Chloroquine allows the secretion of internalized 125I-epidermal growth factor from fibroblasts

Incubation of cells with labelled hormone in the presence of the lysosomotropic agent chloroquine produces an enhanced intracellular accumulation of hormone and receptor. Using a pulse-chase paradigm in which cell surface receptors were labelled with 125I-EGF at 4 degrees C, it was found that when 100 microM chloroquine was present in the 37 degrees C chase medium intact hormone was accumulated in the medium. Without chloroquine, low molecular weight (mw) degradation products were found in the medium. The processes of receptor-mediated endocytosis and subcellular distribution of 125I-EGF-receptor complexes were unchanged by chloroquine. The source of the intact hormone accumulating in the medium was therefore an intracellular compartment(s). The 125I-EGF released from the cells could rebind to surface receptors and be re-internalized; rebinding was inhibited by unlabelled EGF or Concanavalin A in the incubation medium. The concentration of unlabelled EGF required to inhibit rebinding was more than three orders of magnitude greater than the amount of 125I-EGF whose rebinding was inhibited. Thus, the 125I-EGF released from intracellular sites was rebound preferentially over exogenous EGF. The possible pathways for secretion of intact 125I-EGF and mechanisms of its preferential rebinding are discussed.     

Characterization of the rebinding of 125I-epidermal growth factor released from BALB/c-3T3 cells following accumulation in the presence of chloroquine

Lysosomotropic amines, such as chloroquine and methylamine, increase the intracellular accumulation of 125I-EGF by inhibiting lysosomal degradation. It has been shown previously that BALB/c-3T3 cells, prelabeled at 4 degrees C with 125I-EGF for 3 h and subsequently chased at 37 degrees C in the presence of chloroquine, internalized the surface bound 125I-EGF which was subsequently released into the extracellular medium in a high molecular weight form which co-migrated with native 125I-EGF. The secreted 125I-EGF rebound to the cells from which it was released more efficiently than does peptide in the extracellular media. We now show that when the BALB/c-3T3 cells were prelabeled at 37 degrees C for 2 h in the presence of chloroquine, the internalized 125I-EGF released into the medium was in a high molecular weight form which co-migrated with native 125I-EGF and did not rebind anymore efficiently than did peptide in the extracellular media. This lack of rebinding was not due to an alteration in the 125I-EGF molecule since it was still capable of rebinding to naive A431 cells, nor was it due to the exhaustion of EGF receptors on the BALB/c-3T3 cells. The inhibition of rebinding was observed only when the cells were treated with EGF in the presence of chloroquine, and was not due to a general down-regulation of membrane receptors. The differences between the rebinding of 125I-EGF at 4 degrees C and 37 degrees C suggest that EGF may be processed via different pathways in the cell.     

Attenuated processing of epidermal growth factor in the face of marked degradation of transforming growth factor-alpha

T3M4 human pancreatic carcinoma cells avidly bound and internalized 125I-labeled epidermal growth factor (EGF) but did not readily degrade the ligand. Pulse-chase experiments in which the cell-bound radioactivity was allowed to dissociate into the incubation medium in the presence of unlabeled EGF indicated that the majority of the released 125I-EGF consisted of intact EGF and a slightly processed species that readily bound to the cell. Omission of unlabeled EGF during the chase period markedly decreased the amount of radioactivity in the incubation medium, mainly as a result of the rebinding of EGF to the cells. In contrast, T3M4 cells readily degraded 125I-labeled transforming growth factor-alpha (TGF-alpha), and the released radiolabeled products did not rebind to the cells. Both ligands were released from T3M4 cells under acidic conditions, complete dissociation occurring at a pH of 4.5 for EGF, and a pH of 6.5 for TGF-alpha. A 431 human epidermoid carcinoma cells and ASPC-1 human pancreatic carcinoma cells also failed to extensively degrade 125I-EGF, whereas Rat-1 fibroblasts markedly degraded the growth factor. As in the case of T3M4 cells, ASPC-1 cells extensively degraded 125I-TGF-alpha. Degradation of either ligand was blocked by the lysosomotropic compound methylamine in all the tested cell lines. Immunoprecipitation of the EGF receptor with specific polyclonal antibodies and Western blot analysis revealed the anticipated 170-kDa protein in T3M4 cells. Both EGF and TGF-alpha enhanced EGF receptor degradation, but TGF-alpha was less effective than EGF. These findings indicate that in certain cell types EGF and TGF-alpha may be differentially processed.     

Effect of amines on the binding, internalization and degradation of epidermal growth factor and the induction of DNA synthesis in a 3T3 cell culture

The role of intracellular processing of epidermal growth factor (EGF) in the induction of proliferation of quiescent Swiss 3T3 cells was studied using various inhibitors. The number of amines (dansylcadaverine, chloroquine, cystamine, 5-methoxytryptamine) dimethylurea and monensin were shown to block the mitogenic effect of EGF. The majority of these substances while used in concentrations sufficient to inhibit the proliferation do not significantly influence 125I-EGF binding and internalization. The level of EGF degradation was reduced only by chloroquine. The inhibitory effect of amines and monensin on the generation of proliferative signal was supposed to take place at the stages of EGF processing in "specialized" endosomes and in Golgi apparatus.     

Relationship between medium pH and that of the lysosomal matrix as studied by two independent methods.

1. The method of estimating the intralysosomal pH by measuring the distribution of [14C]methylamine in lysosomes isolated from the livers of Triton WR 1339-treated rats has been critically examined. 2. In lysed lysosomes, methylamine is bound to the membrane fragments, but this binding can be completely suppressed by increasing the concentration of monovalent cations in the medium. 3. In intact lysosomes, the binding of [14C]methylamine is only partly inhibited by monovalent cations at 25 degrees C. 4. THe accumulation of [14C]methylamine in intact lysosomes is progressively inhibited as the concentration of methylamine is increased. A similar inhibition of [14C]methylamine accumulation is obtained with NH4Cl. 5. Similar values for the intralysosomal pH were obtained from measurements of the distribution of methylamine, dimethylamine and trimethylamine, which are accumulated in the lysosomes, and of 5,5-dimethyloxazolidinedione-2,4, which is excluded. 6. The breakdown of endocytosed 123I-labelled bovine serum albumin by intact isolated lysosomes is much less sensitive to the pH of the medium than the breakdown of added protein by lysed lysosomes. 7. The intralysosomal pH has been estimated by comparing the rate of breakdown of endocytosed 125I-labelled albumin in intact lysosomes as a function of medium pH with that of added 125I-labelled albumin by lysed lysosomes at different pH values. The values obtained agree well with those calculated from the distribution of [14C]methylamine. 8. Methylamine and NH4Cl inhibit the breakdown of 125I-labelled albumin in intact lysosomes, particularly at high medium pH, but have no effect on the breakdown by lysed lysosomes. 9. It is concluded that a pH difference across the lysosomal membrane (more acidic inside than outside) is maintained by the presence of indiffusible negatively charged groups within the lysosomes, and by the permeation across the lysosomal membrane of protons together with permeant anions (or of OH- in exchange for anions).     

Properties of receptors for epidermal growth factor in detergent solution: Evidence for heterogeneous aggregated states

Between 60% and 100% of epidermal growth factor (EGF) binding activity was recovered from membranes of the A431 human epidermoid carcinoma cell line treated with solutions containing the nonionic detergent Triton X-100. Approximately half of the recovered binding activity was sedimented at low centrifugal forece and hence was operationally insoluble in nonionic detergent solution. Receptors in both the detergent-soluble and -insoluble fractions displayed similar affinities for ¹²⁵I-EGF, and the values were in good agreement with those obtained for receptors in untreated membranes. The receptors in both fractions also formed identical direct linkage complexes with ¹²⁵I-EGF in similar yield, providing no evidence for partitioning of different molecular species of EGF receptors in the detergent-soluble and -insoluble fractions. Gel chromatography of the detergent-soluble membrane fraction on Sepharose 6-B revealed heterogeneity of ¹²⁵I-EGF binding activity; the smallest and most monodisperse peak of activity resolved by this technique was eluted at a Stokes radius of 95 Å. Operationally soluble ¹²⁵I-EGF binding activity also behaved heterogeneously during velocity sedimentation; more than half the activity sedimented more rapidly than the apparently monidisperse, 7S form. An average of less than half the nonionic detergent-solubilized activity recovered from 10 independent membrane preparations behaved as an apparently monodisperse entity. Since a maximum of 60% of ¹²⁵I-EGF binding activity was operationally soluble, less than 25% of the total EGF binding activity was recovered in an apparently monodisperse form. The remaining 75% of the EGF receptors displayed a marked tendency to exist as aggregates in nonionic detergent solutions.     

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.     

Acidic vesicles and the uptake of amines by sea urchin eggs

The radioactive amine [¹⁴C]methylamine is accumulated to a great extent by eggs, with kinetics that are dependent upon temperature (Q₁₀ = 5) and sensitive to metabolic inhibitors. Efflux of [¹⁴C]methylamine from eggs (preloaded with tracer concentrations) is increased immediately after fertilization or NH₄Cl activation. Fluorescent amines (9-aminoacridine (9AA) and acridine orange (AO)) are concentrated in small intracellular compartments, presumably vesicles. The possible role of these vesicles in the accumulation of amines by sea urchin eggs and the activation of the metabolism that ensues is discussed.     

Sodium and potassium compartmentation and transport in the roots of intact lettuce plants

In this report, we consider the accumulation in roots, and transport to the shoot, of Na⁺ and K⁺ in intact lettuce plants (Lactuca sativa cv Black-seeded Simpson). Plants were grown in modified Hoagland medium supplemented with 10 moles NaCl per cubic meter. At this salinity, significant levels of Na⁺ were accumulated in roots and shoots, but there was no reduction in plant growth. Transport characteristics for both Na⁺ and K⁺ were qualitatively similar to those previously reported, for Spergularia marina, indicating that the results obtained with these experimental protocols are not limited to one unconventional experimental plant. The most pronounced difference in transport of the two ions was evident when transport was followed in a chase period after a 10 minute uptake pulse. For Na⁺, there was an initially rapid, but small, loss of label to the medium, and very little movement to the shoot. For K⁺, little label was lost from the plants, but translocation to the shoot proceeded for at least 60 minutes. The transport systems were further distinguished by treating the roots during labeling with 20 micrograms per milliliter cycloheximide. For K⁺, both uptake and translocation were reduced by about 50%. For Na⁺, root accumulation was stimulated more than five-fold, while transport to the shoot was reduced about 20%. Cycloheximide also modified the Na⁺ transport characteristics such that continued translocation occurred during the chase period of pulse-chase studies.     

Evidence for endocytosis-dependent proteolysis in the generation of soluble truncated nerve growth factor receptors by A875 human melanoma cells.

We have identified nerve growth factor receptor (NGFR) on the cell surface and a truncated nerve growth factor receptor (NGFRt) in the conditioned medium of NGFR-negative cells that have been transfected with either the gene or the cDNA for the full-length receptor. By using cell surface iodination or metabolic labeling of A875 human melanoma cells, coupled with immunoprecipitation, we have determined the half-life of the cell-associated receptor to be approximately 7 h. Concomitant with receptor degradation is the accumulation of NGFRt in the extracellular medium. Approximately one-fifth of the labeled receptor can be recovered as the truncated species. These data support the hypothesis that NGFRt is generated by proteolysis of previously intact receptor. Furthermore, although no specific protease inhibitor assayed could affect this processing, NGFR degradation and truncation were retarded by treatment with: 1) the weak base amines, ammonium chloride or methylamine; 2) the carboxylic ionophore, monensin; or 3) the vacuolar ATPase inhibitor, bafilomycin A1, all agents that dissipate endosomal/lysosomal proton gradients via alternate mechanisms. Incubation of cells at 4 degrees C precluded NGFR degradation and truncation. The presence of ligand did not alter the time course of receptor truncation.     

Monensin inhibits receptor-mediated endocytosis of asialoglycoproteins in rat hepatocytes

Isolated rat liver parenchymal cells incubated in the presence of monensin exhibited a reduced uptake of ¹²⁵I-asialofetuin (¹²⁵I-AF). Binding studies indicated that the effect was due to a rapid reduction in the number of active surface receptors for the asialoglycoprotein. Monensin had no effect on receptor internalization, but apparently interrupted the recycling of receptors back to the cell surface. Monensin also inhibited the degradation of ¹²⁵I-AF previously bound to the cells; this inhibition was probably not due to a direct effect on intralysosomal proteolysis, as no lysosomal accumulation of undegraded ligand could be demonstrated in subcellular fractionation studies by means of sucrose gradients. It is more likely that monensin inhibits transfer of the labelled ligand from endocytic vesicles to lysosomes, as indicated by the accumulation of radioactivity in the former and by the ability of monensin to prevent the normally observed time-dependent increase in the buoyant density of endocytic vesicles. Whereas the effect of monensin on binding and uptake of asialofetuin was reversible, the effect on asialofetuin degradation could not be reversed.     

Binding, internalization and intracellular processing of 125I-epidermal growth factor purified by isoelectric focusing

When epidermal growth factor (EGF) which had been extensively purified by HPLC was subjected to iodination with sodium ¹²⁵iodide, 5 major species of differing isoelectric points were produced. Some of these species bound to rat fibroblasts with different affinities but were internalized with equal efficiency. Examination of the internalized ¹²⁵I-labelled molecules revealed processing of all the ¹²⁵I-EGF species to macromolecules with more acidic isoelectric points. The ¹²⁵I-EGF species with a pI of 4.5 corresponded in electrofocusing behavior with intact non-iodinated EGF. Other EGF species probably represented molecules which were covalently modified as a result of the iodination procedure.     

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.     

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.     

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.     

Weakly basic amines inhibit the proteolytic conversion of proalbumin to serum albumin in cultured rat hepatocytes

Effects of weak amines on the proteolytic conversion of proalbumin to serum albumin were studied in primary culture of rat hepatocytes. In control culture proalbumin was converted to serum albumin before discharge into the medium. However, in the presence of chloroquine the conversion to serum albumin was inhibited and proalbumin per se was released into medium. A similar inhibition of the processing was also observed in the presence of other amines such as methylamine and NH4Cl. Thus weak amines mimic the carboxylic ionophore monensin with regard to the effect on proalbumin conversion [Oda & Ikehara (1982) Biochem. Biophys. Res. Commun. 105, 766-772]. Since proteolytic conversion of proalbumin is believed to occur at the Golgi complex, these results suggest that weakly basic amines perturb the Golgi complex in addition to lysosomes and endosomes.     

成纤维细胞表皮生长因子的胞吞和向细胞核转移研究

本文以C3H小鼠胚胎正常成纤维细胞株C3H10T1/2C18(简称NC3H10)及其由氚标记脱氧胸苷(~3H-TdR)恶性转化的细胞株(简称TC 3H 10)为对象,研究了表皮生长因子(EGF)在受体介导下的胞吞和向细胞核转移现象。~(125)I-EGF 与细胞膜上的EGF 受体结合后,胞吞和向细胞核转移呈时间依赖性。两种细胞的EGF 的胞吞和向细胞核转移率接近。但是NC 3 H 10细胞~(125)I-EGF 胞知和向细胞核转移的绝对量明显高于TC 3 H 1 0细胞(p<0.05)。SDS-PAGE 电泳表明向细胞核转移的~(125)I-EGF 是未被降解的完整分子,溶酶体抑制剂NH_4Cl 对~(125)I-EGF 向细胞核转移有明显促进作用(p<0.05)。这些结果表明受体介导下的EGF 胞吞和向细胞核转移可能与EGF 的细胞核内凋节作用密切相关。     

Regulation of epidermal growth factor (EGF) receptor activity during the modulation of protein synthesis.

The capacity of cultured human fibroblasts to bind 125I-labeled epidermal growth factor (EGF) was measured during protein synthesis inhibition and reinitiation. Protein synthesis was inhibited by incubation of human fibroblasts in histidine-free medium supplemented with L-histidinol to produce a stringent amino acid starvation. Under these conditions 125 I-EGF binding activity decreased with a half-life of 14.5 hours. Protein synthesis could be rapidly reinitiated by the addition of L-histidine to human fibroblasts which had been preincubated in histidinol containing media for 36 to 48 hours. 125I-EGF binding activity rapidly increased upon the reinitiation of protein synthesis. In the presence of serum 100% of the original binding capacity was recovered ten hours after the reinitiation or protein synthesis, while 70% of the binding capacity was recovered in 12 hours in serum-free media. The recovery of 125I-EGF binding activity after the reinitiation of protein synthesis, was not blocked by the presence of Actinomycin D, indicating that the messenger RNA for the EGF receptor may accumulate during the period of histidinol-mediated inhibition of protein synthesis. The time course of recovery of 125I-EGF binding activity after the reinitiation of protein synthesis is very similar to that observed during the recovery of receptor activity following "down regulation" of EGF receptor activity. Recovery from down regulation, however, was markedly sensitive to Actinomycin D.     

Kinetics of poliovirus uncoating in HeLa cells in a nonacidic environment.

Lysis of HeLa cells infected with poliovirus revealed intact virus; 135S particles, devoid of VP4 but containing the viral RNA; and 80S empty capsids. During infection the kinetics of poliovirus uncoating showed a continuous decrease of intact virus, while the number of 135S particles and empty shells increased. After 1.5 h of infection conformational transition to altered particles resulted in complete disappearance of intact virions. To investigate the mechanism of poliovirus uncoating, which has been suggested to depend on low pH in endosomal compartments of cells, we used lysosomotropic amines to raise the pH in these vesicles. In the presence of ammonium chloride, however, the kinetics of uncoating were similar to those for untreated cells, whereas in cells treated with methylamine, monensin, or chloroquine, uncoating was merely delayed by about 30 min. This effect could be attributed to a delay of virus entry into cells after treatment with methylamine and monensin, whereas chloroquine stabilized the viral capsid itself. Thus, elevation of endosomal pH did not affect virus uncoating. We therefore propose a mechanism of poliovirus uncoating which is independent of low pH.