Association of p120 ras GAP with endocytic components and colocalization with epidermal growth factor (EGF) receptor in response to EGF stimulation.
Epidermal growth factor (EGF) stimulation of cells induces down-regulation of EGF receptors through receptor-mediated endocytosis. Simultaneously, activated autophosphorylated EGF receptors become associated with the pTyr-binding domains of various intracellular signaling proteins, including those implicated in the regulation of the plasma membrane-associated small G protein p21 Ras. Included among these EGF receptor-binding proteins is the M(r) 120,000 Ras GTPase-activating protein (GAP). Herein we show by subcellular fractionation, immunoprecipitation and Western blotting, indirect immunofluorescence, and double immunogold labeling and electron microscopy that, in response to EGF stimulation of MDCK cells, GAP becomes first plasma membrane localized and subsequently internalized with EGF receptors. In various subcellular membrane fractions examined, EGF receptor and GAP were physically associated. EGF stimulation also induced association of GAP with p190 Rho-GAP and the GAP-p190 complex coimmunoprecipitated from membrane fractions. Recruitment of SH2 domains to activated receptor tyrosine kinases at the plasma membrane is undoubtedly an important mechanism in controlling the subcellular localization and substrate accessibility of signaling proteins. Our results illustrate that another level of signaling protein regulation may occur by virtue of their association with endosomes.
Wang
Z
Z
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Tung
P S
PS
Moran
M F
MF
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002458:Cell Fractionation; D002460:Cell Line / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure; D002462:Cell Membrane / Q000648:ultrastructure; D004285:Dogs; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D020690:GTPase-Activating Proteins; D015151:Immunoblotting; D007150:Immunohistochemistry; D007686:Kidney Tubules, Distal / Q000166:cytology; D008854:Microscopy, Electron; D011233:Precipitin Tests; D011485:Protein Binding / Q000502:physiology; D011506:Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors