Lerdrup Mads, Bruun Silas, Grandal Michael V, Roepstorff Kirstine, Kristensen Malene M, Hommelgaard Anette M, van Deurs Bo
Department of Cellular and Molecular Medicine, University of Copenhagen, The Panum Institute, DK-2200 Copenhagen N, Denmark.
Mol Biol Cell. 2007 Sep;18(9):3656-66. doi: 10.1091/mbc.e07-01-0025. Epub 2007 Jul 11.
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.
ErbB2水平升高与癌症相关，而ErbB2内吞作用受损可能导致其过度表达。因此，有必要了解ErbB2内吞下调的潜在机制。在各种刺激后，ErbB2的C末端可被切割，用格尔德霉素抑制热休克蛋白90（HSP90）后，这种切割伴随着ErbB2的蛋白酶体依赖性内吞作用。然而，C末端切割是否与内吞作用有关尚不清楚。为了研究ErbB2的切割和内吞运输，我们分别将黄色荧光蛋白（YFP）和青色荧光蛋白（CFP）融合到ErbB2的N末端和C末端（YFP-ErbB2-CFP）。在格尔德霉素刺激后，YFP-ErbB2-CFP在非凋亡细胞中以蛋白酶体依赖性方式被切割，并且在早期内体中观察到的切割YFP-ErbB2-CFP的相对量明显大于质膜中的相对量。此外，切割发生在质膜上，与全长ErbB2相比，切割后的ErbB2更有效地被内化和降解。一致地，与全长ErbB2相比，C末端截短的ErbB2也很容易被内吞并在溶酶体中降解。总之，我们认为格尔德霉素导致ErbB2的C末端切割，从而使受体从保留机制中释放出来，并导致ErbB2的内吞作用和溶酶体降解。
