Disulfides modulate RGD-inhibitable cell adhesive activity of thrombospondin.
Thrombospondin (TSP) contains the Arg-Gly-Asp (RGD) sequence that is thought to be important for cell adhesion mediated by several cell-surface integrin receptors. The RGD sequence is located in the type 3 repeat region of TSP that has multiple Ca2+ binding sites and is subject to a complex intramolecular thiol-disulfide isomerization. TSP that we isolated from thrombin-activated human platelets using buffers containing 0.1 mM Ca2+, in which Cys974 is the major labeled cysteine, did not have RGD-inhibitable adhesive activity. However, one of our preparations of TSP and TSP purified following alternative procedures using greater than or equal to 0.3 mM Ca2+ did have RGD-inhibitable adhesive activity. Reduction of TSP with DTT, either before or after adsorption to surfaces, enhanced its adhesive activity. Reduced TSP supported robust cell spreading when coated at concentrations as low as 1 micrograms/ml, whereas "adhesive" TSP not treated with DTT was active at coating concentration of greater than 20 micrograms/ml and supported only modest cell spreading. Lower DTT concentrations were required for enhancement of the adhesive activity of TSP if Ca2+ was chelated with EDTA. Cellular adhesion to DTT-treated TSP was inhibited by RGD-containing peptide and by mAb to a functional site of the alpha v beta 3 integrin. Cell blots of reduced proteolytic fragments of TSP localized the adhesive activity to the RGD-containing type 3 repeat region. These results suggest a novel mechanism for regulation of integrin-ligand interactions in which the ligand can isomerize between inactive and active forms.
Sun
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Department of Medicine, University of Wisconsin, Madison 53706.
Skorstengaard
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Mosher
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002448:Cell Adhesion; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular; D006023:Glycoproteins / Q000378:metabolism; D006868:Hydrolysis; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000276:immunology / Q000378:metabolism; D019030:Receptors, Vitronectin; D019699:Thrombospondins; D019096:Vitronectin
D000911:Antibodies, Monoclonal; D004220:Disulfides; D006023:Glycoproteins; D009842:Oligopeptides; D010980:Platelet Membrane Glycoproteins; D011971:Receptors, Immunologic; D019030:Receptors, Vitronectin; D019699:Thrombospondins; D019096:Vitronectin; C047981:arginyl-glycyl-aspartic acid; D003545:Cysteine