Wang Z, Tung P S, Moran M F
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Cell Growth Differ. 1996 Jan;7(1):123-33.
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.
表皮生长因子（EGF）对细胞的刺激通过受体介导的内吞作用诱导EGF受体下调。同时，活化的自磷酸化EGF受体与各种细胞内信号蛋白的酪氨酸磷酸化结合结构域结合，包括那些参与调节与质膜相关的小G蛋白p21 Ras的蛋白。这些EGF受体结合蛋白中包括分子量为120,000的Ras GTP酶激活蛋白（GAP）。在此我们通过亚细胞分级分离、免疫沉淀和蛋白质印迹、间接免疫荧光以及双免疫金标记和电子显微镜显示，在对MDCK细胞进行EGF刺激后，GAP首先定位于质膜，随后与EGF受体一起内化。在所检测的各种亚细胞膜分级分离物中，EGF受体和GAP在物理上相互关联。EGF刺激还诱导GAP与p190 Rho-GAP结合，并且GAP-p190复合物从膜分级分离物中共免疫沉淀。SH2结构域募集到质膜上活化的受体酪氨酸激酶无疑是控制信号蛋白亚细胞定位和底物可及性的重要机制。我们的结果表明，信号蛋白调节的另一个层面可能是由于它们与内体的结合而发生。
