In vitro endosome-lysosome transfer of dephosphorylated EGF receptor and Shc in rat liver.
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 [(125)I]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 [(125)I]EGF in this compartment decreased by 39%, with an increase in [(125)I]EGF in lysosomes. No transfer of [(125)I]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.
Authier
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F
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. francois.authier@cep.u-psud.fr
Chauvet
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G
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D015151:Immunoblotting; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013696:Temperature; D013997:Time Factors
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D004815:Epidermal Growth Factor; D066246:ErbB Receptors