Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3.
Modification of the cytoplasmic tails of the integrin alpha(IIb)beta(3) plays an important role in the signal transduction in platelets. We searched for proteins that bind to the alpha(IIb) cytoplasmic tail using the yeast two-hybrid assay with a cDNA library of the megakaryocyte-derived cell line and identified a protein, ancient ubiquitous ... protein 1 (Aup1), that is ubiquitously expressed in human cells. Observation of UT7/TPO cells expressing a red fluorescent protein-tagged Aup1 indicated its localization in the cytoplasm. Immunoprecipitation of UT7/TPO cells by an antibody for Aup1 revealed that approximately 40% of alpha(IIb) is complexed with Aup1. Binding study with an alpha(IIb) cytoplasmic tail peptide and glutathione S-transferase-Aup1 fusion protein revealed a low affinity (K(d) = 90 microm). Subsequent yeast two-hybrid assay indicated binding of Aup1 to cytoplasmic tails of other integrin alpha subunits. Binding study with the purified Aup1 and various glutathione S-transferase-alpha(IIb) cytoplasmic tail peptides revealed specific binding of Aup1 to the membrane-proximal sequence (KVGFFKR) that is conserved among the integrin alpha subunits and plays a crucial role in the alpha(IIb)beta(3) inside-out signaling. As Aup1 possesses domains related to signal transduction, these results suggest involvement of Aup1 in the integrin signaling.
Mesh Terms:
Amino Acid Sequence, Blotting, Northern, Carrier Proteins, Cell Membrane, Cytoplasm, DNA, Complementary, Dose-Response Relationship, Drug, Gene Library, Glutathione Transferase, Humans, Integrins, Jurkat Cells, Kinetics, Microscopy, Fluorescence, Molecular Sequence Data, Peptides, Platelet Glycoprotein GPIIb-IIIa Complex, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, RNA, Messenger, Recombinant Fusion Proteins, Signal Transduction, Tumor Cells, Cultured, Two-Hybrid System Techniques
Amino Acid Sequence, Blotting, Northern, Carrier Proteins, Cell Membrane, Cytoplasm, DNA, Complementary, Dose-Response Relationship, Drug, Gene Library, Glutathione Transferase, Humans, Integrins, Jurkat Cells, Kinetics, Microscopy, Fluorescence, Molecular Sequence Data, Peptides, Platelet Glycoprotein GPIIb-IIIa Complex, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, RNA, Messenger, Recombinant Fusion Proteins, Signal Transduction, Tumor Cells, Cultured, Two-Hybrid System Techniques
J. Biol. Chem.
Date: Aug. 09, 2002
PubMed ID: 12042322
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