Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane

摘要

The role of endocytosis in the control of EGF receptor (EGFR) acti vation and cell signaling was explored by using mouse fibroblasts in which dynamin was conditionally depleted. Dynamin is a GTPase shown to play an important role in the control clathrin mediated endocytosis of EGFR and other cell surface receptors. In this report, we demonstrate that EGF binding activity and the display of high and low affinity EGFRs on the cell surface are not affected by dy namin depletion. By contrast, dynamin depletion leads to a strong inhibition of EGFR endocytosis, robust enhancement of EGFR au tophosphorylation and ubiquitination, and slower kinetics of EGFR degradation. Surprisingly, MAPK stimulation induced by either low or high EGF concentrations is not affected by dynamin depletion. While a similar initial Akt response is detected in control or dyna min depleted fibroblasts, a somewhat more sustained Akt stimula tion is detected in the dynamin depleted cells. These experiments demonstrate that dynamin-mediated endocytosis leads to attenua tion of EGFR activation and degradation and that stimulation of the MAPK response and Akt activation are primarily mediated by activated EGFR located in the plasma membrane.