The effect of 1.25 dihydroxyvitamin D3 on binding and internalization of epidermal growth factor in cultures cells. Studies on BT-20 cells using quantitative electron microscope autoradiography

The biological effects of 1.25 (OH)2D3 on epidermal growth receptor (EGF-R) and on EGF internalization were examined in human mammary carcinoma BT-20 cells. In this cell line, with known amplification of the epidermal growth factor receptor gene. EGF was not stimulatory for growth. Biological assay and quantitative EM autoradiography combined with iodinated ligand binding to specific receptors demonstrated that the number of binding sites unit of length of plasma membrane was 2.48-fold higher in treated than in control cells. I-EGF was progressively internalized in a time-and temperature-dependent manner after selective association with the membrane-coated pits. No modification of the time course of I-EGF internalization was noted in the control and in the treated cells, but a different distribution of the labeling in the subcellular compartment was observed in treated cells. In 1.25(OH)2D3-treated batches, the grain density remained low in the receptosomes throughout the experiment, whereas it was high and occurred early in the lysosomes. On the other hand, in control cells, the grain density of the receptosomes was high, whereas it occurred late and was relatively low in the lysosomes. These data suggest that 1.25(OH)2D3 is a regulator of EGF-R level in BT-20 cell line, but it cannot affirmed whether this effect is direct or mediated by other parameters.     

Subcellular structures involved in internalization and degradation of epidermal growth factor

Epidermal Growth Factor (EGF), a small polypeptide which acts as a mitogen for many cell types, has previously been shown to bind to a specific plasma membrane receptor on 3T3 cells. If ¹²⁵I-EGF is bound to 3T3 cells for one hour at 4°C, it remains predominantly associated with the plasma membrane-containing fractions obtained by subjecting cell supernatants to equilibrium sedimentation on sucrose gradients. When binding is followed by a 10-minute incubation at 37°C, over 50% of the ¹²⁵I-EGF is associated with two internal membrane-containing peaks having higher densities than the plasma membrane. After one hour at 37°C, over 80% of the ¹²⁵I-EGF is degraded and removed from the cells. The most rapidly labeled internal peak corresponds in density to brain-coated vesicles (CVs). Antiserum prepared against coated vehicles from brain precipitates the ¹²⁵I-EGF in this peak. In addition, CVs containing ¹²⁵I-EGF can be co-purified from 3T3 cells exposed to ¹²⁵I-EGF, using brain as a carrier. Several lines of evidence suggest that the other ¹²⁵I-EGF-labeled intracellular peak is ¹²⁵I-EGF in lysosomes. These results provide kinetic and biochemical evidence for a unidirectional pathway for EGF catabolism by 3T3 cells. EGF first binds to the plasma membrane bound receptors, is then moved to the cytoplasm in CVs, and finally appears in lysosomes, where it is degraded and released from the cells. Ten-millimolar NH₄Cl blocks lysosomal hydrolysis of EGF almost completely. Subsequently, EGF internalization is inhibited. This finding suggests that the pathway for EGF internalization and degradation is tightly coupled.     

Characterization of binding and receptors for epidermal growth factor in smooth muscle

Summary The mitogenic and differentiation-inducing activities of epidermal growth factor (EGF) in epithelial tissues have been well described. Since non-mitogenic effects of EGF, especially in mesenchymal tissues such as smooth muscle are not well-known (Nanney et al. 1984), we have examined EGF-binding and receptors in smooth muscle from many sites. Specific EGF binding sites were detected by incubating small pieces of tissue with ¹²⁵I-EGF; immunoreactive EGF receptors were detected by immunohistochemistry. In-situ localization of ¹²⁵I-EGF binding sites and immunoreactive EGF receptors of smooth muscle cells in intact mammalian tissues were identical using either ¹²⁵I-EGF autoradiography or anti-EGF receptor antibody in an immunoperoxidase method. Cultured rat aortic smooth muscle also contained specific EGF receptors as detected by their biological response to EGF-binding and internalization of ¹²⁵I-EGF, as well as EGF-stimulated phosphorylation of a 170K protein. The presence of EGF receptors in a well-differentiated smooth muscle cell indicates that EGF may play a physiological, but non-mitogenic role in mammalian tissues in vivo.     

Comparing the epidermal growth factor interaction with four different cell lines: intriguing effects imply strong dependency of cellular context

The interaction of the epidermal growth factor (EGF) with its receptor (EGFR) is known to be complex, and the common over-expression of EGF receptor family members in a multitude of tumors makes it important to decipher this interaction and the following signaling pathways. We have investigated the affinity and kinetics of ¹²⁵I-EGF binding to EGFR in four human tumor cell lines, each using four culturing conditions, in real time by use of LigandTracer®.Highly repeatable and precise measurements show that the overall apparent affinity of the ¹²⁵I-EGF – EGFR interaction is greatly dependent on cell line at normal culturing conditions, ranging from K_D≈200 pM on SKBR3 cells to K_D≈8 nM on A431 cells. The ¹²⁵I-EGF – EGFR binding curves (irrespective of cell line) have strong signs of multiple simultaneous interactions. Furthermore, for the cell lines A431 and SKOV3, gefitinib treatment increases the ¹²⁵I-EGF - EGFR affinity, in particular when the cells are starved. The ¹²⁵I-EGF - EGFR interaction on cell line U343 is sensitive to starvation while as on SKBR3 it is insensitive to gefitinib and starvation.The intriguing pattern of the binding characteristics proves that the cellular context is important when deciphering how EGF interacts with EGFR. From a general perspective, care is advisable when generalizing ligand-receptor interaction results across multiple cell-lines.     

The effects of epidermal growth factor on cell proliferation and prolactin production by GH3 rat pituitary cells

The effects of epidermal growth factor (EGF) were studied in rat pituitary tumor cells, GH₃, grown in serum-supplemented and serum-free chemically defined media. EGF (1 nM) increased the cell number to 132% of the control cultured in the defined medium during a 6-day incubation period, while it decreased the cell number to 60% of the control in the serum-supplemented medium. EGF altered the morphology of the cells grown in the defined medium more markedly to an elongated conformation than that of cells grown in the serum-supplemented medium. EGF also stimulated prolactin (PRL) production by culture in the presence or absence of serum. The effects of the cell density of GH₃ on the action of EGF were shown to appear in two ways. The mitogenic influence of EGF was more effective on, and more responsive to, high-density cells, whereas the stimulatory action on PRL production was less effective on high-density cells. However, the inhibitory effects on cellular growth appeared independently of cell densities. The results obtained with ¹²⁵I-EGF binding experiments indicated that the number of binding sites, affinity, and internalization of EGF receptors were similar in either serum-supplemented or serum-free culture. At low cell density, the number of available ¹²⁵I-EGF binding sites per cell was larger than at high cell density. These results suggested that there was no apparent correlation between EGF binding and its differing effects on the growth of GH₃ cultured in the serum-supplemented and the defined medium.     

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.     

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).     

p-Azidophenylglyoxal-Epidermal Growth Factor: A Photoactivatable Affinity Crosslinking Reagent

Abstract

A photoaffinity derivative of highly purified ¹²⁵I-labelled epidermal growth factor (¹²⁵I-EGF) has been synthesized. The heterobifunctional crosslinking reagent p-azidophenylglyoxal (PAPG) was bound to arginine residues in ¹²⁵I-EGF. PAPG-¹²⁵I-EGF bound to EGF receptors on rat fibroblasts and human A431 epidermoid carcinoma cells in culture. An apparent decreased affinity of PAPG-¹²⁵I-EGF for the EGF receptor is in accord with at least one arginine being at or near the EGF receptor binding site. The PAPG-¹²⁵I-EGF:EGF receptor complexes on rat cells were internalized to the same extent as control EGF:receptor complexes. A431 cells treated with PAPG-¹²⁵I-EGF were irradiated with ultraviolet light and the labelled proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The 3 major labelled proteins had apparent molecular weights ranging from 75,000 to 200,000. Only the labelling of the 200,000-M_r protein was prevented by the addition of excess unlabelled EGF with the PAPG-¹²⁵I-EGF. This molecular weight is in agreement with the reported size of the EGF receptor plus EGF. A protein with apparent molecular weight of 100,000 was labelled by ¹²⁵I-EGF by an unknown mechanism which was dependent on the dose of UV light and blocked by the addition of excess unlabelled EGF.     

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.     

Effects of 12-0-tetradecanoylphorbol-13-acetate (TPA) on rat pheochromocytoma (PC12) cells: Interactions with epidermal growth factor and nerve growth factor

The phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (¹²⁵I-EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent K_i of 11–26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of ³H-choline and ³²P-orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome-associated protein. These effects were also elicited by nerve growth factor (NGF) which, in contrast to the former agents, is a differentiating stimulus for the PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of ¹²⁵I-EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduced by NGF treatment.     

Glucocorticoid-mediated alteration in growth factor binding and action: Analysis of the binding change

The addition of the glucocorticoid analog dexamethasone (DX) to serum-free cultures of human fibroblasts caused a twofold enhancement of the mitogenic response to epidermal growth factor (EGF), although DX by itself was not mitogenic. A basis for this effect was suggested by studies showing that DX also increased the cellular binding of ¹²⁵I-EGF. DX increased the ability of the cells to bind ¹²⁵I-EGF only at low physiological concentrations of this polypeptide. Thus, data from ¹²⁵I-EGF binding to cells incubated without DX produced a linear Scatchard plot, whereas the data from ¹²⁵I-EGF binding to DX-treated cells led to an upwardly curvilinear Scatchard plot. Measurements of ¹²⁵I-EGF association with the cell surface and cytoplasm indicated that this binding change involved an alteration of cell surface EGF receptors. The binding change appeared not to involve negatively cooperative interactions between EGF receptors, nor a change in the number of receptors. The binding alteration could be explained by a model in which DX converted 25–30% of the cell surface EGF receptors to a form having a fourfold increased affinity.     

Intracellular processing of epidermal growth factor and its effect on ligand-receptor interactions

When normal human fibroblasts are brought to a steady state with 125I-labeled epidermal growth factor (125I-EGF), greater than 90% of the radioactivity is intracellular. We investigated this material to determine whether the 125I-EGF is intact or degraded. Our results show that 125I-EGF is rapidly processed after internalization and can be resolved into four peaks by native gel electrophoresis. These different forms were isolated and tested for their ability to bind to cell-surface EGF receptors. The first processed form was fully capable of binding to EGF receptors, but the second processed form could not. The third form was a collection of small degradation products. We calculated that at steady state about 60% of internalized "125I-EGF" was in a form still able to bind to EGF receptors. We then investigated the ability of different reported inhibitors of EGF "degradation" to block the processing of EGF. Although inhibitors of cathepsin B (leupeptin, antipain, N alpha-p-tosyl-L-lysine chloromethyl ketone, and chymostatin) were able to inhibit the release of monoiodotyrosine from treated cells in a time- and concentration-dependent manner, they had little effect on the processing step that apparently inactivates 125I-EGF. In contrast, agents that raised intravesicular pH, such as methylamine and monensin, inhibited the initial steps in EGF processing as well as the later steps. Low temperatures inhibited the transfer of 125I-EGF to the lysosomes and inhibited the conversion of EGF to a nonbindable form, but had little effect on the initial processing. We conclude that the intracellular processing of EGF is a multistep process that is initiated prior to lysosomal fusion, involves cathepsin B activity, and requires an acidic pH. In addition, many of the protease inhibitors that have been utilized to investigate the role of EGF degradation in mitogenesis do not block the conversion of EGF to a form that is apparently unable to interact with its receptor.     

Solubilization of membrane receptor for epidermal growth factor.

The membrane receptor for epidermal growth factor (EGF) has been solubilized from A-431 tumor cells using Triton X-100. Operational criteria used to define solubilization include failure of the binding activity to be pelleted after centrifugation at 90,000 x g for 1.5 hrs and the requirement for polyethylene glycol precipitation to detect ¹²⁵I-EGF: receptor complexes on membrane filters. Properties of the solubilized EGF are characterized and compared to the properties of the particulate receptor. The specific binding capacity of the solubilized EGF receptor was 8.0 picomoles ¹²⁵I-EGF bound per mg protein--approximately 60% of the binding capacity of particulate receptor preparations. Also, solubilization of the EGF receptor resulted in a 10-fold decrease in the affinity of the receptor for ¹²⁵I-EGF.     

Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: The endocytic activation hypothesis

The addition of EGF to cultured murine 3T3 cells produces a decrease in EGF binding activity with concomitant internalization and degradation of the initially bound EGF. When the EGF receptor on cultured 3T3 cells is affinity labeled with high specific activity ¹²⁵I-EGF, and the fate of the affinity labeled EGF-receptor complex determined, the loss in binding activity was accounted for by receptor internalization and subsequent proteolytic processing of the EGF receptor molecules in the lysosomes. Studies of the effects of EGF concentration on EGF binding by cells, EGF-induced receptor internalization and EGF-induced stimulation of ³H-thymidine uptake into cellular DNA show that there is a direct correlation between EGF-induced receptor internalization and EGF-induced stimulation of DNA synthesis, but not between EGF binding and EGF-induced stimulation of DNA synthesis. This correlation is lost at high EGF concentrations, where stimulation of DNA synthesis is suboptimal. Optimal stimulation of DNA synthesis requires a minimum of 6 h of incubation of EGF with cells, and the suboptimal stimulation of DNA synthesis at high EGF concentration is intensified when the period of incubation of EGF with cells is less than 6 h. These data are consistent with a model of hormone signal transmission by Endocytic Activation, wherein the activation of EGF-induced processes requires constant EGF-induced internalization of receptor for a requisite 6–8 h period as an obligatory step in production of “second messenger” in the action of this hormone.     

Epidermal growth factor stimulates fluid phase endocytosis in human fibroblasts through a signal generated at the cell surface

We have investigated the stimulation of fluid phase endocytosis by epidermal growth factor (EGF) in normal human fibroblasts using ¹²⁵I-labeled polyvinylpyrrolidone (¹²⁵I-PVP) as a fluid phase marker. We found that EGF initially induced a thereefold increase in the rate of ¹²⁵I-PVP uptake. This initial burst of fluid uptake terminated within 10 min. Thereafter, the rate of fluie uptake in EGF-treated cells was approximately 40% higher than in control cells. To identify the cellular site of EGF action in stimulating fluid phase endocytosis, we examined the kinetics of the induction of this response as well as the kinetics of cell surface binding and internalization of ¹²⁵I-EGF. Although there was no detectable lag between binding of EGF to the cell surface and its internalization, the kinetics of the two processes were quite different. Significantly, the kinetics of induction of ¹²⁵I-PVP uptake matched the kinetics of binding of ¹²⁵I-EGF to its cell surface receptors, indicating that the signal for the increase in fluid phase endocytosis is generated at the cell surface. To determine if EGF-stimulated fluid phase endocytosis was related to EGF-stimulated endocytosis of its own receptor, we compared the EGF dose dependency and time course of the two processes. Although the stimulated endocytosis of the EGF receptor was not saturable with respect to the concentration of EGF used, the stimulation of fluid phase endocytosis was half maximal at an EGF concentration of 1 ng/ml and saturated at a concentration of 5 ng/ml. Also, the stimulation of fluid phase endocytosis was sevenfold greater initially after adding EGF than after a 30-min continuous incubation with the hormone, whereas the enhanced clearance of the EGF receptor did not change during this time period. We conclude that the EGF-stimulated increase in fluid phase endocytosis is not directly coupled to EGF-stimulated endocytosis of its own receptor but instead to a separate signal generated at the cell surface.     

Ligand-induced desensitization of 125I-epidermal growth factor internalization

The internalization of 125I-epidermal growth factor (EGF) by A431 cells was investigated. Control cells were able to internalize over 80% of receptor-bound 125I-EGF. By contrast, cells treated with EGF before incubation with 125I-EGF internalized only 50% of the surface-bound radioligand. The ligand-induced decrease in 125I-EGF internalization showed a dose response to EGF with half-maximal effect occurring at 3 nM. The alteration in the extent of 125I-EGF internalization did not require extended treatment with high concentrations of the hormone. When the internalization of picomolar versus nanomolar concentrations of EGF were compared, the lower concentrations of 125I-EGF were more completely internalized than the higher concentrations of radioligand. These data are consistent with the hypothesis that occupation of the EGF receptor by hormone rapidly leads to the activation of cellular processes which effectively desensitize the system to further ligand-induced internalization. The decrease in the extent of ligand internalization occurred in cells in which the protein kinase C (Ca2+/phospholipid-dependent enzyme) activity had been down-regulated by prolonged treatment with 12-O-tetradecanoyl-phorbol-13-acetate implying that the desensitization process is independent of protein kinase C. However, the effects of EGF on the extent of hormone internalization could be mimicked by the addition of A23187 and could be prevented by pretreatment of the cells with calmodulin antagonists suggesting the possibility that Ca2+-calmodulin is involved in the regulation of EGF receptor internalization in A431 cells.     

Decreased epidermal growth factor binding in cells growth arrested in G1 by nutrient deficiency

Previous studies have shown that the nontransformed AKR-2B mouse embryo derived cell line may growth arrest by two separate mechanisms in the G₁ phase of the cell cycle-growth factor deficiency arrest (G₀) and low molecular weight nutrient deficiency arrest. An examination of epidermal growth factor (EGF) receptors under the different resting or growth conditions has shown that rapidly growing cells or cells arrested due to growth factor deficiency have the expected amount of ¹²⁵I-EGF binding with approximately 10⁵ receptors per cell being present in G₀ arrested cells. In contrast, cells arrested due to nutrient deficiency show a reduction in ¹²⁵I-EGF binding to 10--20% of that observed under the other conditions. This effect appears to be due to decreased receptor number and not to a change in the affinity of the receptor. Stimulation of DNA synthesis by nutrient replenishment causes a tenfold increase in EGF binding 20 hours later, with some increase in binding being detectable as early as six hours. The increase in binding is inhibited by cycloheximide and actinomycin D. This suggests that new mRNA synthesis as well as increased protein synthesis is required for the increase in EGF binding.     

Nonfunctional epidermal growth factor receptor in cells transformed by Kirsten sarcoma virus

The cell membrane receptor for epidermal growth factor (EGF) appears to be a glycoprotein of Mr 170,000 and mediates the mitogenic and metabolic responses of cells with EGF receptors (EGF-R). Normal rat kidney (NRK) have about 3 X 10(5) EGF-R per cell. Upon transformation of NRK cells by Kirsten sarcoma virus, the transformed derivative (KNRK) loses the ability to bind 125I-EGF. Membranes from NRK and KNRK cells were included in EGF-dependent phosphorylation reactions to search for evidence of the EGF-R. A phosphorylated protein of Mr 170,000 was detected in both NRK and KNRK membranes. The Mr 170,000 protein was identified to be EGF-R by immunoprecipitation with monoclonal antibody to the receptor. Furthermore, two-dimensional peptide mapping using trypsin and chymotrypsin digestions of the iodinated receptors from both NRK and KNRK cells showed essentially identical patterns. These data indicate that the EGF-R is present in KNRK cells with apparently the same protein structure as the NRK counterpart.     

神经节苷脂类抑制BT325细胞系生长机理的初步探讨

用 ¹²⁵I-EGF与人多形成胶质细胞瘤BT325细胞系膜上EGF受体的饱和结合实验, 竞争抑制实验研究GM3,BBG(bovine brain gangliosides)对EGF受体最大结合量, 亲和常数及受体数目的影响; 放射受体法观察EGF-EGFR复合物内吞过程, 测定胞质和培养基中EGF含量.结果表明: BT325细胞质膜上存在高亲和力EGF结合位点,GM3对EGF与其受体的亲和力无明显抑制作用(P＞0.05),但能明显减少其受体的数目(P＜0.05); GM3能明显延长EGF-EGFR复合物内吞过程; GM3处理的胞质中EGF浓度比对照组显著升高(P＜0.05), 培养液中无明显差异,这可能是由于GM3抑制EGF分泌所致.