Association with Membrane Protrusions Makes ErbB2 an Internalization-resistant Receptor

Abstract

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...

Transactivation of the epidermal growth factor receptor in responses to myocardial stress and cardioprotection

“…The preferred ErbB2 dimer partner increases ligand-binding affinity (Karunagaran et al, 1996) and reduces EGF dissociation to prolong signal activation. Heterodimers containing ErbB2 are also more stable and endocytosed at lower rates than other ErbB dimers (Lenferink et al, 1998;Wang et al, 1999;Hommelgaard et al, 2004). Thus, signalling via ErbB1/2 heterodimers may be more powerful and sustained than that arising via ErbB1 homodimers.…”

Lapatinib, a HER2 tyrosine kinase inhibitor, induces stabilization and accumulation of HER2 and potentiates trastuzumab-dependent cell cytotoxicity

“…The cells with higher HER2 expression showed significantly higher trastuzumab-mediated cytolysis (data not shown). The results are expressed as mean of three independent experiments , others have not (Austin et al, 2004;Hommelgaard et al, 2004;Longva et al, 2005). Interestingly, in some of the models that have not observed trastuzumabmediated receptor downregulation (Austin et al, 2004;Longva et al, 2005), there is also a lack of trastuzumabinduced HER2 phosphorylation.…”

“…A key regulator of HER receptor degradation is the E3 ubiquitin ligase c-Cbl (Marmor and Yarden, 2004). Although E3 ubiquitin ligase c-Cbl shows only a marginal effect in ligand-induced HER2 ubiquitination (Wang et al, 1999;Hommelgaard et al, 2004), it does play a role in receptor degradation when c-Cbl is overexpressed or recruited following treatment with anti-HER2 antibodies (Klapper et al, 2000;Wolpoe et al, 2003). However, we cannot rule out that the internalization of HER2 following trastuzumab treatment could be mediated by a kinase-dependent activation of other ubiquitin ligases.…”

Endocytic Down-Regulation of ErbB2 Is Stimulated by Cleavage of Its C-Terminus

“…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).…”

“…After a wash the cells were next incubated with mouse monoclonal Sc08 (Santa Cruz Biotechnology) against the extracellular (N-terminal) part of ErbB2, followed by 5-nm gold-labeled goat anti-mouse antibody (Amersham Biosciences). After a brief fixation and a wash the cells were scraped off and further processed for Epon embedding and electron microscopy (pre-embedding immunogold labeling electron microscopy) as previously described (Hommelgaard et al, 2004). …”