Cell Surface Annexin II Is a High Affinity Receptor for the Alternatively Spliced Segment of Tenascin-C

• Published in: The Journal of cell biology 1994-07, Vol.126 (2), p.539-548
• Main Authors: Chung, Chang Y., Erickson, H. P.
• Format: Article
• Language: English
• Subjects: Alternative Splicing; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Annexin A2 - chemistry; Annexin A2 - isolation & purification; Annexin A2 - metabolism; Annexins; Antibodies; Binding Sites; Biochemistry; Biological and medical sciences; Cattle; Cell adhesion; Cell Adhesion Molecules, Neuronal - genetics; Cell Adhesion Molecules, Neuronal - metabolism; Cell lines; Cells; Cells, Cultured; Cellular biology; Endothelial cells; Endothelium - cytology; Extracellular Matrix Proteins - genetics; Extracellular Matrix Proteins - metabolism; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Glioma; Humans; Kinetics; Lung - chemistry; Molecular Sequence Data; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Neurons; Proteins; Radioligand Assay; Receptors; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - isolation & purification; Receptors, Cell Surface - metabolism; Recombinant Proteins - metabolism; Sequence Analysis; Tenascin; Tumor Cells, Cultured; Ungulates
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.126.2.539

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× 105 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.