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. INTRODUCTIONErbB2, a member of the epidermal growth factor (EGF) receptor (EGFR) family, has no specific ligand, but it is the main heterodimerization partner for the other family members (Sliwkowski et al., 1994;Yarden, 2001;Yarden and Sliwkowski, 2001). The dimerization is mediated by a unique dimerization arm localized to the extracellular (Nterminal) portion of the receptor. Whereas exposure of this arm normally requires ligand binding and reorganization of the exterior portion of ErbB receptors, it is constitutively exposed in ErbB2 (Burgess et al., 2003). Moreover, in contrast to the EGFR, which is rapidly down-regulated upon ligand stimulation (Sorkin and Von Zastrow, 2002), ErbB2 avoids delivery to lysosomes and subsequent proteolysis and has a long half-life at the plasma membrane (Gilboa et al., 1995;Baulida et al., 1996;Baulida and Carpenter, 1997;Waterman et al., 1998;Wang et al., 1999). ErbB2 expression is up-regulated in various cancers. For instance, 20 -30% of breast cancers show strong overexpression, and this correlates with poor prognosis (Slamon et al., 1989;Reese and Slamon, 1997;Eccles, 2001). The oncogenic potential of ErbB2 may in part be explained by its capability to avoid down-regulation together with its pronounced engagement in formation of heterodimers. When overexpressed, ErbB2 causes the other family members to escape the lysosomal pathway upon ligand binding and receptor activation (Yarden, 2001). However, the reason why ErbB2 escapes the lysosomal pathway and remains at the plasma membrane has remained elusive. Basically, two mechanisms could lead to retention of ErbB2 at the plasma membrane: either E...

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The potent oncoprotein and receptor tyrosine kinase ErbB2 is remarkable because it resists efficient downregulation. However, ErbB2 can be downregulated by the HSP-90 inhibitor geldanamycin, but the underlying cellular mechanisms are uncertain. Apparently, delivery of ErbB2 to lysosomes, cleavage of the ErbB2 kinase domain and proteasomal activity are all processes that are involved. Using a non-invasive confocal microscopical assay allowing quantitative analysis of ErbB2 internalization in cell populations, we show that whereas ErbB2 is resistant to internalization in untreated SK-BR-3 cells, geldanamycin stimulates internalization and subsequent degradation in lysosomes. This process depends on proteasomal activity, which is a regulatory upstream event in ErbB2 internalization rather than the actual mechanism of degradation. ErbB2 can be internalized as a full-length protein, thus cleavage of the ErbB2 kinase domain is not a requirement for geldanamycin-stimulated internalization. Moreover, as shown by FRAP (fluorescence recovery after photobleaching) and electron microscopy, geldanamycin induces an increase in the amount of mobile ErbB2 and a redistribution of ErbB2 in the plasma membrane making the receptor accessible to endocytosis. Cells with most ErbB2 endocytosis also have the highest fraction of mobile ErbB2. It is concluded that geldanamycin stimulates internalization of full-length ErbB2 in a proteasome-dependent manner leading to lysosomal degradation.

The role of caveolae in endocytosis is hotly debated. Here, we argue that most caveolae are stable microdomains at the cell surface. Only a small fraction of caveolae is constitutively internalized, leading to a quantitatively minor uptake of ligands and receptors. In addition, we suggest that a more pronounced downregulation of caveolae from the plasma membrane can occur, presumably stimulated by receptor cross-linking and clustering in caveolae. Finally, we propose that future studies dealing with internalization of caveolae should actually document such internalization and include kinetic data. In recent years, increasing attention has been paid to the role of caveolae in various cellular processes, and a plethora of functions have been ascribed to these small, characteristically shaped invaginations of the plasma membrane that are associated with caveolin. However, currently there is still much confusion about the role of caveolae in endocytosis. Supporting a role in endocytosis are several reports on the uptake of cholera toxin (CT) and also studies on SV40 virus internalization (1,2). Against a role in endocytosis are findings from fluorescence recovery after photobleaching (FRAP) studies showing that caveolae are highly immobile structures that do not have a high turnover at the plasma membrane (3). Indeed, caveolin apparently prevents caveolae from pinching off (4,5) and very recent data also show that caveolae are not obligatory for SV40 entry (6). In addition, other recent studies, for instance, on the uptake of prion proteins (7) and CT (8-13) have brought new life to the discussion. A summary of the evidence against caveolae as a major endocytic carrier is shown in Table 1. In this review, we present a unifying working model for caveolar internalization on the basis of available information.

High ErbB2 levels are associated with cancer, and impaired endocytosis of ErbB2 could contribute to its overexpression. Therefore, knowledge about the mechanisms underlying endocytic down-regulation of ErbB2 is warranted. The Cterminus of ErbB2 can be cleaved after various stimuli, and after inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the N-and C-terminus of ErbB2, respectively (YFP-ErbB2-CFP). After geldanamycin stimulation YFP-ErbB2-CFP became cleaved in nonapoptotic cells in a proteasome-dependent manner, and a markedly larger relative amount of cleaved YFP-ErbB2-CFP was observed in early endosomes than in the plasma membrane. Furthermore, cleavage took place at the plasma membrane, and cleaved ErbB2 was internalized and degraded far more efficiently than full-length ErbB2. Concordantly, a C-terminally truncated ErbB2 was also readily endocytosed and degraded in lysosomes compared with full-length ErbB2. Altogether, we suggest that geldanamycin leads to C-terminal cleavage of ErbB2, which releases the receptor from a retention mechanism and causes endocytosis and lysosomal degradation of ErbB2. INTRODUCTIONThe receptor tyrosine kinase ErbB2 is a potent oncoprotein, and a high ErbB2 level can by itself activate ErbB receptors (Worthylake et al., 1999). Concordantly, increased ErbB2 levels can be found in several cancers (Klapper et al., 2000;Yarden, 2001), and high ErbB2 levels are correlated to a worse prognosis for breast cancer patients (Slamon et al., 1987;Hynes and Stern, 1994;De Placido et al., 1998;Pegram et al., 1998). Gene amplification is the most studied mechanisms causing high ErbB2 levels, but also posttranscriptional regulation of ErbB2 plays a role in cancers (Vernimmen et al., 2003;Magnifico et al., 2007). Little attention has been paid to the role of ErbB2 degradation in cancers, although when compromised it also would lead to increased ErbB2 levels and activity. Several studies have shown that endocytic downregulation of ErbB2 is impaired in cancer cells (Sorkin et al., 1993; Baulida et al., 1996;Wang et al., 1999;Hommelgaard et al., 2004;Austin et al., 2004;Longva et al., 2005), and ErbB2 can even transmit this property to the related epidermal growth factor receptor (EGFR; Muthuswamy et al., 1999;Wang et al., 1999;Worthylake et al., 1999;Haslekas et al., 2005). Inhibition of HSP90 (e.g., with geldanamycin [GA]) leads to increased internalization and lysosomal degradation of ErbB2 in a manner depending on proteasomal activity (Tikhomirov and Carpenter, 2000;Austin et al., 2004;Lerdrup et al., 2006). HSP90 inhibition also induces cleavage of the cytoplasmic part of ErbB2, resulting in a transmembrane 135-kDa ErbB2 and short-lived cytoplasmic fragments (Tikhomirov and Carpenter, 2000;Lerdrup et al., 2006). The cleavage oc...