title: Endocytosis separates EGF receptors from endogenous fluorescently labeled HRas and diminishes receptor signaling to MAP kinases in endosomes.
abstract: Signaling from epidermal growth factor receptor (EGFR) to extracellular-stimuli-regulated protein kinase 1/2 (ERK1/2) is proposed to be transduced not only from the cell surface but also from endosomes, although the role of endocytosis in this signaling pathway is controversial. Ras is the only membrane-anchored component in the EGFR-ERK signaling axis, and therefore, its location determines intracellular sites of downstream signaling. Hence, we labeled endogenous H-Ras (HRas) with mVenus fluorescent protein using gene editing in HeLa cells. mVenus-HRas was primarily located at the plasma membrane, and in small amounts in tubular recycling endosomes and associated vesicles. EGF stimulation resulted in fast but transient activation of mVenus-HRas. Although EGF:EGFR complexes were rapidly accumulated in endosomes together with the Grb2 adaptor, very little, if any, mVenus-HRas was detected in these endosomes. Interestingly, the activities of MEK1/2 and ERK1/2 remained high beyond the point of the physical separation of HRas from EGF:EGFR complexes and down-regulation of Ras activity. Paradoxically, this sustained MEK1/2 and ERK1/2 activation was dependent on the active EGFR kinase. Cell surface biotinylation and selective inactivation of surface EGFRs suggested that a small fraction of active EGFRs remaining in the plasma membrane is responsible for continuous signaling to MEK1/2 and ERK1/2. We propose that, under physiological conditions of cell stimulation, EGFR endocytosis serves to spatially separate EGFR-Grb2 complexes and Ras, thus terminating Ras-mediated signaling. However, sustained minimal activation of Ras by a small pool of active EGFRs in the plasma membrane is sufficient for extending MEK1/2 and ERK1/2 activities.
authors: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
authors: Itziar
authors: I
authors: Pinilla-Macua
authors: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
authors: Simon C
authors: SC
authors: Watkins
authors: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 sorkin@pitt.edu.
authors: Alexander
authors: A
authors: Sorkin
mesh_terms: D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*
chemical_list: D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D066246:ErbB Receptors; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)
keywords: EGF receptor; Ras; endocytosis