In vitro endosome‐lysosome transfer of dephosphorylated EGF receptor and Shc in rat liver

FEBS Letters

Authier, François²  Chauvet, Geneviève¹ 

[1] Institut National de la Santé et de la Recherche Médicale U30, Hôpital Necker Enfants-Malades, 75015 Paris, France;Institut National de la Santé et de la Recherche Médicale U510, Faculté de Pharmacie Paris XI, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry, France

关键词: Endocytosis;    Organelle fusion;    Intracellular signaling;    Tyrosine phosphorylation;    EGF;    epidermal growth factor;    EGFR;    epidermal growth factor receptor;    Shc;    src homology 2 (SH2) domain-containing α2 collagen-related;    RTK;    receptor tyrosine kinase;    TCA;    trichloroacetic acid;   

DOI  :  10.1016/S0014-5793(99)01413-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We have studied the endosome-lysosome transfer of internalized epidermal growth factor receptor (EGFR) complexes in a cell-free system from rat liver. Analytical subfractionation of a postmitochondrial supernatant fraction showed that a pulse of internalized [¹²⁵I]EGF was largely associated with a light endosomal fraction devoid of lysosomal markers. After an additional 30 min incubation in vitro in the presence of an ATP-regenerating system, the amount of [¹²⁵I]EGF in this compartment decreased by 39%, with an increase in [¹²⁵I]EGF in lysosomes. No transfer of [¹²⁵I]EGF to the cytosol was detected. To assess the fate of the internalized EGFR protein over the time course of the endo-lysosomal transfer of the ligand, the effect of a saturating dose of native EGF on subsequent lysosomal targeting of the EGFR was evaluated by immunoblotting. A massive translocation of the EGFR to the endosomal compartment was observed in response to ligand injection coincident with its tyrosine phosphorylation and receptor recruitment of the tyrosine-phosphorylated adaptor protein Shc. During cell-free endosome-lysosome fusion, a time-dependent increase in the content of the EGFR and the two 55- and 46-kDa Shc isoforms was observed in lysosomal fractions with a time course superimposable with the lysosomal transfer of the ligand; no transfer of the 66-kDa Shc isoform was detected. The relationship between EGFR tyrosine kinase activity and EGFR sorting in endosomes investigated by immunoblot studies with anti-phosphotyrosine antibodies revealed that endosomal dephosphorylation of EGFR and Shc preceded lysosomal transfer. These results support the view that a lysosomal targeting machinery distinct from the endosomal receptor kinase activity, such as the recruitment of the signaling molecule Shc, may regulate this sorting event in the endosome.

【 授权许可】

Unknown