RECOMBINANT SOLUBLE HUMAN ALPHA(3)BETA(1) INTEGRIN - PURIFICATION, PROCESSING, REGULATION, AND SPECIFIC BINDING TO LAMININ-5 AND INVASIN INA MUTUALLY EXCLUSIVE MANNER

Using insect cells, we expressed large quantities of soluble human int egrin alpha(3)beta(1) ectodomain heterodimers, in which cytoplasmic an d transmembrane domains were replaced by Fos and Jun dimerization moti fs. In direct ligand binding assays, soluble alpha(3)beta(1) specifica lly bound to laminin-5 and laminin-10, but not to laminin-1, laminin-2 , fibronectin, various collagens, nidogen, thrombospondin, or compleme nt factors C3 and C3b. Soluble alpha(3)beta(1) integrin also bound to invasin, a bacterial surface protein, that mediates entry of Yersinia species into the eukaryotic host cell. Invasin completely displaced la minin-5 from the alpha(3)beta(1) integrin, suggesting sterically overl apping or identical binding sites. In the presence of 2 mM Mg2+, alpha (3)beta(1)'s binding affinity for invasin (K-d = 3.1 nM) was substanti ally greater than its affinity for laminin-5 (K-d > 600 nM). Upon addi tion of 1 mM Mn2+, or activating antibody 9EG7, binding affinity for b oth laminin-5 and invasin increased by about 10-fold, whereas the affi nity decreased upon addition of 2 mM Ca2+. Thus, functional regulation of the purified soluble integrin alpha(3)beta(1) ectodomain heterodim er resembles that of wild-type membrane-anchored beta(1) integrins. Th e integrin alpha(3) subunit was entirely cleaved into disulfide-linked heavy and light chains, at a newly defined cleavage site located C-te rminal of a tetrabasic RRRR motif. Within the alpha(3) light chain, al l potential N-glycosylation sites bear N-linked mannose-rich carbohydr ate chains, suggesting an important structural role of these sugar res idues in the stalk-like region of the integrin heterodimer. In conclus ion, studies of our recombinant alpha(3)beta(1) integrin have provided new insights into alpha(3)beta(1) structure, ligand binding function, specificity, and regulation.