1 Division of Molecular and Life Science, Pohang University of Science and Technology, San 31, Hojadong, Pohang, Kyungbuk 790-784, Republic of Korea
2 Department of Physiology, College of Medicine, Pusan National University, Pusan 602-739, Republic of Korea
* Author for correspondence (e-mail: pgs{at}postech.ac.kr)
Accepted 23 March 2004
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Summary |
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Key words: Sorting nexin (SNX), Epidermal growth factor (EGF), EGF receptor (EGFR), Trafficking, Phosphatidylinositol 3-phosphate (PtdIns(3)P)
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Introduction |
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Activated epidermal growth factor (EGF) receptors are rapidly internalized by coated pits, sorted to early endosomes and ultimately degraded in lysosomes by a process known as receptor down-regulation (Sorkin, 2001; Stoscheck and Carpenter, 1984
; Wiley et al., 1991
). This down-regulation is important for terminating signal transduction cascades that lead to cellular growth and proliferation (Wells et al., 1990
). Although internalized receptors are ultimately degraded, the rate of receptor degradation is much slower than the rate of internalization (Wiley et al., 1985
). Accumulated evidence suggests that internalized EGF-EGF receptor (EGFR) complexes may possess the ability to generate cell signaling from endosomes (Wang et al., 2002a
; Burke et al., 2001
). Various signaling molecules that regulate Ras activity, including Grb2, SHC and mSOS are co-internalized with EGFR into endosomes and remain associated with the receptor (Rizzo et al., 2000
; Di Guglielmo et al., 1994
; Sorkin et al., 2000
), suggesting that internalized EGFR may play a role in endosomal signaling. Moreover, it has been reported that the inhibition of EGFR endocytosis in the presence of dominant-negative mutant dynamin decreases ERK activation (Kranenburg et al., 1999
; McPherson et al., 2001
). Together, these data suggest that the endosomal trafficking of activated EGF receptor is essential for its downstream signaling. Although the major events in the endocytosis of EGFR are fairly well understood, the molecular mechanisms underlying endosomal trafficking of EGFR remain poorly characterized.
The sorting nexins (SNXs) are a family of cytoplasmic and membrane-associated proteins that are believed to function in the intracellular trafficking of plasma membrane receptors such as EGFR. Recent studies have revealed that SNXs exert their function through Phox (PX) domain-mediated interaction with phosphatidylinositol (PtdIns) (Cozier et al., 2002; Xu et al.,2001
). Moreover, it has been reported that SNX1 may play a critical role in the down-regulation of EGFR by trafficking from early endosomes to late endosomes/lysosomes (Kurten et al., 1996
; Haft et al., 1998
; Zhong et al., 2002
). Consistent with these results, the PX domain of SNX1 is conserved in several yeast proteins, which participate in membrane trafficking such as Mvp1, Vps5/Grd2 and Grd19. Mvp1 is involved in sorting proteins in the late Golgi for delivery to the vacuole and Vps5/Grd2 and Grd19 are involved in the retrieval of proteins from the late endosome for return to the Golgi apparatus (Ekena and Stevens, 1995
; Horazdovsky et al., 1997
; Northwehr and Hindes, 1997
; Voos and Stevens, 1998
). Given this function of the yeast SNX1 homolog, SNX1 was proposed to target EGFR for lysosomal degradation through the endocytic pathway (Haft et al., 1998
).
In this study, we demonstrate some features of the cellular localization and functions of SNX16. First, SNX16 was localized in early endosomes and specifically interacted with phosphatidylinositol 3-phosphate (PtdIns(3)P). Second, SNX16 was associated with EGFR in an EGF-dependent manner. Third, SNX16 accelerated EGF-induced EGFR down-regulation. These data indicate that SNX16 might regulate EGFR trafficking and be a novel regulator of the EGFR-mediated signaling pathway.
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Materials and Methods |
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Cell culture
COS-7 cells were maintained in DMEM supplemented with 10% heat-inactivated bovine calf serum in a humid atmosphere of 5% CO2:95% O2 at 37°C. Cells were seeded on 6- or 10-cm tissue culture dishes, which were pre-coated with poly-L-lysine (10 µg/ml) and grown for 1-2 days until they reached 50-80% confluence. Cells were then placed in serum-free DMEM for 18 hours before being treated with EGF (10 ng/ml).
east two-hybrid analysis
A bait plasmid was constructed using the PCR fragment encoding the C-terminal region of EGFR (a.a. 712-1210) cloned into pGBT9 vector (Clontech, CA) downstream of the GAL4 binding domain (referred to as pGBT9/C-EGFR). After transformation of yeast host strain HF7c cells with the pGBT9/C-EGFR plasmid, the yeast cells were sequentially transformed with pACT vector (Clontech, CA), which contained rat brain-derived cDNAs downstream of the GAL4 transcriptional activation domain. Selection was performed on Trp-, Leu-, and His-deleted plates. After cotransfecting the yeast cells, a filter color assay was used to assess the transcriptional activity of lacZ (ß-galactosidase), as an indicator of the physical interaction between EGFR and proteins derived from the rat brain cDNA library. The intensity of the color reaction was scored in a semi-quantitative manner by visual inspection.
Plasmid constructs and mutagenesis
The mammalian expression vector for FLAG-epitope tagged wild type rat SNX16 (GenBank accession no. AF305780) was made by PCR. Amplified products were inserted in-frame with the FLAG-epitope tag of pFLAG-CMV-2 (Sigma, SL). The mutation of Tyr145 to Ala, designated SNX16Y145A and the deletion mutant of PX domain (a.a. 105-218), designated SNX16PX were introduced into the FLAG-epitope tagged SNX16 cDNA by PCR-directed mutagenesis.
Cell transfection, immunoprecipitation and immunoblot assay
COS-7 cells were transfected using the liposome-mediated LipofectAMINE transfection (Invitrogen, CA) at a ratio of 1 µg of total plasmid DNA to 6 µl LipofectAMINE in 100 µl of serum free medium. After 4 hours, the medium was changed to DMEM supplemented with 10% heat-inactivated bovine calf serum, and after 36 hours the transfected cells were washed with PBS and treated with lysis buffer A (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM NaF, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM PMSF, 1 µg/ml leupeptin, 5 µg/ml aprotinin). Immunoprecipitation and immunoblotting were performed on lysates scraped from plates with a rubber pipette bulb and the supernatants were obtained after centrifugation at 12,000 g for 15 minutes at 4°C. Cell lysates were then mixed with 5 µg pre-coupled anti-FLAG antibody for 3 hours at 4°C. Immunocomplexes were collected by centrifugation, washed four times with cold lysis buffer A, and solubilized by boiling in 1x Laemmli sample buffer. Proteins were separated in 10% SDS-PAGE and transferred to nitrocellulose membranes. Blocking was performed with TTBS buffer [10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween 20] containing 5% skimmed milk powder. Membranes were then incubated with anti-FLAG and anti-EGFR antibodies for 4 hours at room temperature. Immunoblots were subsequently washed, incubated with horseradish peroxidase-linked secondary antibody for 1 hour at room temperature, washed four times in TTBS buffer and developed by horseradish peroxidase-dependent chemiluminescence (ECL) (Amersham International, UK).
Cellular fractionations
COS-7 cells were grown in DMEM supplemented with 10% BCS at 37°C and 5% CO2. The medium was removed and the dishes washed with ice cold PBS. The cells were scraped in 0.5 ml of homogenization buffer (100 mM MES, pH 6.5, 0.25% sucrose, 1 mM EGTA, 10 mM okadaic acid and protease inhibitor cocktail). The cell suspension was sonicated three times for 10 seconds each, followed by centrifugation at 650 g for 15 minutes to remove intact cells and nuclei. The post-nuclear supernatant (total fraction) was collected and subjected to centrifugation at 100,000 g in a Beckman TLA 100.3 rotor for 40 minutes. The supernatant was collected (cytosol fraction) and the pellet (membrane fraction) was resuspended in lysis buffer A (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM NaF, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM PMSF, 1 µg/ml leupeptin, 5 µg/ml aprotinin, 10 mM okadaic acid), sonicated and centrifuged at 14,000 g for an additional 15 minutes to remove insoluble membrane proteins. The two supernatant fractions were solubilized by boiling in 1x Laemmli sample buffer.
Protein-lipid overlay assay
The protein-lipid overlay assays were based on the procedure previously described (Dowler et al., 2002). Briefly, membrane arrays were generated by spotting the required mass of the indicated phosphoinositides (Cell Signals, Inc) onto a Hybond-C extra membrane. After air drying and blocking with 3% (w/v) fatty acid-free BSA in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.1% (v/v) Tween-20 (TBS), the membrane was incubated overnight at 4°C in the same solution containing 1 µg/ml of the relevant recombinant protein. The membrane was then extensively washed in TBS and incubated with anti-GST antibody. The membrane was washed as before, prior to being incubated with anti-mouse horseradish peroxidase conjugate. Finally, the membrane was thoroughly washed and the proteins associated with the various phosphoinositides were visualized by enhanced chemiluminescence.
Receptor internalization assay
COS-7 cells were cultured to 60-70% confluence prior to being labeled with 1.5 mg/ml N-hydroxy-succinimide-biotin (Pierce, IL). After labeling with NHS-SS-biotin, the cells were incubated at 37°C for the indicated time in the presence or absence of 10 ng/ml EGF. Endocytosis was then stopped by transferring cells back to 4°C. After treating with reducing solution (15.5 mg/ml glutathione, 75 mM NaCl, 75 mM NaOH, and 10% fetal bovine serum) and 5 mg/ml iodoacetamide (Sigma, SL) in phosphate-buffered saline containing 0.8 mM MgCl2, 1.0 mM CaCl2 plus 1% bovine serum albumin, the cells were lysed in TNE (10 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, and 1 mM EDTA). Equal quantities of the cell lysate were used to precipitate the biotinylated proteins with streptavidin beads (Pierce, IL). Biotinylated proteins were eluted from the beads by incubation at 95°C for 5 minutes in Laemmli sample buffer. The amount of receptor bound to the beads was detected by immunoblotting.
Uptake of Rhodamine-conjugated EGF and LysoTracker Red
COS-7 cells expressing GFP-tagged SNX16 were cultured on poly-L-lysine slides. Slides were transferred onto ice, washed once with PBS and incubated at 4°C for 2 hours in a medium containing 10 ng/ml Rhodamine-conjugated EGF or 10 µg/ml LysoTracker Red (Molecular Probes, OR). The cells were then washed with PBS and either processed immediately for fixation or culture medium was added. The slides were incubated for the indicated times at 37°C and then fixed. The cells were then rewashed and blocked for 1 hour with 3% fat-free BSA in PBS. Cells were viewed with a Zeiss LSM 510 confocal microscope equipped with LSM 510 version 2.02 software, an Ar (458 and 488 nm) and a He/Ne (543 and 633 nm) laser.
Reporter gene assay
PC12 cells were grown in poly-L-lysine-coated 24-well plates and transfected with serum responsive element (SRE)-luciferase plasmid using LipofectAMINE reagent (Invitrogen). After 36 hours, the cells were stimulated with EGF (10 ng/ml) for the indicated times. After washing with PBS and lysis, the luciferase activity in 1 µg of lysate was assayed using a luciferase assay kit (Promega, WI) with a luminometer (Labsystems, UK).
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Results |
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To determine whether the PX domain is necessary for the intracellular localization of SNX16, we prepared FLAG-tagged wild type SNX16 (SNX16WT), a PX domain-deleted mutant of SNX16 (SNX16PX) and a point mutant of SNX16 in the PX domain by replacing tyrosine-145 with alanine (SNX16Y145A) (Fig. 1C). Post-nuclear supernatants were divided into cytosol and a membrane particulate fraction from SNX16-transfected COS-7 cells. As assessed by the western blotting of the cell fractions, SNX16WT partitioned into both the membrane particulate and cytosolic fraction, but SNX16
PX and SNX16Y145A were present only in the cytosol fraction (Fig. 1D). To investigate the cellular distribution of SNX16 further, GFP-tagged SNX16WT and SNX16Y145A were expressed in COS-7 cells and their intracellular localizations were analyzed by confocal microscopy. SNX16WT was localized in intracellular vesicles and overlapped with EEA1-positive early endosomes and transferrin receptor (TfR)-positive recycling endosomes. However, SNX16Y145A was diffusely expressed in the cytoplasm, indicating that an intact PX domain structure is essential for the proper targeting of SNX16 to cellular vesicles (Fig. 1E). These results indicate that SNX16 is enriched in early and recycling endosomes and that the PX domain is essential for the cellular localization of SNX16 in endosomal compartments.
PX domain-mediated direct interaction of SNX16 with PtdIns(3)P
To examine whether SNX16 is capable of interacting with phosphoinositides, we conducted a protein-lipid overlay analysis with recombinant SNX16WT and SNX16Y145A proteins. As shown in Fig. 2A, SNX16WT specifically associated with PtdIns(3)P. No significant binding was observed with any other phosphoinositides. However, SNX16Y145A did not associate with PtdIns(3)P, suggesting that the conserved residue in the PX domain of SNX16 is essential for this interaction. Owing to the observed specific binding of the PX domains to PtdIns(3)P, we speculate that PtdIns 3-kinase activity may be involved in the subcellular localization of SNX16. To assess this hypothesis, we examined the effect of wortmannin, a specific inhibitor of PtdIns 3-kinase, on the subcellular localization of SNX16. Treatment with wortmannin abolished the endosomal localization of SNX16, suggesting that the intracellular localization of SNX16 is regulated by PtdIns 3-kinase activity (Fig. 2B). In addition, the association between SNX16 and TfR markedly diminished after wortmannin treatment (Fig. 2C). These results suggest that PtdIns 3-kinase may participate in the association between SNX16 and endosomes.
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Association of SNX16 with epidermal growth factor (EGF) receptor
Previous reports have suggested that other sorting nexins interact with RTKs and play a key role in the RTK-mediated signaling pathway (Haft et al., 1998; Zhong et al., 2002
). In this study, our yeast two-hybrid data suggested that SNX16 interacts with EGFR. To confirm the ability of SNX16 to associate with EGFR, COS-7 cells were transiently transfected with SNX16WT. SNX16 was found to associate with EGFR in a time-dependent manner and this reached a maximum 15 minutes after EGF treatment (Fig. 3A). Next, we examined the colocalization of SNX16 with rhodamine-labeled EGF by confocal microscopy. COS-7 cells expressing GFP-SNXWT were incubated with Rhodamine-labeled EGF at 4°C to allow binding between SNX and EGFR. Cells were then incubated at 37°C and the uptake of bound EGF was examined. Receptor-bound EGF did not enter the cells at 4°C (Fig. 3B), however, incubation of the cells at 37°C allowed endocytosis. Moreover, rhodamine-labeled EGFEGFR complexes were colocalized with SNX16 (Fig. 3B). These results suggest that EGFR travels to endosomal compartments containing SNX16 during endocytosis.
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Effect of SNX16 on the down-regulation of EGFR
Given the association between SNX16 and EGFR, we examined the effect of SNX16 on the trafficking of EGFR in the endosomal compartment. SNX16 potentiated EGF-induced EGFR degradation (Fig. 4A), suggesting that SNX16 may participate in the lysosomal targeting and/or degradation of EGFR. Because EGFR trafficking to endosomes is important in the regulation of receptor-mediated signaling, we examined whether the overexpression of SNX16WT influences EGF-dependent mitogenic-activated protein kinase (MAPK) activation. COS-7 cells transfected with SNX16WT showed a progressive decrease in ERK1/2 phosphorylation after EGF treatment. However, the PX domain-defective mutant showed no difference compared to empty vector-transfected cells.
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To determine whether SNX16 is sequestered in lysosomal compartments of COS-7 cells, we examined the colocalization of SNX16 using Lysotracker Red, a lysosome-directed marker (Fig. 4B). In the absence of EGF, colocalized SNX16-LysoTracker Red signals were weak. However, colocalized signals were much stronger after EGF treatment. These results suggest that SNX16 may be involved in lysosomal targeting. In addition, a comparison of the rates of internalization of EGFR with and without SNX16 overexpression revealed no significant differences (Fig. 4C). Taken together, these results suggest that SNX16 may regulate EGFR targeting to lysosomes, but not ligand-induced receptor internalization.
Effect of SNX16 on SRE-dependent transcription activity
As ERK activation stimulates serum response element (SRE)-dependent transcription (Whitmarsh et al., 1995), we examined the influence of SNX16 on SRE-dependent transcription using an SRE-luciferase reporter gene assay. In vector-transfected cells, EGF-induced SRE activation elicited a threefold increase in luciferase activity and the overexpression of SNX16Y145A also caused a threefold increased response (Fig. 5). However, the overexpression SNX16WT failed to increase SRE-dependent transcriptional activity. These results suggest that SNX16 may negatively regulate EGFR-mediated signaling by potentiating the degradation of EGFR.
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The PX domain is required for the homo-oligomerization of SNX16
The formation of homo- and hetero-oligomers is a common feature of various sorting nexins. Furthermore, the oligomerization of SNXs is critical for their cellular functions (Haft et al., 1998; Kurten et al., 2001
). To determine whether SNX16 can associate with itself, we transfected HA-tagged or Flag-tagged SNX16 in COS-7 cells (Fig. 6). SNX16WT formed homo-oligomers, but the deletion of the PX domain disrupted the oligomerization of SNX16. Furthermore, SNX16Y145A that abrogates vesicular localization (Fig. 1E) and PtdIns(3)P binding (Fig. 2A) did not associate with itself (Fig. 6). These results suggest that the PX domain is a critical determinant of the self-association of SNX16.
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Discussion |
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Knowledge of the intracellular location of SNXs is critical for defining their function. Various PX domain-containing proteins have been localized in early endosomes. Furthermore, previous studies have demonstrated that SNX1 and SNX3 are partly localized in perinuclear vesicles or early endosome, suggesting that SNX1 or SNX3 may be important for vesicular trafficking (Xu et al., 2001; Zhong et al., 2002
). In the present study, we found that SNX16 preferentially localized into early and recycling endosomes (Fig. 1E). Moreover, mutation of the PX domain disrupted the vesicular localization of SNX16, suggesting that the PX domain of SNX16 is essentially required for this process.
Various phosphoinositides are known to regulate diverse cellular signal transduction pathways and/or membrane transport events (Toker, 2002). The cellular level of PtdIns(3)P is regulated by the action of PtdIns 3-kinase, or by the actions of the corresponding lipid phosphatases or lipases (Cantley, 2002
). It has been reported that Vps34p, a Saccharamyces cerevisiae homologue of PtdIns 3-kinase, plays an essential role in membrane transport of PtdIns(3)P from the Golgi apparatus to vacuoles via endosomes (Burda et al., 2002
). In the present study, several lines of evidence suggest that the endosomal localization of SNX16 is dependent on the ability of its PX domain to bind PtdIns(3)P. First, point mutated SNX16 (SNX16Y145A) was incapable of binding to PtdIns(3)P, and did not localize to the endosomal compartments (Fig. 1E and Fig. 2A). Second, treatment with wortmannin, a specific inhibitor of PtdIns 3-kinase, abolished the endosomal localization of SNX16, suggesting that the intracellular localization of SNX16 is regulated by PtdIns 3-kinase activity (Fig. 2B,C). Taken together, these results strongly suggest that the PX domain of SNX16 participates in the association with PtdIns(3)P required for targeting to the endosomal compartments.
SNX1 was cloned by using a yeast two-hybrid screen with a portion of cytoplasmic domain of EGFR as bait (Kurten et al., 1996). In addition, other SNXs such as SNX2 and SNX4 have also been associated with EGFR and PDGFR (Haft et al., 1998
). However, these reports showed that the SNX-receptor interactions were not affected by the activation of the receptors by ligand. Interestingly, we observed that SNX16 specifically associates with EGFR in an EGF-dependent manner (Fig. 3). Furthermore, SNX16 colocalized with the EGF-EGFR complexes in intracellular vesicles. This may be due to EGF-induced redistribution of EGFR to intracellular compartments containing SNX16. Our findings strongly suggest that SNX16 participates in the endosomal trafficking of EGFR by the specific interaction.
SNXs have been shown to regulate the endocytosis of a variety of receptors such as EGFR, protease-activated receptor-1 and low-density lipoprotein receptors (Kurten et al., 1996; Wang et al., 2002b
; Stockinger et al., 2002
). Furthermore, many homologues of SNXs found in yeast have been found to play an essential role in receptor recycling between early endosomes and late endosomes/lysosomes (Horazdovsky et al., 1997
). In mammalian cells, the overexpression of SNX1 was noted to accelerate EGFR degradation, implying a role for this protein in endosomal transport (Kurten et al., 1996
). Consistent with this report, we demonstrate the SNX16 has a function in the degradation of EGFR. Three pieces of evidence support the physiological significance of the function of SNX16 on the regulation of the endosome-to-lysosome trafficking of EGFR. First, SNX16 associates with EGFR in an EGF-induced manner (Fig. 3), and colocalizes with lysosomes after EGF treatment (Fig. 4B). These findings suggest that SNX16 participates in the endosomal trafficking of EGFR. Second, the overexpression of SNX16 accelerates the degradation of EGFR, but point mutation of SNX16 (SNX16Y145A) inhibits EGFR degradation (Fig. 4A), indicating that the PX domain of SNX16 may be critical for the function of SNX16 on EGFR trafficking. Furthermore, the overexpression of SNX16 was found to have no effect on receptor internalization (Fig. 4C), suggesting that SNX16 may be involved in the regulation of the endosome-to-lysosome trafficking of EGFR. Third, the overexpression of SNX16 was found to inhibit serum responsive element (SRE)-dependent transcriptional activity (Fig. 5). Taken together, these results suggest that SNX16 may negatively regulate EGFR-mediated signaling by potentiating the degradation of EGFR.
In yeast, the oligomerization of SNXs is essential for organizing the sorting and recycling of functional complexes (Horazdovsky et al., 1997). Furthermore, in mammalian cells, it has been reported that the homo- and heterodimerization of SNXs is critical for receptor sorting and degradation (Haft et al., 2000
; Kurten et al., 2001
). Moreover, the PX domain of SNX1 was shown to mediate oligomerization with itself and with SNX2 (Haft et al., 1998
). Furthermore, SNX6 and SNX15 also exhibit self-association mediated by the PX domain (Parks et al., 2001
; Phillips et al., 2001
). We observed that the PX domain of SNX16 is essential for its oligomerization (Fig. 6), suggesting that SNX16, which forms the sorting complexes, specifically regulates the function of EGFR and facilitates the degradation of EGFR in an EGF-dependent manner.
In conclusion, this study demonstrates for the first time that SNX16 functions as a sorting cargo in the endosome pathway to lysosomes. Moreover, it establishes a correlation between the ability of SNX16 to bind PtdIns(3)P, the association between SNX16 and the endosomal membrane compartment and its regulation of endosomal function. The endosomal association of SNX16 and the functional regulation of endosomes by SNX16 overexpression depend absolutely on its PtdIns(3)P-binding activity, thus linking the subcellular localization and biochemical properties of SNX16 to its regulation of endosomal function. These findings provide a new insight into how SNX16 might function in protein trafficking in mammalian cells. The challenge now is to elucidate the mechanism by which SNX16 regulates the sorting of EGFR from early endosomes to lysosomes, an event critical for the termination of receptor signaling.
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Acknowledgments |
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