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 sever al cell lines using a radio-ligand binding assay. Specific binding was demonstrated to U-251MG human glioma cells and to a line of bovine ao rtic endothelial cells, but hamster fibroblasts showed no specific bin ding. Recombinant proteins corresponding to specific domains of TN-C w ere 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 re ceptor was annexin II. Annexin II is well characterized as a cytoplasm ic protein, so it was surprising to find it as a presumably extracellu lar receptor for TN-C. To confirm that it was the 35-kD receptor, we o btained purified annexin II and demonstrated its binding to TNfnA-D an d TN-C at nM concentrations. Antibodies to annexin II prominently stai ned the external surface of live endothelial cells and blocked the bin ding 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 cellul ar responses to soluble TN-C in the extracellular matrix