Re-localization of activated EGF receptor and its signal transducers to multivesicular compartments downstream of early endosomes in response to EGF

The rapid internalization of receptor tyrosine kinases after ligand binding has been assumed to be a negative modulation of signal transduction, Howev er, accumulating data indicate that signal transduction from internalized c ell surface receptors also occurs from endosomes, We show that a substantia l fraction of tyrosine-phosphorylated epidermal growth factor receptor (EGF R) and She, Grb2 and Cb1 after internalization relocates from early endosom es to compartments which are negative for the early endosomes, recycling ve sicle markers EEA1 and transferrin in EGF-stimulated cells. These compartme nts contained the multivesicular body and late endosome marker CD63, and th e late endosome and lysosome marker LAMP-1, and showed a multivesicular mor phology, Subcellular fractionation revealed that activated EGFR, adaptor pr oteins and activated ERK 1 and 2 were located in EEA1-negative and LAMP-1-p ositive fractions. Co-immunoprecipitations showed EGFR in complex with both She, Grb2 and Cb1 Treatment with the weak base chloroquine or inhibitors o f lysosomal enzymes after EGF stimulation induced an accumulation of tyrosi ne-phosphorylated EGFR and She in EEA1-negative and CD63-positive vesicles after a 120-min chase period. This was accompanied by a sustained activatio n of ERK 1 and 2, These results suggest that EGFR signaling is not spatiall y restricted to the plasma membrane, primary vesicles and early endosomes, but is continuing from late endocytic trafficking organelles maturing from early endosomes.