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Interaction of Shc with adaptor protein adaptins.
J BIOL CHEM
5265 - 5269.
The role of Shc as a substrate of receptors for growth factors and cytokines is well established. To gain further insight into the function of Shc in signal transduction, we used an affinity method to identify potential Shc-binding proteins, Incubation of bovine brain lysates with a glutathione S-transferase (GST)-Shc fusion protein immobilized on glutathione-Sepharose beads resulted in the binding of cellular proteins of similar to 115, 110, and 100 kDa as well as those of 50 and 17 kDa. Amino acid sequencing of tryptic peptides revealed that the 100-kDa protein was almost identical to beta-adaptin and that the 110- and 115-kDa proteins were almost identical to alpha(A)-adaptin. Using immunoblot analysis, anti-alpha-adaptin antibody recognized several proteins of 100 similar to 115 kDa, and anti-beta-adaptin antibody recognized a 100-kDa protein, suggesting that alpha(A)-, alpha(C)-, and beta-adaptins are bound to the GST-Shc fusion protein. Immunoblot analysis with anti-alpha-adaptin antibody revealed that alpha-adaptin was coimmunoprecipitated with Shc from PC12, KB, and COS cell lysates, suggesting a specific interaction of Shc and adaptins in intact cells. A binding study using mutant GST-Shc fusion proteins revealed that the collagen homologous region (amino acids 233-377) of Shc was required for adaptin binding. Conversely, the collagen homologous region of Shc inhibited the binding of adaptins to GST-Shc. In addition, adaptin was able to bind mutant fusion proteins containing amino acids 233-369, 233-355, 346-369, and 346-355 of Shc, but failed to bind a mutant containing amino acids 233-345, suggesting that amino acids 346-355 (RDLFDMKPFE) in the collagen homologous region of Shc are required for adaptin binding. Thus, this study indicates the specific interaction of Shc with alpha- and beta-adaptin components of plasma membrane adaptor proteins that are thought to be involved in receptor endocytosis.
|Title:||Interaction of Shc with adaptor protein adaptins|
|Open access status:||An open access publication|
|Keywords:||COATED VESICLE PROTEINS, SIGNAL TRANSDUCTION, TYROSINE KINASES, CYTOPLASMIC DOMAIN, INVITRO BINDING, RECEPTOR, CLATHRIN, ASSOCIATION, INTERNALIZATION, IDENTIFICATION|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology|
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