Interaction of type IV collagen with the isolated integrins α1β1 and α2β1

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Abstract
The triple-helical cyanogen-bromide-derived fragment CB3[IV] of collagen IV, located 100 nm from the N-terminus of the molecule, contains the binding sites for the integrins α1β1 and α2β1. To investigate the interaction of these integrins and collagen IV, we performed solid-phase and inhibition assays using as receptor isolated α1β1 and α2β1. The ligands used were the binding-site-bearing trimeric peptide CB3[IV] and its shorter tryptic fragments F1–F4. Using titration curves, in which the binding of soluble receptors to coated ligands and the binding of soluble ligands to coated receptors were analyzed, the binding sites for α1β1 and α2β1 were in different but adjacent areas of CB3[IV]. Triple-helical conformation and distinct primary structures were required for the interaction. Dissociation constants (Ka), for the affinity of integrins for collagen IV, were determined in the 1-nM range in the presence of Mn2+ and Mg2+. In the absence of Mn2+, the Kd values indicated a 30–60-fold decrease in the affinities, which for α2β1 was further reduced by adding Ca2+. In the presence of Ca2+ and Mg2+ the affinity of collagen IV for α1β1 was four-times higher than for α2β1.

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