Echovirus 1 infection depends on biogenesis of novel multivesicular bodies

Non-enveloped picornavirus echovirus 1 (EV1) clusters its receptor α2β1 integrin and causes their internalization and accumulation in α2β1 integrin enriched multivesicular bodies (α2-MVBs). Our results here show that these α2-MVBs are distinct from acidic late endosomes/lysosomes by several criteria: (i) live intra-endosomal pH measurements show that α2-MVBs are not acidic, (ii) they are not positive for the late endosomal marker LBPA or Dil-LDL internalized to lysosomes, and (iii) simultaneous stimulation of epidermal growth factor receptor (EGFR) and α2β1 integrin clustering leads to their accumulation in separate endosomes. EGFR showed downregulation between 15 min and 2 h, whereas accumulation of α2β1 integrin/EV1 led to an increase of integrin fluorescence in cytoplasmic vesicles further suggesting that EV1 pathway is separate from the lysosomal downregulation pathway. In addition, the results demonstrate the involvement of ESCRTs in the biogenesis of α2-MVBs. Overexpression of dominant-negative form of VPS4 inhibited biogenesis of α2-MVBs and efficiently prevented EV1 infection. Furthermore, α2-MVBs were positive for some members of ESCRTs such as Hrs, VPS37A and VPS24 and the siRNA treatment of TSG101, VPS37A and VPS24 inhibited EV1 infection. Our results show that the non-enveloped EV1 depends on biogenesis of novel multivesicular structures for successful infection.     

Transforming growth factor-alpha short-circuits downregulation of the epidermal growth factor receptor

Transforming growth factor-alpha (TGFalpha) is an epidermal growth factor receptor (EGFR) ligand which is distinguished from EGF by its acid-labile structure and potent transforming function. We recently reported that TGFalpha induces less efficient EGFR heterodimerization and downregulation than does EGF (Gulliford et al., 1997, Oncogene, 15:2219-2223). Here we use isoform-specific EGFR and ErbB2 antibodies to show that the duration of EGFR signalling induced by a single TGFalpha exposure is less than that induced by equimolar EGF. The protein trafficking inhibitor brefeldin A (BFA) reduces the duration of EGF signalling to an extent similar to that seen with TGFalpha alone; the effects of TGFalpha and BFA on EGFR degradation are opposite, however, with TGFalpha sparing EGFR from downregulation but BFA accelerating EGF-dependent receptor loss. This suggests that BFA blocks EGFR recycling and thus shortens EGF-dependent receptor signalling, whereas TGFalpha shortens receptor signalling and thus blocks EGFR downregulation. Consistent with this, repeated application of TGFalpha is accompanied by prolonged EGFR expression and signalling, whereas similar application of EGF causes receptor downregulation and signal termination. These findings indicate that constitutive secretion of pH-labile TGFalpha may perpetuate EGFR signalling by permitting early oligomer dissociation and dephosphorylation within acidic endosomes, thereby extinguishing a phosphotyrosine-based downregulation signal and creating an irreversible autocrine growth loop.     

NOK/STYK1 has a strong tendency towards forming aggregates and colocalises with epidermal growth factor receptor in endosomes

Our previous studies showed that the overexpression of Novel Oncogene with Kinase-domain (NOK)/STYK1 led to cellular transformation, tumorigenesis and metastasis. This report characterises the subcellular distribution of NOK in HeLa cells and its localisation in early endosomes. Confocal immunolocalisation studies indicated that NOK had structural subtypes and was distributed into two distinct expression patterns: a dot pattern (DP) and an aggregation pattern (AP). The results of an immunohistochemistry (IHC) analysis of pathological tissues also showed that high expression level of endogenous NOK was expressed in an aggregate-like structure in vivo. Importantly, we found that NOK was localised in endosomes and colocalised with epidermal growth factor receptor (EGFR) in activated endosomal vesicles. However, as the stimulation time increased, NOK and EGFR began to progress through different pathways. EGFR was gradually degraded after treatment with EGF for approximately 20 min, whereas NOK levels were not reduced. This result suggests that NOK mainly plays a role in facilitating the trafficking of EGFR from early endosomes to later endosomes/lysosomes. Taken together, NOK has a strong tendency towards forming aggregates, which may have physiological implications and provide the first evidence that this novel receptor kinase is colocalised with EGFR in endosomes to participate in a post-internalisation step of EGFR.     

Odin (ANKS1A) Modulates EGF Receptor Recycling and Stability

The ANKS1A gene product, also known as Odin, was first identified as a tyrosine-phosphorylated component of the epidermal growth factor receptor network. Here we show that Odin functions as an effector of EGFR recycling. In EGF-stimulated HEK293 cells tyrosine phosphorylation of Odin was induced prior to EGFR internalization and independent of EGFR-to-ERK signaling. Over-expression of Odin increased EGF-induced EGFR trafficking to recycling endosomes and recycling back to the cell surface, and decreased trafficking to lysosomes and degradation. Conversely, Odin knockdown in both HEK293 and the non-small cell lung carcinoma line RVH6849, which expresses roughly 10-fold more EGF receptors than HEK293, caused decreased EGFR recycling and accelerated trafficking to the lysosome and degradation. By governing the endocytic fate of internalized receptors, Odin may provide a layer of regulation that enables cells to contend with receptor cell densities and ligand concentration gradients that are physiologically and pathologically highly variable.     

hSef potentiates EGF-mediated MAPK signaling through affecting EGFR trafficking and degradation

Sef (similar expression to fgf genes) was identified as an effective antagonist of fibroblast growth factor (FGF) in vertebrates. Previous reports have demonstrated that Sef interacts with FGF receptors (FGFRs) and inhibits FGF signaling, however, its role in regulating epidermal growth factor receptor (EGFR) signaling remains unclear. In this report, we found that hSef localizes to the plasma membrane (PM) and is subjected to rapid internalization and well localizes in early/recycling endosomes while poorly in late endosomes/lysosomes. We observed that hSef interacts and functionally colocalizes with EGFR in early endosomes in response to EGF stimulation. Importantly, we demonstrated that overexpression of hSef attenuates EGFR degradation and potentiates EGF-mediated mitogen-activated protein kinase (MAPK) signaling by interfering EGFR trafficking. Finally, our data showed that, with overexpression of hSef, elevated levels of Erk phosphorylation and differentiation of rat pheochromocytoma (PC12) cells occur in response to EGF stimulation. Taken together, these data suggest that hSef plays a positive role in the EGFR-mediated MAPK signaling pathway. This report, for the first time, reveals opposite roles for Sef in EGF and FGF signalings.     

Acute ligand-independent Src activation mimics low EGF-induced EGFR surface signalling and redistribution into recycling endosomes

Src, a non-receptor tyrosine kinase, is a key signal transduction partner of epidermal growth factor (EGF) receptor (EGFR). In human breast cancer, EGFR and Src are frequently over-expressed and/or over-activated. Although reciprocal activation is documented, mechanisms underlying Src:EGFR interactions are incompletely understood. We here exploited ts/v-Src thermo-activation in MDCK monolayers to test whether acute Src activation impacts on signalling and trafficking of non-liganded EGFR. We found that thermo-activation caused rapid Src recruitment to the plasma membrane, concomitant association with EGFR, and its phosphorylation at Y845 and Y1173 predominantly at the cell surface. Like low EGF concentrations, activated Src (i) decreased EGF surface binding without affecting the total EGFR pool; (ii) triggered EGFR endocytosis via clathrin-coated vesicles; (iii) and led to its sequestration in perinuclear/recycling endosomes with avoidance of multivesicular bodies and lysosomal degradation. Combined Src activation and EGF were synergistic for EGFR-Y845 and -Y1173 phosphorylation at some endosomes. We conclude that acute effects of Src in MDCK cells may mimic those of low EGF on EGFR activation and redistribution. Src:EGFR interactions may be sufficient to trigger EGFR activation and might contribute to its local signalling, without requiring either soluble extracellular signal or receptor over-expression.     

The hepatitis C virus non-structural protein NS5A alters the trafficking profile of the epidermal growth factor receptor

Hepatitis C virus (HCV) frequently establishes a persistent infection, leading to chronic liver disease. The NS5A protein has been implicated in this process as it modulates a variety of intracellular signalling pathways that control cell survival and proliferation. In particular, NS5A associates with several proteins involved in the endocytosis of the epidermal growth factor receptor (EGFR) and has been previously shown to inhibit epidermal growth factor (EGF)-stimulated activation of the Ras–Erk pathway by a mechanism that remains unclear. As EGFR signalling involves trafficking to late endosomes, we investigated whether NS5A perturbs EGFR signalling by altering receptor endocytosis. We demonstrate that NS5A partially localizes to early endosomes and, although it has no effect on EGF internalization, it colocalizes with the EGFR and alters its distribution. This redistribution correlates with a decrease in the amount of active EGF–EGFR ligand–receptor complexes present in the late endosomal signalling compartment and also results in a concomitant increase in the total levels of EGFR. These observations suggest that NS5A controls EGFR signalling by diverting the receptor away from late endosomes. This represents a novel mechanism by which a viral protein attenuates cell signalling and suggests that NS5A may perturb trafficking pathways to maintain an optimal environment for HCV persistence.     

Late endosomal traffic of the epidermal growth factor receptor ensures spatial and temporal fidelity of mitogen-activated protein kinase signaling

Mitogen-activated protein kinase (MAPK) signaling is regulated by assembling distinct scaffold complexes at the plasma membrane and on endosomes. Thus, spatial resolution might be critical to determine signaling specificity. Therefore, we investigated whether epidermal growth factor receptor (EGFR) traffic through the endosomal system provides spatial information for MAPK signaling. To mislocalize late endosomes to the cell periphery we used the dynein subunit p50 dynamitin. The peripheral translocation of late endosomes resulted in a prolonged EGFR activation on late endosomes and a slow down in EGFR degradation. Continuous EGFR signaling from late endosomes caused sustained extracellular signal-regulated kinase and p38 signaling and resulted in hyperactivation of nuclear targets, such as Elk-1. In contrast, clustering late endosomes in the perinuclear region by expression of dominant active Rab7 delayed the entry of the EGFR into late endosomes, which caused a delay in EGFR degradation and a sustained MAPK signaling. Surprisingly, the activation of nuclear targets was reduced. Thus, we conclude that appropriate trafficking of the activated EGFR through endosomes controls the spatial and temporal regulation of MAPK signaling.     

Reciprocal priming between receptor tyrosine kinases at recycling endosomes orchestrates cellular signalling outputs

Integration of signalling downstream of individual receptor tyrosine kinases (RTKs) is crucial to fine‐tune cellular homeostasis during development and in pathological conditions, including breast cancer. However, how signalling integration is regulated and whether the endocytic fate of single receptors controls such signalling integration remains poorly elucidated. Combining quantitative phosphoproteomics and targeted assays, we generated a detailed picture of recycling‐dependent fibroblast growth factor (FGF) signalling in breast cancer cells, with a focus on distinct FGF receptors (FGFRs). We discovered reciprocal priming between FGFRs and epidermal growth factor (EGF) receptor (EGFR) that is coordinated at recycling endosomes. FGFR recycling ligands induce EGFR phosphorylation on threonine 693. This phosphorylation event alters both FGFR and EGFR trafficking and primes FGFR‐mediated proliferation but not cell invasion. In turn, FGFR signalling primes EGF‐mediated outputs via EGFR threonine 693 phosphorylation. This reciprocal priming between distinct families of RTKs from recycling endosomes exemplifies a novel signalling integration hub where recycling endosomes orchestrate cellular behaviour. Therefore, targeting reciprocal priming over individual receptors may improve personalized therapies in breast and other cancers.     

rab7 activity affects epidermal growth factor:epidermal growth factor receptor degradation by regulating endocytic trafficking from the late endosome

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family. Ligand (epidermal growth factor or EGF) binding to the EGFR results in the coordinated activation and integration of biochemical signaling events to mediate cell growth, migration, and differentiation. One mechanism the cell utilizes to orchestrate these events is ligand-mediated endocytosis through the canonical clathrin-mediated endocytic pathway. Identification of proteins that regulate the intracellular movement of the EGF.EGFR complex is an important first step in dissecting how specificity of EGFR signaling is conferred. We examined the role of the small molecular weight guanine nucleotide-binding protein (G-protein) rab7 as a regulator of the distal stages of the endocytic pathway. Through the transient expression of activating and inactivating mutants of rab7 in HeLa cells, we have determined that rab7 activity directly correlates with the rate of radiolabeled EGF and EGFR degradation. Furthermore, when inhibitory mutants of rab7 are expressed, the internalized EGF.EGFR complex accumulates in high-density endosomes that are characteristic of the late endocytic pathway. Thus, we conclude that rab7 regulates the endocytic trafficking of the EGF.EGFR complex by regulating its lysosomal degradation.     

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.     

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).     

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.     

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.     

A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells

We have previously shown that overexpression of LIM kinase1 (LIMK1) resulted in a marked retardation of the internalization of the receptor-mediated endocytic tracer, Texas red-labeled epidermal growth factor (EGF) in low-invasive human breast cancer cell MCF-7. We thereby postulate that LIMK1 signaling plays an important role in the regulation of ligand-induced endocytosis of EGF receptor (EGFR) in tumor cells by reorganizing and influencing actin-filament dynamics. In the present study, we further assessed the effect of wild-type LIMK1, a kinase-deficient dominant negative mutant of LIMK1 (DN-LIMK1) and an active, unphosphorylatable cofilin mutant (S3A cofilin) on internalization of EGF-EGFR in MDA-MB-231, a highly invasive human breast cancer cell line. We demonstrate here that a marked delay in the receptor-mediated internalization of Texas red-labeled EGF was observed in the wild-type LIMK1 transfectants, and that most of the internalized EGF staining were accumulated within transferrin receptor-positive early endosomes even after 30 min internalization. In contrast, the expression of dominant-negative LIMK1 mutant rescued the efficient endocytosis of Texas red-EGF, and large amounts of Texas red-EGF staining already reached LIMPII-positive late endosomes/lysosomal vacuoles after 15 min internalization. We further analyzed the effect of S3A cofilin mutant on EGFR trafficking, and found an efficient delivery of Texas red-EGF into late endosomes/lysosomes at 15–30 min after internalization. Taken together, our novel findings presented in this paper implicate that LIMK1 signaling indeed plays a pivotal role in the regulation of EGFR trafficking through the endocytic pathway in invasive tumor cells.     

The SRC homology 2 domain of Rin1 mediates its binding to the epidermal growth factor receptor and regulates receptor endocytosis

Activated epidermal growth factor receptors (EGFRs) recruit intracellular proteins that mediate receptor signaling and endocytic trafficking. Rin1, a multifunctional protein, has been shown to regulate EGFR internalization (1). Here we show that EGF stimulation induces a specific, rapid, and transient membrane recruitment of Rin1 and that recruitment is dependent on the Src homology 2 (SH2) domain of Rin1. Immunoprecipitation of EGFR is accompanied by co-immunoprecipitation of Rin1 in a time- and ligand-dependent manner. Association of Rin1 and specifically the SH2 domain of Rin1 with the EGFR was dependent on tyrosine phosphorylation of the intracellular domain of the EGFR. The recruitment of Rin1, observed by light microscopy, indicated that although initially cytosolic, Rin1 was recruited to both plasma membrane and endosomes following EGF addition. Moreover, the expression of the SH2 domain of Rin1 substantially impaired the internalization of EGF without affecting internalization of transferrin. Finally, we found that Rin1 co-immunoprecipitated with a number of tyrosine kinase receptors but not with cargo endocytic receptors. These results indicate that Rin1 provides a link via its SH2 domain between activated tyrosine kinase receptors and the endocytic pathway through the recruitment and activation of Rab5a.     

Expression of human immunodeficiency virus type 1 gag modulates ligand-induced downregulation of EGF receptor

Many enveloped viruses use the ESCRT proteins of the cellular vacuolar protein sorting pathway for efficient egress from the cell. Recruitment of the ESCRT proteins by human immunodeficiency virus type 1 (HIV-1) Gag is required for HIV-1 particle budding and egress. ESCRT proteins normally function at endosomal membranes, where they facilitate the downregulation of mitogen-activated receptors such as EGF receptor (EGFR) through multivesicular body biogenesis. It is not known whether the Gag-mediated recruitment of ESCRT proteins functionally depletes the pool of these molecules that is available for the downregulation of EGFR. Here we show that the expression of HIV-1 Gag decreases the rate of EGFR downregulation, as assessed by decreases in the rates of (125)I-EGF and EGFR degradation. The effect of Gag was dependent on the presence of the TSG101 binding motif (PTAP) within the Gag C-terminal p6 domain. Cells expressing HIV-1 Gag retained more EGFR in late endosomes. This effect occurred when Gag was expressed alone from a heterologous promoter and when Gag expression was driven by the HIV-1 long terminal repeat within pHXB2DeltaBalD25S, a noninfectious lentiviral vector. Gag-expressing cells exhibited higher levels of activated mitogen-activated protein kinase for longer times after EGF addition than did cells that did not express HIV-1 Gag. These results indicate that HIV-1 Gag can impinge upon the functioning of the cellular vacuolar protein sorting pathway and reveal yet another facet of the intricate effects of HIV-1 infection on host cell physiology.     

Calmodulin regulates intracellular trafficking of epidermal growth factor receptor and the MAPK signaling pathway

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13-mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.     

Regulation of intracellular trafficking of the EGF receptor by Rab5 in the absence of phosphatidylinositol 3-kinase activity

Rab5 and phosphatidylinositol 3-kinase (PI3K) have been proposed to co-regulate receptor endocytosis by controlling early endosome fusion. However, in this report we demonstrate that inhibition of epidermal growth factor (EGF)-stimulated PI3K activity by expression of the kinase-deficient PI3K p110 subunit (p110Δkin) does not block the lysosomal targeting and degradation of the EGF receptor (EGFR). Moreover, inhibition of total PI3K activity by wortmannin or LY294002 significantly enlarges EGFR-containing endosomes and dissociates the early-endosomal autoantigen EEA1 from membrane fractions. However, this does not block the lysosomal targeting and degradation of EGFR. In contrast, transfection of cells with mutant Rab5 S34N or microinjection of anti-Rabaptin5 antibodies inhibits EGFR endocytosis. Our results, therefore, demonstrate that PI3K is not universally required for the regulation of receptor intracellular trafficking. The present work suggests that the intracellular trafficking of EGFR is controlled by a novel endosome fusion pathway that is regulated by Rab5 in the absence of PI3K, rather than by the previously defined endosome fusion pathway that is co-regulated by Rab5 and PI3K.