Journal of Biological Chemistry
Volume 270, Issue 44, 3 November 1995, Pages 26270-26277
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Protein Chemistry and Structure
Regulation of Integrin α5β1-Fibronectin Interactions by Divalent Cations: EVIDENCE FOR DISTINCT CLASSES OF BINDING SITES FOR Mn2+, Mg2+, AND Ca2+(∗)

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Integrin-ligand interactions are known to be dependent on divalent cations, although the precise role of cations in ligand binding is still unclear. Using the interaction between α5β1 and fibronectin as a model system, we have performed a comprehensive analysis of the effects of Mn2+, Mg2+, and Ca2+ on ligand binding. Each cation had distinct effects on the ligand-binding capacity of α5β1: Mn2+ promoted high levels of ligand binding, Mg2+ promoted low levels of binding, and Ca2+ failed to support binding. Studies of the effects of different combinations of cations on ligand binding indicated that the cation-binding sites within α5β1 are not all identical, or of broad specificity, but instead each site shows a distinct preference for one or more cations. Ca2+ strongly inhibited Mn2+-supported ligand binding, but this inhibition was noncompetitive, suggesting that Ca2+ recognizes different cation-binding sites to Mn2+. In contrast, Ca2+ acted as a direct competitive inhibitor of Mg2+-supported ligand binding, implying that Ca2+ can displace Mg2+ from the integrin. However, low concentrations of Ca2+ greatly increased the apparent affinity of Mg2+ for its binding site, suggesting the existence of a distinct high affinity Ca2+-binding site. Taken together, our results imply that the ligand-binding capacity of α5β1 can be regulated in a complex manner through separate classes of binding sites for Mn2+, Mg2+, and Ca2+.

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This work was supported by grants from the Wellcome Trust (to M. J. H. and A. P. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.