COMPETITIVE-BINDING OF VASCULAR CELL-ADHESION MOLECULE-1 AND THE HEPII IIICS DOMAIN OF FIBRONECTIN TO THE INTEGRIN ALPHA-4-BETA-1/

The integrin receptor alpha 4 beta 1 binds to two different ligands, t he extracellular matrix glycoprotein fibronectin and the endothelial c ell surface protein vascular cell adhesion molecule-1 (VCAM-1). Using probes derived from each ligand and a variety of cell adhesion and lig and-receptor binding assays, we have investigated the relationship bet ween the mechanisms of fibronectin and VCAM-1 interaction with alpha 4 beta 1. CS1 peptide, which represents the dominant active site from t he HepII/IIICS recognition domain in fibronectin, was found to inhibit VCAM-1-dependent adhesion in three different assays: MOLT-4 T lymphob lastic leukaemia cell attachment to immobilized recombinant soluble VC AM-1 (rsVCAM-1), MOLT-4 cell attachment to monolayers of VCAM-1-transf ected COS-1 cells, and A375-SM melanoma cell spreading on immobilized rsVCAM-1. Half-maximal inhibition required CS1 concentrations of 1.7-3 .0 mg/ml, some 3-7-fold higher than that needed to autoinhibit adhesio n to CS1-IgG conjugate. Using a more sensitive solid phase receptor-li gand binding assay, CS1 was found to be a potent inhibitor of the bind ing of rsVCAM-1 to alpha 4 beta 1 (half maximal inhibition at 13 mu g/ ml). In agreement with cell-based assays, severalfold lower concentrat ions of CS1 were required to inhibit binding of recombinant HepII/IIIC S region of fibronectin (half-maximal inhibition at 3 mu g/ml). VCAM-1 -alpha 4 beta 1 binding was blocked not only by CS1 peptide but also b y the recombinant HepII/IIICS region of fibronectin. Kinetic analysis of CS1 inhibition of VCAM-1 binding revealed that it was directly comp etitive in nature, indicating that VCAM-1 and fibronectin recognize ei ther identical or spatially overlapping binding sites on alpha 4 beta 1. The implications of these results for the future design of VCAM-1 a ntagonists are discussed.