A comparison of catecholamine-induced internalization of beta-adrenergic receptors and receptor-mediated endocytosis of epidermal growth factor in human astrocytoma cells. Inhibition by phenylarsine oxide

The ligand-induced internalization of beta-adrenergic receptors and the receptor-mediated internalization of epidermal growth factor were blocked, under similar conditions, by phenylarsine oxide (PAO) in human astrocytoma cells (1321N1). The inhibition was not prevented or reversed by monofunctional sulfhydryl agents such as 2-mercaptoethanol or glutathione; however, the inhibitory action of PAO was blocked and reversed by bifunctional thiols such as 2,3-dimercaptoethanol or dithiothreitol. The results are consistent with the interaction of PAO with vicinal sulfhydryl groups to form a stabile ring structure. PAO did not prevent isoproterenol-induced uncoupling (desensitization) of beta-adrenergic receptors even though receptor internalization was completely blocked. The effects of PAO on receptor internalization could not be explained by any action of the trivalent arsenical to lower ATP levels. Ligand binding to both receptors was not detectably altered by PAO under conditions selective for inhibition for endocytosis. The results suggest a common mechanism for internalization of beta-adrenergic receptors and epidermal growth factor by a process that involves vicinal sulfhydryl groups.     

Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization

We have developed a quantitative method to evaluate the interaction between cell surface receptors and the endocytic apparatus. This method exploits occupancy-dependent changes in internalization rates that occur in cells expressing high numbers of receptors. We found that constitutive internalization of the transferrin receptor behaves as a simple, first order process that is unaltered by ligand. Internalization of the epidermal growth factor (EGF) receptor, however, behaves as a saturable, second order process that is induced by receptor occupancy. Internalization of EGF receptors occurs through at least two distinct pathways: a low capacity pathway that has a relatively high affinity for occupied receptors, and a low affinity pathway that has a much higher capacity. The high affinity pathway was observed in all cells having receptors with intrinsic tyrosine kinase activity. Mutant EGF receptors lacking kinase activity could not utilize the high affinity pathway and were internalized only through the low affinity one. Mutated receptors with decreased affinity for kinase substrates were also internalized at decreased rates through the high affinity, inducible pathway. In the case of vitellogenin receptors in Xenopus oocytes, occupied receptors competed more efficiently for internalization than empty ones. Insulin increased the endocytic capacity of oocytes for vitellogenin receptors. Similarly, serum increased the capacity of the inducible pathway for EGF receptors in mammalian cells. These data are consistent with a model of internalization in which occupied receptors bind to specific cellular components that mediate rapid internalization. Ligand-induced internalization results from an increase in the affinity of occupied receptors for the endocytic apparatus. Hormones can also indirectly regulate endocytosis by increasing the number of coated pits or their rate of internalization. The ability to dissect receptor-specific effects from cell-specific ones should be very useful in investigating the molecular mechanisms of receptor mediated endocytosis.     

Epidermal growth factor receptor internalization through clathrin-coated pits requires Cbl RING finger and proline-rich domains but not receptor polyubiquitylation

Cbl proteins have been implicated in the regulation of endocytic trafficking of epidermal growth factor receptor. However, the precise role of Cbl in epidermal growth factor receptor endocytosis is not defined. To directly visualize Cbl in cells and perform structure-function analysis of Cbl's role in epidermal growth factor receptor internalization, a yellow fluorescent protein-fusion of c-Cbl was constructed. Upon epidermal growth factor receptor activation, Cbl-yellow fluorescent protein moved with epidermal growth factor receptor to clathrin-coated pits and endosomes. Localization of Cbl-yellow fluorescent protein to these endocytic organelles was dependent on a proline-rich domain of c-Cbl that interacts with Grb2 as shown by fluorescence resonance energy transfer microscopy. In contrast, direct binding of Cbl to phosphotyrosine 1045 of the epidermal growth factor receptor was required for epidermal growth factor receptor polyubiquitination, but was not essential for Cbl-yellow fluorescent protein localization in epidermal growth factor receptor-containing compartments. These data suggest that the binding of Cbl to epidermal growth factor receptor through Grb2 is necessary and sufficient for Cbl function during clathrin-mediated endocytosis. Overexpression of c-Cbl mutants that are capable of Grb2 binding but defective in linker/RING finger domain function severely inhibited epidermal growth factor receptor internalization. The same dominant-negative mutants of Cbl did not block epidermal growth factor receptor recruitment into coated pits but retained receptors in coated pits, thus preventing receptor endocytosis and transport to endosomes. These data suggest that the linker and RING finger domain of Cbl may function during late steps of coated vesicle formation. We propose that the RING domain of Cbl facilitates endocytosis either by epidermal growth factor receptor monoubiquitylation or by ubiquitylation of proteins associated with the receptor.     

Inhibitors of transglutaminase prevent agonist-mediated internalization of beta-adrenergic receptors

In frog erythrocytes, desensitization of beta-adrenergic receptors is characterized by a decrease in the beta-receptor recognition sites in the plasma membrane and a concomitant increase in the number of this receptor's binding sites in the cytosol. We have documented that this redistribution of the receptor recognition sites reflects the internalization of the surface-bound beta-adrenergic receptors. The present study was addressed to examine whether transglutaminase plays a role in the agonist-mediated internalization of beta-adrenergic receptor recognition sites. Pretreatment of cells with methylamine was found to decrease the internalization and the loss of membrane-bound beta-adrenergic receptors induced by isoproterenol. Methylamine appears to be equally potent in inhibiting transglutaminase activity and in preventing internalization and the receptor loss. The effect of methylamine on soluble and on membrane-bound beta-adrenergic receptors is due to a change in Bmax rather than Kd of these binding sites. Among eight inhibitors of transglutaminase tested, the rank order potency for blocking the enzyme can be correlated with that for preventing the receptor loss and receptor internalization. Moreover, these drug effects on beta-adrenergic receptors are unrelated to the inhibition of isoproterenol-sensitive adenylate cyclase or the binding of [3H]dihydroalprenolol to beta-receptors. These result may lend credence to the view that transglutaminase participates in the internalization and the decrease of membrane-bound receptors during desensitization of beta-adrenergic receptors.     

Transplanted LDL and mannose-6-phosphate receptor internalization signals promote high-efficiency endocytosis of the transferrin receptor.

The internalization signals of several constitutively recycling receptors have recently been identified as regions of four or six amino acids that include an aromatic residue, usually tyrosine. Here, we show that transplanted signals from the low density lipoprotein receptor (LDLR) and cation-independent mannose-6-phosphate receptor (Man-6-PR) promote rapid internalization of the transferrin receptor (TR), directly establishing that recognition signals are interchangeable, self-determined structural motifs and that signals from type I membrane proteins are active in a type II receptor. We also show that the chemical and spatial patterns of critical residues in both four- and six-residue internalization motifs are consistent with a tight turn structure. A six-residue LDLR signal is needed for activity in TR, suggesting that an amino-terminal aromatic side chain is obligatory. In contrast, the carboxy-terminal aromatic side chain in the TR signal can be replaced by a large hydrophobic residue. Thus, internalization signals apparently require an aromatic amino-terminal residue and either an aromatic or large hydrophobic carboxy-terminal residue rather than a conserved tyrosine per se. Consistent with this conclusion, predicted internalization signals from the poly-Ig receptor, YSAF, and asialoglycoprotein receptor (ASGPR) subunit H1, YQDL, also promote internalization of TR.     

Large-scale screening assay to measure epidermal growth factor internalization

Recently, we showed that the internalization of the epidermal growth factor (EGF) receptor is inhibited by hydrogen peroxide (H(2)O(2)) in human fibroblasts. In order to test the effect of various stress conditions on receptor internalization and to test a variety of antioxidants in their capacity to prevent or reduce the H(2)O(2)-induced inhibition of internalization, a screening assay was developed to measure the internalization in 96-well plates. In this assay, cells are exposed to biotin-conjugated EGF and the amount of internalized EGF is detected with horseradish peroxidase-conjugated streptavidin. We show that the results obtained by this new assay are comparable with those from internalization studies performed with radioactive labeled EGF. Therefore, the cellular internalization assay as presented here is a reliable method to measure EGF receptor internalization. Moreover, because elaborate processing of the cells is not required, the assay is a relatively fast and inexpensive method to study ligand-induced internalization in 96-well plates and thereby is suitable for large-scale screening of compounds or conditions interfering with this internalization.     

The asialoglycoprotein receptor internalizes and recycles independently of the transferrin and insulin receptors

Receptor-mediated endocytosis of specific ligands is mediated through clustering of receptor-ligand complexes in coated pits on the cell surface, followed by internalization of the complex into endocytic vesicles. We show that internalization of asialoglycoprotein by HepG2 hepatoma cells is accompanied by a rapid (t1/2 = 0.5-1 min) depletion of surface asialoglycoprotein receptors. This is followed by a rapid (t1/2 = 2-4 min) reappearance of surface receptors; most of these originate from endocytosed cell-surface receptors. The loss and reappearance of asialoglycoprotein receptors is specific, and depends on prebinding of ligand to its receptor. HepG2 cells also contain abundant receptors for both insulin and transferrin. Endocytosis of asialoglycoprotein and its receptor has no effect on the number of surface binding sites for transferrin or insulin. We conclude that binding of asialoglycoprotein to its surface receptor triggers a rapid and specific endocytosis of the receptor-ligand complex, probably due to a clustering in clathrin-coated pits or vesicles.     

Relationship between an altered membrane form and a low affinity form of the beta-adrenergic receptor occurring during catecholamine-induced desensitization. Evidence for receptor internalization

We have investigated the relationship between the catecholamine-induced occurrence in 1321N1 human astrocytoma cells of beta-adrenergic receptors that exhibit low apparent affinity for hydrophilic ligands in short-time assays with intact cells and a population of beta-adrenergic receptors that migrate in a light vesicle fraction on sucrose density gradients. Pretreatment of cells with concanavalin A prevents the generation of both of these forms of the receptor during incubation with agonists but does not prevent the agonist-induced decrease in isoproterenol-stimulated cyclic AMP production that also occurs during desensitization. Selective labeling of the low affinity beta-receptors with 125I-pindolol followed by centrifugation on sucrose density gradients revealed that all of the receptors in the light vesicle fraction from desensitized cells were of the low affinity type, but that a portion of the low affinity receptors also migrated in a heavier sucrose fraction together with the plasma membrane. In contrast, in control cells, no low affinity receptors were present in the heavy sucrose fractions. The agonist-induced occurrence of these various forms of the beta-adrenergic receptor can be explained on the basis of current models of desensitization involving agonist-induced internalization of beta-adrenergic receptors.     

Tyrosine kinase-defective insulin receptors undergo insulin-induced microaggregation but do not concentrate in coated pits

Biologically active colloid-gold complexes were used to compare ligand-induced microaggregation, redistribution, and internalization of insulin receptors on Rat 1 fibroblasts expressing wild type (HIRc) or tyrosine kinase-defective (HIR A/K1018) human insulin receptors. Insulin-like growth factor I (IGF I) and alpha 2-macroglobulin receptors also were compared. On both cell types, all four unoccupied receptor types occurred predominantly as single receptors. Ligand binding caused receptor microaggregation. Microaggregation of wild type or kinase-defective insulin receptors or IGF I receptors was not different. alpha 2-Macroglobulin receptors formed larger microaggregates. Compared to wild type insulin or IGF I receptors, accumulation of kinase-defective insulin receptor microaggregates in endocytic structures was decreased, and the size of microaggregates in coated pits was significantly smaller. As a result, receptor-mediated internalization of gold-insulin by HIR A/K1018 cells was less than 6% of the cell-associated particles compared to approximately 60% of the particles in HIRc cells. On HIR A/K1018 cells, alpha 2-macroglobulin and IGF I were internalized via coated pits demonstrating that those structures were functional. These results suggest that: 1) ATP binding, receptor autophosphorylation, and activation of receptor kinase activity are not required for receptor microaggregation; 2) receptor microaggregation per se is not sufficient to cause ligand-induced receptor-mediated internalization or the biological effects of insulin; and 3) autophosphorylation of the beta-subunit or activation of the receptor kinase activity is required for the insulin-induced concentration of occupied receptors in coated pits.     

Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling

Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.     

Multiple pathways for ligand internalization in rat hepatocytes. I: Effects of anoxia, phenylarsine oxide and monensin.

It has been suggested that there are multiple pathways for the cellular internalization of insulin. To investigate these pathways we have examined the effects of three perturbations of endocytosis on the insulin internalization process and have compared these effects with those obtained using an asialoglycoprotein, asialofetuin (Afet), and epidermal growth factor (EGF). Freshly isolated hepatocytes were incubated with radiolabeled ligands and internalization measured under conditions of anoxia to deplete cellular ATP, in the presence of phenylarsine oxide (PAO) to inhibit endocytosis, and in the presence of monensin to interfere with endosomal acidification. Afet internalization essentially was blocked by all three treatment processes, while insulin internalization was inhibited approximately 40% in the presence of anoxia, and 54% in the presence of PAO. Monensin exhibited differential effects on internalization of high and low insulin concentrations. The effects of the treatment processes on EGF internalization were intermediate to those seen with Afet and insulin. These results suggest that insulin and EGF utilize routes of internalization exhibiting different energy requirements that may correspond to coated pit, non-coated pit, and fluid-phase internalization pathways. The observations with Afet internalization remain consistent with utilization of the coated pit pathway.     

Kinetics of tyrosine phosphorylation and internalization of human EGF receptors overexpressed in NIH 3T3 fibroblasts

Binding of epidermal growth factor (EGF) to cells rapidly induces tyrosine phosphorylation of its receptor which is followed by its internalization and dephosphorylation. The kinetics of these processes differs widely in time from minutes to hours according to cell types. In this paper we analyzed EGF receptor phosphorylation and down-regulation in NIH 3T3 cells transfected with the recombinant hEGF-R cDNA which express 4 X 10(5) receptors/cell. In the presence of EGF receptor phosphorylation reached a maximum after 1 min and was then maintained for about 1 h, while during this time the number of EGF-binding sites was reduced to 40% of the initial number. Detailed analysis of the fate of a population of receptors previously activated and autophosphorylated at 4 degrees C, after warming to 37 degrees C in the absence of the ligand, showed that internalization of the cell surface-associated EGF and dephosphorylation of the receptor were rapid (t1/2 15 min) and followed a similar kinetics. Our data indicate that at any given time only a fraction of the total cell surface receptors is phosphorylated on tyrosine and that dephosphorylation occurs at the cell surface or very rapidly after internalization. In addition the data also suggest that a certain recycling of previously internalized receptors may occur in these cells during EGF treatment.     

Endosomes,receptor tyrosine kinase internalization and signal transduction

Upon the binding of insulin or epidermal growth factor to their cognate receptors on the liver parenchymal plasmalemma, signal transduction and receptor internalization are near co-incident. Indeed, the rapidity and extent; of ligand mediated receptor internalization into endosomes in liver as well as other organs predicts that signal transduction is regulated at this intracellular locus. Although internalization has been thought as a mechanism to attenuate ligand mediated signal transduction responses, detailed studies of internalized receptors in isolated liver endosomes suggest an alternative scenario whereby selective signal transduction pathways can be accessed at this locus.     

Up-regulated EGF receptors undergo to rapid internalization and ubiquitin-dependent degradation in human cancer cells exposed to 8-Cl-cAMP

8-Cl-cAMP, a cAMP analogue that antagonizes type I cAMP-dependent protein kinase, is a novel anti-tumor agent presently under investigation in clinical trials. Herein we report the effects of this agent on epidermal growth factor receptor expression and degradation in human KB cancer cells. Exposure to 10 microM 8-Cl-cAMP for 48 h induced a 65% increase in epidermal growth factor receptor surface expression while the receptor synthesis was 22-fold enhanced. Analysis of epidermal growth factor-dependent receptor internalization in 8-Cl-cAMP-treated cells showed a higher endocytosis rate as well as an accelerated degradation which occurred together with an increased receptor ubiquitination. The enhanced degradation of epidermal growth factor receptor correlated with the lack of epidermal growth factor-induced proliferation and mitogen-activated protein kinase stimulation. The disregulation of epidermal growth factor receptor internalization and ubiquitin-dependent degradation could underlay a new mechanism of the anti-tumor activity of 8-Cl-cAMP suggesting its combination with agents that disrupt epidermal growth factor receptor signalling.     

Regulation of epidermal growth factor receptor internalization by G protein-coupled receptors

The epidermal growth factor (EGF) receptor (EGFR) plays a central role in regulating cell proliferation, differentiation, and migration. Cellular responses to EGF are dependent upon the amount of EGFR present on the cell surface. Stimulation with EGF induces sequestration of the receptor from the plasma membrane and its subsequent downregulation. Recently, internalization of the EGFR was also shown to be required for mitogenic signaling via the activation of MAP kinases. Therefore, mechanisms regulating internalization of the EGFR represent an important facet for the control of cellular response. Here, we demonstrate that EGFR is removed from the cell surface not only following stimulation with EGF, but also in response to stimulation of G protein-coupled lysophosphatidic acid (LPA) and beta2 adrenergic (beta2AR) receptors. Using a FLAG epitope-tagged EGFR to quantitate receptor internalization, we show that incubation with EGF, LPA, or isoproterenol (ISO) causes the time-dependent loss of cell surface EGFR. Internalization of EGFR by these ligands involves the tyrosine kinase activity of the receptor itself and c-Src, as well as the GTPase activity of dynamin. Unexpectedly, we find that internalization of the EGFR by EGF is dependent upon Gbetagamma and beta-arrestin proteins; expression of minigenes encoding the carboxyl terminii of the G protein-coupled receptor kinase 2, or beta-arrestin1, attenuates LPA-, ISO-, and EGF-mediated internalization of EGFR. Thus, G protein-coupled receptors can control the function of the EGFR by regulating its endocytosis.     

Phosphorylation of the epidermal growth factor receptor at threonine 654 inhibits ligand-induced internalization and down-regulation

To assess the functional significance of phosphorylation of the epidermal growth factor (EGF) receptor at Thr654, we compared the effects of 12-O-tetradecanoyl-13-acetate (TPA) on ligand-induced internalization and down-regulation between wild-type and mutant receptors that contain an alanine substitution at position 654. Activation of protein kinase C with TPA blocked EGF-induced internalization and down-regulation of Thr654 receptors and inhibited in vivo tyrosine kinase activity by 80%. TPA did not inhibit transferrin receptor internalization or constitutive EGF receptor internalization, suggesting that protein kinase C activation inhibits only the ligand-induced process. Inhibition by TPA of induced internalization, down-regulation, and kinase activity required threonine at position 654 since full-length Ala654 EGF receptors were significantly resistant to TPA inhibition of these ligand-induced activities. However, C'-terminal truncation further enhanced this resistance to TPA inhibition. The EGF-dependent internalization of kinase-inactive receptors truncated at residue 1022 was also impaired by TPA in Thr654 receptors, but not in Ala654 receptors, indicating that phosphorylation at Thr654 also interferes with tyrosine kinase-independent receptor activities. We conclude that the dominant regulatory effect of protein kinase C on the EGF receptor is mediated through phosphorylation at Thr654 which effectively inactivates the receptor. The submembrane region of the EGF receptor appears to regulate transmission of conformational information from the extracellular ligand-binding site to the cytoplasmic kinase and regulatory domains.     

Hydrogen peroxide reversibly inhibits epidermal growth factor (EGF) receptor internalization and coincident ubiquitination of the EGF receptor and Eps15.

Recently, we demonstrated that hydrogen peroxide (H2O2) inhibits the internalization of the epidermal growth factor (EGF) receptor and the EGF-induced mono-ubiquitination of EGF receptor pathway substrate clone #15 (Eps15) in fibroblasts. In addition, it was suggested that EGF receptor internalization might be inhibited by H2O2 by inhibition of ubiquitination of proteins involved in endocytosis. Here, we show that H2O2 also inhibits the poly-ubiquitination of the EGF receptor in fibroblasts. Furthermore, recovery of the cells resulted in re-establishment of ubiquitination of both the EGF receptor and Eps15 and coincided with restoration of internalization of those receptors that had bound EGF in the presence of H2O2. In addition, EGF receptor internalization was inhibited by the sulphydryl reagent N-ethylmaleimide (NEM), indicating that intact SH groups might be required for receptor-mediated endocytosis. Furthermore, H2O2 rapidly induced an increase in the cellular ratio of GSSG:GSH (oxidized glutathione:reduced glutathione) and removal of H2O2 resulted in a fast restoration of the ratio of GSSG:GSH. Therefore, these results suggest a relation between the inhibition of internalization ubiquitination and an increase in GSSG:GSH ratio, which strengthens the hypothesis that H2O2 inhibits EGF receptor internalization by an inhibition of ubiquitination of proteins involved in EGF receptor-mediated endocytosis.     

Ligand-induced internalization and increased cell calcium are mediated via distinct structural elements in the carboxyl terminus of the epidermal growth factor receptor.

Signals that can mediate ligand-induced receptor internalization and calcium regulation are present in a 48-amino acid "calcium-internalization" domain in the C' terminus of the epidermal growth factor (EGF) receptor. The basis of calcium and internalization regulation signalled by this 48-amino acid sequence was analyzed using deletion and substitution mutant receptors. Cells expressing truncated receptors containing either the NH2- or COOH-terminal portion of the 48-residue domain displayed high affinity EGF-dependent endocytosis and receptor down-regulation. These endocytosis-competent EGF receptor mutants that lacked any autophosphorylation site were unable to increase the concentration of intracellular calcium. To investigate the role of self-phosphorylation in EGF-induced calcium mobilization, phenylalanine was substituted for the single autophosphorylated tyrosine residue in this region of an internalization-competent truncated receptor. The receptor-mediated calcium response was abolished, while ligand-dependent receptor internalization was unimpaired. These results demonstrate that EGF-dependent receptor endocytosis and calcium mobilization are separate events. Tyrosine self-phosphorylation is required for increased [Ca2+]i, while structural features distinct from autophosphorylation are required for receptor internalization.     

Specific factors are required for kinase-dependent endocytosis of insulin receptors.

Mouse B82 cells that support high affinity saturable endocytosis of epidermal growth factor receptors (EGFR) exhibited only low rates of nonsaturable internalization of insulin receptors (InsR). To investigate the defect in endocytosis of InsR in B82 cells, we examined the role of sequence motifs and tyrosine kinase, the two receptor components shown to be required for efficient saturable endocytosis of InsR in Rat 1 cells. Placement of residues encoded by exon 16 of the InsR onto an EGFR truncated to residue 958 restored EGF-induced internalization of this mutant receptor indicating that the sequence codes in exon 16 are recognized by B82 cells. To determine whether the kinase function could be provided in trans, a B82 cell expressing both receptors was established. EGF-activated EGFR kinase was not able to restore insulin-dependent rapid endocytosis to InsR. However, fusion of untransfected Rat1 cells with InsR-expressing B82 cells enabled rapid endocytosis of InsR, indicating that the internalization defect can be complemented. These results indicate that, although internalization codes can function in the context of other receptors, activation of tyrosine kinase receptors requires an additional specific component.     

Hydrogen peroxide inhibits epidermal growth factor receptor internalization in human fibroblasts

Several cellular signal transduction cascades are affected by oxidative stress. In this study, the effect of hydrogen peroxide (H2O2) on the endocytosis of the epidermal growth factor (EGF) receptor was investigated. Exposure of HER14 cells to H2O2 resulted in a concentration-dependent inhibition of EGF receptor internalization. Binding studies demonstrated that this H2O2-induced inhibition in internalization was not due to altered binding of EGF to its receptor. Addition of H2O2 at different time points during internalization showed that EGF receptor internalization was rapidly reduced, suggesting that one of the first steps in the internalization process is inhibited. In addition, H2O2 inhibited the internalization of a different receptor, the chicken hepatic lectin receptor, in a concentration-dependent manner as well. Treatment of cells with another inducer of oxidative stress, cumene hydroperoxide, also resulted in a decreased internalization. Finally, we showed that H2O2 inhibited EGF-induced mono-ubiquitination of the EGF receptor pathway substrate clone 15, a process that normally occurs during EGF receptor endocytosis. These results clearly show that oxidative stress interferes with EGF signaling.