intact A431 cells was studied. We detected epidermal growth factor (EGF) induced tyrosine phosphorylation of EGF-R not occupied with ligand. Cell monolayers were subjected to irradiation after incubation with photoreactive derivative of EGF and uncoupled EGF was extracted by acidic treatment. Subsequent immunoprecipitation with antiphosphotyrosine antibodies resulted in precipitation of both EGF-R complexes with EGF and EGF-R with unoccupied ligand-binding site. The fact of precipitation of EGF-R with unoccupied ligand-binding site in conjunction with our finding of rapid dephosphorylation of EGF-R after EGF extraction by acidic treatment, strongly supports the interpretation that cross-phosphorylation of EGF-R may take place in intact cells.     

The relationship between epidermal growth factor receptors and the terminal differentiation of A431 carcinoma cells

Escherichia coli harboring the gene coding for human interleukin-2 (IL-2) produced methionyl IL-2 (Met-IL-2) having an additional methionine residue at the amino terminus as well as IL-2 starting with the amino terminal alanine. IL-2 and Met-IL-2 were copurified from a cell-free extract. It was difficult to separate these two molecular species from each other because of the similarities of their physico-chemical characteristics. We found that the isoelectric points of IL-2 and Met-IL-2 were slightly but significantly different and succeeded in separating these two molecular species by utilizing the difference of their isoelectric points. The isoelectric points of IL-2 and Met-IL-2 thus obtained were determined to be 7.7 and 7.5, respectively. The in vitro specific activities of these two species were the same and similar to that of natural human IL-2 derived from peripheral blood lymphocytes.     

Vaccinia virus growth factor stimulates tyrosine protein kinase activity of A431 cell epidermal growth factor receptors.

Infection of A431 cells with vaccinia virus, or exposure to a mitogenic polypeptide secreted by vaccinia virus-infected cells, induces tyrosine phosphorylation of epidermal growth factor receptors.     

Single-molecule analysis of epidermal growth factor binding on the surface of living cells

Global cellular responses induced by epidermal growth factor (EGF) receptor (EGFR) occur immediately with a less than 1% occupancy among tens of thousands of EGFR molecules on single cell surface. Activation of EGFR requires the formation of a signaling dimer of EGFR bound with a single ligand to each molecule. How sufficient numbers of signaling dimers are formed at such low occupancy rate is still not known. Here, we have analyzed the kinetics of EGF binding and the formation of the signaling dimer using single-molecule imaging and mathematical modeling. A small number of EGFR on the cell surface formed dimeric binding sites, which bound EGF two orders of magnitude faster than the monomeric binding sites. There was a positive cooperative binding of EGF to the dimeric binding sites through a newly discovered kinetic intermediate. These two mechanisms facilitate the formation of signaling dimers of EGF/EGFR complexes.     

Epidermal growth factor treatment of A431 cells alters the binding capacity and electrophoretic mobility of the cytoskeletally associated epidermal growth factor receptor

The epidermal growth factor (EGF) receptor interacts with structural elements of A431 cells and remains associated with the cytoskeleton following extraction with nonionic detergents. Extraction of cells with 0.15% Triton X-100 resulted in detection of only approximately 40% of the EGF binding sites on the cytoskeleton. If the cells were exposed to EGF prior to extraction, approximately twofold higher levels of low-affinity EGF binding sites were detected. The difference in number of EGF binding sites was not a consequence of differences in numbers of EGF receptors associated with the cytoskeleton; equal amounts of 35S-labeled receptor were immunoprecipitated from the cytoskeletons of both control and EGF-treated cells. The effect of EGF pretreatment on binding activity was coincident with a change in the mobility of the receptor from a doublet of Mr approximately 160-180 kDa to a single sharp band at 180 kDa. The alteration in receptor mobility was not a simple consequence of receptor phosphorylation in that the alteration was not reversed by alkaline phosphatase treatment, nor was the shift produced by treatment of the cells with phorbol ester. The two EGF receptor species demonstrated differential susceptibility to V8 proteinase digestion. The EGF-induced 180 kDa species was preferentially digested by the proteinase relative to the 160 kDa species, indicating that EGF binding results in a conformational change in the receptor. The EGF-mediated preservation of binding activity and altered conformation may be related to receptor oligomerization.     

Partition of epidermal growth factor receptors on freeze-fractured plasma membranes of A431 cells is affected by the ligand

The fracture immunolabel technique, which permits assessment of the partition of transmembrane proteins with the inner or outer leaflets of the freeze-fractured membrane, was used to analyze the behavior on fracture of epidermal growth factor (EGF) receptors over the plasma membranes of A431 cells. The receptors partition mainly with the outer leaflet of the freeze-fractured plasma membranes, whereas they become associated with the inner leaflet when they are occupied by the ligand. This modified partition is even more evident after receptor clustering induced by incubation with EGF at 37 degrees C. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases the number of receptors over both inner and outer leaflets. An effect similar to that induced by the ligand is obtained when receptor aggregation is achieved using anti-receptor monoclonal antibodies (MAb). The modified partition therefore indicates receptor activation and appears to be a consequence of receptor cross-linking rather than to reflect a conformational change of the receptor molecule. Parallel immunolabeling with anti-phosphotyrosine antibodies of freeze-fractured EGF-treated A431 cells reveals that the receptors, when activated, are associated only with the inner leaflet of the plasma membrane.     

Transforming growth factor beta modulates phosphorylation of the epidermal growth factor receptor and proliferation of A431 cells.

Transforming growth factor beta (TGF-beta) increased the phosphorylation of the epidermal growth factor (EGF) receptor and inhibited the growth of A431 cells. Incubation with TGF-beta induced maximal EGF receptor phosphorylation to levels 1.5-fold higher than controls. Phosphorylation increased more prominently (4-5-fold) on tyrosine residues as determined by phosphoamino acid analysis and antiphosphotyrosine antibody immunoblotting. The kinase activity of EGF receptor was also elevated 2.5-fold when cells were cultured in the presence of TGF-beta. The antiproliferative effect of TGF-beta on A431 cells was accompanied by prolongation of G0-G1 phase and by morphological changes. TGF-beta augmented the growth inhibition of A431 cells which could be induced by EGF. In parallel, the specific EGF-induced increase in total phosphorylation of the EGF receptor was also augmented in the presence of TGF-beta. In cells cultured with TGF-beta, the phosphorylation of EGF receptor tyrosines induced by 20-min exposure to EGF was further increased 2-3-fold, suggesting additive effects upon receptor phosphorylation. EGF receptor activation by TGF-beta is characterized by kinetics quite distinct from that induced by EGF and therefore appears to take place through an independent mechanism. The TGF-beta-induced elevation in the phosphorylation of the EGF receptor may have a role in the augmented growth inhibition of A431 cells observed in the presence of EGF and TGF-beta.     

Regulation of protein breakdown by epidermal growth factor in A431 cells

Addition of epidermal growth factor (EGF) to cultures of A431 human epidermoid carcinoma cells produces an increase in the rate of intracellular protein breakdown that cannot be accounted for by increased proteolysis in lysates from EGF-treated cells. In support of this observation, inhibition of protein synthesis with cycloheximide does not reduce the EGF response in cell monolayers. On the other hand, inhibitors of lysosomal proteolytic function such as leupeptin, vinblastine and especially the weak base, ammonia, are able to block the ability of EGF to increase protein breakdown. Additional results suggest that the EGF effect is mediated via a stimulation of autophagy. First, the autophagocytosis inhibitor, 3-methyladenine, reduces the EGF response, and second, the ability of insulin to inhibit protein breakdown by preventing the formation of autophagic vacuoles is overcome by EGF. Moreover, the actions of inhibitors and competing hormones are similar to those reported for glucagon, a hormone known to increase autophagy. The EGF response on protein breakdown persists for at least 6 h after thorough washing of the A431 monolayers. This result contrasts with the rapid reversal of EGF effects in other cell lines. Examination of the fate of bound EGF in cells washed and incubated for 2 h at 37 degrees C shows that some 500-fold more EGF per mg protein is retained on the surface of A431 cells compared to AG2804-transformed fibroblasts, a difference which probably explains the unusual persistence of the EGF effect on protein breakdown.     

The effect of the lysosomotropic agent primaquine on the endocytosis of the epidermal growth factor receptors in A431 cells]

It has been shown elsewhere that the epidermal growth factor (EGF) in A431 cells can recycle in receptor-bound state (Teslenko et al., 1987; Sorkin et al., 1989, 1991). Present study deals with the action of primaquine, a lysosomotropic agent, on EGF-receptor complexes (EGF-RC). By the method of indirect immunofluorescence with anti-EGF-R monoclonal antibody it is found that following a 1 h incubation of cells at 37 degrees C in the presence of EGF a bright staining of endosomes appears in the intranuclear region, while after incubation of the cells at 4 degrees only margins of cells are stained. Such a pattern of fluorescence is peculiar of endocytosis in A431 cells. When the cells were incubated in the presence of a 0.3 mM primaquine for 1 h, the immunostaining is changed: bright compact spot in the para-Golgi region appeared. The effect of primaquine is reversible. When the cells after preincubation with EGF were incubated in the absence of EGF for 3 h at 37 degrees C, the staining of cell margins could be observed again, demonstrating the recycling of EGF-RC. Under similar conditions of cell incubation, but in the presence of primaquine, the staining of the para-Golgi region was not changed. In the experiments with 125I-EGF it was shown that intracellular accumulations of 125I-EGF were maintained when the cells were incubated in the presence of 0.3 mM primaquine. It is concluded that primaquine inhibits the recycling of EGF-R in A431 cells.     

Tumor necrosis factor regulates tyrosine phosphorylation on epidermal growth factor receptors in A431 carcinoma cells: evidence for a distinct mechanism.

Previous studies of tumor necrosis factor (TNF) action on tumor cells revealed a possible role for tyrosine phosphorylation of epidermal growth factor (EGF) receptor in the growth-regulatory activities of this cytokine (N. J. Donato, G. E. Gallick, P. A. Steck, and M. G. Rosenblum, J. Biol. Chem., 264: 20474-20481, 1989). EGF receptor immunoprecipitated from [32P] phosphate-equilibrated A431 cells demonstrated that TNF treatment resulted in both a time- and concentration-dependent stimulation of EGF receptor phosphorylation, which was maximal (approximately 3-fold) after 10-20 min of TNF exposure (10 nM). Incubation of A431 cells with an equivalent concentration of EGF resulted in similar stimulation of EGF receptor phosphorylation, albeit at different phosphotyrosine levels. Antiphosphotyrosine immunoblot analysis confirmed these results but suggested that the extent and kinetics of TNF-induced tyrosine phosphorylation were distinct from those obtained in EGF-treated cells. Resolution of tryptic phosphopeptides from EGF receptor demonstrated that TNF-induced phosphorylation of EGF receptor was similar, but not identical, to profiles obtained from EGF-treated cells and distinct when compared to the actions of phorbol ester. Unlike EGF, TNF was unable to directly stimulate EGF receptor tyrosine kinase activity in membranes prepared from A431 cells. In addition, TNF treatment had no significant effect on either the high- or low-affinity ligand-binding sites on EGF receptor and did not alter the kinetics or extent of ligand-induced internalization of EGF receptors. However, EGF receptor biosynthesis was consistently increased upon prolonged treatment with TNF (4-12 h). Our results suggest that TNF regulates both phosphorylation and biosynthesis of EGF receptor in a manner distinct from that of both EGF and phorbol ester, and studies of the differential phosphorylation of EGF receptor may aid in understanding the molecular mode of TNF action.