Recombinant Soluble Human α3β1Integrin: Purification, Processing, Regulation, and Specific Binding to Laminin-5 and Invasin in a Mutually Exclusive Manner

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Abstract
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 (Kd = 3.1 nM) was substantially greater than its affinity for laminin-5 (Kd > 600 nM). Upon addition of 1 mM Mn2+, or activating antibody 9EG7, binding affinity for both laminin-5 and invasin increased by about 10-fold, whereas the affinity decreased upon addition of 2 mM Ca2+. Thus, functional regulation of the purified soluble integrin α3β1 ectodomain heterodimer resembles that of wild-type membrane-anchored β1 integrins. The integrin α3 subunit was entirely cleaved into disulfide-linked heavy and light chains, at a newly defined cleavage site located C-terminal of a tetrabasic RRRR motif. Within the α3 light chain, all potential N-glycosylation sites bear N-linked mannose-rich carbohydrate chains, suggesting an important structural role of these sugar residues in the stalk-like region of the integrin heterodimer. In conclusion, studies of our recombinant α3β1 integrin have provided new insights into α3β1 structure, ligand binding function, specificity, and regulation.

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