ERK2 SIGNALING FROM INTERNALIZED EPIDERMAL GROWTH-FACTOR RECEPTOR IN BROKEN A431 CELLS

Activation of extracellular signal-regulated kinase 2 (ERK2) signallin g from epidermal growth factor receptors (EGFRs) is widely assumed to originate in the plasma membrane. Using an in vitro assay, we investig ated whether EGF/EGFR complexes internalised by endocytosis in A431 ce lls can initiate signalling in the ERE;? pathway. At 0 degrees C, bind ing of EGF induced tyrosine phosphorlyation of EGFR and, when the cell s were subsequently broken by scraping and warmed in the presence of e xogenous cytosol, activation of ERK2 occurred. At 0 degrees C, washes with pH 4.5 media reversed EGF binding, tyrosine phosphorylation and E RK-2 activation in exogenous cytosol, providing conditions in which si gnalling from the cell surface and internalised EGFRs could he disting uished. When cells containing internalised EGF/EGFR complexes were fir st washed in low pH media at 0 degrees C and then broken and incubated in exogenous cytosol, substantial activation of ERK2 occurred. This a ctivation reached a maximum after a 5-min internalisation and was almo st completely prevented by incubation in 0.45 M sucrose, a known inhib itor of receptor-mediated endocytosis. These data are consistent with activation of the ERK2 signalling pathway by internalised EGRFs situat ed in endosomal compartments. Our observation that EGFR tyrosine depho sphorylation is incomplete above pH 5.5 suggests that signalling is in itiated in early endosomes. (C) 1998 Elsevier Science Inc.