CELL-BINDING DOMAIN OF ENDOTHELIAL-CELL THROMBOSPONDIN - LOCALIZATION TO THE 70-KDA CORE FRAGMENT AND DETERMINATION OF BINDING CHARACTERISTICS

Endothelial and other cell types synthesize thrombospondin (TSP), secrete into their culture medium, and incorporate it into their extracellular matrix. TSP is a large multifunctional protein capable of specific interactions with other matrix components, as well as with cell surfaces, and can modulate cell adhesion to the extracellular matrix. With the aim of understanding the mechanism by which TSP exerts its effect on cell adhesion, we studied the interaction of endothelial cell TSP (EC-TSP) with three different cell types: endothelial cells, granulosa cells, and myoblasts. We find that endothelial cells specifically bind radiolabeled EC-TSP with a K(d) of 25 nM, and the number of binding sites is 2.6 x 10(6)/cell. Binding is not inhibitable by the cell-adhesion peptide GRGDS, indicating that the cell-binding site of EC-TSP is not in the RGD-containing domain. Localization of the cell-binding site was achieved by testing two chymotryptic fragments representing different regions of the TSP molecule, the 70-kDa core fragment and the 27-kDa N-terminal fragment, for their ability to bind to the cells. Cell-binding capacity was demonstrated by the 70-kDa fragment but not by the 27-kDa fragment. Binding of both intact [I-125]EC-TSP and of the I-125-labeled 70-kDa fragment was inhibited by unlabeled TSP, heparin, fibronectin (FN), monoclonal anti-TSP antibody directed against the 70-kDa fragment (B7-3), and by full serum, but not by heparin-absorbed serum or the cell-adhesion peptide GRGDS. The 70-kDa fragment binds to endothelial cells with a K(d) of 47 nM, and the number of binding sites is 5.0 x 10(6)/cell. Rat granulosa cells, which synthesize and secrete TSP, can also bind both intact EC-TSP and the 70-kDa fragment with binding features very similar for the two ligands. That is, the binding of both ligands is inhibited to a similar extent by unlabeled EC-TSP, by the monoclonal antibody B7-3, and by FN, but not by the GRGDS peptide. In the case of granulosa cells, heparin did not inhibit binding of EC-TSP or the 70-kDa fragment. Granulosa cells bind intact EC-TSP and the 70-kDa fragment with dissociation constants of 1.8 and 38 nM, respectively, indicating a significant reduction in affinity for TSP upon fragmentation. The number of binding sites on granulosa cells is 1.7 x 10(5)/cell for the intact protein and 2.5 x 10(5)/cell for the 70-kDa fragment. In contrast to the TSP-synthesizing endothelial and granulosa cells, primary rat myoblasts, which do not synthesize TSP, fail to bind EC-TSP. Our observations suggest that (a) TSP synthesis correlates with the ability of a given cell type to bind TSP and (b) EC-TSP binding to the TSP-synthesizing cell is mediated by a domain contained in the 70-kDa core fragment. The mechanism of binding, however, may vary from one cell type to another, as suggested by the differential effect of heparin on TSP binding by endothelial and granulosa cells.