Activation of the epidermal growth factor (EGF) receptor induces formation of EGF receptor- and Grb2-containing clathrin-coated pits

In HeLa cells depleted of adaptor protein 2 complex (AP2) by small interfering RNA (siRNA) to the mu2 or alpha subunit or by transient overexpression of an AP2 sequestering mutant of Eps15, endocytosis of the transferrin receptor (TfR) was strongly inhibited. However, epidermal growth factor (EGF)-induced endocytosis of the EGF receptor (EGFR) was inhibited only in cells where the alpha subunit had been knocked down. By immunoelectron microscopy, we found that in AP2-depleted cells, the number of clathrin-coated pits was strongly reduced. When such cells were incubated with EGF, new coated pits were formed. These contained EGF, EGFR, clathrin, and Grb2 but not the TfR. The induced coated pits contained the alpha subunit, but labeling density was reduced compared to control cells. Induction of clathrin-coated pits required EGFR kinase activity. Overexpression of Grb2 with inactivating point mutations in N- or C-terminal SH3 domains or in both SH3 domains inhibited EGF-induced formation of coated pits efficiently, even though Grb2 SH3 mutations did not block activation of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K). Our data demonstrate that EGFR-induced signaling and Grb2 are essential for formation of clathrin-coated pits accommodating the EGFR, while activation of MAPK and PI3K is not required.     

Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits

Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitin-interacting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.     

Specific Grb2-mediated interactions regulate clathrin-dependent endocytosis of the cMet-tyrosine kinase

Lysosomal degradation of the receptor-tyrosine kinase cMet requires receptor ubiquitination by the E3 ubiquitin ligase Cbl followed by clathrin-dependent internalization. A role for Cbl as an adaptor for cMet internalization has been previously reported. However, the requirement for Cbl ubiquitin ligase activity in this process and its mode of recruitment to cMet has yet to be determined. Cbl can directly bind cMet at phosphotyrosine 1003 or indirectly via Grb2 to phosphotyrosine 1356 in the multisubstrate binding domain of cMet. The direct binding of Cbl with cMet is critical for receptor degradation and not receptor internalization. Here we show a strict requirement for Grb2 and the ubiquitin ligase activity of Cbl for cMet endocytosis. Receptor internalization was impaired by small interfering RNA depletion of Grb2, overexpression of dominant negative Grb2 mutants, and point mutations in the cMet multisubstrate docking site that inhibits the direct association of Grb2 with cMet. The requirement for Grb2 was specific and did not involve the multiadaptor Gab1. cMet internalization was impaired in cells expressing an ubiquitin ligase-deficient Cbl mutant or conjugation-deficient ubiquitin but was unaffected in cells expressing a Cbl mutant that is unable to bind cMet directly. Expression of a Cbl-Grb2 chimera rescued impaired cMet endocytosis in cells depleted of endogenous Grb2. These results indicate that the ubiquitin ligase activity of Cbl is critical for clathrin-dependent cMet internalization and suggest a role for Grb2 as an intermediary linking Cbl ubiquitin ligase activity to this process.     

Effect of tyrosine kinase inhibitors on clathrin-coated pit recruitment and internalization of epidermal growth factor receptor

Several inhibitors of epidermal growth factor receptor (EGFR) kinase and Src family kinases (SFK) were employed to study the role of these kinases in EGFR internalization through clathrin-coated pits. The EGFR kinase-specific compound PD158780 substantially diminished EGFR internalization. PP2, an inhibitor of SFK, had a moderate effect on EGFR internalization in several types of cells, including cells lacking SFK, indicating that the inhibition of endocytosis by PP2 is mediated by kinases other than SFK. In contrast, SU6656, a more specific inhibitor of SFK, did not affect EGFR internalization. To examine what stage of internalization requires receptor kinase activity, we established a quantitative assay based on three-dimensional fluorescence microscopy that measures co-localization of an EGF-rhodamine conjugate and a fluorescently tagged clathrin adaptor protein complex, AP-2. Interestingly, recruitment of EGFR into coated pits did not require physiological temperature because the maximal accumulation of EGFR in coated pits was observed at 4 degrees C. Pretreatment of the cells with PD158780 prevented EGFR recruitment into coated pits, whereas the inhibitor did not block the internalization of receptors that had first been allowed to enter the coated pits at 4 degrees C. These data demonstrate that the activation of receptor kinase is essential for the initial, coated pit recruitment step of endocytosis.     

Epidermal growth factor receptor internalization through clathrin-coated pits requires Cbl RING finger and proline-rich domains but not receptor polyubiquitylation

Cbl proteins have been implicated in the regulation of endocytic trafficking of epidermal growth factor receptor. However, the precise role of Cbl in epidermal growth factor receptor endocytosis is not defined. To directly visualize Cbl in cells and perform structure-function analysis of Cbl's role in epidermal growth factor receptor internalization, a yellow fluorescent protein-fusion of c-Cbl was constructed. Upon epidermal growth factor receptor activation, Cbl-yellow fluorescent protein moved with epidermal growth factor receptor to clathrin-coated pits and endosomes. Localization of Cbl-yellow fluorescent protein to these endocytic organelles was dependent on a proline-rich domain of c-Cbl that interacts with Grb2 as shown by fluorescence resonance energy transfer microscopy. In contrast, direct binding of Cbl to phosphotyrosine 1045 of the epidermal growth factor receptor was required for epidermal growth factor receptor polyubiquitination, but was not essential for Cbl-yellow fluorescent protein localization in epidermal growth factor receptor-containing compartments. These data suggest that the binding of Cbl to epidermal growth factor receptor through Grb2 is necessary and sufficient for Cbl function during clathrin-mediated endocytosis. Overexpression of c-Cbl mutants that are capable of Grb2 binding but defective in linker/RING finger domain function severely inhibited epidermal growth factor receptor internalization. The same dominant-negative mutants of Cbl did not block epidermal growth factor receptor recruitment into coated pits but retained receptors in coated pits, thus preventing receptor endocytosis and transport to endosomes. These data suggest that the linker and RING finger domain of Cbl may function during late steps of coated vesicle formation. We propose that the RING domain of Cbl facilitates endocytosis either by epidermal growth factor receptor monoubiquitylation or by ubiquitylation of proteins associated with the receptor.     

Epidermal growth factor receptor efficiently activates mitogen-activated protein kinase in HeLa cells and Hep2 cells conditionally defective in clathrin-dependent endocytosis

Epidermal growth factor (EGF)-induced signaling was investigated in cells conditionally defective in clathrin-dependent endocytosis by overexpression of K44A dynamin in HeLa cells and potassium depletion in Hep2 cells. Overexpression of mutant dynamin disrupts high-affinity EGF-EGF receptor (EGFR) interaction (T. Ringerike, E. Stang, L. E. Johannessen, D. Sandnes, F. O. Levy, and I. H. Madshus, 1998, J. Biol. Chem. 273, 16639-16642). However, the EGFR substrates Shc and c-Cbl were as efficiently tyrosine phosphorylated in endocytosis-deficient HeLa cells exhibiting only low-affinity EGFRs as in HeLa cells with intact endocytosis and with both high- and low-affinity EGFRs. Both Raf and mitogen-activated protein kinase (MAPK) were activated to the same extent and with the same kinetics. HeLa cells distributed equally in the cell cycle regardless of EGFR internalization. Upon potassium depletion of Hep2 cells, EGF-induced EGFR endocytosis was inhibited. However, the EGFR and MAPK were efficiently activated by EGF in both the absence and the presence of clathrin-dependent endocytosis. The EGFR was weakly tyrosine phosphorylated by potassium depletion even in the absence of EGF, and this activation resulted in detectable activation of MAPK. Our results demonstrate that internalization of EGFR by clathrin-dependent endocytosis is not required for activation of MAPK.     

Endocytosis and intracellular trafficking of ErbBs

This review article describes the pathways and mechanisms of endocytosis and post-endocytic sorting of the EGF receptor (EGFR/ErbB1) and other members of the ErbB family. Growth factor binding to EGFR accelerates its internalization through clathrin-coated pits which is followed by the efficient lysosomal targeting of internalized receptors and results in receptor down-regulation. The role of EGFR interaction with the Grb2 adaptor protein and Cbl ubiquitin ligase, and receptor ubiquitination in the clathrin-dependent internalization and sorting of EGFR in multivesicular endosomes is discussed. Activation and phosphorylation of ErbB2, ErbB3 and ErbB4 also results in their ubiquitination. However, these ErbBs are internalized and targeted to lysosomes less efficiently than EGFR. When overexpressed endocytosis-impaired ErbBs may inhibit the internalization and degradation of EGFR.     

Endocytosis and intracellular trafficking of ErbBs

This review article describes the pathways and mechanisms of endocytosis and post-endocytic sorting of the EGF receptor (EGFR/ErbB1) and other members of the ErbB family. Growth factor binding to EGFR accelerates its internalization through clathrin-coated pits which is followed by the efficient lysosomal targeting of internalized receptors and results in receptor down-regulation. The role of EGFR interaction with the Grb2 adaptor protein and Cbl ubiquitin ligase, and receptor ubiquitination in the clathrin-dependent internalization and sorting of EGFR in multivesicular endosomes is discussed. Activation and phosphorylation of ErbB2, ErbB3 and ErbB4 also results in their ubiquitination. However, these ErbBs are internalized and targeted to lysosomes less efficiently than EGFR. When overexpressed endocytosis-impaired ErbBs may inhibit the internalization and degradation of EGFR.     

Polyubiquitination of the epidermal growth factor receptor occurs at the plasma membrane upon ligand-induced activation

We have previously shown that, although overexpression of mutant dynamin inhibits clathrin-dependent endocytosis and disrupts high affinity binding of epidermal growth factor (EGF) to the EGF receptor (EGFR), it does not inhibit ligand-induced translocation of the EGFR into clathrin-coated pits. In the present study, we demonstrate that, upon ligand binding and incubation at 37 degrees C, the EGFR was polyubiquitinated regardless of overexpression of mutant dynamin. In cells not overexpressing mutant dynamin, the EGFR was rapidly internalized and deubiquitinated. In cells being endocytosis-deficient, due to overexpression of mutant dynamin, however, the EGFR was upon prolonged chase first found in deeply invaginated coated pits, and then eventually moved out of the coated pits and back onto the smooth plasma membrane. Polyubiquitination occurred equally efficiently in cells with or without intact clathrin-dependent endocytosis, while the kinetics of ubiquitination and deubiquitination was somewhat different. We further found that the EGF-induced ubiquitination of Eps15 occurred both in the absence and presence of endocytosis with the same kinetics as polyubiquitination of the EGFR, but that the EGF-induced monoubiquitination of Eps15 was somewhat reduced upon overexpression of mutant dynamin. Our data show that EGF-induced polyubiquitination of the EGFR occurs at the plasma membrane.     

A chimeric pre-ubiquitinated EGF receptor is constitutively endocytosed in a clathrin-dependent, but kinase-independent manner

The roles of EGF receptor (EGFR) kinase activity and ubiquitination in EGFR endocytosis have been controversial. The adaptor protein and ubiquitin ligase Cbl has reportedly been required. Consistently, we now report that siRNA-mediated knock-down of c-Cbl and Cbl-b significantly slowed clathrin-dependent internalization of activated wild-type (wt) EGFR by inhibiting recruitment of the EGFR to clathrin-coated pits. However, a chimeric protein consisting of wt-EGFR, a C-terminal linker and four linearly connected ubiquitins was found to interact with Eps15 and epsin 1 and to be constitutively endocytosed in a clathrin-dependent manner. Interestingly, endocytosis of this fusion protein did not require binding of EGF. Nor was kinase activity required, and the fusion protein was endocytosed in the presence of an EGFR kinase inhibitor, which efficiently counteracted tyrosine phosphorylation. This demonstrates that ubiquitination over-rides the requirement for kinase activity in recruitment of the EGFR to clathrin-coated pits.     

Relationships between EGFR signaling-competent and endocytosis-competent membrane microdomains

Membrane microdomains, the so-called lipid rafts, function as platforms to concentrate receptors and assemble the signal transduction machinery. Internalization, in most cases, is carried out by different specialized structures, the clathrin-coated pits. Here, we show that several endocytic proteins are efficiently recruited to morphologically identified plasma membrane lipid rafts, upon activation of the epidermal growth factor (EGF) receptor (EGFR), a receptor tyrosine kinase. Analysis of detergent-resistant membrane fractions revealed that the EGF-dependent association of endocytic proteins with rafts is as efficient as that of signaling effector molecules, such as Grb2 or Shc. Finally, the EGFR, but not the nonsignaling transferrin receptor, could be localized in nascent coated pits that almost invariably contained raft membranes. Thus, specialized membrane microdomains have the ability to assemble both the molecular machineries necessary for intracellular propagation of EGFR effector signals and for receptor internalization.     

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

Mutation of the binding site for Cbl (Tyr1045) in the EGF receptor (EGFR) results in impaired ubiquitination but does not affect EGFR internalization. However, the Y1045F mutation resulted in strongly decreased degradation of the EGFR, as well as efficient recycling of EGFR to the plasma membrane. Significantly, more wild-type EGFR than Y1045F EGFR was found localizing to multivesicular late endosomes. Ubiquitination of the EGFR was in HeLa cells inhibited both upon overexpressing the N-terminal part of Cbl and upon overexpressing a double mutant Grb2 incapable of interacting with Cbl and thereby being incapable of indirectly recruiting Cbl to the EGFR. Collectively, these data suggest that the ubiquitination resulting from direct binding of Cbl to pTyr1045 of the EGFR is critical for lysosomal sorting of the EGFR in contrast to ubiquitination resulting from Grb2-mediated binding of Cbl to the EGFR.     

Endocytosis of epidermal growth factor receptor regulated by Grb2-mediated recruitment of the Rab5 GTPase-activating protein RN-tre

The Grb2 adaptor protein is best known for its role in signaling to the small GTPase p21(ras), mediated through its interaction with the SOS guanine nucleotide exchange factor. Here, we demonstrate that Grb2 also signals to Rab5, a small GTPase that plays a key role in early endocytic trafficking. Grb2 functions through association with RN-tre, a GTPase-activating protein for Rab5. Grb2 and RN-tre associate both in vitro and in vivo, with interaction mediated by both SH3 domains of Grb2 and extended proline-rich sequences in RN-tre. Association between Grb2 and RN-tre is constitutive and occurs independently of Eps8, a previously identified binding partner of RN-tre. Epidermal growth factor (EGF) stimulates recruitment of RN-tre to the EGF receptor (EGFR) in a Grb2-dependent manner. Grb2 and the EGFR are internalized and co-localized in endocytic vesicles in response to EGF. Overexpression of RN-tre blocks the internalization of both proteins, consistent with its function as a negative regulator of Rab5 and endocytosis. Strikingly, RN-tre does not block EGF-induced internalization of a Grb2 mutant deficient in RN-tre binding. These results 1) suggest that the ability of RN-tre to inhibit internalization of the EGFR requires Grb2-mediated binding to the receptor and 2) identify Grb2 as a critical regulator of Rab5 and EGFR endocytosis.     

Grb2 mediates negative regulation of stem cell factor receptor/c-Kit signaling by recruitment of Cbl

Aberrant activation of c-Kit is involved in a number of human diseases including cancers and leukemias. Certain receptor tyrosine kinases, such as epidermal growth factor receptor, have been shown to indirectly recruit Cbl through the adapter protein Grb2, leading to receptor ubiquitination and degradation. In order to study the role of Grb2 in c-Kit degradation, a series of mutations of the Grb2 binding sites in c-Kit were generated (Y703F, Y936F, and Y703F/Y936F). Since other signal transduction molecules are also known to bind Y703 and Y936, the more selective asparagine-to-alanine (N-to-A) mutants N705A, N938A, and N705A/N938A were generated. We could clearly demonstrate that binding of Grb2 was dependent on intact phosphorylation sites Y703 and Y936. Furthermore, we could demonstrate the presence of Cbl in a complex with Grb2 and c-Kit. Thus, Grb2 is able to indirectly recruit Cbl to c-Kit. In the N-to-A mutants, Cbl phosphorylation was strongly reduced, which correlated with reduced ubiquitination of c-Kit as well as decreased internalization and degradation of the receptor. Taken together, we have demonstrated that, in addition to its role in positive signaling via the Ras/Erk pathway, Grb2 mediates c-Kit degradation through recruitment of Cbl to c-Kit, leading to ubiquitination of c-Kit followed by internalization and degradation.     

Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference

To identify proteins that participate in clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR), 13 endocytic proteins were depleted in HeLa cells using highly efficient small interfering RNAs that were designed using a novel selection algorithm. The effects of small interfering RNAs on the ligand-induced endocytosis of EGFR were compared with those effects on the constitutive internalization of the transferrin receptor. The knock-downs of clathrin heavy chain and dynamin produced maximal inhibitory effects on the internalization of both receptors. Depletion of alpha, beta2, or micro2 subunits of AP-2 reduced EGF and transferrin internalization rates by 40-60%. Down-regulation of several accessory proteins individually had no effect on endocytosis but caused significant inhibition of EGF and transferrin endocytosis when the homologous proteins were depleted simultaneously. Surprisingly, knockdown of clathrin-assembly lymphoid myeloid leukemia protein, CALM, did not influence transferrin endocytosis but considerably affected EGFR internalization. Thus, CALM is the second protein besides Grb2 that appears to play a specific role in EGFR endocytosis. This study demonstrates that the efficient gene silencing by rationally designed small interfering RNA can be used as an approach to functionally analyze the entire cellular machineries, such as the clathrin-coated pits and vesicles.     

Growth factor receptor binding protein 2-mediated recruitment of the RING domain of Cbl to the epidermal growth factor receptor is essential and sufficient to support receptor endocytosis

Knockdown of growth factor receptor binding protein 2 (Grb2) by RNA interference strongly inhibits clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR). To gain insights into the function of Grb2 in EGFR endocytosis, we have generated cell lines in which endogenous Grb2 was replaced by yellow fluorescent protein (YFP)-tagged Grb2 expressed at the physiological level. In these cells, Grb2-YFP fully reversed the inhibitory effect of Grb2 knockdown on EGFR endocytosis and, moreover, trafficked together with EGFR during endocytosis. Overexpression of Grb2-binding protein c-Cbl did not restore endocytosis in Grb2-depleted cells. However, EGFR endocytosis was rescued in Grb2-depleted cells by chimeric proteins consisting of the Src homology (SH) 2 domain of Grb2 fused to c-Cbl. The "knockdown and rescue" analysis revealed that the expression of Cbl-Grb2/SH2 fusions containing RING finger domain of Cbl restores normal ubiquitylation and internalization of the EGFR in the absence of Grb2, consistent with the important role of the RING domain in EGFR endocytosis. In contrast, the carboxy-terminal domain of Cbl, when attached to Grb2 SH2 domain, had 4 times smaller endocytosis-rescue effect compared with the RING-containing chimeras. Together, the data suggest that the interaction of Cbl carboxy terminus with CIN85 has a minor and a redundant role in EGFR internalization. We concluded that Grb2-mediated recruitment of the functional RING domain of Cbl to the EGFR is essential and sufficient to support receptor endocytosis.     

Protease-activated receptor-1 down-regulation: a mutant HeLa cell line suggests novel requirements for PAR1 phosphorylation and recruitment to clathrin-coated pits

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly activated by a proteolytic mechanism, then internalized and degraded in lysosomes. The latter is critical for temporal fidelity of thrombin signaling. Toward understanding PAR1 down-regulation, we first investigated the pathway of PAR1 internalization. Activated PAR1 was rapidly recruited to clathrin-coated pits, where it colocalized with transferrin receptor (TfnR). Dominant-negative dynamin and clathrin hub mutants both blocked PAR1 internalization. Blockade of PAR1 internalization with dynamin K44A also inhibited activation-dependent PAR1 degradation. Thus activated PAR1 internalizes via clathrin-coated pits together with receptors that recycle and is then sorted away from such receptors and delivered to lysosomes. In the course of these studies we identified a mutant HeLa cell line, designated JT1, that was defective in PAR1 internalization. PAR1 signaled robustly in JT1 cells but was not phosphorylated or recruited to clathrin-coated pits after activation. Internalization of TfnR was intact in JT1 cells and internalization of beta(2)-adrenergic receptor, a GPCR that internalizes and recycles, was present but perhaps reduced. Taken together, these studies suggest that PAR1 is internalized in a dynamin- and clathrin-dependent manner like TfnR and beta(2)-adrenergic receptor but requires a distinct gene product for recruitment into this pathway.     

Single-molecule diffusion study of activated EGFR implicates its endocytic pathway

In this work, we have imaged the lateral diffusion of activated epidermal growth factor receptor (EGFR) on cell membrane for studying its internalization pathway. After EGF activation, the mobility of individual EGFR molecules was measured and compared with that in the cells disrupted of clathrin-coated pits and caveolae, the two endocytosis-competent membrane microdomains. The results implicated that activated EGFR molecules associated with clathrin-coated pits but not caveolae at low doses of EGF, whereas they were located in these two domains at high EGF doses. It provided supporting evidence for the occurrence of both clathrin-dependent and caveolae-dependent EGFR endocytosis.     

Cell surface ceramide controls translocation of transferrin receptor to clathrin-coated pits

Transferrin receptor mediates internalization of transferrin with bound ferric ions through the clathrin-dependent pathway. We found that binding of transferrin to the receptor induced rapid generation of cell surface ceramide which correlated with activation of acid, but not neutral, sphingomyelinase. At the onset of transferrin internalization both ceramide level and acid sphingomyelinase activity returned to their basic levels. Down-regulation of acid sphingomyelinase in cells with imipramine or silencing of the enzyme expression with siRNA stimulated transferrin internalization and inhibited its recycling. In these conditions colocalization of transferrin with clathrin was markedly reduced. Simultaneously, K⁺ depletion of cells which interfered with the assembly of clathrin-coated pits inhibited the uptake of transferrin much less efficiently than it did in control conditions. The down-regulation of acid sphingomyelinase activity led to the translocation of transferrin receptor to the raft fraction of the plasma membrane upon transferrin binding. The data suggest that lack of cell surface ceramide, generated in physiological conditions by acid sphingomyelinase during transferrin binding, enables internalization of transferrin/transferrin receptor complex by clathrin-independent pathway.     

Association with membrane protrusions makes ErbB2 an internalization-resistant receptor

In contrast to the epidermal growth factor (EGF) receptor, ErbB2 is known to remain at the plasma membrane after ligand binding and dimerization. However, why ErbB2 is not efficiently down-regulated has remained elusive. Basically, two possibilities exist: ErbB2 is internalization resistant or it is efficiently recycled after internalization. By a combination of confocal microscopy, immunogold labeling electron microscopy, and biochemical techniques we show that ErbB2 is preferentially associated with membrane protrusions. Moreover, it is efficiently excluded from clathrin-coated pits and is not seen in transferrin receptor-containing endosomes. This pattern is not changed after binding of EGF, heregulin, or herceptin. The exclusion from coated pits is so pronounced that it cannot just be explained by lack of an internalization signal. Although ErbB2 is a raft-associated protein, the localization of ErbB2 to protrusions is not a result of raft binding. Also, an intact actin cytoskeleton is not required for keeping ErbB2 away from coated pits. However, after efficient cross-linking, ErbB2 is removed from protrusions to occur on the bulk membrane, in coated pits, and in endosomes. These data show that ErbB2 is a remarkably internalization-resistant receptor and suggest that the mechanism underlying the firm association of ErbB2 with protrusions also is the reason for this resistance.