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 it 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 Kd 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 [125I]EC-TSP and of the 125I-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 Kd of 47 nM, and the number of binding sites is 5.0 x 10(6)/cell.(ABSTRACT TRUNCATED AT 250 WORDS)
Dardik
R
R
Department of Polymer Research, Weizmann Institute of Science, Rehovot, Israel.
Lahav
J
J
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D018955:CD36 Antigens; D002417:Cattle; D002448:Cell Adhesion; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005260:Female; D006107:Granulosa Cells / Q000166:cytology / Q000378:metabolism; D066298:In Vitro Techniques; D008970:Molecular Weight; D009132:Muscles / Q000166:cytology / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D016024:Receptors, Cytoadhesin / Q000378:metabolism; D019699:Thrombospondins
D018955:CD36 Antigens; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D016024:Receptors, Cytoadhesin; D019699:Thrombospondins