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Using insect cells, we expressed large quantities of soluble human integrin α₃β₁ ectodomain heterodimers, in which cytoplasmic and transmembrane domains were replaced by Fos and Jun dimerization motifs. In direct ligand binding assays, soluble α₃β₁ specifically bound to laminin-5 and laminin-10, but not to laminin-1, laminin-2, fibronectin, various collagens, nidogen, thrombospondin, or complement factors C3 and C3b. Soluble α₃β₁ integrin also bound to invasin, a bacterial surface protein, that mediates entry of Yersinia species into the eukaryotic host cell. Invasin completely displaced laminin-5 from the α₃β₁ integrin, suggesting sterically overlapping or identical binding sites. In the presence of 2 mM Mg²⁺, α₃β₁'s binding affinity for invasin (K_d = 3.1 nM) was substantially greater than its affinity for laminin-5 (K_d > 600 nM). Upon addition of 1 mM Mn²⁺, or activating antibody 9EG7, binding affinity for both …