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.     

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.     

Endocytosis and intracellular transport of epidermal growth factor after destruction of the actin cytoskeleton by cytochalasin B

Endocytosis of the epidermal growth factor (EGF) was investigated in three cell lines--A431, 3T6 and Swiss 3T3--after their incubation with cytochalasin B (CB). CB was introduced into culture medium (10 mkg/ml) 1.5-2 hours before addition of 125I-EGF (20-40 ng/ml). The label uptake rate was measured after a 35-40 minutes incubation of cells with 125I-EGF. It appeared that disorganization of microfilamentous network caused by CB exerted no influence on the binding of EGF to the surface membrane receptors and its internalization. Nevertheless, the experiments performed on A431 cells using a fluorescent label--rhodamine--bound to EGF (EGF-R) indicate that CB, though not influencing the initial steps of endocytosis, inhibits the next step--the intracellular transport of EGF-receptor complexes from the trans-Golgi region to lysosomes. As was shown elsewhere (Barkan, Nikol'sky, 1986), CB inhibits the mitogenic effect of EGF on resting Swiss 3T3 cells. So, the process of EGF-receptor uptake and delivery to the trans-Golgi region is evidently not enough to stimulate the cell proliferation; next steps of transport and degradation of ligand-receptor complexes are presumably needed.     

The recycling of EGF-receptor complexes in A431 cells

As was demonstrated elsewhere (L. V. Teslenko et al., 1987), the epidermal growth factor (EGF) can recycle after internalization by A431 cells in membrane-bound state. In the present study, direct evidence on recycling of EGF-receptor complexes is presented using a covalently crosslinking reagent. The recycling was shown to occur via peripheral endosomes as well as through para-Golgi endosomes. It was found that among EGF degradation inhibitors tested only primaquine (300 microM) was able to decrease significantly the rate of recycling. The lowering of the temperature to 17 degrees C led to blocking the EGF degradation as well as to inhibiting the recycling. The data obtained suggested that the recycling of EGF-receptor complexes is relatively independent of their degradation.     

Effect of an vacuolar proton pump inhibitor bafilomycin A1 on the intracellular processing of markers for receptor-mediated and liquid phase endocytosis

By means of subcellular fractionation in density Percoll gradients, immunoblotting and immunofluorescense, the effect of BafA1 on endocytosis of EGF-receptor complexes and horse radish peroxidase (HRP) in A431, HER14 and HC11 cell lines was studied. It was shown that the pretreatment of all used cell lines with BafA1 completely inhibited EGF degradation, but did not interfere with the delivery of significant portion of EGF-receptor complexes to late endosomes and lysosomes and transition of the receptor to juxtranuclear region. At the same time, BafA1 was found to dramatically inhibit the delivery of fluid phase marker HRP to late endosomes of A431 cells. The BafA1 effect on endocytosis of high concentrations of EGF was similar to that on HRP endocytosis. Regulatory mechanisms of early-to-late endosomal compartment transition are discussed.     

Demonstration of an intact complex of epidermal growth factor and its receptor in the endosomes of A-431 cells

The compartmentalization of the epidermal growth factor (EGF) receptors in A-431 cells was studied using centrifugation of the microsomal fraction of these cells in continuous Percoll gradient. The existence of an intact (non-degraded) EGF receptor in plasma membrane and endosome fraction was demonstrated by electrophoretic analysis of in vitro phosphorylated Percoll fractions. No phosphorylated receptor was revealed in lysosomal fraction by this method. The existence of non dissociated EGF-receptor complexes in intracellular compartments 30 minutes after the start of internalization was proven using a synthesized photoreactive labeled EGF derivative (125I-EGF-SANAH). The removing of pH gradient in organellar membranes by 10 mkM of monensin did not affect dissociation from its receptor. The data obtained proved the existence of non-dissociated and non-degraded EGF-receptor complexes in the endosomal compartment of A-431 cells.     

Effect of synthetic proteasomal inhibitor MG132 on dynamics of EGF-receptor complexes endocytosis in A431 cells

The effect of proteasomal activity suppression induced by MG132, a synthetic proteasomal inhibitor of EGF-receptor complexes endocytosis in human epidermoid carcinoma A431 cell line, was studied. Using subcellular fractionation in 17% Percoll gradient, it was demonstrated that the addition of MG132 to the cells 15 min following stimulation of EGF endocytosis resulted in a slight accumulation of 125I-EGF in early endosomes, and in much more significant accumulation of the labeled growth factor in late endosomes/lysosomes, as compared to untreated cells. The release of 125I-EGF degradation products into the incubation medium was significantly (3-12-fold) inhibited in the presence of MG132. At the same time biochemical analysis has demonstrated that the EGF receptor itself is not a direct target of proteasomes, since it is revealed as a full-length protein with native mol. mass (170 kDa) in fractions of early and late endosomes and lysosomes. Possible mechanisms of the MG132 effect on intracellular processing of EGF-receptor complexes are discussed.     

Dimerization of internalized epidermal growth factor receptors.

Binding of epidermal growth factor (EGF) to cell surface EGF receptors initiates the formation of the receptor homodimers that can be detected by covalent cross-linking in intact cells or in detergent-solubilized cell extracts. Low pH dissociation of EGF from surface receptors results in immediate monomerization of receptor dimers. Using chemical cross-linking during mild permeabilization or cell solubilization, we have detected dimers of internalized EGF receptors in human carcinoma A-431 cells and transfected NIH 3T3 cells that express human EGF receptors. The percentage of internalized cross-linked receptor dimers was similar to that observed for surface EGF receptors. Furthermore, at the time of maximal accumulation of EGF-receptor complexes within the endosomal compartment (10-15 min of incubation at 37 degrees C), both the dimeric and monomeric forms of the EGF receptor are tyrosine-phosphorylated to the same extent as surface dimer and monomer species. In transfected NIH 3T3 cells, the level of dimerized and internalized kinase-negative EGF receptors was not different from that observed for wild-type receptors. These data suggest that for some time after internalization EGF does not dissociate from its receptor and indicate that a receptor conformation is preserved intracellularly that allows maintenance of receptor-receptor interactions and tyrosine kinase activity.     

Association of ganglioside-protein conjugates into cell and Sendai virus. Requirement for the HN subunit in viral fusion

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.     

Lateral diffusion of epidermal growth factor complexed to its surface receptors does not account for the thermal sensitivity of patch formation and endocytosis

The patching and endocytosis of EGF (epidermal growth factor) bound to A-431 cells (a human epidermoid carcinoma line) are temperature-sensitive processes which are completely inhibited at 4 degrees C. Receptor-mediated endocytosis generally occurs through coated regions, and EGF bound to its membrane receptor must diffuse laterally to these points of internalization. In this work we investigated the thermal sensitivity of the lateral diffusion of EGF receptor complexes and the thermal sensitivity of the patching and endocytosis of the hormone receptor complexes. Using the fluorescence photobleach recovery technique, we measured the lateral diffusion coefficients of a fluorescent derivative of EGF as a function of temperature. The lateral diffusion coefficient (D) increased gradually from 2.8 X 10(-10) cm2/s at 5 degrees C to 8.5 X 10(-10) cm2/s at 37 degrees C, and no phase transition was detected. Neither was a phase transition detected when we measured the diffusion coefficient of fluorescent lipid probes over this temperature range. From a calculation of the collision frequency of the occupied EGF receptors with coated regions using our measured values of D at 5 and 37 degrees C, we conclude that diffusion is not the rate-limiting step for either endocytosis or patching.     

The phosphorylation and tyrosine kinase activity of an internalized complex of epidermal growth factor and its receptor

A study was made of the functional state of the epidermal growth factor (EGF)--receptor complexes in A-431 cells. Conditions of surface bound EGF extraction were selected which allow to consider the intracellular EGF--receptor complexes only. A procedure of high efficient and specific immunoprecipitation of tyrosyl-phosphorylated EGF receptors was developed. It is shown that the dissociation of EGF--receptor complexes leads to receptor dephosphorylation due to a rapid and reversible inactivation of EGF receptor tyrosine kinase. The internalized receptor is found to be tyrosyl-phosphorylated and to retain tyrosine kinase for at least an hour after the internalization. The dynamics of dissociation, degradation and dephosphorylation of EGF--receptor complexes has been estimated. The rates of these processes prove to be almost negligible for the first 2.5 hours after internalization.     

Subcellular characterization of the endocytosis of small oligomers of mouse immunoglobulin G in murine macrophages

To characterize the internalization and degradation of model immune complexes in murine macrophages, the endocytosis of well-defined radiolabeled IgG dimers and heavy oligomers (5 to 7 IgG molecules per complex), which were covalently cross-linked at the antigen-combining site, was studied. Of those heavy oligomers which were bound to the cell at 4 degrees C, 50 to 60% (400,000 molecules of IgG) were internalized within 30 min at 37 degrees C and, subsequently, were completely degraded over a period of 3 hr. Low pH had little effect on the dissociation of the oligomer from its receptor. The degradation of oligomers was markedly inhibited when macrophages were treated with monensin, a proton ionophore which raises organelle pH. Because this treatment did not prevent the delivery of oligomer into the lysosome, the transport of a soluble complex of IgG from the cell surface to the lysosome was not a pH-dependent event. On the other hand, 25 to 30% (50,000 molecules) of those dimers capable of binding to the cell entered the macrophage, but only 5000 molecules were degraded. When macrophages were studied by using density gradient centrifugation, within 15 min, heavy oligomers were found in a vesicle which sedimented at a density between that of the plasma membrane and lysosome. The density of this vesicle was similar to that of endosomes studied in other receptor-ligand systems. Heavy oligomers were within lysosomes shortly thereafter. Incubation of cells at 18 degrees C prevented the appearance of heavy oligomer within the lysosomes and resulted in the concentration of oligomers within an intracellular compartment of a density slightly heavier than that of plasma membrane. At 37 degrees C, dimers sedimented in a similar region of the gradient. But unlike heavy oligomers, dimers never entered lysosomes. These data suggest that the degree of Fc receptor clustering induced by oligomers of IgG influenced the intracellular fate of the ligand.     

The role of Src-kinase in the regulation of endocytosis of EGF-receptor complexes. Distribution of clathrin after stimulation of EGR endocytosis in various cell lines during inhibition of Src-kinase activity

A distribution of EGF receptor and clathrin during EGF endocytosis in A431, HER14, WT and PURO cell lines was studied by indirect immunofluorescence. Though the initial distribution of EGF-receptors on A431 and HER14 cells was somewhat different, the late stages of endocytosis proceeded equally and were marked by formation of bright spots in the juxtanuclear region characteristic of the late endosomes. The Src-family kinase inhibitor CGP77675 had no influence on the dynamics of receptor endocytosis at the immunofluorescent level in both cell lines. Stimulation of EGF-receptor endocytosis in A431 cells did not also result in any redistribution of clathrin in the areas where the majority of EGF-receptors are localized, i.e. in the lateral plasma membrane both in the control cells and under CGP77675 treatment. Clathrin in A431, WT and PURO cells demonstrated even a punctuated pattern throughout the cytoplasm with some accumulation in the juxtanuclear region. This distribution depended neither on the absence or presence of Src activity nor on EGF addition. The data obtained indicate that 1) EGF-receptors do not serve as the initiation sites during clathrin coated pit assembly; 2) Src-kinase activation does not result in significant clathrin redistribution in the plasma membrane, and its influence on EGF endocytosis can be considered as a secondary effect.     

Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system

This study was conducted to determine how extraordinarily high numbers of epidermal growth factor receptors (EGF-R) affected the binding and internalization of EGF in the transformed cell line A431. I found that at low EGF concentrations, the kinetics of binding behaved as a nonsaturable, first-order process showing no evidence of multiple-affinity classes of receptors. However, EGF dissociation rates were strongly dependent on the degree of receptor occupancy in both intact cells and isolated membranes. This occupancy-dependent dissociation appears to be due to diffusion-limited binding. EGF-induced receptor internalization was rapid and first order when the absolute number of occupied receptors was below 4 x 10(3) min-1. However, at higher occupancies the specific internalization rate progressively declined to a final limiting value of 20% normal. The saturation of EGF-R endocytosis was specific since internalization of transferrin receptors was not affected by high concentrations of either transferrin or EGF. Saturation of EGF-R endocytosis probably involves a specific component of the endocytic pathway since fluid phase endocytosis increased coordinately with EGF-R occupancy. I conclude that there are several aspects of EGF-R dynamics on A431 cells are neither similar to the behavior of EGF-R in other cell types nor similar to the reported behavior of other hormone receptors. Although A431 cells have an extraordinary number of EGF-R, they do not seem to have corresponding levels of at least two other crucial cell surface components: one that mediates EGF-induced rapid receptor internalization and one that attenuates EGF-induced membrane responses. These factors, in addition to the presence of diffusion-limited binding at low EGF concentrations, are probably responsible for the appearance of multiple-affinity classes of receptors in this cell type.     

Primary amines do not prevent the endocytosis of epidermal growth factor into 3T3 fibroblasts

Various amines block the degradation of endocytosed epidermal growth factor (EGF) without affecting the binding of the hormone to its surface receptors. However, studies based on fluorescence microscopy demonstrate that amines block the internalization of alpha 2-macroglobulin and EGF by preventing it from clustering in clathrin coated pits. In order to resolve this controversy we have studied in detail the effect of various amines on the localization and processing of fluorescent and radiolabelled EGF. We have explored the effect of amines on EGF binding and localization, receptor mobility, membrane fluidity, receptor down regulation, hormone degradation and release of degradative products as a function of time and temperature. Our conclusions are as follows. 1. Primary amines prevent the formation of visible patches of fluorescent EGF and alpha 2-macroglobulin on the cell surface at least for 15 min, thus increasing the diffusion coefficients and the mobile fraction of EGF-receptor complexes on the cell surface. 2. Amines do not block the endocytosis of EGF and alpha 2-macroglobulin. On most cells fluorescent EGF and alpha 2-macroglobulin are clustered and endocytosed within 30-45 min at 37 degrees C. 3. Amines do not effect the internalization of 125I-labelled-EGF and the down regulation of EGF receptors. 4. Amines block the degradation of the endocytosed EGF.     

Isolation and characteristics of monoclonal antibodies to the external domain of the EGF receptor in human A431 epidermoid carcinoma

The monoclonal antibody to the epidermal growth factor (EGF) receptor was generated after fusion of PAI myeloma cells with immunized BALB/c mouse spleen cells, using intact A431 epidermoid carcinoma cells as an immunogen. The antibody, denoted 5A9, is an IgG, which recognizes a protein with molecular mass 170 kDa during immunoblot analysis, immunoprecipitates phosphoprotein with molecular mass 170 kDa from the membrane preparations of A431 cells, and, according to immunofluorescence experiments, is distributed in the cell similar to the EGF-rhodamine conjugate. It is concluded that the produced antibodies are specific to EGF-receptor. At the same time the 5A9 (50 nM) do not compete with EGF for binding with high and low affinity receptors. They fail to induce internalization of the EGF-receptor and do not exert influence on intracellular degradation of EGF-receptor. Monoclonal antibodies 5A9 are also unable to inhibit the EGF-induced protein kinase activity of the receptor and do not stimulate protein kinase activity by themselves. Thus, the prepared monoclonal antibodies can be used to register the EGF-receptor cellular localization without affecting biologic activity of the receptor.     

EGF induces receptor down-regulation with no receptor recycling in KB cells

Several ligands, including epidermal growth factor (EGF), have been found to negatively modulate or down-regulate their specific plasma membrane receptors. Using both 125I-EGF and a monoclonal antibody against the EGF-receptor (EGF-R1), we studied the down-regulation of the EGF-receptor in the human adenocarcinoma cell line KB. The results presented here demonstrate that incubating KB cells at 37 degrees C with EGF rapidly decreases the number of plasma membrane EGF-receptors. In addition, there is a concomitant rise of equal magnitude in the number of EGF molecules taken up. The latter result argues strongly that there is negligible recycling of the EGF-receptor in KB cells and that the major portion of internalized EGF-receptor complexes are transported to lysosomes and subsequently degraded. The fate of the EGF-receptor is markedly different from that of receptors not subject to down-regulation. The biochemical signals that operate to regulate such diverse receptor traffic in cells remains to be elucidated.     

Visualization of epidermal growth factor receptor in cryosections of cultured A431 cells by immuno-gold labeling

Cryo-ultramicrotomy in combination with immuno-gold labeling has been demonstrated to present a powerful tool in the visualization of extra- and intracellular located antigens. We have applied this method to localize epidermal growth factor (EGF) receptor in cultured A431 human epidermoid carcinoma cells. However, both the labeling efficiency, maintenance of antigenicity, and the recognizability of the ultrastructure in cryosections are highly dependent upon the fixation procedures. Using 125I-EGF or a consecutive labeling with a monoclonal anti EGF-receptor antibody, rabbit-anti-mouse antibody and 125I-protein A, it was shown that maintenance of antigenicity was optimal using 2% paraformaldehyde as a fixative, whereas under these conditions also the recognizability of ultrastructure was sufficient. After appropriate fixation and labeling, gold particles were observed associated with various regions of the plasma membrane, including coated pits, and with various types of vesicles, including coated vesicles, intracellular vesicular membranes, multi-vesicular bodies and lysosomes. The results indicate that this method allows a visualization of EGF-receptors and resolution of the EGF-receptor processing pathway at the electron microscopic level, independent of the internalization process of labeled ligands.     

Endocytosis of EGF-receptor complexes at various cell cycle stages

Parameters of EGF-receptor complex endocytosis have been studied in the early and late G1 phase and in mitosis. As a model, mouse mammary epithelial cells HC11 were used, whose growth depends on EGF presence in the medium. The Scatchard analysis has demonstrated that the surface receptors are represented by two receptor populations: 4800 high affinity (KD = 10(-11) M) receptors, and 73,000 low affinity (KD = 4.10(-9) M) receptors. Incubation of cells with the growth factor (5 ng/ml) resulted in a decrease in 125I-EGF binding, with its level being low until entering the S-phase. Under these conditions, receptors disposed on the plasma membrane presented a homogeneous population (KD = 8.10(-11) M, 14,000 receptors per cell). No reliable difference was revealed between the EGF-receptor complexes, internalized in early and late G1 phases, in respect to the internalization rate, level of recycling, degradation, and dynamics of compartmentalization. However, endocytosis of EGF-receptor complexes was found to be completely blocked in mitosis at the stage of internalization.     

Uptake and transport of mannosylated ligands by alveolar macrophages. Studies on ATP-dependent receptor-ligand dissociation

During endocytosis, mannosylated ligands enter vesicles which have a density intermediate between that of the plasma membrane and secondary lysosomes. Mannosylated ligands are transferred from these vesicles to lysosomes. A solubilization-precipitation assay was used to study the dissociation of mannosylated ligands from their receptor. In whole cells dissociation was rapid (t 1/2 (37 degrees C) = 8 min) and took place before delivery of the ligand to lysosomes. Receptor-ligand dissociation within membrane vesicles, washed free of cytosol, could be induced by addition of ATP and GTP but not ADP. Receptor-ligand dissociation caused by manipulating the pH of the vesicles suggested that the pH within endosomes was lowered to 5.5 by addition of ATP. Dissociation was blocked by proton ionophores and Zn2+, but was unaffected by inhibitors of the F1, Fo-ATPase or the Na+,K+-ATPase. Dissociation did not require Na+ or K+ and was blocked by anion transport inhibitors. Dissociation was slowed in the absence of permeant anions (Cl-). Receptor-ligand complexes within vesicles isolated as early as 2 min following ligand internalization responded to addition of ATP. The results suggest that receptor-ligand dissociation in endosomes requires ATP, possibly to power endosomal acidification via an ATP-dependent proton pump. Dissociation is enhanced in the presence of permeant anions, suggesting the involvement of an anion channel or carrier.