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ABSTRACT: 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 C-terminus 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.
Molecular Biology of the Cell 10/2007; 18(9):3656-66. · 4.94 Impact Factor
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06/2006: pages 69 - 89; , ISBN: 9783527608072
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ABSTRACT: 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.
Journal of Cell Science 02/2006; 119(Pt 1):85-95. · 6.11 Impact Factor
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ABSTRACT: 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.
Traffic 10/2005; 6(9):720-4. · 4.92 Impact Factor
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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.
Molecular Biology of the Cell 05/2004; 15(4):1557-67. · 4.94 Impact Factor
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ABSTRACT: The function of caveolae is hotly debated. It now seems clear that caveolae are stable membrane domains that are kept in place by the actin cytoskeleton. However, this stability can be perturbed, leading to caveolar internalization. Caveolae are important in the regulation of various signaling processes, such as nitric oxide activity, and in cholesterol efflux and cholesterol-ester uptake. Caveolin deficiency particularly affects the cardiovascular system and the lungs but, because the knockout mice are viable, none of the proposed functions appears to be essential. Rather than having a specific function, caveolae might be considered to be multifunctional organelles with a physiological role that varies depending on cell type and cellular needs.
Trends in Cell Biology 03/2003; 13(2):92-100. · 12.35 Impact Factor
