| Endocytosis is an essential process required for functions including nutrient uptake, membrane recycling, and signal transduction. In comparison to the clathrin mediated pathway, clathrin-independent pathways are poorly understood. New work is beginning to reveal a picture of multiple non-clathrin pathways, including an Arf6 associated pathway and a pathway leading to GPI-anchored protein enriched early endosomes (GEECs).;ErbB2 internalization did not require tyrosine kinase activity and did not occur by caveolae as SKBr3 cells lack detectable caveolae. The receptor colocalized with cholera toxin, a GPI-anchored protein and a fluid tracer, and was often found in ring shaped or tubular structures consistent with features of the GEEC pathway. The GEEC pathway was first described in Chinese Hamster Ovary (CHO) cells and thought to be specific for GPI-anchored proteins. We found that ErbB2 colocalized with GPI-anchored proteins Thy1 and PLAP soon after internalization in CHO cells. Surprisingly, ErbB2 and Thy1 also colocalized substantially with chimeric fusion proteins of PLAP containing transmembrane and cytosolic domains, proteins expected to be excluded from the GEEC pathway. Combined with other data from the lab, these results suggest that this pathway is not specific for GPI-anchored proteins, but may instead represent a bulk internalization pathway.;After internalization, ErbB2 was transported to the interior of early endosomes and then to late endosomes and lysosomes. Chloroquine retarded degradation of a truncated form of ErbB2. ErbB2 accumulated in vesicles containing constitutively-active Arf6Q67L only in the absence of geldanamycin. In geldanamycin-treated cells expressing Arf6Q67L, ErbB2 was transported to early and late endosomes, and degraded at the same rate as in untransfected cells.;The EGF receptor family member ErbB2 is overexpressed in 25-30% of human breast cancers. While ErbB2 is normally highly concentrated on the cell surface, the ansamycin antibiotic geldanamycin induces ubiquitination, internalization and degradation of the receptor. Here, we demonstrated that ErbB2 was internalized through a non-clathrin pathway in geldanamycin-treated SKBr3 human breast cancer cells. Internalization was not inhibited by dominant negative forms of Eps15 or dynamin, or by the clathrin inhibitor chlorpromazine. ErbB2 did not colocalize with transferrin or clathrin during initial internalization. |
