Shc mediates ligand-induced internalization of epidermal growth factor receptors.
In order to clarify the physiological relevance of the interaction between Shc and adaptins, components of plasma membrane-coated pit adaptor complex AP2, we investigated the role of Shc in ligand-induced endocytosis of epidermal growth factor (EGF) receptors. In vitro peptide binding assay showed that alpha-adaptin bound to the wild-type peptide corresponding to amino acids 346-355 of Shc, RDLFDMKPFE, but not to the mutant peptide in which both phenylalanines at 349 and 354 were substituted for alanines (FA). Using adenovirus vectors carrying a herpes simplex virus epitope-tagged 52-kDa wild-type Shc and Shc FA, we examined the interaction between Shc, AP2, and EGF receptors in intact cells. Alpha-adaptin bound to wild-type Shc in an EGF-dependent manner, whereas EGF-dependent association of alpha-adaptin with Shc FA was markedly reduced. In addition, EGF increased the amount of alpha-adaptin coprecipitated with EGF receptors in cells expressing wild-type Shc but not Shc FA. These results suggest that EGF stimulates Shc-AP2 complex formation and association of Shc-AP2 complexes with EGF receptors. Internalization assay showed that (125)I-EGF internalization was reduced in cells overexpressing Shc FA. Immunofluorescence study showed that punctate staining along the plasma membrane border as well as punctate pattern characteristic of cytoplasmic vesicles near the plasma membrane was enhanced in cells expressing wild-type Shc. These results suggest, therefore, the implication of Shc in ligand-induced endocytosis of EGF receptors in intact cells.
Sakaguchi
K
K
Second Department of Internal Medicine, Kobe University School of Medicine, Kobe, 650-0017, Japan.
Okabayashi
Y
Y
Kasuga
M
M
D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D007457:Iodine Radioisotopes; D008024:Ligands; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011506:Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1
D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D007457:Iodine Radioisotopes; D008024:Ligands; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010446:Peptide Fragments; D011506:Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D004815:Epidermal Growth Factor; D066246:ErbB Receptors