Cell surface annexin II is a high affinity receptor for the alternatively spliced segment of tenascin-C.
We have investigated the binding of soluble tenascin-C (TN-C) to several cell lines using a radioligand binding assay. Specific binding was demonstrated to U-251MG human glioma cells and to a line of bovine aortic endothelial cells, but hamster fibroblasts showed no specific binding. Recombinant proteins corresponding to specific domains of TN-C were used to map the binding site(s) in TN-C. The alternatively spliced segment (TNfnA-D) inhibited the binding of native TN-C most strongly, and itself bound to glioma and endothelial cells. Scatchard analysis of TNfnA-D binding indicated 2-5 x 10(5) binding sites per cell, with an apparent 2 nM dissociation constant. The cell surface receptor for TNfnA-D was identified as a 35-kD protein on the basis of blot binding assays and affinity chromatography of membrane extracts on native TN-C and TNfnA-D columns. Protein sequencing indicated that this 35-kD receptor was annexin II. Annexin II is well characterized as a cytoplasmic protein, so it was surprising to find it as a presumably extracellular receptor for TN-C. To confirm that it was the 35-kD receptor, we obtained purified annexin II and demonstrated its binding to TNfnA-D and TN-C at nM concentrations. Antibodies to annexin II prominently stained the external surface of live endothelial cells and blocked the binding of TNfnA-D to the cells. Thus annexin II appears to be a receptor for the alternatively spliced segment of TN-C, and may mediate cellular responses to soluble TN-C in the extracellular matrix.
Chung
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Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
Erickson
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HP
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D017306:Annexin A2 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D015816:Cell Adhesion Molecules, Neuronal / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004727:Endothelium / Q000166:cytology; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D005910:Glioma; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000737:chemistry; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D019063:Tenascin; D014407:Tumor Cells, Cultured
D017306:Annexin A2; D015816:Cell Adhesion Molecules, Neuronal; D016326:Extracellular Matrix Proteins; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D019063:Tenascin