Endosomal signalling of epidermal growth factor receptors contributes to EGF-stimulated cell cycle progression in primary hepatocytes.

Agonist-induced internalisation of receptors may lead to the formation of signalling endosomes. There is little evidence relating to whether this occurs to native receptors in non-transformed cells, and no previous studies asking whether this endosomal signalling can promote cell cycle progression in non-transformed cells. We investigated the hypothesis that in primary hepatocytes clathrin-dependent epidermal growth factor (EGF)-induced internalisation of the EGF receptor leads to signalling from endosomal EGF-EGF receptor complexes which may support EGF-stimulated cell cycle progression. We used EGF-stimulation of rat hepatocytes followed by confocal microscopy, and Western blots for phosphoproteins. [(3)H]thymidine incorporation into DNA was used as a indicator of progression to S-phase. Confocal microscopy demonstrated co-internalisation of EGF, EGF receptors and transferrin into endosomes. Internalisation of EGF/EGF receptor/transferrin was blocked by expression of dominant-negative dynamin, but not by the tyrosine kinase inhibitor AG 1478. Dominant-negative dynamin expression reduced EGF-stimulated extracellular signal-related kinase and Akt signalling, but increased tyrosine phosphorylated EGF receptor. EGF-stimulated cell cycle progression requires stimulation of EGF receptors during an initial period (e.g. 1h) and also later during a 24h incubation. EGF receptor internalisation in the presence of AG 1478 followed by removal of the inhibitor resulted in signalling from internalised EGF receptors that is sufficient for the initial stimulation to provide progression to S-phase of the cell cycle. These observations on hepatocytes characterise, for the first time in non-transformed cells, endosomal signalling from internalised EGF receptors, and provide evidence that this endosomal signalling may support the early phase of EGF-stimulated cell cycle progression.