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Shc mediates ligand-induced internalization of epidermal growth factor receptors
K Sakaguchi1, Y Okabayashi, M Kasuga
1Second Department of Internal Medicine, Kobe University School of Medicine, Kobe, 650-0017, Japan.
Abstract:
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.
Insights
Shc protein facilitates epidermal growth factor (EGF) receptor endocytosis by interacting with the AP2 complex. This interaction is crucial for EGF receptor internalization and cellular response.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Shc proteins are known to interact with components of the AP2 complex.
- Adaptins, including alpha-adaptin, are key players in the formation of coated pits during endocytosis.
- Epidermal growth factor (EGF) receptors are internalized upon ligand binding, a process mediated by endocytosis.
Purpose of the Study:
- To investigate the physiological role of the Shc-adaptin interaction in EGF receptor endocytosis.
- To elucidate the mechanism by which Shc influences EGF receptor internalization.
Main Methods:
- In vitro peptide binding assays to assess Shc-alpha-adaptin interaction.
- Use of adenovirus vectors to express wild-type and mutant Shc proteins in cells.
- Co-precipitation assays to study protein-protein interactions in intact cells.
- Internalization assays to quantify EGF receptor uptake.
- Immunofluorescence microscopy to visualize protein localization and endocytosis patterns.
Main Results:
- Alpha-adaptin binds to wild-type Shc in an EGF-dependent manner, with reduced binding to a mutant Shc (Shc FA).
- EGF stimulation enhances the association of alpha-adaptin with Shc and promotes the coprecipitation of alpha-adaptin with EGF receptors.
- Overexpression of Shc FA reduces EGF internalization, while wild-type Shc enhances punctate staining indicative of endocytosis.
- Shc facilitates the formation of Shc-AP2 complexes and their association with EGF receptors, promoting endocytosis.
Conclusions:
- Shc plays a significant role in ligand-induced endocytosis of EGF receptors.
- The interaction between Shc and the AP2 complex is critical for efficient EGF receptor internalization.
- EGF signaling modulates Shc-AP2 complex formation and its subsequent association with EGF receptors, driving endocytosis.