Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Jul 9.
Published in final edited form as: J Cell Sci. 2008 Sep 2;121(Pt 19):3155–3166. doi: 10.1242/jcs.020404

Clathrin-Independent Endocytosis of ErbB2 in Geldanamycin-Treated Human Breast Cancer Cells

Daniel J Barr 1, Anne G Ostermeyer-Fay 1, Rachel A Matundan 1, Deborah A Brown 1,*
PMCID: PMC2707784  NIHMSID: NIHMS92795  PMID: 18765569

Summary

The EGF receptor family member ErbB2 in is commonly overexpressed in human breast cancer cells and correlates with poor prognosis. Geldanamycin (GA) induces ErbB2 ubiquitination, intracellular accumulation, and degradation. Whether GA stimulates ErbB2 internalization is controversial. We found that ErbB2 was internalized constitutively at a rate that was not affected by GA in SKBr3 breast cancer cells. Instead, GA altered endosomal sorting, causing transport of ErbB2 to lysosomes for degradation. In contrast to earlier work, we found that ErbB2 internalization occurred by a clathrin-independent, tyrosine kinase-independent pathway that was not caveolar, as SKBr3 cells lack caveolae. Like cargo of the GEEC (GPI-anchored protein-enriched early endosomal compartment) pathway, internalized ErbB2 colocalized with cholera toxin B subunit (CTxB), GPI-anchored proteins, and fluid after internalization, and was often seen in short tubules or large vesicles. However, in contrast to the GEEC pathway in other cells, internalization of ErbB2 and fluid in SKBr3 cells did not require Rho family GTPase activity. ErbB2 accumulated in vesicles containing constitutively active Arf6Q67L only without GA; Arf6Q67L did not slow transport to lysosomes in GA-treated cells. Further characterization of this novel clathrin-, caveolae-, and Rho-family-independent endocytic pathway may suggest new strategies for down-regulation of ErbB2 in breast cancer.

Keywords: non-clathrin endocytosis, membrane traffic, receptor tyrosine kinase, receptor down-regulation, Arf6, Rho family GTPase, HER2

Introduction

ErbB2 is in the ErbB family of receptor tyrosine kinases, which also includes the epidermal growth factor receptor (EGFR or ErbB1) (Yarden and Sliwkowski, 2001). ErbB2 is the preferred heterodimerization partner of the other ErbB family members and enhances their signaling, although it has no known ligand of its own (Graus-Porta et al., 1997; Lenferink et al., 1998). ErbB2 is overexpressed in 20–30% of human breast cancers and is associated with poor prognosis (Slamon et al., 1987). Thus, ErbB2 down-regulation is an important goal in treatment of breast cancer.

Unlike other ErbB proteins, ErbB2 binds constitutively to the chaperone Hsp90 (Xu et al., 2001). The ansamycin antibiotic geldanamycin (GA) releases Hsp90 from its client proteins, destabilizing them (Hohfeld et al., 2001; Richter and Buchner, 2001). Following release of Hsp90 from ErbB2, both Hsp70 and the co-chaperone and E3 ubiquitin ligase CHIP are recruited to the receptor (Citri et al., 2004; Xu et al., 2002; Zhou et al., 2003). CHIP ubiquitinates ErbB2, leading to its intracellular accumulation and degradation with a t1/2 of about 2 hours. The GA analog 17-allylamino, 17-demethoxygeldanamycin is in clinical trials for treatment of ErbB2-dependent breast cancer (Sharp and Workman, 2006).

The step(s) at which GA affects ErbB2 endocytic transport remain controversial. One group found that cytoplasmic domain interactions normally stabilize ErbB2 at the plasma membrane and make it resistant to internalization (Hommelgaard et al., 2004; Lerdrup et al., 2007). They found that GA overcame this effect and stimulated ErbB2 internalization (Lerdrup et al., 2006). By contrast, a second group found that ErbB2 was constitutively internalized, but normally recycled efficiently so that most of the protein was on the plasma membrane at steady state (Austin et al., 2004). In their hands, GA did not affect internalization of ErbB2, but altered sorting in endosomes, reducing recycling and leading to accumulation inside multivesicular bodies (MVBs) (Austin et al., 2004).

Early reports suggested that after release of Hsp90, ErbB2 is degraded by proteasomes, rather than in lysosomes (Citri et al., 2002; Mimnaugh et al., 1996; Way et al., 2004). However, later work suggested that ErbB2 is transported through early and late endosomes for degradation in lysosomes. Internalized ErbB2 colocalizes with transferrin (Tf), and is present in internal vesicles inside MVBs, suggesting transport to lysosomes for degradation (Austin et al., 2004).

ErbB2 degradation was initially reported to be insensitive to lysosomal inhibitors (Citri et al., 2002; Mimnaugh et al., 1996; Way et al., 2004). However, later work showed that a cytoplasmic domain fragment is cleaved from Erb2 in GA-treated cells, rendering the protein undetectable by antibodies previously used on blots (Tikhomirov and Carpenter, 2000; Tikhomirov and Carpenter, 2001). Use of an extracellular domain-specific antibody showed that lysosomal inhibitors stabilize a clipped 135 kDa form of ErbB2 in GA-treated cells (Tikhomirov and Carpenter, 2000). Proteasome inhibitors were later shown to retard ErbB2 degradation in GA-treated cells indirectly, by inhibiting internalization (Lerdrup et al., 2006). Even intact ErbB2 was found to be internalized and degraded in lysosomes upon GA treatment (Lerdrup et al., 2006).

In this paper, we examined internalization of ErbB2 in GA-treated breast cancer cells. We found that GA did not affect the rate of ErbB2 internalization. In contrast to a previous report (Austin et al., 2005), we found that ErbB2 was internalized by a clathrin-independent pathway. ErbB2 was internalized by a similar pathway in transfected COS-7 cells, showing the generality of this finding.

Cells exhibit a number of clathrin-independent endocytic pathways (Conner and Schmid, 2003; Kirkham and Parton, 2005). Endocytosis in caveolae requires caveolin-1, dynamin, and tyrosine kinase activity (Kirkham and Parton, 2005). A similar pathway found in cells that lack caveolin-1 also requires tyrosine kinase activity (Damm et al., 2005). CHO cells internalize GPI-anchored proteins, but not transmembrane proteins, into GPI-anchored protein-enriched early endosomal compartments (GEECs) that also contain fluid-phase markers (Sabharanjak et al., 2002; Kalia et al., 2006; Chadda et al., 2007; Kumari and Mayor, 2008).

Cholera toxin B subunit (CTxB) binds the ganglioside GM1 and can be internalized by several different means, including the clathrin-mediated and caveolar pathways (Lencer and Saslowsky, 2005; Sandvig and Van Deurs, 2002). At least in some cells, most CTxB is taken up into clathrin- and caveolin-independent carriers (CLICs) with a distinctive tubular and ring-like morphology (Kirkham and Parton, 2005). These also contain internalized GPI-anchored proteins and fluid, suggesting that they are the same as GEECs (Kirkham et al., 2005).

In contrast to CHO cells, Hela cells do not have a dedicated pathway for internalization of GPI-anchored proteins. In these cells, both GPI-anchored and transmembrane proteins follow a non-clathrin, non-caveolar endocytic pathway regulated by Arf6 (Naslavsky et al., 2003; Naslavsky et al., 2004). Constitutively active Arf6Q67L causes cargo of this pathway to accumulate in enlarged vacuoles (Naslavsky et al., 2003; Naslavsky et al., 2004). The swollen vacuoles induced by Arf6Q67L were proposed to represent an intermediate compartment, upstream of early endosomes (Naslavsky et al., 2003). Arf6Q67L slows exit from this compartment, leading to swelling. As expected from this model, Arf6Q67L blocked normal transport to Rab5-positive early endosomes and then on to lysosomes, and also blocked degradation of cargo that accumulated in the Arf6Q67L-positive vacuoles (Naslavsky et al., 2003).

By contrast, the GEEC pathway in CHO cells is not regulated by Arf6 (Kalia et al., 2006). However, both the Arf6-regulated and GEEC pathways rapidly merge with the “classical” clathrin-mediated pathway at the level of Rab5-positive early endosomes (Kalia et al., 2006; Naslavsky et al., 2003).

Several clathrin-independent endocytic pathways require Rho-family GTPase activity (Mayor and Pagano, 2007). For instance, RhoA is required for uptake of interleukin-2 receptor in T cells (Lamaze et al., 2001) and of albumin in CHO cells (Cheng et al., 2006). Cdc42 is required for efficient fluid-phase uptake in immature dendritic cells (Garrett et al., 2000) and for the GEEC pathway in CHO cells (Chadda et al., 2007; Sabharanjak et al., 2002).

Here, we showed that in SKBr3 cells both with and without GA, ErbB2 was internalized via a non-clathrin, non-caveolar pathway together with a GPI-anchored protein, CTxB, and fluid. In GA-treated cells, ErbB2 was then transported to lysosomes for degradation. Although ErbB2 accumulated in Arf6Q67L-positive vacuoles in the absence of drug treatment, Arf6Q67L did not slow ErbB2 transport to lysosomes in GA-treated cells. Endocytosis of ErbB2 and fluid in SKBr3 cells did not require Rho family GTPase function.

Results

ErbB2 internalization in GA-treated SkBr3 cells is independent of clathrin

Immunofluorescence microscopy (IF) showed high levels of ErbB2 on the surface of SKBr3 cells (Fig. 1A), as reported previously (Austin et al., 2004; Hommelgaard et al., 2004). Also as reported (Austin et al., 2004; Hommelgaard et al., 2004), fluorescein-conjugated surface-bound anti-ErbB2 antibodies (Fl-anti-ErbB2) did not greatly affect ErbB2 localization (Fig. 1B). By contrast, after 2–3 hours GA treatment, ErbB2 was abundant in intracellular puncta, while surface localization was reduced (Fig. 1C).

Fig. 1.

Fig. 1

Open in a new tab

Effect of bound antibodies, GA, and CPZ on ErbB2 localization in SKBr3 cells. (A) ErbB2 in fixed, permeabilized cells detected by IF. (B) Cells were warmed for 2 hours after binding Fl-anti-ErbB2 before fixation. (C) Cells were treated with GA for 2 hours before detecting ErbB2 by IF. (D,E) Cells were pre-treated for 45 minutes at 37°C with GA, with (D) or without (E) 12 µg/ml CPZ, before binding anti-ErBb2 antibodies and AF-594-Tf for 1 hour on ice and warming for 2 minutes with the same drugs. Cells were acid-washed and processed for IF, detecting ErbB2 with the AF-488 Zenon mouse IgG labeling kit. (D,E) ErbB2, left; Tf, center; merged images, right. Scale bar; 10 µm. (F,G) Internalization of biotinylated Tf (F) or biotinylated anti-ErbB2 antibodies (G) was measured by CELISA after treatment with GA (circles) or both GA and CPZ (squares). Values shown are the mean +/− s.e.m. of 3 experiments.

To determine the role of the clathrin pathway in ErbB2 endocytosis in GA-treated cells, we first used chlorpromazine (CPZ), a cationic amphiphile that inhibits this pathway (Wang et al., 1993). CPZ efficiently inhibited uptake of AF-594-Tf, but did not block ErbB2 internalization (Fig. 1D, 1E). After CPZ treatment, structures containing internalized ErbB2 sometimes had a tubular morphology (D. J. Barr, unpublished) that may have resulted from the ability of CPZ to induce membrane curvature (Lange and Steck, 1984). A CELISA assay showed that CPZ inhibited uptake of biotinylated Tf (Fig. 1F), but did not affect the internalization of biotinylated anti-ErbB2 antibodies (Fig. 1G).

We next determined whether ErbB2 colocalized with markers of the clathrin pathway soon after internalization. Most ErbB2-positive puncta did not label for rhodamine-conjugated Tf (Rh-Tf) (Fig. 1, Fig. 2A,E,H). By contrast, internalized EGFR and Tf colocalized extensively with each other (Fig. 2B,H). ErbB2 did not co-localize significantly with clathrin (Fig. 2C,F), while EGFR did (Fig. 2D,G). ErbB2-positive structures often had a distinctive morphology, captured most clearly in favorable epifluorescence images (Fig. 2A,E): either short tubules, or round structures with visible lumens. This image also showed that ErbB2-positive structures remained closer to the plasma membrane than Rh-Tf-positive structures after 5 minutes of internalization. These results showed that ErbB2 did not colocalize with markers of the clathrin-dependent internalization pathway soon after internalization in GA-treated cells, and suggested that it was internalized by a different mechanism.

Fig. 2.

Fig. 2

Open in a new tab

Localization of internalized ErbB2, Rh-Tf, EGFR, and clathrin. SKBr3 cells were pre-treated with GA for 1 hour before binding Fl-anti-ErbB2 (A,C,E,F) or Fl-anti-EGFR (B,D,G) and warming for 5 minutes, with Rh-Tf in A,B,E, acid washing, and IF. (C,D) Clathrin heavy chain (CHC) was detected by IF. (E–G) high-magnification views of boxed regions in A,C,D respectively. Right-hand panels in A–D and bottom panels in E–G show merged images. (A) epifluorescence images; all other panels show maximum intensity projections of deconvolved Z-stacks. Scale bars; 10 µm. Bar in B applies to panels B–D. (H) Quantitation of colocalization of Rh-Tf with ErbB2 or EGFR, in cells treated as in A,B, except that internalization was for 2 minutes.

Eps15 associates with the clathrin coat, and is required for clathrin-mediated uptake (Conner and Schmid, 2003). Dynamins mediate endocytic vesicle scission and are required for both clathrin-mediated and caveolar endocytosis (Conner and Schmid, 2003). Dominant-negative forms of Eps15 and dynamin-1 had similar effects in GA-treated SKBr3 cells: Rh-Tf uptake was inhibited, while ErbB2 internalization was still detected (Fig. 3A,B). Results were quantitated (Fig. 3C,D) by counting cells scored positive or negative for internalization of ErbB2 and Tf.

Fig. 3.

Fig. 3

Open in a new tab

Dominant-negative forms of Eps15 and dynamin inhibit internalization of Rh-Tf but not ErbB2. SKBr3 cells transfected with EGFP-DN-Eps15 (A,C) or HA-DN-dynamin-1 (B,D) were pretreated with GA for 2 hours before binding unlabeled anti-ErbB2 antibodies (A,C) or Fl-anti-ErbB2 antibodies (B,D), warmed for 30 minutes with Rh-Tf, fixed and permeabilized. (A,B) EGFP-DN-Eps15 (A, green) or HA-DN-dynamin (B, blue) are shown with Rh-TF and ErbB2 in deconvolved images, each from a Z-stack of a field in which one cell expressed DN-Eps15 (A) or DN-dynamin (B). ErbB2 was detected with AF-350 goat anti-mouse antibodies (A,C; blue) or by fluorescein fluorescence (B,D; green). Scale bar; 10 µm. (C,D) ErbB2 and Rh-Tf internalization in cells expressing EGFP-DN-Eps15 (C) or DN-dynamin (D), and untransfected cells on the same coverslips. Cells showing at least three intracellular puncta were scored positive. Numbers shown are averages of two experiments (counting at least 100 transfected and 100 untransfected cells in each experiment) that varied by <10%.

Together, these results showed that ErbB2 was internalized primarily by a clathrin-independent mechanism in GA-treated SKBr3 cells. We next compared ErbB2 internalization to other clathrin-independent endocytic pathways.

ErbB2 internalization does not require tyrosine kinase activity

As SKBr3 cells do not express caveolin-1 and lack caveolae (Hommelgaard et al., 2004), ErbB2 cannot be internalized by caveolar endocytosis in these cells. However, a “caveolar-like” pathway, followed by cargoes normally internalized in caveolae, can exist in cells that lack caveolae. Endocytosis by this pathway is inhibited by the tyrosine kinase inhibitor genistein (Damm et al., 2005; Sharma et al., 2004). We next determined whether ErbB2 internalization was sensitive to genistein. As a positive control, we verified that genistein inhibited EGF-induced stimulation of tyrosine kinase activity and internalization of tyrosine-phosphorylated substrates. SKBr3 cells express EGFR, though at lower levels than ErbB2 (Beerli et al., 1995). In untreated serum-starved cells (D. J. Barr, unpublished) and in cells treated with GA alone (Fig. 4A, middle), anti-phosphotyrosine staining was usually dim and largely restricted to the plasma membrane. However, after EGF treatment, internal puncta stained brightly with anti-phosphotyrosine antibodies (Fig. 4B, middle). As reported (Haslekås et al., 2005; Wang et al., 1999), EGF did not alter the distribution of ErbB2 (Fig. 4B, top). As expected, both ErbB2 and tyrosine-phosphorylated substrates were internalized in cells treated with EGF and GA together (Fig. 4C). Genistein blocked tyrosine phosphorylation in cells treated with EGF and GA, but ErbB2 internalization remained robust (Fig. 4D). CELISA analysis showed that genistein did not inhibit, and in fact slightly stimulated, ErbB2 internalization (Fig. 4E). This is consistent with a report that ErbB2 internalization in GA-treated cells does not require its tyrosine kinase activity (Xu et al., 2001), and also shows that no other tyrosine kinase is required.

Fig. 4.

Fig. 4

Open in a new tab

Genistein inhibits EGF-stimulated tyrosine kinase activity but not ErbB2 internalization. SKBr3 cells were serum-starved overnight, treated as described for individual panels, fixed, and permeabilized. Cells were treated with: (A) GA for 2 hours; (B) 100 ng/ml EGF for 10 minutes; (C) GA for 2 hours, with 100 ng/ml EGF added for the last 10 minutes; and (D) 100 µg/ml genistein for 1 hour, then GA added for another 2 hours, and 100 ng/ml EGF added for the last 10 minutes. Deconvolved images from Z-stacks are shown. ErbB2 (green, top) and P-Tyr (red, middle) were detected by IF. Bottom; merged images. Scale bar; 10 µm. (E) SKBr3 cells were pre-treated with GA (circles) or GA + 100 µg/ml genistein (squares) for 45 minutes before binding biotinylated anti-ErbB2 antibodies and warming for 0–5 minutes. Internalized antibodies were quantitated by CELISA. Values shown are the mean +/− s.e.m. of 3 experiments.

ErbB2 colocalizes with AF-594-CTxB, GPI-anchored proteins, and a fluid phase marker immediately after internalization

To determine whether ErbB2 was internalized by a GEEC/CLIC-like pathway (Kalia et al., 2006; Kirkham and Parton, 2005; Sabharanjak et al., 2002), we examined markers of those pathways. After 5 minutes of uptake in GA-treated SKBr3 cells, internalized ErbB2 colocalized extensively with co-internalized AF-594-CTxB, GPI-anchored placental alkaline phosphatase (PLAP), and the fluid phase marker FluoroRuby dextran (Fig. 5A–D). Quantitation of the colocalization of ErbB2 with CTxB, PLAP, and Thy1 (another GPI-anchored protein) is shown in Fig. 5E. Slightly higher colocalization of ErbB2 with Thy1 than with PLAP may result from more efficient acid stripping of anti-Thy1 than anti-PLAP antibodies from the cell surface.

Fig. 5.

Fig. 5

Open in a new tab

Internalized ErbB2 colocalizes with AF-594-CTxB, GPI-anchored proteins, and dextran in GA-treated SKBr3 cells. Cells were pre-treated with GA 1 hour, subjected to antibody and/or toxin binding, warmed for 5 minutes, acid-stripped, and fixed. (A) Fl-anti-ErbB2 and AF-594-CTxB (0.5 µg/ml) were bound to cells. A merged maximum intensity projection image of a deconvolved Z-stack is shown (ErbB2, green; AF-594-CTxB, red). Asterisks indicate region shown enlarged in B (ErbB2, left; AF-594-CTxB, middle; merged image, right). (C) Fl-anti-PLAP Fab fragments and Rh-anti-ErbB2 antibodies were bound to cells. A deconvolved image from a Z-stack, showing part of the edge of one cell, is shown. ErbB2, left; PLAP, middle; merged image, right. (D) Fl-anti-ErbB2 was bound to cells, which were warmed with 1 mg/ml FluoroRuby dextran. An epifluorescence image, showing part of the edge of one cell, is shown. ErbB2, left; dextran, middle; merged image, right. Scale bars; A, 10 µm; D (applies to B–D), 5 µm. (E) Colocalization of ErbB2 and CTxB, or ErbB2 and PLAP, in cells treated as in A–C (except that internalization was for 2 minutes) was quantitated. To measure colocalization of ErbB2 and Thy1.1, SKBr3 cells transfected with Thy1.1 were treated with GA 1 hour. Fl-anti-ErbB2 and AF594-anti-Thy1 Fab fragments were bound on ice, and cells warmed for 2 minutes. Residual surface-bound antibodies were acid-stripped before fixation, visualization, and quantitation.

These observations suggest that ErbB2 is internalized by a pathway similar to the GEEC pathway (Kalia et al., 2006; Kirkham et al., 2005; Sabharanjak et al., 2002). Also in common with the GEEC pathway, the PI(3) kinase inhibitor LY294002 did not inhibit ErbB2 internalization (Fig. S1 in supplementary material).

GA does not affect ErbB2 internalization

We next wanted to determine whether ErbB2 was internalized by the same pathway in the absence of GA. ErbB2 internalized without GA for 2 or 5 minutes was indistinguishable from that internalized in drug-treated cells (D. J. Barr, unpublished). As in GA-treated cells (Fig. 5), newly-internalized ErbB2 colocalized with PLAP, CTxB, and dextran in cells that were not treated with the drug (Fig. 6A–D). Furthermore, in agreement with a previous report (Austin et al., 2004), the CELISA assay showed that GA did not affect the rate of ErbB2 internalization (Fig. 6E). We conclude that GA does not affect the rate of ErbB2 internalization or the endocytic pathway followed.

Fig. 6.

Fig. 6

Open in a new tab

Internalized ErbB2 colocalizes with CTxB, GPI-anchored proteins, and dextran in SKBr3 cells without GA. (A–D) Cells were subjected to antibody and/or toxin binding on ice for 1 hour, warmed for 2 minutes, acid-stripped, and fixed. (A) Fl-anti-ErbB2 and AF-594-CTxB (0.5 µg/ml) were bound to cells. (B) Fl-anti-PLAP Fab fragments and Rh-anti-ErbB2 antibodies were bound. (C) Fl-anti-ErbB2 was bound to cells, which were warmed with 1 mg/ml FluoroRuby dextran. (D) Fl-anti-ErbB2 and Rh-Tf were bound. (A–D) Deconvolved images from Z-stacks are shown. ErbB2, left; CTxB, PLAP, dextran, or Tf, middle; merged images, right. Scale bar; 10 µm. (E) Internalization of biotinylated anti-ErbB2 antibodies was measured by CELISA in cells treated with (squares) or without (circles) GA. Values shown are the mean +/− s.e.m. of 3 experiments.

Internalization of ErbB2 and fluid in SKBr3 cells does not require Rho-family GTPase activity

We used C. difficile toxin B, a Rho family GTPase inhibitor (Jank et al., 2007), to determine whether a member of this family regulated fluid-phase uptake or ErbB2 endocytosis in SKBr3 cells. Growth factors stimulate macropinocytosis and fluid-phase uptake through activation of Rac (Bryant et al., 2007; Ridley and al, 1992; Schnatwinkel et al., 2004). To avoid possible confounding effects of growth-factor-stimulated macropinocytosis, we performed these studies in serum-starved cells. Newly-internalized ErbB2 colocalized with fluid, PLAP, and CTxB in serum-starved cells, showing that serum starvation did not alter ErbB2’s internalization pathway (Fig. S2 in supplementary material).

As expected, C. difficile toxin B completely abolished stress fiber formation, a process that requires RhoA function (Pellegrin and Mellor, 2007) (Fig. 7A). The toxin also drastically altered cell morphology, presumably by inhibiting Rho family proteins that regulate the actin cytoskeleton (Fig. 7B,C). (Cell-surface ErbB2 was visualized in these cells after dextran internalization, serving as a marker of cell shape.) Nevertheless, the toxin did not greatly affect internalization of ErbB2 or a fluid-phase marker, as judged by fluorescence microscopy and biochemical internalization assays (Fig. 7B–E). As expected from this result, dominant-negative forms of cdc42 and RhoA did not block ErbB2 internalization (Fig. S3 in supplementary material). We conclude that Rho family proteins are not required for ErbB2 internalization in SKBr3 cells. This property distinguishes the internalization pathway followed by ErbB2, fluid, and GPI-anchored proteins in these cells from the GEEC pathway described in CHO cells (Sabharanjak et al., 2002).

Fig. 7.

Fig. 7

Open in a new tab

C. difficile toxin B does not inhibit internalization of ErbB2 or fluid in SKBr3 cells. (A) SKBr3 cells grown for 48 hours on poly-Lys-coated coverslips were left untreated (Con) or treated 2 hours with 0.5 µg/ml C. difficile toxin B (Tox), fixed, permeabilized and incubated with rhodamine phalloidin (4 U/ml). Stress fibers were seen in about half of the control cells and <1% of treated cells. (B,C) Serum-starved SKBr3 cells on poly-Lys-coated coverslips were left untreated (B) or treated 2 hours with 0.5 µg/ml C. difficile toxin B (C) before addition of FluoroRuby dextran (1 mg/ml) for 10 minutes. After fixation, surface morphology was visualized with anti-ErbB2 antibodies and green secondary antibodies. Scale bar; 10 µm. (D,E) Internalization of biotinylated anti-ErbB2 antibodies (D) or biotinylated BSA (E) was measured by CELISA in serum-starved SKBr3 cells treated with GA (D, circles), GA + C. difficile toxin B (D, squares), C. difficile toxin alone (E, squares), or left untreated (E, circles) as described in Methods. Where appropriate, cells were pre-incubated with C. difficile toxin B (0.5 µg/ml) for 2 hours at 37°C with addition of GA for the last 45 minutes. Values shown are the mean +/− s.e.m. of 3 experiments.

ErbB2 is transported to early and late endosomes and is degraded in lysosomes in GA-treated cells

ErbB2 colocalized significantly with Rh-Tf at long internalization times (Fig. 8A). This suggested that ErbB2 merged with the classical endocytic pathway following internalization. Consistently, and in agreement with earlier work (Austin et al., 2004), after 2 hours GA treatment, ErbB2 colocalized with the early endosome markers EEA1 and Rab5 (Fig. 8B,C), and accumulated inside enlarged endosomes present in cells expressing constitutively active Rab5Q79L (Stenmark et al., 1994) (Fig. 8D). ErbB2 was sometimes detected inside structures surrounded with EEA1 in an irregular form (Fig. 8B). These structures are probably the same as immature CD63-negative MVBs, surrounded by Tf-positive tubules, in which ErbB2 was detected in the interior vesicles by electron microscopy after 3 hours GA treatment (Austin et al., 2004). The irregular appearance of EEA1 may reflect its localization in these tubules. After prolonged GA treatment, ErbB2 also colocalized with the late endosomal markers GFP-Rab7 and CD63 (Fig. 9). Austin et al. reported little colocalization of ErbB2 with late endosome markers after 3 hours GA treatment (Austin et al., 2004). This might have resulted from rapid degradation following delivery to lysosomes. As expected, colocalization of ErbB2 with LAMP1 was enhanced when lysosomal proteases were inhibited with leupeptin (Fig. 9D).

Fig. 8.

Fig. 8

Open in a new tab

ErbB2 is delivered to early endosomes after GA treatment. SKBr3 cells were left untransfected (A,B) or transiently transfected with GFP-Rab5 (C) or GFP-Rab5Q79L (D), and then treated with GA for 2 hours (with Rh-Tf added for the last 30 minutes in A), fixed, and permeabilized. (A–D) Left panels; ErbB2, detected with polyclonal antibodies. Center panels: (A) Rh-Tf fluorescence; (B) endogenous EEA1; (C,D) GFP fluorescence. Right panels; merged images. (A) deconvolved image from a Z stack; (B–D) epifluorescence images. Scale bars; A, 10 µm; D (applies to B–D), 5 µm.

Fig. 9.

Fig. 9

Open in a new tab

ErbB2 is delivered to late endosomes and lysosomes after GA treatment. After transient expression of GFP-Rab7 (A only), SKBr3 cells were treated with GA for 5 hours (together with 0.1 mg/ml leupeptin, D only), fixed, and permeabilized. Left panels; ErbB2 was detected with polyclonal (A,B) or monoclonal (C,D) antibodies and appropriate secondary antibodies. Middle panels: (A) Rab7 (GFP fluorescence); (B) endogenous CD63; (C,D) endogenous LAMP1. Right panels; merged images. Epifluorescence images are shown. Scale bar; 5 µm.

Lysosomotropic amines like chloroquine raise the pH of endosomes and lysosomes and induce their swelling. Some groups have found that these compounds inhibit transport from early to late endosomes (Gu and Gruenberg, 2000), while others have found no such effect (Vonderheit and Helenius, 2005). ErbB2 colocalized with CD63 and LAMP1 in cells treated cells GA and chloroquine (Fig. S4 in supplementary material), showing that transport of ErbB2 to late endosomes and lysosomes did not require acidic luminal pH in these organelles. Together, these results showed that after internalization by a clathrin-independent pathway, ErbB2 is transported to early and late endosomes and then to lysosomes for degradation.

To test this possibility further, we examined ErbB2 degradation in GA-treated SKBr3 cells by Western blotting. Consistent with an earlier report (Tikhomirov and Carpenter, 2000), a fragment of about 135 kDa that reacted with extracellular domain-specific anti-ErbB2 antibodies accumulated in lysates of cells treated with GA and chloroquine (Fig. 10A, arrow). Full-length ErbB2 also appeared to be stabilized under these conditions. Including both full-length and 135 kDa forms of the protein in the quantitation, we found that chloroquine significantly slowed ErbB2 degradation (Fig. 8B). Thus, consistent with earlier results (Lerdrup et al., 2006; Tikhomirov and Carpenter, 2000), at least a major fraction of ErbB2 is degraded in lysosomes.

Fig. 10.

Fig. 10

Open in a new tab

GA-induced ErbB2 degradation is sensitive to chloroquine. SKBr3 cells were incubated with GA with or without CQ for the times indicated and lysed. Proteins were separated by SDS-PAGE and transferred to membranes for Western blotting and detection of ErbB2. (A) Western blots. Top, GA alone; bottom, GA and chloroquine (CQ). Arrow; ca. 135 kDa ErbB2 fragment. (B) Bands were quantitated by scanning densitometry and plotted as % of 0-time signal remaining at each time.

ErbB2 accumulates in vesicles containing constitutively-active Arf6-Q67L only in the absence of GA

A non-clathrin-mediated endocytic pathway regulated by Arf6 has been described (Naslavsky et al., 2003; Naslavsky et al., 2004). Constitutively-active Arf6Q67L causes cargo of this pathway to accumulate in Arf6Q67L-positive endosomes, which are often enlarged. To determine whether ErbB2 followed this pathway, we expressed HA-tagged Arf6-Q67L in SKBr3 cells, treated cells with GA, and visualized Arf6-Q67L and ErbB2. ErbB2 did not accumulate in Arf6-Q67L-positive endosomes (Fig. 11A). Instead, ErbB2 partially colocalized with GFP-Rab5 and GFP-Rab7, showing that it reached early and late endosomes even in the presence of Arf6-Q67L (Fig. 11B,C). By contrast, ErbB2 accumulated in enlarged Arf6-Q67L-positive endosomes, rather than Rab5- or Rab7-positive endosomes, when cells were not treated with GA (Fig. 11D–F).

Fig. 11.

Fig. 11

Open in a new tab

ErbB2 accumulates in Arf-Q67L-positive endosomes only without GA. (A–F) SKBr3 cells were transfected with Arf6-Q67L, alone (A,D) or with GFP-Rab5 (B,E) or GFP-Rab7 (C,F). Fl-anti-ErbB2 (A,D) or unlabeled anti-ErbB2 antibodies (B,C,E,F) were bound for 1 hour before cells were warmed for 2 hours with (A–C) or without (D–F) GA. Internalized anti-ErbB2 was detected in fixed and permeabilized cells by Fl-anti-ErbB2 fluorescence (A,D) or with Texas red goat-anti-mouse antibodies (B,C,E,F). Although Arf6Q67L was not visualized in B,C,E, or F, vacuoles characteristic of Arf6-Q67L expression were seen. Scale bar; 10 µm. (G,H) COS-7 cells were transfected with ErbB2 alone or together with Arf6-Q67L as indicated. Cells were incubated with GA for the indicated times and solubilized in gel loading buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose. ErbB2 and Arf6-Q67L were detected by immunoblotting as described in Methods. (H) A representative Arf6-Q67L blot is shown, demonstrating expression in co-transfected cells (+Arf6-Q67L), but not in cells expressing ErbB2 alone (−Arf6-Q67L).

We wanted to determine whether Arf6-Q67L affected degradation of ErbB2. However, the transfection efficiency of SKBr3 cells was too low for this. An alternate approach was to express ErbB2 in COS-7 cells, either alone or together with Arf6-Q67L. COS-7 cells express some endogenous ErbB2, detectable in our hands by Western blotting but not by IF. Western blotting showed that transfected ErbB2 was at least 5–10 times more abundant than endogenous ErbB2. Furthermore, IF analysis showed that at least 80% of cells in co-transfected dishes that expressed ErbB2 also expressed Arf6-Q67L. Thus, this system was feasible for determining the effect of Arf6-Q67L on ErbB2 degradation.

Before doing this, we characterized ErbB2 internalization and trafficking in transfected GA-treated COS-7 cells (Fig. S5 in supplementary material). After 2 minutes internalization, most ErbB2 did not colocalize with newly-internalized AF-594-Tf (Fig. S5A, top). Instead, ErbB2 colocalized well with internalized AF-594-CTxB, Thy1, and dextran at this time (Fig. S5A, bottom 3 rows). Dominant-negative dynamin inhibited internalization of AF-594-Tf but not ErbB2 (Fig. S5B). After prolonged GA treatment, ErbB2 colocalized with early and late endosome markers (Fig. S5C). We conclude that ErbB2 followed similar endocytic pathways in COS-7 and SKBr3 cells after GA treatment.

Arf6Q67L did not affect ErbB2 degradation in GA-treated co-transfected COS-7 cells (Fig. 11G,H). Together, these results suggest that following ErbB2 internalization, Arf6Q67L inhibited recycling (the normal fate of the protein in the absence of GA), but did not affect degradative transport of ubiquitinated ErbB2 through early and late endosomes to lysosomes in GA-treated cells.

Discussion

GA does not affect ErbB2 internalization

Austin et al. reported that ErbB2 is internalized constitutively, at the same rate with and without GA treatment, and that GA affects ErbB2 trafficking only in endosomes (Austin et al., 2004). They found that in control cells, ErbB2 recycles to the plasma membrane following internalization, while GA induces sorting of ErbB2 into vesicles that bud into the interior of MVBs. Our results agree with these findings. By contrast, another group reported that ErbB2 is resistant to internalization (Hommelgaard et al., 2004), and that GA overcomes this resistance to facilitate endocytosis (Lerdrup et al., 2006; Lerdrup et al., 2007).

The discrepancy between these results may stem methodological differences. van Deurs and colleagues examined cells by IF after 2–4 hours of GA treatment (Hommelgaard et al., 2004; Lerdrup et al., 2006; Lerdrup et al., 2007). Because they saw internal ErbB2-positive puncta in these cells, but not in control cells, they concluded that ErbB2 underwent endocytosis only after GA treatment. However, by examining cells after short internalization times, Austin et al. showed that ErbB2 is endocytosed constitutively (Austin et al., 2004). Nevertheless, because recycling to the surface is efficient, little internalized ErbB2 is seen at steady state. Examining endocytosis at early times may be especially important for proteins like ErbB2, which may be internalized more slowly than cargo of the clathrin pathway (Fig. 2A,E). The balance between internalization and recycling may be tilted more heavily in favor of recycling for ErbB2 than for TfR, making it difficult to see internalized protein at steady state.

ErbB2 is internalized by a clathrin-independent pathway

In contrast to our findings, Austin et al. reported that several clathrin pathway inhibitors affected ErbB2 uptake (Austin et al., 2005). We do not know the explanation for this difference. Austin et al. assayed for inhibition of internalization by binding labeled anti-ErbB2 antibodies to cells, warming for 3 hours with or without inhibitors, and then quantitating internalized antibodies. Effects seen after this prolonged internalization time might have resulted from inhibition of recycling, rather than of initial internalization. In fact, prolonged CPZ treatment caused accumulation of both Tf and ErbB2 in enlarged endosomes, suggesting that recycling was inhibited (D. J. Barr, unpublished).

Why EGFR but not ErbB2 is targeted to clathrin-coated pits

Because EGFR and ErbB2 are very similar, it may seem surprising that ErbB2 is not targeted to clathrin-coated pits. Recent findings on trafficking of EGFR and other ErbB family members can help explain this finding.

EGFR internalization does not require the clathrin adaptor AP2 (Hinrichsen et al., 2003; Motley et al., 2003), but requires Grb2 (Jiang et al., 2003; Wang and Moran, 1996) and c-Cbl or cbl-b (Ettenberg et al., 1999; Levkowitz et al., 1999). c-Cbl binds tyrosine-phosphorylated EGFR both directly, through its SH2 domain (Galisteo et al., 1995; Levkowitz et al., 1999), and indirectly, via Grb2 (Fukazawa et al., 1996; Jiang et al., 2003; Meisner and Czech, 1995), and ubiquitinates EGFR.

c-Cbl binds only to tyrosine-phosphorylated EGFR. By contrast, internalization of ErbB2 in GA-treated cells does not require tyrosine phosphorylation. Furthermore, ErbB family members other than EGFR do not recruit c-Cbl even when activated (Levkowitz et al., 1996; Muthuswamy et al., 1999). Even activated EGFR-ErbB2 heterodimers fail to bind c-Cbl, probably because ErbB2 is unable to phosphorylate the c-Cbl binding site on EGFR (Muthuswamy et al., 1999). This could explain why EGFR is the only ErbB family member that is rapidly down-regulated upon activation (King et al., 1988; Lenferink et al., 1998; Stern et al., 1986; Waterman et al., 1998), and why the cytoplasmic domains of ErbB2-4 are internalization impaired (Baulida et al., 1996; Sorkin et al., 1993; Waterman et al., 1999). As expected, heterodimerization with ErbB2 inhibits EGFR down-regulation following ligand binding (Haslekås et al., 2005; Lenferink et al., 1998; Lidke et al., 2004; Muthuswamy et al., 1999; Wang et al., 1999).

It is not clear how c-Cbl stimulates EGFR internalization. According to one model, ubiquitinated EGFR is recognized by ubiquitin-binding domains of proteins associated with the clathrin coat (de Melker et al., 2004; Fallon et al., 2006; Haglund et al., 2003; Stang et al., 2004). Alternatively, ubiquitination may target EGFR for clathrin-independent internalization (Sigismund et al., 2005). By contrast, other workers have proposed that ubiquitination is not required for EGFR internalization (Duan et al., 2003), and that the essential role of c-Cbl in EGFR internalization is independent of ubiquitination (Huang et al., 2006; Jiang and Sorkin, 2003; Soubeyran et al., 2002). In fact, EGFR Lys-mutants showing 70–80% reduced ligand-stimulated ubiquitination are internalized normally (Huang et al., 2006). A ubiquitin-independent role of c-Cbl in EGFR internalization would probably involve a complex of c-Cbl, CIN85, and endophilin reported to be required for EGFR endocytosis (Soubeyran et al., 2002). In any case, an essential role for c-Cbl in efficient EGFR internalization seems clear. The failure of ErbB2 to bind c-Cbl could explain why it is not targeted to clathrin-coated pits.

If ubiquitination signals EGFR internalization, then ubiquitination of ErbB2 by CHIP could play a similar role - possibly targeting ErbB2 for clathrin-independent internalization, as reported for EGFR (Sigismund et al., 2005). However, in contrast to that report, we found that ErbB2 internalization did not occur via caveolae in SKBr3 cells. Furthermore, the finding that GA did not affect ErbB2’s internalization rate argues against a role for ubiquitination in internalization.

Relation of ErB2 endocytosis to other non-clathrin pathways

ErbB2 internalization in SKBr3 cells did not occur via caveolae, as these cells lack caveolae (Hommelgaard et al., 2004). Furthermore, unlike caveolar endocytosis (Henley et al., 1998) and also unlike a pathway used to internalize the interleukin-2 receptor in lymphocytes (Lamaze et al., 2001), ErbB2 internalization did not require dynamin. ErB2 internalization differed from a “caveolar-like” pathway that operates in cells lacking caveolae (Damm et al., 2005) in not requiring tyrosine kinase activity.

ErbB2 internalization was similar to the GEEC/CLIC pathway (Kalia et al., 2006; Kirkham et al., 2005; Sabharanjak et al., 2002) in not requiring clathrin, dynamin, or caveolae. In addition, newly-internalized ErbB2 colocalized with GPI-anchored proteins, CTxB, and a fluid phase marker. Structures containing newly-internalized ErbB2 often had the distinctive appearance of CLICs. Nevertheless, in SKBr3 cells, internalization of ErbB2 and a fluid-phase marker did not require Rho family GTPase activity. Independence of Rho-family GTPases in this GEEC-like pathway may reflect cell type differences, possibly including the transformed state of SKBr3 cells.

ErbB2 accumulated in Arf6-Q67L-positive endosomes in control cells, in common with cargo of the Arf6-regulated pathway. However, after GA treatment, ErbB2 was transported normally to lysosomes for degradation. Arf6Q67L may prevent internalized ErbB2 from recycling, causing it to accumulate in swollen vacuoles in the absence of GA, but not inhibit ubiquitin-based sorting into MVBs and downstream transport lysosomes. Our finding contrasts with an earlier report that Arf6-Q67L inhibited transport of MHC Class I protein and CD59 to lysosomes for degradation (Naslavsky et al., 2004). The difference may result from the fact that ErbB2, unlike the markers examined in the earlier study, was ubiquitinated.

How ErbB2 might be targeted for internalization

We do not know what targets ErbB2 for internalization by the pathway we have described. A clue may come from the characteristics of the GEEC/CLIC pathway(s) (Kalia et al., 2006; Kirkham et al., 2005; Sabharanjak et al., 2002). Both GPI-anchored proteins and CTxB have a high affinity for lipid rafts, or membrane microdomains in the liquid-ordered phase. ErbB2 can be enriched in detergent-resistant membranes, an indication of high raft affinity (Hommelgaard et al., 2004; Nagy et al., 2002; Yang et al., 2004; Zhou and Carpenter, 2001; Zurita et al., 2004). Thus, a subset of transmembrane proteins with high raft affinity may be targeted to the GEEC/CLIC pathway, and to the GEEC-like pathway followed by ErbB2 in SKBr3 cells.

Downstream trafficking of ErbB2

As reported previously (Austin et al., 2004), ErbB2 was transported to EEA1-positive early endosomes after internalization. The earlier workers also found ErbB2 in vesicles inside MVBs in GA-treated cells, suggesting transport to degradative compartments (Austin et al., 2004). However, they noted that ErbB2-positive MVBs retained immature characteristics such as recycling tubules even after prolonged GA treatment. They were also surprised to see poor colocalization of ErbB2 with the late endosome marker CD63 (Austin et al., 2004). By contrast, we saw good colocalization of ErbB2 with CD63 and LAMP1, especially after treatment with leupeptin or chloroquine. ErbB2 degradation was inhibited by chloroquine when blots were probed with extracellular-domain antibodies, confirming previous work (Tikhomirov and Carpenter, 2000; Tikhomirov and Carpenter, 2001) and suggesting that the apparent chloroquine insensitivity reported initially (Citri et al., 2002; Mimnaugh et al., 1996; Way et al., 2004) resulted from inability of blotting antibodies to detect a clipped form of the protein.

In summary, we showed that after GA treatment, ErbB2 is internalized by a non-clathrin, non-caveolar pathway, and then merges with the classical endocytic pathway for transport to lysosomes and degradation.

Materials and methods

Cells and transfection

SKBr3 human breast cancer cells and COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium with 10% iron-supplemented calf serum (JRH) and penicillin/streptomycin. Cells were transiently transfected with Lipofectamine 2000 (Invitrogen) and examined 1–2 days post-transfection, except for cells expressing Arf6-Q67L, which were examined 14–16 hours post-transfection.

Plasmids

GFP-Eps15 mutant ΔE95/295 (Benmerah et al., 1999) was from A. Benmerah (Institut Cochin, Paris, France). Tetracycline-inducible dominant negative (K44A) HA-dynamin-1 (Damke et al., 1994), co-expressed with pTet-Off (Clontech), was from J. Pessin (Albert Einstein University, Bronx NY). HA-Arf6-Q67L (Hernández-Deviez et al., 2004) in pCB7 (Frank et al., 1998) was from J. Casanova (University of Virginia, Charlottesville, VA). pBC12/PLAP was described (Berger et al., 1987). EGFP-wild type and mutant (Q79L) Rab5 (Volpicelli et al., 2001) were from A. Levey (Emory University, Atlanta GA). GFP Rab7 (Guignot et al., 2004) was from C. Roy (Yale University, New Haven, CT). 3xHA- wild type and dominant-negative RhoA and cdc42 plasmids (T19N and T17N respectively) were from University of Missouri - Rolla cDNA Resource Center (www.cDNA.org). Mouse Thy1.1 (Zhang et al., 1991) was from J. Rose (Yale University, New Haven, CT). pEGFP-2xFYVE (Petiot et al., 2003) was from W. Maltese (University of Toledo, Toledo, OH). Human ErbB2 in pcDNA3 was from L. Neckers (NIH).

Antibodies, fluorescent compounds, and other reagents

Anti-ErbB2 antibodies: for IF, monoclonal antibodies 4D5 (purified from supernatant of hybridoma cells (ATCC) grown in an Integra Biosciences CELLine two-compartment bioreactor, from Microbiology International (Frederick, MD)), or 9G6.10 or N28 (for acid-stripping experiments) from LabVision were used for cell-surface detection. Rabbit polyclonal anti-ErbB2 antibodies (DakoCytomation USA) or monoclonal antibodies l were used on fixed/permeabilized cells. LabVision anti-ErbB2 antibody #20 was used on blots. Other mouse monoclonal antibodies: anti-HA tag from Applied Biological Materials; anti-phosphotyrosine from Upstate Biological; anti-epidermal growth factor receptor (EGFR) #3 from LabVision; anti-EEA1 and anti-Thy1 (OX-7) from BD Biosciences; and anti-CD63 from the Developmental Studies Hybridoma Bank, University of Iowa. Rabbit polyclonal antibodies: anti-clathrin heavy chain (Simpson et al., 1996) from M. S. Robinson (University of Cambridge, Cambridge, UK); anti-HA tag from Santa Cruz Biotechnology; anti-LAMP-1 from Affinity Bioreagents; and anti-PLAP from DakoCytomation USA. Anti-PLAP Fab fragments were prepared using immobilized papain and anti-Thy-1.1 Fab fragments using immobilized ficin (Pierce) using supplier’s instructions. Cleavage was verified by Western blotting. Fluorescein was conjugated to anti-ErbB2 and anti-EGFR antibodies and to anti-PLAP Fab fragments; rhodamine to human transferrin (Tf); AF594 to anti-Thy1 Fab fragments, and biotin to anti-ErbB2 antibodies, Tf, and bovine serum albumin (BSA) using N-hydroxysuccinimide-linked fluorescein, rhodamine AF594, or PEO4-biotin (Pierce), using conditions recommended by the supplier. Dichlorotriazinylaminofluorescein-goat anti-mouse IgG, fluorescein-goat anti-rabbit IgG, Texas red-goat anti-mouse IgG, Texas red-goat anti-rabbit IgG, and horseradish peroxidase-goat anti-mouse IgG were from Jackson Immunoresearch Laboratories. Alexa Fluor (AF)-350-goat anti-mouse and anti-rabbit IgGs, AF-594-cholera toxin B subunit (AF-594-CTxB), AF-680-goat anti-mouse IgG and -goat anti-rabbit IgG used for fluorescent detection of bands on blots, FluoroRuby™ dextran (10,000 MW), and AF-594-Tf and rhodamine-phalloidin were from Molecular Probes, Invitrogen. Where indicated, the AF-488 Zenon Mouse IgG labeling kit (Invitrogen), consisting of fluorescently-tagged Fab fragments of goat antibodies directed against the Fc portion of mouse IgG, was used to detect anti-ErbB2 antibodies. Other reagents: GA (used at 5 µM) was from the Drug Synthesis and Chemistry Branch, National Cancer Institute (Bethesda, MD); EGF from EMD Biosciences; Clostridium difficile toxin B from Tech Lab; LY294002 from Cayman Chemicals; and peroxidase-conjugated streptavidin polymer from Sigma Aldrich or Fitzgerald Industries. Other reagents were from Sigma Aldrich.

Fluorescence microscopy

Fluorescence microscopy was as described (Ostermeyer et al., 2004) except that for detection of LAMP-1, fixed cells were permeabilized with PBS containing 0.5% saponin and then treated for 10 minutes at room temperature with phosphate-buffered saline (PBS; 150 mM NaCl, 20 mM phosphate buffer, pH 7.4) containing 0.1% sodium borohydride and 0.1% saponin. 0.1% saponin was included in all further incubations.

For internalization assays, antibodies (2 µg/ml) were bound to cells at 4°C for 1 hour before starting internalization by addition of pre-warmed media. Residual surface-bound antibodies were stripped with acid (100 mM Gly, 50 mM KCl, 20 mM magnesium acetate pH 2.3), using 3 washes of 3 minutes each. Fluorescent Tf (35 µg/ml) was either bound to cells with antibodies, or included in the media during warming, as indicated.

Images shown were captured and processed by epifluorescence microscopy as described (Ostermeyer et al., 2001; Ostermeyer et al., 2004) or by deconvolution microscopy using a Zeiss Axiovert 200 deconvolution microscope and processing images with Axiovision software (version 4.4). To acquire Z stacks, 25–35 serial images were recorded at 350 nm intervals along the Z-axis using a 63x or 100x oil immersion objective. Out of plane fluorescence was removed by deconvolution using the inverse filter algorithm or a modification of the constrained iterative algorithm. Images shown are representative sections from deconvolved Z- stacks or maximum intensity projections of Z stacks.

Colocalization analysis was performed on images obtained with a Zeiss LSM 5 Pascal confocal laser-scanning microscope using ImageJ (http://rsb.info.nih.gov/ij) and the JaCoP plug-in (Bolte and Cordelières, 2006). Manders coefficients M1 and M2, reflecting channel1/channel2 overlap and channel2/channel1 overlap respectively, are reported. Pilot experiments were performed to determine effective maximum and minimum colocalization values. Maximal M1 and M2 values (colocalization of internalized Fl-anti-ErbB2 and Texas red-goat anti-mouse secondary antibodies) ranged from 0.7–0.8. Minimal values (colocalization of early endosomes visualized with AF-594-Tf internalized for 10 minutes and the Golgi, visualized with anti-GM130 and dichlorotriazinylaminofluorescein-goat anti-mouse IgG) ranged from 0.05–0.20.

Cell-based ELISA (CELISA) to measure ErbB2, Tf, or BSA internalization

SKBr3 cells seeded in 35 mm dishes the day before the assay (3 × 105 cells/dish) were pretreated with GA and/or other drugs for 45 minutes except as noted. Biotinylated anti-ErbB2 antibodies (15 µg/ml) were bound for 2 hours at 4°C. After washing with Hanks’ balanced salt solution (Invitrogen), prewarmed media was added for various times. Internalization was stopped by washing with ice-cold Hanks’ balanced salt solution and transferring dishes to ice. To mask residual surface-bound antibodies, cells were incubated with streptavidin (40 µg/ml) for 1 hour at 4°C. After washing, cells were paraformaldehyde-fixed, permeabilized, and blocked as for IF. Cells were then incubated with peroxidase-conjugated streptavidin polymer (1 µg/ml in PBS with 0.05% Tween 20) for 1 hour at room temperature. After washing, SureBlue Reserve tetramethylbenzidine (TMB) substrate (KPL, Gaithersburg, MD) was added for 10 sec, before stopping the reaction with TMB stop solution and measuring absorbance (450 nm) in a spectrophotometer. Except as noted, background (signal in control dishes left on ice) was subtracted from all values. Assays for biotinylated Tf (75 µg/ml) or BSA (5 mg/ml) uptake were the same except they were not pre-bound, but added to media for internalization. 10-fold excess unlabeled Tf reduced biotinylated-Tf signal to background after a 20-minute uptake (A. G. Ostermeyer-Fay, unpublished).

Other methods

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transfer to polyvinylidene difluoride, Western blotting, and detection by enhanced chemiluminescence were as described (Arreaza et al., 1994). Bands were scanned and quantitated using NIH Image. For Fig. 11G,H, blots were incubated with AF680-secondary antibodies and detected and quantitated with the Odyssey-Infrared Imaging System (LI-COR Biosciences).

Supplementary Material

Fig. S1
Fig. S2
Fig. S3
Fig. S4
Fig. S5
Supp.Fig.Legs

Acknowledgements

We thank A. Benmerah, J. Casanova, A. Levey, W. Maltese, L. Neckers, J. Pessin, J. Rose, and C. Roy for plasmids, M. S. Robinson for anti-clathrin antibodies, and R. Haltiwanger and L. Listenberger for reading the manuscript. This work was supported by grant GM47897 (to D. A. B.) from the National Institutes of Health.

Abbreviations used

AF

Alexa Fluor

BSA

bovine serum albumin

CELISA

cell-based enzyme linked immunosorbent assay

CLIC

clathrin-independent carrier

CPZ

chlorpromazine

CTxB

cholera toxin B subunit

EGFR

epidermal growth factor receptor

Fl-anti-ErbB2

fluorescein-conjugated anti-ErbB2 antibodies

GA

geldanamycin

GEEC

GPI-anchored protein-enriched early endosome

GPI

glycosyl phosphatidylinositol

IF

immunofluorescence microscopy

MVBs

multivesicular bodies

PBS

phosphate-buffered saline

PLAP

placental alkaline phosphatase

Rh-Tf

rhodamine-conjugated Tf

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

Tf

transferrin.

References

  1. Arreaza G, Melkonian KA, LaFevre-Bernt M, Brown DA. Triton X-100-resistant membrane complexes from cultured kidney epithelial cells contain the Src-family protein tyrosine kinase p62yes. J. Biol. Chem. 1994;269:19123–19127. [PubMed] [Google Scholar]
  2. Austin CD, De Mazière AM, Pisacane PI, van Dijk SM, Eigenbrot C, Sliwkowski MX, Klumperman J, Scheller RH. Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin. Mol. Biol. Cell. 2004;15:5268–5282. doi: 10.1091/mbc.E04-07-0591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Austin CD, Wen X, Gazzard L, Nelson C, Scheller RH, Scales SJ. Oxidizing potential of endosomes and lysosomes limits intracellular cleavage of disulfide-based antibody–drug conjugates. Proc. Natl. Acad. Sci. USA. 2005;102:17987–17992. doi: 10.1073/pnas.0509035102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baulida J, Kraus MH, Alimandi M, DiFiore PP, Carpenter G. All ErbB receptors other than the epidermal growth factor receptor are endocytosis impaired. J. Biol. Chem. 1996;271:5251–5257. doi: 10.1074/jbc.271.9.5251. [DOI] [PubMed] [Google Scholar]
  5. Beerli RR, Graus-Porta D, Woods-Cook K, Chen X, Yarden Y, Hynes NE. Neu differentiation factor activation of ErbB-3 and ErbB-4 is cell specific and displays a differential requirement for ErbB-2. Mol. Cell. Biol. 1995;15:6496–6505. doi: 10.1128/mcb.15.12.6496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Benmerah A, Bayrou M, Cerf-Bensussan N, Dautry-Varsat A. Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J. Cell Sci. 1999;112:1303–1311. doi: 10.1242/jcs.112.9.1303. [DOI] [PubMed] [Google Scholar]
  7. Berger J, Howard AD, Gerber L, Cullen BR, Udenfriend S. Expression of active, membrane-bound human placental alkaline phosphatase by transfected simian cells. Proc. Natl. Acad. Sci. USA. 1987;84:4885–4889. doi: 10.1073/pnas.84.14.4885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bolte S, Cordelières FP. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 2006;224:213–232. doi: 10.1111/j.1365-2818.2006.01706.x. [DOI] [PubMed] [Google Scholar]
  9. Bryant DM, Kerr MC, Hammond LA, Joseph SR, Mostov KE, Teasdale RD, Stow JL. EGF induces macropinocytosis and SNX1-modulated recycling of E-cadherin. J. Cell Sci. 2007;120:1818–1828. doi: 10.1242/jcs.000653. [DOI] [PubMed] [Google Scholar]
  10. Chadda R, Howes MT, Plowman SJ, Hancock JF, Parton RG, Mayor S. Cholesterol-sensitive Cdc42 activation regulates actin polymerization for endocytosis via the GEEC pathway. Traffic. 2007;8:702–717. doi: 10.1111/j.1600-0854.2007.00565.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cheng ZJ, Singh RD, Sharma DK, Holicky EL, Hanada K, Marks DL, Pagano RE. Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements. Mol. Biol. Cell. 2006;17:3197–3210. doi: 10.1091/mbc.E05-12-1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Citri A, Alroy I, Lavi S, Rubin C, Xu W, Grammatikakis N, Patterson C, Neckers L, Fry DW, Yarden Y. Drug-induced ubiquitylation and degradation of ErbB receptor tyrosine kinases: implications for cancer therapy. EMBO J. 2002;21:2407–2417. doi: 10.1093/emboj/21.10.2407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Citri A, Kochupurakkal BS, Yarden Y. The Achilles heel of ErbB-2/HER2: regulation by the Hsp90 chaperone machine and potential for pharmacological intervention. Cell Cycle. 2004;3:51–60. [PubMed] [Google Scholar]
  14. Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature. 2003;422:37–44. doi: 10.1038/nature01451. [DOI] [PubMed] [Google Scholar]
  15. Damke H, Baba T, Warnock DE, Schmid SL. Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J. Cell Biol. 1994;127:915–934. doi: 10.1083/jcb.127.4.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Damm EM, Pelkmans L, Kartenbeck J, Mezzacasa A, Kurzchalia T, Helenius A. Clathrin- and caveolin-1–independent endocytosis: entry of simian virus 40 into cells devoid of caveolae. J. Cell Biol. 2005;168:477–488. doi: 10.1083/jcb.200407113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. de Melker AA, van der Horst G, Borst J. Ubiquitin ligase activity of c-Cbl guides the epidermal growth factor receptor into clathrin-coated pits by two distinct modes of Eps15 recruitment. J. Biol. Chem. 2004;279:55465–55473. doi: 10.1074/jbc.M409765200. [DOI] [PubMed] [Google Scholar]
  18. Duan L, Miura Y, Dimri M, Majumder B, Dodge IL, Reddi AL, Ghosh A, Fernandes N, Zhou P, Mullane-Robinson K, et al. Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis. J. Biol. Chem. 2003;278:28950–28960. doi: 10.1074/jbc.M304474200. [DOI] [PubMed] [Google Scholar]
  19. Ettenberg SA, Keane MM, Nau MM, Frankel M, Wang LM, Pierce JH, Lipkowitz S. cbl-b inhibits epidermal growth factor receptor signaling. Oncogene. 1999;18:1855–1866. doi: 10.1038/sj.onc.1202499. [DOI] [PubMed] [Google Scholar]
  20. Fallon L, Bélanger CML, Corera AT, Kontogiannea M, Regan-Klapisz E, Moreau F, Voortman J, Haber M, Rouleau G, Thorarinsdottir T, et al. A regulated interaction with the UIM protein Eps15 implicates parkin in EGF receptor trafficking and PI(3)K–Akt signalling. Nat. Cell Biol. 2006;8:834–842. doi: 10.1038/ncb1441. [DOI] [PubMed] [Google Scholar]
  21. Frank SR, Hatfield JC, Casanova JE. Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP-ribosylation factor nucleotide exchange factor ARNO. Mol. Biol. Cell. 1998;9:3133–3146. doi: 10.1091/mbc.9.11.3133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fukazawa T, Miyake S, Band V, Band H. Tyrosine phosphorylation of Cbl upon epidermal growth factor (EGF) stimulation and its association with EGF receptor and downstream signaling proteins. J. Biol. Chem. 1996;271:14554–14559. doi: 10.1074/jbc.271.24.14554. [DOI] [PubMed] [Google Scholar]
  23. Galisteo ML, Dikic I, Batzer AG, Langdon WY, Schlessinger J. Tyrosine phosphorylation of the c-cbl proto-oncogene protein product and association with epidermal growth factor (EGF) receptor upon EGF stimulation. J. Biol. Chem. 1995;270:20242–20245. doi: 10.1074/jbc.270.35.20242. [DOI] [PubMed] [Google Scholar]
  24. Garrett WS, Chen LM, Kroschewski R, Ebersold M, Turley S, Trombetta S, Galán JE, Mellman I. Developmental control of endocytosis in dendritic cells by cdc42. Cell. 2000;102:325–334. doi: 10.1016/s0092-8674(00)00038-6. [DOI] [PubMed] [Google Scholar]
  25. Graus-Porta D, Beerli RR, Daly JM, Hynes NE. ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J. 1997;16:1647–1655. doi: 10.1093/emboj/16.7.1647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gu F, Gruenberg J. ARF1 regulates pH-dependent COP functions in the early endocytic pathway. J. Biol. Chem. 2000;275:8154–8160. doi: 10.1074/jbc.275.11.8154. [DOI] [PubMed] [Google Scholar]
  27. Guignot J, Caron E, Beuzon C, Bucci C, Kagan J, Roy C, Holden DW. Microtubule motors control membrane dynamics of Salmonella-containing vacuoles. J. Cell Sci. 2004;117:1033–1045. doi: 10.1242/jcs.00949. [DOI] [PubMed] [Google Scholar]
  28. Haglund K, Sigismund S, Polo S, Szymkiewicz I, Di Fiore PP, Dikic I. Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation. Nat. Cell Biol. 2003;5:461–466. doi: 10.1038/ncb983. [DOI] [PubMed] [Google Scholar]
  29. Haslekås C, Breen K, Pedersen KW, Johannessen LE, Stang E, Madshus IH. The inhibitory effect of ErbB2 on epidermal growth factor-induced formation of clathrin-coated pits correlates with retention of epidermal growth factor receptor-ErbB2 oligomeric complexes at the plasma membrane. Mol. Biol. Cell. 2005:5832–5842. doi: 10.1091/mbc.E05-05-0456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Henley JR, Kruegger EWA, Oswald BJ, McNiven MA. Dynamin-mediated internalization of caveolae. J. Cell Biol. 1998;141:85–99. doi: 10.1083/jcb.141.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hernández-Deviez DJ, Roth MG, Casanova JE, Wilson JM. ARNO and ARF6 regulate axonal elongation and branching through downstream activation of phosphatidylinositol 4-phosphate 5-kinase alpha. Mol. Biol. Cell. 2004;15:111–120. doi: 10.1091/mbc.E03-06-0410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hinrichsen L, Harborth J, Andrees L, Weber K, Ungewickell EJ. Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells. J. Biol. Chem. 2003;278:45160–45170. doi: 10.1074/jbc.M307290200. [DOI] [PubMed] [Google Scholar]
  33. Hohfeld J, Cyr DM, Patterson C. From the cradle to the grave: molecular chaperones that may choose between folding and degradation. EMBO Rep. 2001;2:885–890. doi: 10.1093/embo-reports/kve206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Hommelgaard AM, Lerdrup M, van Deurs B. Association with membrane protrusions makes ErbB2 an internalization-resistant receptor. Mol. Biol. Cell. 2004;15:1557–1567. doi: 10.1091/mbc.E03-08-0596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Huang F, Kirkpatrick D, Jiang X, Gygi S, Sorkin A. Differential regulation of EGF receptor internalization and degradation by multiubiquitination within the kinase domain. Mol. Cell. 2006;21:737–748. doi: 10.1016/j.molcel.2006.02.018. [DOI] [PubMed] [Google Scholar]
  36. Jank T, Giesemann T, Astories K. Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function. Glycobiology. 2007;17:15R–22R. doi: 10.1093/glycob/cwm004. [DOI] [PubMed] [Google Scholar]
  37. Jiang X, Huang F, Marusyk A, Sorkin A. Grb2 regulates internalization of EGF receptors through clathrin-coated pits. Mol. Biol. Cell. 2003;14:858–870. doi: 10.1091/mbc.E02-08-0532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Jiang X, Sorkin A. Epidermal growth factor receptor internalization through clathrin-coated pits requires Cbl RING finger and proline-rich domains but not receptor polyubiquitylation. Traffic. 2003;4:529–543. doi: 10.1034/j.1600-0854.2003.t01-1-00109.x. [DOI] [PubMed] [Google Scholar]
  39. Kalia M, Kumari S, Chadda R, Hill MM, Parton RG, Mayor S. Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3′-kinase–dependent machinery. Mol. Biol. Cell. 2006;17:3689–3704. doi: 10.1091/mbc.E05-10-0980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. King CR, Borrello I, Bellot F, Comoglio P, Schlessinger J. EGF binding to its receptor triggers a rapid tyrosine phosphorylation of the ErbB-2 protein in the mammary tumor cell line SK-BR-3. EMBO J. 1988;7:1647–1651. doi: 10.1002/j.1460-2075.1988.tb02991.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Kirkham M, Fujita A, Chadda R, Nixon SJ, Kurzchalia TV, Sharma DK, Pagano RE, Hancock JF, Mayor S, Parton RG. Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles. J. Cell Biol. 2005;168:465–476. doi: 10.1083/jcb.200407078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Kirkham M, Parton RG. Clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers. Biochim. Biophys. Acta. (Mol. Cell Res.) 2005;1745:273–286. doi: 10.1016/j.bbamcr.2005.06.002. [DOI] [PubMed] [Google Scholar]
  43. Kumari S, Mayor S. ARF1 is directly involved in dynamin-independent endocytosis. Nat. Cell Biol. 2008;10:30–41. doi: 10.1038/ncb1666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Lamaze C, Dujeancourt A, Baba T, Lo CG, Benmerah A, Dautry-Varsat A. Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway. Mol. Cell. 2001;7:661–671. doi: 10.1016/s1097-2765(01)00212-x. [DOI] [PubMed] [Google Scholar]
  45. Lange Y, Steck TL. Mechanism of red blood cell acanthocytosis and echinocytosis in vivo. J. Membr. Biol. 1984;77:153–159. doi: 10.1007/BF01925863. [DOI] [PubMed] [Google Scholar]
  46. Lencer WI, Saslowsky D. Raft trafficking of AB(5) subunit bacterial toxins. Biochim. Biophys. Acta. (Mol. Cell Res.) 2005;1746:314–321. doi: 10.1016/j.bbamcr.2005.07.007. [DOI] [PubMed] [Google Scholar]
  47. Lenferink AEG, Pinkas-Kramarski R, van de Poll MLM, van Vugt MJH, Klapper LN, Tzahar E, Waterman H, Sela M, van Zoelen EJJ, Yarden Y. Differential endocytic routing of homo- and hetero-dimeric ErbB tyrosine kinases confers signaling superiority to receptor heterodimers. EMBO J. 1998;17:3385–3397. doi: 10.1093/emboj/17.12.3385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Lerdrup M, Hommelgaard AM, Grandal M, van Deurs B. Geldanamycin stimulates internalization of ErbB2 in a proteasome-dependent way. J. Cell Sci. 2006;119:85–95. doi: 10.1242/jcs.02707. [DOI] [PubMed] [Google Scholar]
  49. Lerdrup M, Bruun S, Grandal MV, Roepstorff K, Kristensen MM, Hommelgaard AM, van Deurs B. Endocytic down-regulation of ErbB2 is stimulated by cleavage of its C-terminus. Mol. Biol. Cell. 2007;18:3656–3666. doi: 10.1091/mbc.E07-01-0025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Levkowitz G, Klapper LN, Tzahar E, Freywald A, Sela M, Yarden Y. Coupling of the c-Cbl protooncogene product to Erb-1/EGF-receptor but not to other ErbB proteins. Oncogene. 1996;12:1117–1125. [PubMed] [Google Scholar]
  51. Levkowitz G, Waterman H, Ettenberg SA, Katz M, Tsygankov AY, Alroy I, Lavi S, Iwai K, Reiss Y, Ciechanover A, et al. Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1. Mol. Cell. 1999;4:1029–1040. doi: 10.1016/s1097-2765(00)80231-2. [DOI] [PubMed] [Google Scholar]
  52. Lidke DS, Nagy P, Heintzmann R, Arndt-Jovin DJ, Post JN, Grecco HE, Jares-Erijman EA, Jovin TM. Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction. Nat. Biotechnol. 2004;22:198–203. doi: 10.1038/nbt929. [DOI] [PubMed] [Google Scholar]
  53. Mayor S, Pagano RE. Pathways of clathrin-independent endocytosis. Nat. Rev. Mol. Cell Biol. 2007;8:603–612. doi: 10.1038/nrm2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Meisner H, Czech MP. Coupling of the proto-oncogene product c-Cbl to the epidermal growth factor receptor. J. Biol. Chem. 1995;270:25332–25335. doi: 10.1074/jbc.270.43.25332. [DOI] [PubMed] [Google Scholar]
  55. Mimnaugh EG, Chavany C, Neckers L. Polyubiquitination and proteasomal degradation of the p185c-ErbB-2 receptor protein-tyrosine kinase induced by geldanamycin. J. Biol. Chem. 1996;271:22796–22801. doi: 10.1074/jbc.271.37.22796. [DOI] [PubMed] [Google Scholar]
  56. Motley A, Bright NA, Seaman MNJ, Robinson MS. Clathrin-mediated endocytosis in AP-2–depleted cells. J. Cell Biol. 2003;162:909–918. doi: 10.1083/jcb.200305145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Muthuswamy SK, Gilman M, Brugge JS. Controlled dimerization of ErbB receptors provides evidence for differential signaling by homo- and heterodimers. Mol. Cell. Biol. 1999;19:6845–6857. doi: 10.1128/mcb.19.10.6845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Nagy P, Vereb G, Sebestyén Z, Horváth G, Lockett SJ, Damjanovich S, Park JW, Jovin TM, Szöllosi J. Lipid rafts and the local density of ErbB proteins influence the biological role of homo- and heteroassociations of ErbB2. J. Cell Sci. 2002;115:4251–4262. doi: 10.1242/jcs.00118. [DOI] [PubMed] [Google Scholar]
  59. Naslavsky N, Weigert R, Donaldson JG. Convergence of non-clathrin- and clathrin-derived endosomes involves Arf6 inactivation and changes in phosphoinositides. Mol. Biol. Cell. 2003;14:417–431. doi: 10.1091/mbc.02-04-0053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Naslavsky N, Weigert R, Donaldson JG. Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements. Mol. Biol. Cell. 2004;15:3542–3552. doi: 10.1091/mbc.E04-02-0151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Ostermeyer AG, Paci JM, Zeng Y, Lublin DM, Munro S, Brown DA. Accumulation of caveolin in the endoplasmic reticulum redirects the protein to lipid storage droplets. J. Cell Biol. 2001;152:1071–1078. doi: 10.1083/jcb.152.5.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Ostermeyer AG, Ramcharan LT, Zeng Y, Lublin DM, Brown DA. Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets. J. Cell Biol. 2004;164:69–78. doi: 10.1083/jcb.200303037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Pellegrin S, Mellor H. Actin stress fibres. J. Cell Sci. 2007;120:3491–3499. doi: 10.1242/jcs.018473. [DOI] [PubMed] [Google Scholar]
  64. Petiot A, Faure J, Stenmark H, Gruenberg J. PI3P signaling regulates receptor sorting but not transport in the endosomal pathway. J. Cell Biol. 2003;162:971–979. doi: 10.1083/jcb.200303018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Richter K, Buchner J. Hsp90: Chaperoning signal transduction. J. Cell Physiol. 2001;188:281–290. doi: 10.1002/jcp.1131. [DOI] [PubMed] [Google Scholar]
  66. Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992;70:401–410. doi: 10.1016/0092-8674(92)90164-8. [DOI] [PubMed] [Google Scholar]
  67. Sabharanjak S, Sharma P, Parton RG, Mayor S. GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Dev. Cell. 2002;2:411–423. doi: 10.1016/s1534-5807(02)00145-4. [DOI] [PubMed] [Google Scholar]
  68. Sandvig K, Van Deurs B. Transport of protein toxins into cells: pathways used by ricin, cholera toxin and Shiga toxin. FEBS Lett. 2002;529:49–53. doi: 10.1016/s0014-5793(02)03182-4. [DOI] [PubMed] [Google Scholar]
  69. Schnatwinkel C, Christoforidis S, Lindsay MR, Uttenweiler-Joseph S, Wilm M, Parton RG, Zerial M. The Rab5 effector Rabankyrin-5 regulates and coordinates different endocytic mechanisms. PLoS Biol. 2004;2:E261. doi: 10.1371/journal.pbio.0020261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Sharma DK, Brown JC, Choudhury A, Peterson TE, Holicky E, Marks DL, Simari R, Parton RG, Pagano RE. Selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol. Mol. Biol. Cell. 2004;15:3114–3122. doi: 10.1091/mbc.E04-03-0189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Sharp S, Workman P. Inhibitors of the HSP90 molecular chaperone: Current status. Adv. Cancer Res. 2006;95:323–348. doi: 10.1016/S0065-230X(06)95009-X. [DOI] [PubMed] [Google Scholar]
  72. Sigismund S, Woelk T, Puri C, Maspero E, Tacchetti C, Transidico P, Di Fiore PP, Polo S. Clathrin-independent endocytosis of ubiquitinated cargos. Proc. Natl. Acad. Sci. USA. 2005;102:2760–2765. doi: 10.1073/pnas.0409817102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Simpson F, Bright NA, West MA, Newman LS, Darnell RB, Robinson MS. A novel adaptor-related protein complex. J. Cell Biol. 1996;133:749–760. doi: 10.1083/jcb.133.4.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235:177–182. doi: 10.1126/science.3798106. [DOI] [PubMed] [Google Scholar]
  75. Sorkin A, DiFiore PP, Carpenter G. The carboxyl terminus of epidermal growth factor/erbB2 chimerae is internalization impaired. Oncogene. 1993;8:3021–3028. [PubMed] [Google Scholar]
  76. Soubeyran P, Kowanetz K, Szymkiewicz I, Langdon WY, Dikic I. Cbl-CIN85-endophilin complex mediates ligand-induced downregulation of EGF receptors. Nature. 2002;416:183–187. doi: 10.1038/416183a. [DOI] [PubMed] [Google Scholar]
  77. Stang E, Blystad FD, Kazazic M, Bertelsen V, Brodahl T, Raiborg C, Stenmark H, Madshus IH. Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits. Mol. Biol. Cell. 2004;15:3591–3604. doi: 10.1091/mbc.E04-01-0041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Stenmark H, Parton RG, Steele-Mortimer O, Lutcke A, Gruenberg J, Zerial M. Inhibition of Rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBO J. 1994;13:1287–1296. doi: 10.1002/j.1460-2075.1994.tb06381.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Stern DF, Heffernan PA, Weinberg RA. p185, a product of the neu proto-oncogene, is a receptorlike protein associated with tyrosine kinase activity. Mol. Cell Biol. 1986;6:1729–1740. doi: 10.1128/mcb.6.5.1729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Tikhomirov O, Carpenter G. Geldanamycin induces ErbB-2 degradation by proteolytic fragmentation. J. Biol. Chem. 2000;275:26625–26631. doi: 10.1074/jbc.M003114200. [DOI] [PubMed] [Google Scholar]
  81. Tikhomirov O, Carpenter G. Caspase-dependent cleavage of ErbB-2 by geldanamycin and staurosporin. J. Biol. Chem. 2001;276:33675–33680. doi: 10.1074/jbc.M101394200. [DOI] [PubMed] [Google Scholar]
  82. Volpicelli LA, Lah JJ, Levey AI. Rab5-dependent trafficking of the m4 muscarinic acetylcholine receptor to the plasma membrane, early endosomes, and multivesicular bodies. J. Biol. Chem. 2001;276:47590–47598. doi: 10.1074/jbc.M106535200. [DOI] [PubMed] [Google Scholar]
  83. Vonderheit A, Helenius A. Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes. PLoS Biol. 2005;3:e233. doi: 10.1371/journal.pbio.0030233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  84. Wang LH, Rothberg KG, Anderson RGW. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J. Cell Biol. 1993;123:1107–1117. doi: 10.1083/jcb.123.5.1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. Wang Z, Moran MF. Requirement for the adapter protein GRB2 in EGF receptor endocytosis. Science. 1996;272:1935–1939. doi: 10.1126/science.272.5270.1935. [DOI] [PubMed] [Google Scholar]
  86. Wang Z, Zhang L, Yeung TK, Chen X. Endocytosis deficiency of epidermal growth factor (EGF) receptor-ErbB2 heterodimers in response to EGF stimulation. Mol. Biol. Cell. 1999;10:1621–1636. doi: 10.1091/mbc.10.5.1621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Waterman H, Alroy I, Strano S, Seger R, Yarden Y. The C-terminus of the kinase-defective neuregulin receptor ErbB-3 confers mitogenic superiority and dictates endocytic routing. EMBO J. 1999;18:3348–3358. doi: 10.1093/emboj/18.12.3348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. Waterman H, Sabanai B, Geiger B, Yarden Y. Alternative intracellular routing of ErbB receptors may determine signaling potency. J. Biol. Chem. 1998;273:13819–13827. doi: 10.1074/jbc.273.22.13819. [DOI] [PubMed] [Google Scholar]
  89. Way TD, Kao MC, Lin JK. Apigenin induces apoptosis through proteasomal degradation of HER2/neu in HER2/neu-overexpressing breast cancer cells via the phosphatidylinositol 3-kinase/Akt-dependent pathway. J. Biol. Chem. 2004;279:4479–4489. doi: 10.1074/jbc.M305529200. [DOI] [PubMed] [Google Scholar]
  90. Xu W, Marcu M, Yuan X, Mimnaugh E, Patterson C, Neckers L. Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu. Proc. Natl. Acad. Sci. USA. 2002;99:12847–12852. doi: 10.1073/pnas.202365899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  91. Xu W, Mimnaugh E, Rosser MF, Nicchitta C, Marcu M, Yarden Y, Neckers L. Sensitivity of mature ErbB2 to geldanamycin is conferred by its kinase domain and is mediated by the chaperone protein Hsp90. J. Biol. Chem. 2001;276:3702–3708. doi: 10.1074/jbc.M006864200. [DOI] [PubMed] [Google Scholar]
  92. Yang XL, Xiong WC, Mei L. Lipid rafts in neuregulin signaling at synapses. Life Sci. 2004;75:2495–2504. doi: 10.1016/j.lfs.2004.04.036. [DOI] [PubMed] [Google Scholar]
  93. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat. Rev. Mol. Cell. Biol. 2001;2:127–137. doi: 10.1038/35052073. [DOI] [PubMed] [Google Scholar]
  94. Zhang F, Crise B, Su B, Hou Y, Rose JK, Bothwell A, Jacobson K. Lateral diffusion of membrane-spanning and glycosylphosphatidylinositol-linked proteins: Toward establishing rules governing the lateral mobility of membrane proteins. J. Cell Biol. 1991;115:75–84. doi: 10.1083/jcb.115.1.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  95. Zhou P, Fernandes N, Dodge IL, Reddi AL, Rao N, Safran H, DiPetrillo TA, Wazer DE, Band V, Band H. ErbB2 degradation mediated by the co-chaperone protein CHIP. J. Biol. Chem. 2003;278:13829–13837. doi: 10.1074/jbc.M209640200. [DOI] [PubMed] [Google Scholar]
  96. Zhou W, Carpenter G. Heregulin-dependent translocation and hyperphosphorylation of ErbB-2. Oncogene. 2001;20:3918–3920. doi: 10.1038/sj.onc.1204517. [DOI] [PubMed] [Google Scholar]
  97. Zurita AR, Crespo PM, Koritschoner NP, Daniotti JL. Membrane distribution of epidermal growth factor receptors in cells expressing different gangliosides. Eur. J. Biochem. 2004;271:2428–2437. doi: 10.1111/j.1432-1033.2004.04165.x. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1
NIHMS92795-supplement-Fig__S1.pdf (1,021.4KB, pdf)
Fig. S2
NIHMS92795-supplement-Fig__S2.pdf (1.6MB, pdf)
Fig. S3
NIHMS92795-supplement-Fig__S3.pdf (1.1MB, pdf)
Fig. S4
NIHMS92795-supplement-Fig__S4.pdf (650.8KB, pdf)
Fig. S5
NIHMS92795-supplement-Fig__S5.pdf (239.7KB, pdf)
Supp.Fig.Legs
NIHMS92795-supplement-Supp_Fig_Legs.pdf (79.9KB, pdf)

RESOURCES