共查询到20条相似文献,搜索用时 15 毫秒
1.
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. 相似文献
2.
Intracellular processing of epidermal growth factor and its effect on ligand-receptor interactions 总被引:7,自引:0,他引:7
H S Wiley W VanNostrand D N McKinley D D Cunningham 《The Journal of biological chemistry》1985,260(9):5290-5295
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. 相似文献
3.
We have investigated the cellular fate of epidermal growth factor (EGF) in KB cells and a variant, KB-R2A, that was isolated based on its resistance to diphtheria toxin and subsequently was shown to be resistant to infection by RNA viruses (Moehring and Moehring, 1972, Infect. Immunity. 6:487-492). Both cell lines bind 125I-EGF and internalize the cell-bound hormone at the same rate. However, when the degradation of internalized 125I-EGF was measured by the release of low molecular weight (mw) hydrolysis products into the medium, the toxin-resistant KB-R2A cells degraded the hormone at a drastically reduced rate; 50% and 3% of the cell-bound 125I-EGF was degraded and released by 80 min in the KB and KB-R2A cells, respectively. To investigate the fate of cell-associated EGF prior to release into the medium, the radioactivity in extracts of cells labeled with 125I-EGF was fractionated by native gel electrophoresis. In KB cells three peaks of radioactivity other than native 125I-EGF were resolved. Time course and subcellular fractionation studies showed that the first processed product appeared while the hormone was located in the endocytic vesicles and the appearance of the other two peaks correlated with the arrival of the hormone in the lysosomal compartment. KB-R2A cells also produced the first intermediate but they produced only very low amounts of the other two peaks. These studies show that endocytic vesicles in both cell lines contain enzymes capable of processing EGF prior to the arrival of the hormone in the lysosomes and show that the KB-R2A cells have a lesion that prevents the complete degradation of the hormone. We propose that the KB-R2A cell line has a defective mechanism for the intracellular processing of a number of ligands that are internalized by the process of receptor-mediated endocytosis and that this defect is located beyond the initial endocytic step. 相似文献
4.
Recycling of epidermal growth factor-receptor complexes in A431 cells: identification of dual pathways 总被引:5,自引:2,他引:3
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A Sorkin S Krolenko N Kudrjavtceva J Lazebnik L Teslenko A M Soderquist N Nikolsky 《The Journal of cell biology》1991,112(1):55-63
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. 相似文献
5.
L A Casciola-Rosen C A Renfrew A L Hubbard 《The Journal of biological chemistry》1992,267(17):11856-11864
We used a combination of subcellular fractionation and lactoperoxidase-mediated iodination to examine the polypeptide compositions of three hepatocyte endocytic compartments: early endosomes, late endosomes, and lysosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase which binds specifically to asialoglycoprotein receptors was perfused through isolated rat livers at 37 degrees C. Subcellular fractions enriched in various endocytic compartments were then isolated by differential and isopycnic centrifugation, and the lactoperoxidase moiety of the internalized conjugate was used to catalyze the iodination of lumenal-facing proteins. The 125I profiles of early and late endosomes were strikingly similar after gel electrophoresis. Using immunoprecipitation, we directly identified and compared the relative amounts of the Na+,K(+)-ATPase and several different acid hydrolases and membrane receptors in all three fractions. The asialoglycoprotein receptor and the low density lipoprotein related protein were approximately nine times more abundant in early endosomes than late endosomes, suggesting that they recycle from early endosomes. In addition, cathepsin D, but not cathepsin L, beta-glucuronidase, and lgp 120, was detected in early endosomes; however, all of these molecules were detected in lysosomes. Our findings provide strong evidence that early endosomes mature into late endosomes and that there is either selective delivery or selective retention of hydrolases at discrete points in the endocytic pathway. 相似文献
6.
Recycling of epidermal growth factor in A431 cells 总被引:3,自引:0,他引:3
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. 相似文献
7.
Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics 总被引:22,自引:18,他引:4
We have used biochemical and morphological techniques to demonstrate that hepatocytes in the perfused liver bind, internalize, and degrade substantial amounts of murine epidermal growth factor (EGF) via a receptor-mediated process. Before ligand exposure, about 300,000 high-affinity receptors were detectable per cell, displayed no latency, and co-distributed with conventional plasma membrane markers. Cytochemical localization using EGF coupled to horseradish peroxidase (EGF-HRP) revealed that the receptors were distributed along the entire sinusoidal and lateral surfaces of hepatocytes. When saturating concentrations of EGF were perfused through a liver at 35 degrees C, ligand clearance was biphasic with a rapid primary phase of 20,000 molecules/min per cell that dramatically changed at 15-20 min to a slower secondary phase of 2,500 molecules/min per cell. During the primary phase of uptake, approximately 250,000 molecules of EGF and 80% of the total functional receptors were internalized into endocytic vesicles which could be separated from enzyme markers for plasma membranes and lysosomes on sucrose gradients. The ligand pathway was visualized cytochemically 2-25 min after EGF-HRP internalization and a rapid transport from endosomes at the periphery to those in the Golgi apparatus-lysosome region was observed (t 1/2 approximately equal to 7 min). However, no 125I-EGF degradation was detected for at least 20 min. Within 30 min after EGF addition, a steady state was reached which lasted up to 4 h such that (a) the rate of EGF clearance equaled the rate of ligand degradation (2,500 molecules/min per cell); (b) a constant pool of undegraded ligand was maintained in endosomes; and (c) the number of accessible (i.e., cell surface) receptors remained constant at 20% of initial values. By 4 h hepatocytes had internalized and degraded 3 and 2.3 times more EGF, respectively, than the initial number of available receptors, even in the presence of cycloheximide and without substantial loss of receptors. All of these results suggest that EGF receptors are internalized and that their rate of recycling to the surface from intracellular sites is governed by the rate of entry of ligand and/or receptor into lysosomes. 相似文献
8.
Dileucine-based motifs have been shown to regulate endosomal sorting of a number of membrane proteins. Previously, we have shown that the dileucine motif Leu(679), Leu(680) in the juxtamembrane domain of the human epidermal growth factor receptor is involved in the endosome-to-lysosome transport of ligand-receptor complexes. Substitution of alanine residues for Leu(679), Leu(680) led to a reduction in ligand-induced receptor degradation without affecting internalization. In the current study, we have further characterized ligand-dependent intracellular sorting of EGF receptors containing a L679A, L680A. Immunocytochemical studies reveal that although mutant receptors redistribute from the cell surface to transferrin receptor-positive endocytic vesicles similar to wild-type following ligand stimulation, their accumulation in Lamp-1-positive late endosomes/lysosomes is retarded compared to wild-type. Kinetic analysis of (125)I-EGF trafficking shows that reduced accumulation of internalized mutant receptors in Lamp-1-positive vesicles is due to rapid recycling of ligand-receptor complexes from early endocytic compartments. In addition, the fraction of intracellular (125)I-EGF that is transported to late endocytic compartments in cells with mutant receptors is not as efficiently degraded as it is in cells with wild-type receptors. Furthermore, wild-type receptors in endocytic vesicles isolated by Percoll gradient fractionation are more resistant to in vitro digestion with proteinase K than mutant receptors. We propose that mutant receptors interact inefficiently with lysosomal sorting machinery, leading to their increased recycling. Our results are consistent with a model in which the Leu(679), Leu(680) signal facilitates sequestration of ligand-receptor complexes into internal vesicles of multivesicular endosome-to-lysosome transport intermediates. 相似文献
9.
Receptor-bound somatostatin and epidermal growth factor are processed differently in GH4C1 rat pituitary cells 总被引:3,自引:0,他引:3
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GH4C1 cells, a clonal strain of rat pituitary tumor cells, have high-affinity, functional receptors for the inhibitory hypothalamic peptide somatostatin (SRIF) and for epidermal growth factor (EGF). In this study we have examined the events that follow the initial binding of SRIF to its specific plasma membrane receptors in GH4C1 cells and have compared the processing of receptor-bound SRIF with that of EGF. When cells were incubated with [125I-Tyr1]SRIF at temperatures ranging from 4 to 37 degrees C, greater than 80% of the specifically bound peptide was removed by extraction with 0.2 M acetic acid, 0.5 M NaCl, pH 2.5. In contrast, the subcellular distribution of receptor-bound 125I-EGF was temperature dependent. Whereas greater than 95% of specifically bound 125I-EGF was removed by acid treatment after a 4 degrees C binding incubation, less than 10% was removed when the binding reaction was performed at 22 or 37 degrees C. In pulse-chase experiments, receptor-bound 125I-EGF was transferred from an acid-sensitive to an acid-resistant compartment with a half-time of 2 min at 37 degrees C. In contrast, the small amount of [125I-Tyr1]SRIF that was resistant to acid treatment did not increase during a 2-h chase incubation at 37 degrees C. Chromatographic analysis of the radioactivity released from cells during dissociation incubations at 37 degrees C showed that greater than 90% of prebound 125I-EGF was released as 125I-tyrosine, whereas prebound [125I-Tyr1]SRIF was released as a mixture of intact peptide (55%) and 125I-tyrosine (45%). Neither chloroquine (0.1 mM), ammonium chloride (20 mM), nor leupeptin (0.1 mg/ml) increased the amount of [125I-Tyr1]SRIF bound to cells at 37 degrees C. Furthermore, chloroquine and leupeptin did not alter the rate of dissociation or degradation of prebound [125I-Tyr1]SRIF. In contrast, these inhibitors increased the amount of cell-associated 125I-EGF during 37 degrees C binding incubations and decreased the subsequent rate of release of 125I-tyrosine. The results presented indicate that, as in other cell types, EGF underwent rapid receptor-mediated endocytosis in GH4C1 cells and was subsequently degraded in lysosomes. In contrast, SRIF remained at the cell surface for several hours although it elicits its biological effects within minutes. Furthermore, a constant fraction of the receptor-bound [125I-Tyr1]SRIF was degraded at the cell surface before dissociation. Therefore, after initial binding of [125I-Tyr1]SRIF and 125I-EGF to their specific membrane receptors, these peptides are processed very differently in GH4C1 cells. 相似文献
10.
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. 相似文献
11.
K. A. Kondratov A. L. Chernorudskiy A. P. Amosova E. S. Kornilova 《Cell and Tissue Biology》2009,3(5):454-460
In the work, the effect of tyrphostin AG1478, a specific inhibitor of the receptor tyrosine kinase, on the behavior of an
internalized EGF receptor at different stages upon the stimulation of endocytosis has been analyzed. It was found that tyrphostin
added 30 min after the stimulation of endocytosis resulted in recycling of a significant portion of 125I-EGF onto the cell surface. This portion decreased with time. EGF-receptor complexes, which are recycled under the action
of AG1478, did not dissociate, possibly due to the ability of tyrphostin AG1478 to initiate receptor oligomerization in the
absence of ligand and, therefore, probably affect dissociation constants. It was found that only a portion of the EGF receptor
localized in early endosomes was able to recycle upon TK inhibition. The addition of inhibitor 30 and 60 min after the stimulation
of endocytosis resulted in a decrease in the labeled EGF degradation. At early stages, internalized EGF-receptor complexes
were mostly blocked in early endosomes, while, at late stages, their accumulation occurred in incompletely matured late endosomes.
These data indicate that there is the late endocytic stage transition that depends on the receptor TK. Furthermore, the addition
of tyrphostin after 90 min of endocytosis did not lead to a decrease, but rather an increase in degradation, which indicates
the existence of mechanisms that create a temporal window during which receptor TK can carry out functions that are not directly
connected with endocytosis. 相似文献
12.
Ligand-mediated autophosphorylation activity of the epidermal growth factor receptor during internalization 总被引:7,自引:2,他引:5
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《The Journal of cell biology》1989,109(6):2751-2760
The association of EGF with its receptor in endosomes isolated from rat liver homogenates was assessed biochemically by polyethylene glycol precipitation and morphologically by electron microscope radioautography. The proportion of receptor-bound ligand in endosomes at 15 min after the injection of doses of 0.1 and 1 microgram EGF/100 g body weight was 57%. This value increased to 77% for the dose of 10 micrograms EGF injected. Quantitative electron microscope radioautography carried out on endosomes isolated at 15 min after the injection of 10 micrograms 125I-EGF demonstrated that most radiolabel was over the endosomal periphery thereby indicating that ligand- receptor complexes were in the bounding membrane but not in intraluminal vesicles of the content. EGF receptor autophosphorylation activity during internalization was evaluated in plasmalemma and endosome fractions. This activity was markedly but transiently reduced on the cell surface shortly after the administration of saturating doses of EGF. The same activity, however, was augmented and prolonged in endosomes for up to 30 min after EGF injection. The transient desensitization of cell surface activity was not due to prior in vivo phosphorylation since receptor dephosphorylation in vitro failed to restore autophosphorylation activity. Transient desensitization of cell surface autophosphorylation activity coincided with a diminished capacity for endocytosis of 125I-EGF with endocytosis returning to normal after the restoration of cell surface autophosphorylation activity. The inhibition of cell surface autophosphorylation activity and the activation of endosomal autophosphorylation activity coincident with downregulation suggest that EGF receptor traffic is governed by ligand-regulated phosphorylation activity. 相似文献
13.
Thyrotropin releasing hormone action in pituitary cells. Protein kinase C-mediated effects on the epidermal growth factor receptor 总被引:1,自引:0,他引:1
Thyrotropin releasing hormone (TRH) causes phosphatidylinositol bisphosphate hydrolysis to form inositol trisphosphate and diacylglycerol. Since diacylglycerol activates protein kinase C (Ca2+/phospholipid-dependent enzyme), this enzyme may be involved in mediating the physiological response to TRH. Activation of protein kinase C leads to phosphorylation of receptors for epidermal growth factor (EGF) and decreased EGF affinity. The present study examined the effect of TRH on EGF binding to intact GH4C1 rat pituitary tumor cells to test whether TRH activates protein kinase C. Cells were incubated with TRH at 37 degrees C and specific 125I-EGF binding was then measured at 4 degrees C. 125I-EGF binding was decreased by a 10-min treatment with 0.1-100 nM TRH to 30-40% of control in a dose-dependent manner. 125I-EGF binding was not altered if cells were incubated at 4 degrees C, although TRH receptors were saturated or in a variant pituitary cell line without TRH receptors. TRH (10 min at 37 degrees C) decreased EGF receptor affinity but caused little change in receptor density, 125I-EGF internalization, or degradation. When cells were incubated continuously with TRH, there was a recovery of 125I-EGF binding after 24 h. Incubation with the protein kinase C activating phorbol ester TPA caused an immediate (less than 10 min) profound (greater than 85%) decrease in 125I-EGF binding followed by partial recovery at 24 h. Maximally effective doses of TRH and TPA decreased EGF receptor affinity with half-times of 3 min. EGF treatment (5 min) caused an increase in the tyrosine phosphate content of several proteins; prior incubation with TRH resulted in a small decline in the EGF response. GH4C1 cells were incubated with 500 nM TPA for 24 h in order to down-regulate protein kinase C. Protein kinase C depletion was confirmed by immunoblots and the effects of TRH and TPA on 125I-EGF binding were tested. TRH and TPA were both much less effective in cells pretreated with phorbol esters. TRH increased cytoplasmic pH measured with an intracellularly trapped pH sensitive dye after mild acidification with nigericin. This TRH response is presumed to be the result of protein kinase C-mediated activation of the amiloride-sensitive Na+/H+ exchanger and was blunted in protein kinase C-depleted cells. All of these results are consistent with the view that TRH acts rapidly in the intact cell to activate protein kinase C and that a consequence of this activation is EGF receptor phosphorylation and Na+/H+ exchanger activation. 相似文献
14.
Proteolytic cleavage of ricin A chain in endosomal vesicles. Evidence for the action of endosomal proteases at both neutral and acidic pH 总被引:3,自引:0,他引:3
Macrophages actively internalize macromolecules into endosomal vesicles containing proteases. The plant toxin, ricin A chain delivered into this pathway by receptor-mediated endocytosis, was found to be exquisitely sensitive to cleavage by these proteases. Proteolytic fragments of ricin A chain were generated within cells as early as 2-3 min after internalization. Toxin proteolysis was initiated in early endosomal vesicles, and transport to lysosomes was not required. As endosomes transit the cell, their lumenal pH drops from neutral to acidic. Previous studies in macrophages had suggested that endosomal proteolysis is dependent on vesicle acidification. Isolated endosomal vesicles containing ricin A chain catalyzed the cleavage of this protein in vitro; however, proteolysis was observed at both neutral and acidic pH. Experiments using isolated endosomes demonstrated that both cysteine and aspartyl proteases were responsible for the cleavage of ricin A chain. The cysteine protease, cathepsin B, catalyzed toxin proteolysis in endosomes between pH 4.5 and 7.0 while aspartyl protease activity was maximal below pH 5.5. Radiolabeling the lumenal contents of macrophage endosomes confirmed that both the cysteine protease, cathepsin B, and the aspartyl protease, cathepsin D, were present in these vesicles. These proteases were not present on the plasma membrane but were found in early endosomes indicating they are derived from an intracellular source. The presence of proteases with different pH optima in early endosomes suggests that processing in these vesicles may be regulated by changes in endosomal pH. This result represents an important difference in protein processing in endosomes versus lysosomes and provides new insights into the function of endosomal proteases. 相似文献
15.
Rabbit alveolar macrophages rapidly internalize and degrade mannosylated bovine serum albumin (125I-mannose-BSA). Trichloroacetic acid-soluble degradation products appear in the cells as early as 6 min after uptake at 37 degrees C, and in the extracellular medium after 10 min. Incubation of endocytic vesicles containing this ligand in isotonic buffers at pH 7.4 + ATP resulted in intravesicular proteolysis, which was inhibited by monensin, nigericin, or ammonium chloride. At pH 5.0, degradation proceeded rapidly and was abolished by lysis of the vesicles with 0.1% Triton X-100. Readdition of lysosomes to the incubation mixture did not increase the rate of prelysosomal degradation. Proteolysis of 125I-mannose-BSA was optimal at pH 4.5, and inhibited by low concentrations of the cathepsin D inhibitor pepstatin A. After subcellular fractionation of the macrophages on Percoll gradients, 125I-mannose-BSA sedimented with prelysosomal vesicles and was not transported to secondary lysosomes. Addition of pepstatin A to extracellular medium during internalization of prebound 125I-mannose-BSA partially inhibited degradation of ligand, and resulted in transfer of undegraded 125I-mannose-BSA to lysosomes after 20 min. Using 125I-bovine serum albumin as a substrate for the protease in the presence of 0.1% Triton X-100, we have shown that as much as 36% of the total pepstatin A-sensitive activity sediments with nonlysosomal membranes. After intraendosomal iodination using lactoperoxidase, a labeled protease was isolated by affinity chromatography on pepstatin-agarose. The labeled protease, which had a subunit size of 46 kDa, was detected in endocytic vesicles after 5 min of internalization. These results suggest that a cathepsin D-like protease is responsible for the degradation of 125I-mannose-BSA in macrophages, and that this ligand is degraded in a prelysosomal vesicle. 相似文献
16.
Endosome-lysosome fusion at low temperature 总被引:5,自引:0,他引:5
Based on an initial study (Dunn, W. A., Hubbard, A. L., and Aronson, Jr., N. N. (1980) J. Biol. Chem. 255, 5971-5978), low temperature is often used to selectively inhibit fusion between endosomes and lysosomes. Here we have tried to characterize the nature of this inhibition. In addition to endocytic contents markers, we have used a covalent membrane marker to measure the interaction between endosomes and lysosomes over extended periods of time at low temperature. Mouse macrophage cells (P388D1) and human skin fibroblasts were enzymatically labeled with radioactive galactose to provide a covalent marker for plasma-membrane glycoconjugates. Subsequent endocytic membrane traffic for 24 h at 16 degrees C resulted in a significant transfer of membrane marker, as well as of endocytic contents marker, to high density lysosomes, as observed by subcellular fractionation. The kinetics of this transfer have been analyzed for macrophages using the membrane marker, horseradish peroxidase as fluid-phase, and iodinated acetyl low density lipoprotein as receptor-mediated endocytic contents marker. Transfer to lysosomes occurred only about 6 h after application of the respective marker at 16 degrees C. When transfer to lysosomes was initiated by 15 min preincubation at 37 degrees C, subsequent cooling to 16 degrees C did not inhibit ongoing transfer which continued with the same kinetics as when observed after the lag phase. These results show that low temperature delays an unidentified pre-fusion step, but does not inhibit endosome-lysosome fusion as such. 相似文献
17.
The hepatic binding and uptake kinetics of epidermal growth factor: studies with isolated rat hepatocytes 总被引:1,自引:0,他引:1
Hepatocytes are known to bind and internalize a variety of small molecular weight proteins by a process known as receptor-mediated endocytosis (RME). The purpose of this investigation was to characterize the binding and uptake kinetics of a small protein known to be taken up by the liver by RME, epidermal growth factor (EGF), using suspensions of freshly isolated rat hepatocytes. Rat hepatocytes accumulated 125I-EGF (90 pM) in a temperature-dependent fashion. Isolated hepatocytes incubated at 37 degrees C with 125I-EGF began to release a TCA-soluble radiolabeled material into the incubation medium with a lag period of 20 min. EGF uptake by isolated hepatocytes was linear for only 60 seconds and displayed saturation kinetics (apparent Km of 4 nM and a Vmax of 105 fM/min/10(6) cells). Hepatocytes incubated at 4 degrees C bound, but did not internalize, EGF. Under these conditions, EGF binding was saturable at concentrations above 8 nM. A Scatchard analysis revealed that the average number of receptors per hepatocyte was 7.7 X 10(4) with a dissociation constant of 2.6 nM. These data demonstrate that freshly isolated hepatocytes are capable of binding, internalizing and metabolizing EGF and thus are a good model to study RME of small molecular weight proteins. 相似文献
18.
Inhibition of the binding of 125I-labelled epidermal growth factor to mouse cells by a mitogen in goat mammary secretions. 总被引:9,自引:6,他引:3
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Pre-colostrum and colostrum from goats cause a marked inhibition of the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The ability of these secretions to inhibit 125I-EGF binding is closely correlated with the ability to stimulate DNA synthesis in quiescent 3T3 cell cultures, suggesting that goat mammary secretions may contain an EGF-related mitogen. However, the material in colostrum which inhibits 125I-EGF binding to Swiss 3T3 cells is a basic protein with Mr greater than 20000 and is thus quite different from mouse and human EGF. Furthermore, the colostral-mediated inhibition of 125I-EGF binding, although rapid and apparently competitive, differs from the inhibition of binding induced by native, unlabelled EGF. Thus, the inhibitory effect of colostrum is markedly decreased when the assay temperature is shifted from 37 degrees C to 4 degrees C whereas unlabelled EGF is an effective competitive inhibitor at both 37 degrees C and 4 degrees C. Incubation of cells with EGF causes a reduction in cell surface EGF receptors whereas exposure to colostrum does not induce down-regulation of the EGF receptor. Our results suggest that the colostral factor does not bind directly to EGF receptors but inhibits 125I-EGF binding by an indirect mechanism which involves a temperature-sensitive step. 相似文献
19.
D G Kay W H Lai M Uchihashi M N Khan B I Posner J J Bergeron 《The Journal of biological chemistry》1986,261(18):8473-8480
The rat liver epidermal growth factor (EGF) receptor was assessed for EGF-dependent autophosphorylation as well as phosphorylation of a defined exogenous substrate in purified plasmalemma and Golgiendosome fractions isolated from rat liver homogenates. While EGF-dependent kinase activity was readily detected in plasmalemma the corresponding activity in Golgi-endosome fractions required detergent. Consequent to the systemic injection of EGF in vivo, the majority (approximately 60%) of receptor as evaluated by 125I-EGF binding was rapidly lost (T 1/2 approximately 8 min) from the plasmalemma and correspondingly accumulated in the Golgi-endosome fraction in a dose-dependent manner. Electron microscope radioautography of 125I-EGF uptake into Golgi-endosome fractions identified internalization into lipoprotein-filled vesicles of heterogenous size and shape but not into stacked saccules of the Golgi apparatus. Evaluation of receptor kinase activity in plasmalemma fractions isolated at various times after EGF injection in vivo showed more rapid loss of EGF-dependent autophosphorylation activity (T 1/2 approximately 10 s) than of receptor content (T 1/2 approximately 8 min). In contrast to the EGF receptor kinase of the plasmalemma fraction, kinase activity accumulating in endosomes was activated, i.e. maximally stimulated, in the absence of EGF or Triton X-100 in vitro. Furthermore, following the peak time of accumulation of EGF receptor kinase in endosomes (5-15 min) EGF-dependent autophosphorylation activity and EGF receptor content were lost more slowly (T 1/2 approximately 27 and 87 min for the loss of autophosphorylation activity and receptor content, respectively). The rapidity of translocation of activated EGF receptor into endosomes (30 s) and the dose response to low levels (1 microgram) of EGF injected are consistent with a physiological role for internalized EGF receptor kinase activity. 相似文献
20.
Diacylglycerol treatment rapidly decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells 总被引:6,自引:0,他引:6
The synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol (OAG) and phorbol esters activate protein kinase C in intact cells. We report here that OAG inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The inhibition was detected as early as 1 min after treatment at 37 degrees C and persisted for at least 120 min. The effect of OAG was reversed upon removal of this diacylglycerol. Detailed Scatchard analysis of 125I-EGF binding to Swiss 3T3 cells at 4 degrees C after a 1 h incubation with a saturating dose of OAG at 37 degrees C, demonstrates that this OAG pretreatment does not change the apparent number of EGF receptors but causes a marked decrease in their apparent affinity for the ligand. Prolonged treatment (40 h) of the cells with phorbol dibutyrate (PBt2) which causes a marked decrease in the number of phorbol ester binding sites and in the activity of protein kinase C, prevented the inhibition of 125I-EGF binding by both PBt2 and OAG. The results support the possibility that protein kinase C plays a role in the transmodulation of the EGF receptor in intact cells. 相似文献