UBE4B Protein Couples Ubiquitination and Sorting Machineries to Enable Epidermal Growth Factor Receptor (EGFR) Degradation

The signaling of plasma membrane proteins is tuned by internalization and sorting in the endocytic pathway prior to recycling or degradation in lysosomes. Ubiquitin modification allows recognition and association of cargo with endosomally associated protein complexes, enabling sorting of proteins to be degraded from those to be recycled. The mechanism that provides coordination between the cellular machineries that mediate ubiquitination and endosomal sorting is unknown. We report that the ubiquitin ligase UBE4B is recruited to endosomes in response to epidermal growth factor receptor (EGFR) activation by binding to Hrs, a key component of endosomal sorting complex required for transport (ESCRT) 0. We identify the EGFR as a substrate for UBE4B, establish UBE4B as a regulator of EGFR degradation, and describe a mechanism by which UBE4B regulates endosomal sorting, affecting cellular levels of the EGFR and its downstream signaling. We propose a model in which the coordinated action of UBE4B, ESCRT-0, and the deubiquitinating enzyme USP8 enable the endosomal sorting and lysosomal degradation of the EGFR.     

Recruitment of Uev1B to Hrs-containing endosomes and its effect on endosomal trafficking

Endocytosis of signaling receptors, such as epidermal growth factor receptor (EGFR), tightly controls the signal transduction process triggered by ligand activation of these receptors. To identify new regulators of the endocytic trafficking of EGFR an RNA interference screen was performed for genes involved in ubiquitin conjugation and down-regulation of EGFR. The screen revealed that small interfering RNAs (siRNAs) that target the conserved ubiquitin-binding domain Uev1 increased down-regulation of EGFR. Since these siRNAs simultaneously targeted multiple genes containing a Uev1 domain, we analyzed the role of these gene products by overexpressing individual Uev1-related proteins. This analysis revealed that overexpression of Uev1A (UBE2V1) has no effect on the degradation of EGFR:EGF complexes. In contrast, overexpression of Uev1B (TMEM189-UBE2V1 isoform 2) slowed the degradation of EGF:receptor complexes. The Uev1B protein was found to strongly colocalize and associate with ubiquitin and Hrs in endosomes. Moreover, overexpression of Uev1B abrogated the ability of Hrs to colocalize with EGFR. The B-domain of Uev1B, and not the UEV-domain, was mainly responsible for the observed phenotypes suggesting the presence of a novel endosomal targeting sequence within the B-domain. Together, the data show that elevated levels of Uev1B protein in cells lead to decreased efficiency of endosomal sorting by associating with ubiquitinated proteins and Hrs.     

CIN85 regulates ubiquitination and degradative endosomal sorting of the EGF receptor

CIN85 has been demonstrated to interact with a number of proteins involved in endocytosis and intracellular sorting. However, the exact functional role of CIN85 in endocytosis remains unclear. We have investigated whether CIN85 plays a role in EGF-induced EGF receptor (EGFR) internalization, as previously suggested, or whether CIN85 is rather involved in endosomal sorting of the EGFR. When over-expressing a dominant negative interfering CIN85 mutant consisting of three SH3 domains only, we found that internalization of EGF was inhibited. However, when knocking down CIN85 by RNAi, the EGF–EGFR uptake appeared similar to in control cells. Furthermore, in CIN85 depleted cells, EGF-induced ubiquitination of the EGFR was decreased, and degradation of EGF–EGFR complexes was delayed. Our data further demonstrated that depletion of CIN85 increased the recycling of EGF, suggesting that CIN85 plays a role in endosomal sorting of the ubiquitinated EGFR. Our data also demonstrated that CIN85 was constitutively associated with Hrs, and this strengthens the hypothesis of a functional role of CIN85 in endosomal EGFR sorting.     

LAPTM4B is a PtdIns(4,5)P2 effector that regulates EGFR signaling,lysosomal sorting,and degradation

Lysosomal degradation is essential for the termination of EGF‐stimulated EGF receptor (EGFR) signaling. This requires EGFR sorting to the intraluminal vesicles (ILVs) of multi‐vesicular endosomes (MVEs). Cytosolic proteins including the ESCRT machineries are key regulators of EGFR intraluminal sorting, but roles for endosomal transmembrane proteins in receptor sorting are poorly defined. Here, we show that LAPTM4B, an endosomal transmembrane oncoprotein, inhibits EGF‐induced EGFR intraluminal sorting and lysosomal degradation, leading to enhanced and prolonged EGFR signaling. LAPTM4B blocks EGFR sorting by promoting ubiquitination of Hrs (an ESCRT‐0 subunit), which inhibits the Hrs association with ubiquitinated EGFR. This is counteracted by the endosomal PIP kinase, PIPKIγi5, which directly binds LAPTM4B and neutralizes the inhibitory function of LAPTM4B in EGFR sorting by generating PtdIns(4,5)P₂ and recruiting SNX5. PtdIns(4,5)P₂ and SNX5 function together to protect Hrs from ubiquitination, thereby promoting EGFR intraluminal sorting. These results reveal an essential layer of EGFR trafficking regulated by LAPTM4B, PtdIns(4,5)P₂ signaling, and the ESCRT complex and define a mechanism by which the oncoprotein LAPTM4B can transform cells and promote tumor progression.     

Differential Effects of EGFR Ligands on Endocytic Sorting of the Receptor

Endocytic downregulation is a pivotal mechanism turning off signalling from the EGF receptor (EGFR). It is well established that whereas EGF binding leads to lysosomal degradation of EGFR, transforming growth factor (TGF)-α causes receptor recycling. TGF-α therefore leads to continuous signalling and is a more potent mitogen than EGF. In addition to EGF and TGF-α, five EGFR ligands have been identified. Although many of these ligands are upregulated in cancers, very little is known about their effect on EGFR trafficking.
We have compared the effect of six different ligands on endocytic trafficking of EGFR. We find that, whereas they all stimulate receptor internalization, they have very diverse effects on endocytic sorting. Heparin-binding EGF-like growth factor and Betacellulin target all EGFRs for lysosomal degradation. In contrast, TGF-α and epiregulin lead to complete receptor recycling. EGF leads to lysosomal degradation of the majority but not all EGFRs. Amphiregulin does not target EGFR for lysosomal degradation but causes fast as well as slow EGFR recycling. The Cbl ubiquitin ligases, especially c-Cbl, are responsible for EGFR ubiquitination after stimulation with all ligands, and persistent EGFR phosphorylation and ubiquitination largely correlate with receptor degradation.     

The signaling and transformation potency of the overexpressed HER2 protein is dependent on the normally-expressed EGFR

Human epidermal growth factor receptor 2 (HER2) belongs to the EGFR family of receptor tyrosine kinases that comprises four members. As opposed to the other family members, HER2 does not require ligand binding for activation. Hence, HER2 molecules can undergo spontaneous dimerization, autophosphorylation and activation of downstream signaling pathways especially under conditions of overexpression, a commonly encountered phenomenon in breast cancer. In this study, we sought to investigate the mechanism by which HER2 musters signaling and transformation potency. We show that HER2 overexpression per se induces a significant increase in basal mitogenic and cell survival signaling, which was augmented by EGF stimulation. Inhibition of the normally expressed EGFR significantly suppressed the ability of overexpressed HER2 to induce enhanced signaling and cell transformation, suggesting that HER2 requires the EGFR and potentially other members to maximize its signaling and transformation potency. The novel observation revealed by prolonged EGF stimulation studies was the biphasic signaling pattern in the presence of HER2 overexpression that suggested the induction of a short-circuited mechanism, permitting sustained signaling. Our results further show that the short-circuited signaling was due to the re-shuttling of internalized receptor molecules to the Rab11-positive recycling endosomes, while suppressing channeling to the LAMP1-positive lysosome-targeting endosomes. Therefore, HER2's oncogenicity is dependent, not only on its constitutively active nature, but also on its ability to muster collaborative signaling from family members through modulation of ligand-induced receptor regulation.     

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.     

Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking

A ubiquitin-binding endosomal protein machinery is responsible for sorting endocytosed membrane proteins into intraluminal vesicles of multivesicular endosomes (MVEs) for subsequent degradation in lysosomes. The Hrs-STAM complex and endosomal sorting complex required for transport (ESCRT)-I, -II and -III are central components of this machinery. Here, we have performed a systematic analysis of their importance in four trafficking pathways through endosomes. Neither Hrs, Tsg101 (ESCRT-I), Vps22/EAP30 (ESCRT-II), nor Vps24/CHMP3 (ESCRT-III) was required for ligand-mediated internalization of epidermal growth factor (EGF) receptors (EGFRs) or for recycling of cation-independent mannose 6-phosphate receptors (CI-M6PRs) from endosomes to the trans-Golgi network (TGN). In contrast, both Hrs and ESCRT subunits were equally required for degradation of both endocytosed EGF and EGFR. Whereas depletion of Hrs or Tsg101 caused enhanced recycling of endocytosed EGFRs, this was not the case with depletion of Vps22 or Vps24. Depletion of Vps24 instead caused a strong increase in the levels of CI-M6PRs and a dramatic redistribution of the Golgi and the TGN. These results indicate that, although Hrs-STAM and ESCRT-I, -II and -III have a common function in degradative protein sorting, they play differential roles in other trafficking pathways, probably reflecting their functions at distinct stages of the endocytic pathway.     

Recycling of the epidermal growth factor receptor is mediated by a novel form of the clathrin adaptor protein Eps15

Levels of the epidermal growth factor receptor (EGFR) at the cell surface are tightly regulated by a complex endocytic machinery. Following internalization, EGFR is either recycled back to the cell surface or transported to the late endosome/lysosome for degradation. Currently, the molecular machinery that regulates this sorting pathway is only partially defined. Eps15 (EGFR pathway substrate 15) is an endocytic adaptor protein that is well known to support clathrin-mediated internalization of EGFR at the plasma membrane. Using RT-PCR, we have identified a novel short form of Eps15 (Eps15S) from rat liver that lacks the 111 C-terminal amino acids present in the traditional Eps15 form. The goal of this study was to define the functional role of the novel Eps15S form in EGFR trafficking. Overexpression of a mutant form of Eps15S (Eps15S ΔEH2/EH3) did not block EGFR internalization but reduced its recycling to the cell surface. After knockdown of all Eps15 forms, re-expression of Eps15S significantly reduced EGFR degradation while promoting recycling back to the cell surface. In contrast, re-expression of Eps15 did not potentiate receptor recycling. Furthermore, overexpression of the mutant Eps15S substantially reduced cell proliferation, linking EGFR recycling to downstream mitogenic effects. Finally, we found that Eps15S is localized to the Rab11-positive recycling endosome that is disrupted in cells expressing the Eps15S mutant, leading to an accumulation of the EGFR in early endosomes. These findings suggest that distinct forms of Eps15 direct EGFR to either the late endosome/lysosome for degradation (Eps15) or to the recycling endosome for transit back to the cell surface (Eps15S).     

Recycling of EGFR and ErbB2 is associated with impaired Hrs tyrosine phosphorylation and decreased deubiquitination by AMSH

ErbB receptors play an important role in normal cellular growth, differentiation and development, but overexpression or poor downregulation can result in enhanced signaling and cancerous growth. ErbB signaling is terminated by clathrin-dependent receptor-mediated endocytosis, followed by incorporation in multi-vesicular bodies and subsequent degradation in lysosomes. In contrast to EGFR, ErbB2 displays poor ligand-induced downregulation and enhanced recycling, but the molecular mechanisms underlying this difference are poorly understood. Given our previous observation that both EGFR and an EGFR-ErbB2 chimera undergo Cbl-mediated K63-polyubiquitination, we investigated in the present study whether activation of the EGFR and the EGFR-ErbB2 chimera is associated with tyrosine phosphorylation of the ESCRT-0 complex subunit Hrs and AMSH-mediated deubiquitination. EGF stimulation of the EGFR resulted in efficient Hrs tyrosine phosphorylation and deubiquitination by the K63-polyubiquitin chain-specific deubiquitinating enzyme AMSH. In contrast, EGF activation of EGFR-ErbB2 showed significantly decreased Hrs tyrosine phosphorylation and deubiquitination by AMSH. To test whether this phenotype is the result of endosomal recycling, we induced recycling of the EGFR by stimulation with TGFα. Indeed, even though TGFα-stimulation of EGFR is associated with efficient ligand-stimulated K63-polyubiquitination, we observed that Hrs tyrosine phosphorylation as well as AMSH-mediated deubiquitination is significantly reduced under these conditions. Using various EGFR-ErbB2 chimeras, we demonstrate that enhanced recycling, decreased Hrs tyrosine phosphorylation and decreased AMSH mediated deubiquitination of EGFR-ErbB2 chimeras is primarily due to the presence of ErbB2 sequences or the absence of EGFR sequences C-terminal to the Cbl binding site. We conclude that endosomal recycling of the EGFR and ErbB2 receptors is associated with significantly impaired tyrosine phosphorylation of the ESCRT-0 subunit Hrs as well as decreased deubiquitination by AMSH, which is consistent with the finding that recycling receptors are not efficiently incorporated in the MVB pathway.     

The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor

The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.     

Endosomal signaling of epidermal growth factor receptor stimulates signal transduction pathways leading to cell survival

In spite of intensified efforts to understand cell signaling from endosomes, there is no direct evidence demonstrating that endosomal signaling is sufficient to activate signal transduction pathways and no evidence to demonstrate that endosomal signaling is able to produce a biological outcome. The lack of breakthrough is due in part to the lack of means to generate endosomal signals without plasma membrane signaling. In this paper, we report the establishment of a system to specifically activate epidermal growth factor (EGF) receptor (EGFR) when it endocytoses into endosomes. We treated cells with EGF in the presence of AG-1478, a specific EGFR tyrosine kinase inhibitor, and monensin, which blocks the recycling of EGFR. This treatment led to the internalization of nonactivated EGF-EGFR complexes into endosomes. The endosome-associated EGFR was then activated by removing AG-1478 and monensin. During this procedure we did not observe any surface EGFR phosphorylation. We also achieved specific activation of endosome-associated EGFR without using monensin. By using this system, we provided original evidence demonstrating that (i) the endosome can serve as a nucleation site for the formation of signaling complexes, (ii) endosomal EGFR signaling is sufficient to activate the major signaling pathways leading to cell proliferation and survival, and (iii) endosomal EGFR signaling is sufficient to suppress apoptosis induced by serum withdrawal.     

Endocytosis of the dermatan sulfate proteoglycan decorin utilizes multiple pathways and is modulated by epidermal growth factor receptor signaling

Human skin fibroblasts efficiently internalize the matrikine decorin by receptor-mediated endocytosis, however, very little is known about its intracellular trafficking routes up to lysosomal degradation. In an in vitro system measuring uptake and degradation of [(35)S]sulfate-labeled decorin, endocytosis was blocked by 46% when clathrin assembly/disassembly was inhibited using chlorpromazine. Pharmacological inhibition of EGF receptor signaling caused 34% reduction of decorin uptake, whereas inhibition of the IGF receptor had no effect. Using confocal immunofluorescence microscopy, we determined that only about 5-10% of internalized decorin colocalized with the EGFR. Thus, uptake depends on EGFR signaling rather than trafficking along the same pathway. Decorin passes through early endosomes towards trafficking to lysosomes, since more than 50% of decorin colocalized with EEA1. Moreover, inhibition of endosomal fusion by wortmannin caused a profound inhibition of decorin endocytosis. Overexpression of the clathrin-binding Hrs protein, which has previously been shown to inibit EGFR degradation blocked the degradation of decorin. Cholesterol depletion by filipin inhibited uptake of decorin by 34%, however, nearly no intracellular colocalization was found between decorin and caveolin-1. The combined use of filipin and chlorpromazine had an additive inhibitory effect on decorin endocytosis. Moreover, chlorpromazine diverted decorin from the chlorpromazine-sensitive pathway to an alternative uptake route. The CD44/hyaluronan pathway was excluded as an endocytic route for decorin. Our observations indicate that decorin is taken up by more than one endocytic pathway. Of note, lipid-raft-dependent EGFR signaling modulates decorin uptake, suggesting the presence of a potential feedback regulation mechanism for desensitization of signaling events mediated by decorin.     

Recognising the signals for endosomal trafficking

The endosomal compartment is a major sorting station controlling the balance between endocytic recycling and lysosomal degradation, and its homeostasis is emerging as a central factor in various neurodegenerative diseases such as Alzheimer's and Parkinson's. Membrane trafficking is generally coordinated by the recognition of specific signals in transmembrane protein cargos by different transport machineries. A number of different protein trafficking complexes are essential for sequence-specific recognition and retrieval of endosomal cargos, recycling them to other compartments and acting to counter-balance the default endosomal sorting complex required for transport–mediated degradation pathway. In this review, we provide a summary of the key endosomal transport machineries, and the molecular mechanisms by which different cargo sequences are specifically recognised.     

Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.     

Sorting Out the Sorting Functions of Endosomes in Arabidopsis

In animals, sorting of membrane proteins following their internalization from the plasma membrane (PM) by endocytosis occurs through a series of different endosomal compartments. In plants, how and where these sorting events take place is still poorly understood and our current view of the endocytic pathway still largely relies on analogies made from the animal system. However, extensive differences seem to exist between animal and plant endosomal functions, as exemplified by the role of the trans-Golgi network (TGN) as an early endosomal compartment in plants or the functional diversification of conserved sorting complexes. By using the Arabidopsis root tip as a reference model, we and other have begun to shed light on the complexity of the plant endocytic pathways. Notably, we have recently characterized the functions of an endosomal compartment, the SNX1-endosomes, also referred to as the prevacuolar compartment (PVC) or multivesicular bodies (MVB), in the sorting of different cargo proteins, including two related auxin-efflux carriers, PIN1 and PIN2. We have shown that routing decisions take place at this endosomal level, such as the sorting of PIN2 toward the lytic vacuole for degradation or PIN1 toward the PM for recycling.Key Words: Arabidopsis, intracellular trafficking, endocytic recycling, endosomes, MVB, PVC, VPS29, SNX, PIN, cell polarity     

c-Src trafficking and co-localization with the EGF receptor promotes EGF ligand-independent EGF receptor activation and signaling

c-Src is a non-receptor tyrosine kinase that associates with both the plasma membrane and endosomal compartments. In many human cancers, especially breast cancer, c-Src and the EGF receptor (EGFR) are overexpressed. Dual overexpression of c-Src and EGFR correlates with a Src-dependent increase in activation of EGFR, and synergism between these two tyrosine kinases increases the mitogenic activity of EGFR. Despite extensive studies of the functional interaction between c-Src and EGFR, little is known about the interactions in the trafficking pathways for the two proteins and how that influences signaling. Given the synergism between c-Src and EGFR, and the finding that EGFR is internalized and can signal from endosomes, we hypothesized that c-Src and EGFR traffic together through the endocytic pathway. Here we use a regulatable c-SrcGFP fusion protein that is a bona fide marker for c-Src to show that c-Src undergoes constitutive macropinocytosis from the plasma membrane into endocytic compartments. The movement of c-Src was dependent on its tyrosine kinase activity. Stimulation of cells with EGF revealed that c-Src traffics into the cell with activated EGFR and that c-Src expression and kinase activity prolongs EGFR activation. Surprisingly, even in the absence of EGF addition, c-Src expression induced activation of EGFR and of EGFR-mediated downstream signaling targets ERK and Shc. These data suggest that the synergy between c-Src and EGFR also occurs as these two kinases traffic together, and that their co-localization promotes EGFR-mediated signaling.     

Suppressors of T-cell receptor signaling Sts-1 and Sts-2 bind to Cbl and inhibit endocytosis of receptor tyrosine kinases

The ubiquitin (Ub) ligase Cbl plays a critical role in attenuation of receptor tyrosine kinase (RTK) signaling by inducing ubiquitination of RTKs and promoting their sorting for endosomal degradation. Herein, we describe the identification of two novel Cbl-interacting proteins, p70 and Clip4 (recently assigned the names Sts-1 and Sts-2, respectively), that inhibit endocytosis of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor. Sts-1 and Sts-2 contain SH3 domains that interacted with Cbl, Ub-associated domains, which bound directly to mono-Ub or to the EGFR/Ub chimera as well as phosphoglycerate mutase domains that mediated oligomerization of Sts-1/2. Ligand-induced recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization, reduction in the number of EGFR-containing endocytic vesicles, and subsequent block of receptor degradation followed by prolonged activation of mitogenic signaling pathways. On the other hand, interference with Sts-1/Sts-2 functions diminished ligand-induced receptor degradation, cell proliferation, and oncogenic transformation in cultured fibroblasts. We suggest that Sts-1 and Sts-2 represent a novel class of Ub-binding proteins that regulate RTK endocytosis and control growth factor-induced cellular functions.     

Inhibitory regulation of EGF receptor degradation by sorting nexin 5

Endosomal trafficking of EGF receptor (EGFR) upon stimulation is a highly regulated process during receptor-mediated signaling. Recently, the sorting nexin (SNX) family has emerged as an important regulator in the membrane trafficking of EGFR. Here, we report the identification of a novel interaction between two members of the family, SNX1 and SNX5, which is mediated by the newly defined BAR domain of both SNXs. We have also shown that the PX domain of SNX5 binds specifically to PtdIns other than to PtdIns(3)P. Furthermore, the BAR domain but not the PX domain of SNX5 is sufficient for its subcellular membrane association. Functionally, overexpression of SNX5 inhibits the degradation of EGFR. This process appears to be independent of its interaction with SNX1. However, overexpression of SNX1 is able to attenuate the effect of SNX5 on EGFR degradation, suggesting the two proteins may play antagonistic roles in regulating endosomal trafficking of the receptor.     

Rab4 affects both recycling and degradative endosomal trafficking

The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen-1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.