Identification of an alpha(3)beta(1) integrin recognition sequence in thrombospondin-1

A synthetic peptide containing amino acid residues 190-201 of thrombospondi n-l (TSP1) promoted adhesion of MDA-MB-435 breast carcinoma cells when immo bilized and inhibited adhesion of the same cells to TSP1 when added in solu tion. Adhesion to this peptide was enhanced by a beta(1) integrin-activatin g antibody, Mn2+, and insulin-like growth factor I and was inhibited by an alpha(3)beta(1) integrin function-blocking antibody. The soluble peptide in hibited adhesion of cells to the immobilized TSP1 peptide or spreading on i ntact TSP1 but at the same concentrations did not inhibit attachment or spr eading on type Iv collagen or fibronectin, Substitution of several residues in the TSP1 peptide with Ala residues abolished or diminished the inhibito ry activity of the peptide in solution, but only substitution of Arg-198 co mpletely inactivated the adhesive activity of the immobilized peptide. The essential residues for activity of the peptide as a soluble inhibitor are A sn-196, Val-197, and Arg-198, but flanking residues enhance the inhibitory activity of this core sequence, either by altering the conformation of the active sequence or by interacting with the integrin, This functional sequen ce is conserved in all known mammalian TSP1 sequences and in TSP1 from Xeno pus laevis, The TSP1 peptide also inhibited adhesion of MDA-MB-435 cells to the laminin-l peptide GD6, which contains a potential integrin-recognition sequence Asn-Leu-Arg and is derived from a similar position in a pentraxin module. Adhesion studies using recombinant TSP1 fragments also localized b eta 1 integrin-dependent adhesion to residues 175-242 of this region, which contain the active sequence.