Interaction of Shc with adaptor protein adaptins.
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 approximately 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 alphaA-adaptin. Using immunoblot analysis, anti-alpha-adaptin antibody recognized several proteins of 100 approximately 115 kDa, and anti-beta-adaptin antibody recognized a 100-kDa protein, suggesting that alphaA-, alphaC-, 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.
Okabayashi
Y
Y
Second Department of Internal Medicine, Kobe University School of Medicine, Kobe 650, Japan.
Sugimoto
Y
Y
Totty
N F
NF
Hsuan
J
J
Kido
Y
Y
Sakaguchi
K
K
Gout
I
I
Waterfield
M D
MD
Kasuga
M
M
D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033966:Adaptor Protein Complex beta Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005910:Glioma; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured; D018909:src Homology Domains
C439123:AP2A1 protein, Bos taurus; D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033966:Adaptor Protein Complex beta Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000906:Antibodies; D017931:DNA Primers; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C439124:adaptor protein complex 2, alpha 2 subunit