Epidermal growth factor receptor degradation: an alternative view of oncogenic pathways

Positive regulation of epidermal growth factor receptor signalling is related to many human malignancies. Besides overexpression and gain of function mutations, the escape from negative regulation through an increase in epidermal growth factor receptor stability has evolved as yet another key factor contributing to enhanced receptor activity. Intensive research over the past years has provided considerable evidence concerning the molecular mechanisms which provide epidermal growth factor receptor degradation. c-Cbl mediated ubiquitination, endocytosis via clathrin-coated pits, endosomal sorting and lysosomal degradation have become well-investigated cornerstones. Recent findings on the interdependency of the endosomal sorting complexes required for transport in multivesicular body sorting, stress the topicality of receptor tyrosine kinase downregulation. Here, we review the degradation pathway of the epidermal growth factor receptor, following the receptor from ligand binding to the lysosome and illustrating different modes of oncogenic deregulation.     

An integrated model of epidermal growth factor receptor trafficking and signal transduction

Endocytic trafficking of many types of receptors can have profound effects on subsequent signaling events. Quantitative models of these processes, however, have usually considered trafficking and signaling independently. Here, we present an integrated model of both the trafficking and signaling pathway of the epidermal growth factor receptor (EGFR) using a probability weighted-dynamic Monte Carlo simulation. Our model consists of hundreds of distinct endocytic compartments and approximately 13,000 reactions/events that occur over a broad spatio-temporal range. By using a realistic multicompartment model, we can investigate the distribution of the receptors among cellular compartments as well as their potential signal transduction characteristics. Our new model also allows the incorporation of physiochemical aspects of ligand-receptor interactions, such as pH-dependent binding in different endosomal compartments. To determine the utility of this approach, we simulated the differential activation of the EGFR by two of its ligands, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). Our simulations predict that when EGFR is activated with TGF-alpha, receptor activation is biased toward the cell surface whereas EGF produces a signaling bias toward the endosomal compartment. Experiments confirm these predictions from our model and simulations. Our model accurately predicts the kinetics and extent of receptor downregulation induced by either EGF or TGF-alpha. Our results suggest that receptor trafficking controls the compartmental bias of signal transduction, rather than simply modulating signal magnitude. Our model provides a new approach to evaluating the complex effect of receptor trafficking on signal transduction. Importantly, the stochastic and compartmental nature of the simulation allows these models to be directly tested by high-throughput approaches, such as quantitative image analysis.     

Endosomal receptor kinetics determine the stability of intracellular growth factor signalling complexes

There is an emerging paradigm that growth factor signalling continues in the endosome and that cell response to a growth factor is defined by the integration of cell surface and endosomal events. As activated receptors in the endosome are exposed to a different set of binding partners, they probably elicit differential signals compared with when they are at the cell surface. As such, complete appreciation of growth factor signalling requires understanding of growth factor-receptor binding and trafficking kinetics both at the cell surface and in endosomes. Growth factor binding to surface receptors is well characterized, and endosomal binding is assumed to follow surface kinetics if one accounts for changes in pH. Yet, specific binding kinetics within the endosome has not been examined in detail. To parse the factors governing the binding state of endosomal receptors we analysed a whole-cell mathematical model of epidermal growth factor receptor trafficking and binding. We discovered that the stability of growth factor-receptor complexes within endosomes is governed by three primary independent factors: the endosomal dissociation constant, total endosomal volume and the number of endosomal receptors. These factors were combined into a single dimensionless parameter that determines the endosomal binding state of the growth factor-receptor complex and can distinguish different growth factors from each other and different cell states. Our findings indicate that growth factor binding within endosomal compartments cannot be appreciated solely on the basis of the pH-dependence of the dissociation constant and that the concentration of receptors in the endosomal compartment must also be considered.     

B cell receptors and complement receptors target the antigen to distinct intracellular compartments

The processing of exogenous Ags is an essential step for the generation of immunogenic peptides that will be presented to T cells. This processing relies on the efficient intracellular targeting of Ags, because it depends on the content of the compartments in which Ags are delivered in APCs. Opsonization of Ags by the complement component C3 strongly enhances their presentation by B cells and increases their immunogenicity in vivo. To investigate the role of C3 in the targeting of Ags, we compared the intracellular traffic of proteins internalized by complement receptor (CR) and B cell receptor (BCR) in B lymphocytes. Whereas both receptors are able to induce efficient Ag presentation, their intracellular pathways are different. CR ligand is delivered to compartments containing MHC class II molecules (MHC-II) but devoid of transferrin receptor and Lamp-2, whereas BCR rapidly targets its ligand toward Lamp-2-positive, late endosomal MHC-II-enriched compartments through intracellular vesicles containing transferrin receptor. CR and BCR are delivered to distinct endocytic pathways, and the kinetic evolution of the protein content of these pathways is very different. Both types of compartments contain MHC-II, but CR-targeted compartments receive less neosynthesized MHC-II than do BCR-targeted compartments. The targeting induced by CR toward compartments that are distinct from BCR-targeted compartments probably participates in C3 modulation of Ag presentation.     

Triacylglycerol hydrolysis in isolated hepatic endosomes.

Three endosomal compartments including the compartment for uncoupling receptor and ligand (CURL), multivesicular bodies (MVB), and a putative recycling fraction (retrosomes) were isolated from rat liver homogenates fifteen minutes after a bolus injection of very low density lipoprotein (VLDL) was delivered into a femoral vein. Assays for enzyme markers indicate a minimal contamination with either lysosomes or Golgi. The increase in specific activity of the radiolabeled ligand (VLDL) during the isolation procedure from homogenate to MVB, demonstrates a 200-250-fold purification of this organelle. All three fractions have the ability to catabolize triacylglycerol substrate both as triolein and as VLDL triacylglycerol. Furthermore, incubation of isolated endosomes following injection of endogenously labeled VLDL demonstrate their ability to hydrolyze VLDL triacylglycerol in situ. Three distinct lipolytic pH optima were found at pH 5.5, 7.1, and 8.6. The effects of serum, MgCl2, CaCl2, NaCl, sodium dodecyl sulfate, bile acids, and antibody to hepatic triacylglycerol lipase on the individual endosome fractions demonstrated distinct lipolytic activities in the different compartments. Results indicate that both an endosomal neutral lipase as well as hepatic triacylglycerol lipase make a significant contribution to lipolytic processing of endocytosed lipoproteins prior to their resecretion of further processing in hepatic lysosomes.     

Aging Affects Epidermal Growth Factor Receptor Phosphorylation and Traffic Kinetics

With increasing donor age, cultured human fibroblasts express fewer epidermal growth factor receptors and display decreased mitogenic responsiveness to epidermal growth factor. To determine age-associated differences in epidermal growth factor receptor phosphorylation and traffic kinetics, we studied in fibroblasts derived from donors of different ages autophosphorylation of the receptor after ligand binding and trafficking of the receptor–ligand complexes. We now report an age-associated delay in the rate of receptor phosphorylation after epidermal growth factor stimulation. Furthermore, receptor/ligand trafficking is affected by aging. There is an age-associated decrease and delay in the number of occupied receptors that are transported intracellularly and in their rate of clearance from the plasma membrane. Our data show that aging affects receptor/ligand activation and processing and suggest that the decreased cellular mitogenic response with aging may be, at least in part, the result of decrements in receptor activation and processing.     

Involvement of multiple subcellular compartments in intracellular processing of epidermal growth factor

The intracellular translocation and processing of epidermal growth factor (EOF) in 3T3 cells has been studied utilizing Percoll density gradients. EGF is internalized and rapidly becomes associated with two types of intracellular compartments. The extent to which EGF is delivered to these two compartments is apparently regulated depending upon the cell's physiological condition. In growth medium, an increased proportion of EGF is taken up into a Golgi-like element. Uptake through this pathway correlates with a decrease in degradation of the ligand. In the absence of scrum and amino acids, an increased proportion of EGF is taken up into a component which has a density of 1.05. Uptake through this pathway correlates with increased degradation of the ligand. The ligand taken up through both pathways is transferred to dense vesicles which comigrate with lysosomes. In the presence of growth medium, however, dense vesicles containing EGF can be shown to be lysosomal enzyme-deficient upon further fractionation. In addition, in the presence of serum, a portion of the internalized EGF is apparently released from the cells, intact, and then re-bound. The processes described may be important in the production of a mitogenic response and the ability of cells to self-regulate their responsiveness to the growth factor.     

Botulinum neurotoxin A and an engineered derivate targeted secretion inhibitor (TSI) A enter cells via different vesicular compartments

Botulinum neurotoxins (BoNTs) are highly potent multi-domain proteins, responsible for botulism in animals and humans. The modular structural organization of BoNTs has led to the development of novel engineered bio-therapeutic proteins called targeted secretion inhibitors (TSIs). We report here that botulinum neurotoxin A (BoNT/A) and a TSI/A in which the neuronal binding domain of BoNT/A has been substituted by an epidermal growth factor (EGF) ligand, named EGFR-targeted TSI/A, exploit different routes to gain entry in the same in vitro neuroblastoma cell system, SiMa cells. We found that the EGF ligand conferred the affinity to the EGFR-targeted TSI/A at the EGF receptor when compared to an untargeted TSI/A and also the ability to internalize into the cells and cleave its cytosolic target protein SNAP-25. Using high content analysis we found that both BoNT/A and the EGFR-targeted TSI/A enter the cell in a concentration-dependent manner and in compartments which are able to translocate the proteins into the cytosol within 4?h. The EGFR-targeted TSI/A internalized into a compartment which gave a punctate staining pattern by immunofluorescence and partially overlapped with structures positive for the early endosomal marker EAA1; whereas BoNT/A did not internalize into a punctate compartment but did so in an acidifying compartment consistent with local synaptic vesicle recycling. These findings show that the BoNT/A translocation domain, common to both BoNT/A and the EGFR-targeted TSI/A, is a versatile tool for cytosolic delivery from distinct intracellular vesicular compartments.     

Small GTPases in vesicle trafficking

Plant small GTPases belonging to the Rop, Arf, and Rab families are regulators of vesicle trafficking. Rop GTPases regulate actin dynamics and modulate H(2)O(2) production in polar cell growth and pathogen defence. A candidate Rop GDP to Rop GTP exchange factor (RopGEF) SPIKE1 is involved in the morphogenesis of leaf epidermal cells. The ArfGEF GNOM regulates the endosomal recycling of the PIN proteins, which are involved in polar auxin transport. Intracellular localisation of small GTPases and functional studies using dominant mutant versions of Arf and Rab GTPases are defining novel plant-specific membrane compartments, especially those that participate in endosomal vesicle trafficking.     

Delta/notch-like epidermal growth factor (EGF)-related receptor, a novel EGF-like repeat-containing protein targeted to dendrites of developing and adult central nervous system neurons

We have identified a novel epidermal growth factor (EGF)-like repeat-containing single-pass transmembrane protein that is specifically expressed in the developing and mature central nervous system. Sequence analysis revealed that the 10 EGF-like repeats in the extracellular domain are closely related to those of the developmentally important receptor Notch and its ligand Delta. We thus named the molecule Delta/Notch-like EGF-related receptor (DNER). DNER protein is strongly expressed in several types of post-mitotic neurons, including cortical and hippocampal pyramidal neurons, cerebellar granule cells, and Purkinje cells. DNER protein is localized to the dendritic plasma membrane and endosomes and is excluded from the axons, even when overexpressed. The tyrosine-based sorting motif in the cytoplasmic domain is required for dendritic targeting of DNER. Direct in vivo binding of DNER to the coat-associated protein complex AP-1 strongly suggests that DNER undergoes AP-1-dependent sorting to the somatodendritic compartments from the trans-Golgi network and subsequent passage through the endosomal system.     

EGFR-dependent phosphorylation of leucine-rich repeat kinase LRRK1 is important for proper endosomal trafficking of EGFR

Ligand-induced activation of the epidermal growth factor receptor (EGFR) initiates trafficking events that relocalize the receptors from the cell surface to intracellular endocytic compartments. We recently reported that leucine-rich repeat kinase 1 (LRRK1) is involved in the trafficking of EGFR from early to late endosomes. In this study, we demonstrate that EGFR regulates the kinase activity of LRRK1 via tyrosine phosphorylation and that this is required for proper endosomal trafficking of EGFR. Phosphorylation of LRRK1 at Tyr-944 results in reduced LRRK1 kinase activity. Mutation of LRRK1 Tyr-944 (Y944F) abolishes EGF-stimulated tyrosine phosphorylation, resulting in hyperactivation of LRRK1 kinase activity and enhanced motility of EGF-containing endosomes toward the perinuclear region. The compartments in which EGFR accumulates are mixed endosomes and are defective in the proper formation of intraluminal vesicles of multivesicular bodies. These results suggest that feedback down-regulation of LRRK1 kinase activity by EGFR plays an important role in the appropriate endosomal trafficking of EGFR.     

Inhibition of endosomal insulin-like growth factor-I processing by cysteine proteinase inhibitors blocks receptor-mediated functions

The receptor for the type 1 insulin-like growth factor (IGF-I) has been implicated in cellular transformation and the acquisition of an invasive/metastatic phenotype in various tumors. Following ligand binding, the IGF-I receptor is internalized, and the receptor.ligand complex dissociates as the ligand is degraded by endosomal proteinases. In the present study we show that the inhibition of endosomal IGF-I-degrading enzymes in human breast and murine lung carcinoma cells by the cysteine proteinase inhibitors, E-64 and CA074-methyl ester, profoundly altered receptor trafficking and signaling. In treated cells, intracellular ligand degradation was blocked, and although the receptor and two substrates, Shc and Insulin receptor substrate, were hyperphosphorylated on tyrosine, IGF-I-induced DNA synthesis, anchorage-independent growth, and matrix metalloproteinase synthesis were inhibited. The results suggest that ligand processing by endosomal proteinases is a key step in receptor signaling and function and a potential target for therapy.     

Multiparametric image analysis reveals role of Caveolin1 in endosomal progression rather than internalization of EGFR

Endosomes constitute a central layer in the regulation of growth factor signaling. We applied flow cytometry, confocal microscopy and automated image quantification to define the role of Caveolin1 (Cav1) in epidermal growth factor (EGF) receptor (i) internalization and (ii) endosomal trafficking. Antisense-downregulation of Cav1 did not affect internalization of EGF:EGFR-complexes from the plasma membrane. Instead, Cav1-knockdown had a profound effect on endosomal trafficking and caused a shift in EGF vesicle distribution towards Rab7-negative compartments at late timepoints. Moreover, image quantification with single-endosome resolution revealed that EGF:Cav1-complexes undergo a maturation pattern reminiscent of late endosomes. Our data suggest a model in which Caveolin1 acts upon EGF endosomes internalized via the Clathrin-pathway and functions at the transition from early to late endosomes.     

Intracellular receptor/ligand sorting based on endosomal retention components

Endocytosed molecules are sorted in endosomes to different cellular destinations (e.g., to lysosomes or to the plasma membrane). Diverse endosomal sorting results have been reported for different ligands and receptors in a variety of cell types, but the general principles governing these sorting outcomes are not well understood. For example, we observed a wide range of sorting outcomes with the epidermal growth factor (EGF)/receptor system in fibroblasts using several members of the EGF family and site-directed ligand and receptor mutants. In this article we describe a mechanistic mathematical model of endosomal sorting based on the hypothesis that receptors may be selectively retained by the endosomal sorting apparatus and that this process may be modulated by receptor occupancy. Our results show that this single mechanism can account for the wide variety of observed sorting outcomes. By providing a conceptual framework for understanding endosomal sorting, this model not only helps interpret our experimental results for the EGF/receptor system, but also provides some insight into the principles governing sorting. For example, the model predicts that the influence of selective endosomal retention of receptor/ligand complexes is seen in deviations of ligand sorting outcomes from pure fluid phase sorting behavior. Furthermore, the model suggests that selective endosomal retention of complexes within endosomes gives rise to three sorting regimes characterized by distinguishable qualitative trends in the dependence of ligand sorting fractions on intracellular ligand concentrations. (c) 1996 John Wiley & Sons, Inc.     

Evidence for a role of SNX16 in regulating traffic between the early and later endosomal compartments

Sorting nexins (SNXs) are a growing family of proteins characterized by the presence of a PX domain. The PX domain mediates membrane association by interaction with phosphoinositides. The SNXs are generally believed to participate in membrane trafficking, but information regarding the function of individual proteins is limited. In this report, we describe the major characteristics of one member, SNX16. SNX16 is a novel 343-amino acid protein consisting of a central PX domain followed by a potential coiled-coil domain and a C-terminal region. Like other sorting nexins, SNX16 associates with the membrane via the PX domain which interacts with the phospholipid phosphatidylinositol 3-phosphate. We show via biochemical and cellular studies that SNX16 is distributed in both early and late endosome/lysosome structures. The coiled-coil domain is necessary for localization to the later endosomal structures, as mutant SNX16 lacking this domain was found only in early endosomes. Trafficking of internalized epidermal growth factor was also delayed by this SNX16 mutant, as these cells showed a delay in the segregation of epidermal growth factor in the early endosome for its delivery to later compartments. In addition, the coiled-coil domain is shown here to be important for homo-oligomerization of SNX16. Taken together, these results suggest that SNX16 is a sorting nexin that may function in the trafficking of proteins between the early and late endosomal compartments.     

The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.

The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles.     

Trafficking of the ErbB receptors and its influence on signaling

Although members of the ErbB receptor family are found predominantly at the cell surface, these receptors undergo constant cycling between the plasma membrane and the endosomal compartment. In the absence of an activating ligand, these receptors are slowly internalized (t(1/2) approximately 30 min) but are quickly recycled. The constitutive degradation rate of the epidermal growth factor (EGF) receptor (EGFR) is slower than other ErbB family members and only the EGFR appears to alter its trafficking pattern in response to ligand binding. This altered pattern is characterized by accelerated internalization and enhanced lysosomal targeting. Ligand-regulated trafficking of the EGFR is mediated by a series of motifs distributed through the cytoplasmic domain of the receptor that are exposed by a combination of activation-mediated conformation changes and the binding of proteins such as Grb2. As a consequence of induced internalization, most EGFR signaling occurs within endosomes whereas signaling by the other members of the ErbB family appear to be generated predominantly from the cell surface. Overexpression of ErbB family members can disrupt normal receptor trafficking by driving heterodimerization of receptors with disparate trafficking patterns. Because different ErbB receptor substrates are localized in different cellular compartments, disrupted trafficking could be an important factor in the altered signaling patterns observed as a consequence of receptor overexpression.     

Negative regulation of epidermal growth factor signaling by selective proteolytic mechanisms in the endosome mediated by cathepsin B

We have investigated the relevant protease activity in rat liver, which is responsible for most of the receptor-mediated epidermal growth factor (EGF) degradation in vivo. EGF was sequentially cleaved by endosomal proteases at a limited number of sites, which were identified by high performance liquid chromatography and mass spectrometry. EGF proteolysis is initiated by hydrolysis at the C-terminal Glu(51)-Leu(52) bond. Three additional minor cleavage sites were identified at positions Arg(48)-Trp(49), Trp(49)-Trp(50), and Trp(50)-Glu(51) after prolonged incubation. Using nondenaturating immunoprecipitation and cross-linking procedures, the major proteolytic activity was identified as that of the cysteine protease cathepsin-B. The effect of injected EGF on subsequent endosomal EGF receptor (EGFR) proteolysis was further evaluated by immunoblotting. Using endosomal fractions prepared from EGF-injected rats and incubated in vitro, the EGFR was lost with a time course superimposable with the loss of phosphotyrosine content. The cathepsin-B proinhibitor CA074-Me inhibited both in vivo and in vitro the endosomal degradation of the EGFR and increased the tyrosine phosphorylation states of the EGFR protein and the molecule SHC within endosomes. The data, therefore, describe a unique pathway for the endosomal processing of internalized EGF receptor complexes, which involves the sequential function of cathepsin-B through selective degradation of both the ligand and receptor.     

In vitro endosome-lysosome transfer of dephosphorylated EGF receptor and Shc in rat liver

We have studied the endosome-lysosome transfer of internalized epidermal growth factor receptor (EGFR) complexes in a cell-free system from rat liver. Analytical subfractionation of a postmitochondrial supernatant fraction showed that a pulse of internalized [(125)I]EGF was largely associated with a light endosomal fraction devoid of lysosomal markers. After an additional 30 min incubation in vitro in the presence of an ATP-regenerating system, the amount of [(125)I]EGF in this compartment decreased by 39%, with an increase in [(125)I]EGF in lysosomes. No transfer of [(125)I]EGF to the cytosol was detected. To assess the fate of the internalized EGFR protein over the time course of the endo-lysosomal transfer of the ligand, the effect of a saturating dose of native EGF on subsequent lysosomal targeting of the EGFR was evaluated by immunoblotting. A massive translocation of the EGFR to the endosomal compartment was observed in response to ligand injection coincident with its tyrosine phosphorylation and receptor recruitment of the tyrosine-phosphorylated adaptor protein Shc. During cell-free endosome-lysosome fusion, a time-dependent increase in the content of the EGFR and the two 55- and 46-kDa Shc isoforms was observed in lysosomal fractions with a time course superimposable with the lysosomal transfer of the ligand; no transfer of the 66-kDa Shc isoform was detected. The relationship between EGFR tyrosine kinase activity and EGFR sorting in endosomes investigated by immunoblot studies with anti-phosphotyrosine antibodies revealed that endosomal dephosphorylation of EGFR and Shc preceded lysosomal transfer. These results support the view that a lysosomal targeting machinery distinct from the endosomal receptor kinase activity, such as the recruitment of the signaling molecule Shc, may regulate this sorting event in the endosome.     

Variation of hydrophobicity of human urinary epidermal growth factor

Epidermal growth factor is present in human urine in large amounts, but its biological significance is not known. The results of this study indicate that the predominant 6000-dalton form of epidermal growth factor in human urine is divided by hydrophobic interaction chromatography into four fractions; only 3% of the total 6000-dalton epidermal growth factor coeluted with the biosynthetic epidermal growth factor and the rest was separated into three different peaks. These different forms may lack one or two amino or carboxy terminal amino acids from the 53 amino acids present in epidermal growth factor, or they may be products of deamidation or oxidation of amino acid(s). Further knowledge of these micromodifications of epidermal growth factor secreted in urine may reveal the origin and function of epidermal growth factor in humans.